We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists

4,100+

Open access books available

116,000+

International authors and editors

120M+

Downloads

Our authors are among the

Top 1%

most cited scientists

12.2%

Contributors from top 500 universities

Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI)

## Interested in publishing with us? Contact book.department@intechopen.com

Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com

## **Meet the editor**

Dr Christian Joseph R. Cumagun is a professor of plant pathology at the Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños (UPLB). He obtained his BSc in Agriculture and MSc in Plant Pathology from UPLB, and his Doctorate in Agricultural Sciences (magna cum laude) from the University of Hohenheim, Germany. Dr Cumagun received

a pre-doctoral fellowship at the University of Copenhagen, Denmark and postdoctoral fellowship at Kobe University, Japan. He has published more than 30 articles in national and international peer-reviewed journals. Among his awards are: The Outstanding Young Men (TOYM) in Agricultural Sciences, from the Junior Chamber International, Philippines, and the Outstanding Young Scientist (OYS), from the National Academy of Science and Technology (NAST), Philippines. He is currently appointed a Young Affiliate of The Academy of Sciences for the Developing World (TWAS), based in Trieste, Italy.

Contents

**Preface IX** 

Chapter 1 **Taxonomic Review of and Development of a Lucid** 

M. Mahamuda Begum, Teresita U. Dalisay

Chapter 2 **General Description of** *Rhizoctonia* **Species Complex 41** 

Chapter 3 **Phytobacterial Type VI Secretion System – Gene Distribution, Phylogeny, Structure and Biological Functions 53**  Panagiotis F. Sarris, Emmanouil A. Trantas, Nicholas Skandalis, Anastasia P. Tampakaki, Maria Kapanidou, Michael Kokkinidis,

**Effector Proteins in** *Magnaporthe oryzae* **117** 

**A Potential Target for Disease Control 131** 

**Pathogenic Fungi and Fungicide Resistance 151**  Nieves Capote, Ana María Pastrana, Ana Aguado

Kenichi Ikeda, Kanako Inoue, Hiroko Kitagawa, Hiroko Meguro,

and Christian Joseph R. Cumagun

Genhua Yang and Chengyun Li

and Nickolas J. Panopoulos

Jing Yang and Chengyun Li

Saki Shimoi and Pyoyun Park

Chapter 7 **Molecular Tools for Detection of Plant** 

and Paloma Sánchez-Torres

**Responses in Cut Flowers 85** 

Chapter 5 **Functional Identification of Genes Encoding** 

Chapter 6 **The Role of the Extracellular Matrix (ECM) in Phytopathogenic Fungi:** 

Chapter 4 **Novel Elicitors Induce Defense** 

Anastasios I. Darras

**Key for Philippine Cercosporoids and Related Fungi 1** 

### Contents

#### **Preface** XI


X Contents



### Preface

Plant pathology is an applied science which deals with the nature, causes and control of plant diseases in agriculture and forestry. The vital role of plant pathology in attaining food security and food safety for the world cannot be overemphasized.

The book begins with taxonomy of fungal pathogens using Lucid key, a multi-access computer based key for identification for Cercosporoids and a comprehensive review of *Rhizoctonia solani* anastomosis groups and subgroups in Chapters 1 and 2 respectively. Correct identification of the disease causing agent is a prerequisite to any successful plant disease management.

Recent advances in molecular genetics and biology have provided clues as to how plant pathogens act as elicitors to induce defence reactions on the plant. The phytobacterial type VI secretion system, specific only for Gram negative bacteria is a known virulence factor of plant pathogenic bacteria. For an environmentally sound management strategy, novel elicitors from *Botrytis cinerea* have shown potential disease control in cut flower production. The rice blast pathogen *Magnaporthe oryzae* is known to encode effector proteins which were functionally identified to elucidate the pathogenicity mechanism of the fungus. Fungal adhesion to host cells, a crucial stage for the initiation of infection, can be prevented by treatment with enzymes or gelatinolytic bacteria. These topics are discussed in Chapters 3-6.

Molecular tools have greatly enhanced our capability to detect and diagnose plant pathogens. Chapter 7 describes the different molecular techniques developed including detection of fungicide resistance. Chapter 8 covers the quantification of *Fusarium* biomass by PCR and ELISA, a direct tool that does not require the time consuming isolation and culture of the fungus.

Integrated disease management is the best approach for a sustainable crop protection. Chapters 9 deals with available disease management strategies for gray mold of castor bean while Chapter 10 focuses specifically on the effect of nutrition and soil properties on pathogens of tropical trees. Integrated disease management program for *Fusarium* in banana and *Pseudomonas* in olive trees are described in Chapter 11 and 12. Chapter

#### XII Preface

13 deals with disease resistance, considered to be the most effective, economical and environmentally-friendly plant disease management strategy.

I thank the authors for their invaluable contributions to this book.

**Dr. Christian Joseph R. Cumagun,**  Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, Philippines

## **Taxonomic Review of and Development of a Lucid Key for Philippine Cercosporoids and Related Fungi**

M. Mahamuda Begum, Teresita U. Dalisay and Christian Joseph R. Cumagun *Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, Philippines* 

#### **1. Introduction**

The genus *Mycosphaerella* Johanson, contains more than 3000 names (Aptroot 2006), and has been linked to more than 30 well-known anamorphic genera (Crous 2006a and 2006b). It has a worldwide distribution from tropical and subtropical to warm and cool regions (Crous 1998; Crous et al. 2000 and 2001). *Mycosphaer*ella, however, has been associated with at least 27 different coelomycete or hyphomycete anamorph genera (Kendrick and DiCosmo, 1979), 23 of which were accepted by Crous et al. (2000). More than 3000 names have already been published in *Cercospora* (Pollack, 1987). The genus *Cercospora* Fresen., which is one of the largest genera of hyphomycetes, has been linked to *Mycosphaerella* teleomorphs (Crous et al., 2000). *Cercospora* was first monographed by Chupp (1954), who accepted 1419 species. Subsequent workers such as, F.C. Deighton, B.C. Sutton and U. Braun divided *Cercospora* in to almost 50 different genera which are morphologically similar and distinct with each other (Crous and Braun, 2003).

Cercosporoid fungi are a collective term for a group of fungi belonging, to the genus *Cercospora* and its allied genera, namely *Pseudocercospora*, *Passalora*, *Asperisporium, Corynespora, Cladosporium.* Differences among them are based mainly on a combination of characters that include the structure of conidiogenous loci (scars) and hila, presence or absence of pigmentation and ornamentation in conidiophores and conidia, geniculate or non-geniculate conidiophore, and rare presence of additional or unique features such as knotty appearance of conidiophores.

*Cercospora* Fresen. is one of the largest genera of Hyphomycetes. Saccardo (1880) defined *Cercospora* as having brown conidiophores and vermiform, brown, oliivaceous or rarely subhyaline conidia. Deighton continuously studied the *Cercospora* species (Deighton, 1967a, 1967b, 1971, 1973, 1974, 1976, 1979, 1983, and 1987) and reclassified numerous species into several allied genera based mainly on two distinct taxonomic categories: thickened conidial scars occur in the *Cercospora* and in allied genera such as*, Passalora* and *Stenella,* while unthickened scars are characteristics of the genera *Pseudocercospora* and *Stigmina.*

Taxonomic Review of and Development

**2. Genus** *Cercospora*

*Cercospora* Fresen. (Crous and Braun, 2003).

**Type species:** *Cercospora penicillata* (Ces.) Fresen.

darkened (Crous and Braun, 2003).

of a Lucid Key for Philippine Cercosporoids and Related Fungi 3

to the development of morphological Lucid key for their identification. For this purpose, field collections were conducted from 2007 to 2009. Microscopic studies on the association of Cercosporoid species to the diseased leaves were carried out at the Postharvest Pathology Lab, Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños (UPLB). The field collection was confined mostly within UPLB campus, particularly propagation farm and medicinal plant gene bank, vegetable farm of the Crop Science Cluster, UPLB, the production farm at the Jamboree site, production farm at International

1. Conidiogenous loci inconspicuous or subdenticulate, but always unthickened and not darkened or subconspicuous, i.e., unthickened, but somewhat refractive or rarely very slightly darkened, or only outer rim slightly darkened and refractive (visible as minute rings)-------------------------------------------------------------------------------------------*Pseudocercospora*  1. Conidiogenous loci conspicuous, i.e., thickened and darkened throughout, only with a minute central pore-----------------------------------------------------------------------------------------------2 2. with verruculose superficial secondary mycelium; conidia amero- to scolecosporous, mostly verruculose-----------------------------------------------------*Stenella* 2. If superficial secondary mecelium present, hyphae smooth or almost so------------3 3. Conidia hyaline or subhyaline, scolecosporous, acicular, obclavate-cylindrical, filiform, usually pluriseptate---------------------------------------------------------------------------------*Cercospora*  3. Conidia pigmented or, if subhyaline, conidia non-scolecosporus, ellipsoid-ovoid, short cylindrical, fusoid and only few septa-----------------------------------------------------------*Passalora* 4. Conidiogenous loci protuberant, with a central convex part (dome) surrounded by a raised periclinal rim, conidia in long, often branched chains or solitary, smooth to verruculose ------------------------------------------------------------------------*Cladosporium*  4. Conidiogenous loci conspicuously thickened, conidia non-scolecosporous, ellipsoid-ovoid, short subcylindrical, aseptate or only with few septa-------------------- -------------------------------------------------------------------------------------------*Asperisporium*  5. Conidia contain from 4-20 pseudo-crosswalls (pseudosepta), the base of the conidium (hilum) conspicuously thickened------------------------------------------------------------- *Corynespora*  5. Conidia without septa or with one or a few transverse septa, conidiophores apical portion

Rice Research Institute (IRRI), and some residential gardens in Los Baños, Laguna.

sometimes branched------------------------------------------------------------------- *Periconiella*

whether the collection is considered a first record or has already been reported.

Further descriptions of the genera and species belonging to the genus, as they were associated from the collections were presented. The last column of the table indicates

*Stromata* lacking to well developed, subhyaline to usually pigmented; *conidiophores* mononematous, macronematous, solitary to fasciculate, arising from internal hyphae or stomata, erect, continuous to pluriseptate, subhyaline to pigmented; *conidiogenous loci* conspicuous, thickened and darkened, planate; *conidia* solitary to catenate, scolecosporous, obclavate, cylindrical, filiform, acicular, hyaline, smooth or almost so, hila thickened and

**Key to Cercosporoid Genera** (Crous and Braun, 2003).

Cercosporoid fungi are commonly associated with leaf spots (Wellman, 1972) ranging from slight, diffuse discolorations to necrotic spots or leaf blight (Shin and Kim, 2001). Cercosporoid fungi can also cause necrotic lesions on flowers, fruits, bracts, seeds and pedicels of numerous hosts in most climatic regions (Agrios, 2005). They are responsible for great damages to beneficial plants. Furthermore, other than important pathogens of major agricultural crops such as cereals, vegetables, ornamentals, forest trees, grasses and many others species are also known to be hyperparasitic to other plant pathogenic fungi (Goh and Hsieh, 1989).Cercosporoid fungi are known to cause some of the economically important diseases worldwide. One of the most important and common diseases associated with this fungus is the black sigatoka caused by *Mycosphaerella fijiensis* which was first discovered and considered to have caused epidemics in the Valley of Fiji (Stakman and Harrar, 1957).

The Cercosporoid fungi of Philippines are insufficiently known. There have been no comprehensive studies on this group of fungi in Philippines. Welles (1924 and 1925) worked with physiological behavior of Philippine Cer*cosporas* on artificial media and the extent of parasitism. There were 87 species of *Cercospora* reported in the Philippines from 1937 onwards (Quimio and Abilay, 1977). Teodoro (1937) had enumerated 65 species of *Cercospora* in the Philippines. In most cases, however, the causal species have only been cited but not characterized. No attempt was made to determine the host range of each of the species. Naming of the species was based mainly on Chupp's monograph (Chupp, 1953), which together with Vasudeva's book (Vasudeva, 1963) book on Indian *Cercosporae,* as the main reference books used by Quimio and Abilay (1977).

Identification of fungal plant pathogens is commonly done using one of several wellillustrated dichotomous keys by Ellis (1971 and 1976), Sutton (1980), Hanlin (1990), and Barnett & Hunter (1998). Multi-access keys for identifying biological agents are very useful, especially for the non-specialist, as it is not necessary to be able to detect all of the fine distinctions usually found in dichotomous keys. The disadvantage of those printed keys is that they require the user to be able to scan a series of tables of numbers and select those that are common to the specimen being examined (Michaelides *et al.* 1979; Sutton 1980). This task is ideally suited to computers. The Lucid system developed by the Centre for Biological Information Technology (CBIT), University of Queensland (Norton 2000) allows development of multi-access computer-based keys that can also incorporate graphics and text. The result is a very powerful tool. Although some keys have previously been developed for fungi using Lucid, they have generally been for specific groups such as rainforest fungi of Eastern Australia (Young 2001). The main purpose in developing these Lucid identification systems has been to contribute to taxonomic capacity building in two ways - by enabling identification keys to be easily developed and by increasing the availability and usefulness of these keys by making them available on CD or via the Internet. A Lucid was used for identifying genera for identifying genera of plant pathogenic Cercosporoid fungi of Philippine crops. The key was comprised of many characters, which has the potential for being rather cumbersome. For simplicity, the characters were placed in groups and states relating to the structures like the morphology of conidiophores, the stromata, conidia, and fruiting bodies and the names of host family and genus.

The primary objective of this study was to identify Cercosporoid fungi of the Philippines, use recent taxonomic information to amend or rename species, formulate taxonomic keys, and develop Lucid key for identification. An existing computer based software was applied

Cercosporoid fungi are commonly associated with leaf spots (Wellman, 1972) ranging from slight, diffuse discolorations to necrotic spots or leaf blight (Shin and Kim, 2001). Cercosporoid fungi can also cause necrotic lesions on flowers, fruits, bracts, seeds and pedicels of numerous hosts in most climatic regions (Agrios, 2005). They are responsible for great damages to beneficial plants. Furthermore, other than important pathogens of major agricultural crops such as cereals, vegetables, ornamentals, forest trees, grasses and many others species are also known to be hyperparasitic to other plant pathogenic fungi (Goh and Hsieh, 1989).Cercosporoid fungi are known to cause some of the economically important diseases worldwide. One of the most important and common diseases associated with this fungus is the black sigatoka caused by *Mycosphaerella fijiensis* which was first discovered and considered to have caused epidemics in the Valley of Fiji (Stakman and Harrar, 1957).

The Cercosporoid fungi of Philippines are insufficiently known. There have been no comprehensive studies on this group of fungi in Philippines. Welles (1924 and 1925) worked with physiological behavior of Philippine Cer*cosporas* on artificial media and the extent of parasitism. There were 87 species of *Cercospora* reported in the Philippines from 1937 onwards (Quimio and Abilay, 1977). Teodoro (1937) had enumerated 65 species of *Cercospora* in the Philippines. In most cases, however, the causal species have only been cited but not characterized. No attempt was made to determine the host range of each of the species. Naming of the species was based mainly on Chupp's monograph (Chupp, 1953), which together with Vasudeva's book (Vasudeva, 1963) book on Indian *Cercosporae,* as the

Identification of fungal plant pathogens is commonly done using one of several wellillustrated dichotomous keys by Ellis (1971 and 1976), Sutton (1980), Hanlin (1990), and Barnett & Hunter (1998). Multi-access keys for identifying biological agents are very useful, especially for the non-specialist, as it is not necessary to be able to detect all of the fine distinctions usually found in dichotomous keys. The disadvantage of those printed keys is that they require the user to be able to scan a series of tables of numbers and select those that are common to the specimen being examined (Michaelides *et al.* 1979; Sutton 1980). This task is ideally suited to computers. The Lucid system developed by the Centre for Biological Information Technology (CBIT), University of Queensland (Norton 2000) allows development of multi-access computer-based keys that can also incorporate graphics and text. The result is a very powerful tool. Although some keys have previously been developed for fungi using Lucid, they have generally been for specific groups such as rainforest fungi of Eastern Australia (Young 2001). The main purpose in developing these Lucid identification systems has been to contribute to taxonomic capacity building in two ways - by enabling identification keys to be easily developed and by increasing the availability and usefulness of these keys by making them available on CD or via the Internet. A Lucid was used for identifying genera for identifying genera of plant pathogenic Cercosporoid fungi of Philippine crops. The key was comprised of many characters, which has the potential for being rather cumbersome. For simplicity, the characters were placed in groups and states relating to the structures like the morphology of conidiophores, the

stromata, conidia, and fruiting bodies and the names of host family and genus.

The primary objective of this study was to identify Cercosporoid fungi of the Philippines, use recent taxonomic information to amend or rename species, formulate taxonomic keys, and develop Lucid key for identification. An existing computer based software was applied

main reference books used by Quimio and Abilay (1977).

to the development of morphological Lucid key for their identification. For this purpose, field collections were conducted from 2007 to 2009. Microscopic studies on the association of Cercosporoid species to the diseased leaves were carried out at the Postharvest Pathology Lab, Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños (UPLB). The field collection was confined mostly within UPLB campus, particularly propagation farm and medicinal plant gene bank, vegetable farm of the Crop Science Cluster, UPLB, the production farm at the Jamboree site, production farm at International Rice Research Institute (IRRI), and some residential gardens in Los Baños, Laguna.

#### **Key to Cercosporoid Genera** (Crous and Braun, 2003).

1. Conidiogenous loci inconspicuous or subdenticulate, but always unthickened and not darkened or subconspicuous, i.e., unthickened, but somewhat refractive or rarely very slightly darkened, or only outer rim slightly darkened and refractive (visible as minute rings)-------------------------------------------------------------------------------------------*Pseudocercospora*  1. Conidiogenous loci conspicuous, i.e., thickened and darkened throughout, only with a minute central pore-----------------------------------------------------------------------------------------------2 2. with verruculose superficial secondary mycelium; conidia amero- to scolecosporous, mostly verruculose-----------------------------------------------------*Stenella* 2. If superficial secondary mecelium present, hyphae smooth or almost so------------3 3. Conidia hyaline or subhyaline, scolecosporous, acicular, obclavate-cylindrical, filiform, usually pluriseptate---------------------------------------------------------------------------------*Cercospora*  3. Conidia pigmented or, if subhyaline, conidia non-scolecosporus, ellipsoid-ovoid, short cylindrical, fusoid and only few septa-----------------------------------------------------------*Passalora* 4. Conidiogenous loci protuberant, with a central convex part (dome) surrounded by a raised periclinal rim, conidia in long, often branched chains or solitary, smooth to verruculose ------------------------------------------------------------------------*Cladosporium*  4. Conidiogenous loci conspicuously thickened, conidia non-scolecosporous, ellipsoid-ovoid, short subcylindrical, aseptate or only with few septa-------------------- -------------------------------------------------------------------------------------------*Asperisporium*  5. Conidia contain from 4-20 pseudo-crosswalls (pseudosepta), the base of the conidium (hilum) conspicuously thickened------------------------------------------------------------- *Corynespora*  5. Conidia without septa or with one or a few transverse septa, conidiophores apical portion sometimes branched------------------------------------------------------------------- *Periconiella*

Further descriptions of the genera and species belonging to the genus, as they were associated from the collections were presented. The last column of the table indicates whether the collection is considered a first record or has already been reported.

#### **2. Genus** *Cercospora*

*Cercospora* Fresen. (Crous and Braun, 2003).

*Stromata* lacking to well developed, subhyaline to usually pigmented; *conidiophores* mononematous, macronematous, solitary to fasciculate, arising from internal hyphae or stomata, erect, continuous to pluriseptate, subhyaline to pigmented; *conidiogenous loci* conspicuous, thickened and darkened, planate; *conidia* solitary to catenate, scolecosporous, obclavate, cylindrical, filiform, acicular, hyaline, smooth or almost so, hila thickened and darkened (Crous and Braun, 2003).

**Type species:** *Cercospora penicillata* (Ces.) Fresen.

Taxonomic Review of and Development

*Begonia* sp. (Begonia) lacking

*Capsicum annum*  (Chili)

*Cucurbita moschata*  (Squash)

lacking

lacking

*Cercospora begoniae*

*Cercospora capsici* 

*Cercospora citrullina* 

of a Lucid Key for Philippine Cercosporoids and Related Fungi 5

2-5 in a fascicle or borne singly, pale to very pale brown in colour, paler and more narrow towards the apex, straight or

geniculate, septate, truncate at the apex, scars conspicuously thickened 30-180 x

5-15 in a fascicle, pale to olivaceous brown, straight to slightly curved, not branched, mildly geniculate, acicular to filiform, straight to mildly curved, scars conspicuously thickened, 30-140 x

3-5 µm

3-6 µm

4-6 µm

2-5 in a fascicle, pale to olivaceous brown, straight to slightly bent or curved, geniculate, multiseptate, simple, occasionally swollen at some points, subtruncate at the apex, scars conspicuously thickened, 35-250 x

**Ref. Coll. Accession No.** 

> CALP <sup>11676</sup>FR

CALP

CALP 11675 FR

<sup>11693</sup>AR

hyaline, acicular, straight to mildly

curved, indistinctly multiseptate, acute at the apex, truncate at the base, hilum conspicuously thickend, 50-260 x

2.5-4µm

hyaline, solitary, acicular to filiform, straight to mildly curved, multiseptate, apex-acute to subacute, base truncate or obconically truncate, hilum thickened, 50-170 x 3-4.5 µm

hyaline, solitary, acicular/ cylindroobclavate, straight-curved, multiseptate, apex-subacute to obtuse, basesubtruncate or rounded, hilum thickened and darkened, 20-200 x 2-3.5 µm

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

In the present study, 48 *Cercospora* diseases were reported. Among them, 20 species were now considered under a compound species *Cercospora apii s. lat.* (Table 1) and 28 under *Cercospora s. str.* which is host specific with a host range confined to species of a single host genus or some allied host genera of a single family (Table 2).The reported genus *Cercospora* was introduced by Fresenius with *Cercospora penicillata* as the type species. Since then over 1000 species were reported and characterized and were compiled in the book "Monograph of the Genus *Cercospora*'' by Chupp (1953). He proposed a broad concept for the genus, simply noting whether hila were thickened or not, and if conidia were pigmented or not, single or in chains. Recently Crous & Braun (2003) recognized four true cercosporoid genera, viz. *Cercospora*, *Pseudocercospora* Speg., *Passalora* Fr. and *Stenella* Syd., and several other morphologically similar genera, based on molecular sequence analyses and a reassessment of morphological characters. They represented a compilation of more than 3000 names that have been published or proposed in *Cercospora*, of which 659 are presently recognized in this genus, with a further 281 being referred to *C.apii s.lat*. They amended the species *C. apii* and it is now a compound species, referred to as *C. apii s.lat.* It infects hundreds of plant species. *Cercospora apii s.lat*. is characterized by having solitary to fasciculate, usually long, brown, septate conidiophores with conspicuously thickened and darkened conidiogenous loci and long, acicular, hyaline, pluriseptate conidia formed singly. *Cercospora s.str* is characterized by having stromata, with numerous, densely arranged rather short conidiophores, small conidiogenous loci, and obclavate-cylindrical conidia with truncate base (Table 2).


In the present study, 48 *Cercospora* diseases were reported. Among them, 20 species were now considered under a compound species *Cercospora apii s. lat.* (Table 1) and 28 under *Cercospora s. str.* which is host specific with a host range confined to species of a single host genus or some allied host genera of a single family (Table 2).The reported genus *Cercospora* was introduced by Fresenius with *Cercospora penicillata* as the type species. Since then over 1000 species were reported and characterized and were compiled in the book "Monograph of the Genus *Cercospora*'' by Chupp (1953). He proposed a broad concept for the genus, simply noting whether hila were thickened or not, and if conidia were pigmented or not, single or in chains. Recently Crous & Braun (2003) recognized four true cercosporoid genera, viz. *Cercospora*, *Pseudocercospora* Speg., *Passalora* Fr. and *Stenella* Syd., and several other morphologically similar genera, based on molecular sequence analyses and a reassessment of morphological characters. They represented a compilation of more than 3000 names that have been published or proposed in *Cercospora*, of which 659 are presently recognized in this genus, with a further 281 being referred to *C.apii s.lat*. They amended the species *C. apii* and it is now a compound species, referred to as *C. apii s.lat.* It infects hundreds of plant species. *Cercospora apii s.lat*. is characterized by having solitary to fasciculate, usually long, brown, septate conidiophores with conspicuously thickened and darkened conidiogenous loci and long, acicular, hyaline, pluriseptate conidia formed singly. *Cercospora s.str* is characterized by having stromata, with numerous, densely arranged rather short conidiophores, small conidiogenous loci,

and obclavate-cylindrical conidia with truncate base (Table 2).

lacking

present

*Cercospora amaranti* 

*Cercospora anonae* 

*Amaranthus viridis*  (Amaranth)

*Anona squamosa*  (Sugar apple)

**Species Host Stromata Conidiophores Conidia** 

2-15 in a divergent fascicle, pale to olivaceous brown, multiseptate, not branched, straight

dense fascicle, pale to olivaceous brown, almost uniform in colour and width, not geniculate, slightly branched, septate, large scar present, 20-180 x 2-5 µm

to slightly geniculate, scars conspicuously thickened, 35-200 x

4-5.5 µm

**Ref. Coll. Accession No.** 

CALP

CALP

<sup>11734</sup>AR

<sup>11707</sup>FR

hyaline, acicular, smooth walled, straight, basetruncate, apex-

hilum thickened and darkened, 40- 200 x 2-4.5 µm

hyaline, solitary, acicular, straight to curved, septate, base obconically truncate, tip acute, hilum thickened and darkened, 40-200 x 1.5-3 µm

acute,

**Status of collection**


Taxonomic Review of and Development

*Euphorbia heterophylla* (Milk weed)

*Impatiens balsamina*  (Balsam plant)

*Dhalia variabilis*  (Dahlia)

*Cercospora euphorbiae* 

*Cercospora fukushiana* 

*Cercospora grandissima* 

of a Lucid Key for Philippine Cercosporoids and Related Fungi 7

2-5 in a fascicle, pale to olivaceous brown, straight to mildly curved, sometimes branched, rarely geniculate,

multiseptate, large conidial scars at the subtruncate tip, 25-100 x 4.5-6 µm

divergent fascicle, pale olivaceous brown in colour, apex subtruncate, 1-4 septate, rarely branched, straight to flexuous or geniculate, scars medium sized and thickened, 40-150 x

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

11677 AR

<sup>11678</sup>AR

<sup>11724</sup>FR

hyaline, solitary, cylindrical to acicular, straight to mildly curved, multiseptate, obconically truncate base, obtuse tip, hilum thickened and darkened, 40-120

x 3-4.5 µm

curved, indistinctly multiseptate, acute to subacute at the apex, truncate at the base, hilum conspicuously thickend, 40-250 x

3-4 µm

x 2-4 µm

Hyaline, solitary, acicular, straight to slightly curved, multiseptate, apex-acute, basetruncate, hilum thickened and darkened, 50-250

hyaline, acicular, straight to mildly

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

lacking

present

lacking

3-4 µm

x 4-5 µm

1-10 in a fascicle, pale to medium brown, straight or mildly geniculate with thickened scars, subtruncate at the apex,60-100


2-15 in a fascicle, pale to very pale brown, straight to mildly geniculate, multiseptate, scars conspicuously thickened, 20-180 x

4-5 µm

2-5 in a fascicle, pale olivaceous brown, width irregular, straight to slightly curved, mildly geniculate, septate, conidial scars conspicuous and thickened, 40- 250 x 4-5.5 µm

10-30 in a

divergent fascicle, brown to dark brown at the base and apical portion rather paler, straight to mildly geniculate,

multiseptate, scars

large and conspicuously thickened, 40-150x

5-6.5 µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

<sup>11710</sup>AR

<sup>11728</sup>FR

<sup>11688</sup>AR

hyaline, solitary,

acicular, multiseptate, apex- acute to subacute, basesubtruncate or rounded, hilum thickened, 45-190

x 2-4 µm

hilum

conspicuously thickened and darkened, 45-200 x 3.5-5 µm

hyaline, acicular/ cylindro-bclavate, straight - curved, multiseptate, apex- subacute, base-subtruncate to truncate, hilum thickened and darkened, 50-250 x 2-3.5 µm

hyaline, solitary, acicular to obclavato-

cylindric, straight to mildly curved, multiseptate, obtuse apex, truncate base,

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

lacking

lacking

present

*Cercospora citrullina* 

*Cercospora citrullina* 

*Cercospora cruenta* 

*Luffa cylindrica*  (Sponge gourd)

*Phaseolus lunatus*  (Lima bean)

*Momordica charantia*  (Bitter gourd)


Taxonomic Review of and Development

*Ipomoea aquatica*  (Kangkong)

*Lagenaria vulgari*  (Bottle gourd)

*Laportea crenulata*  (Laportea)

*Cercospora ipomoeae*

*Cercospora lagenariae* 

*Cercospora laporticola* 

of a Lucid Key for Philippine Cercosporoids and Related Fungi 9

arise singly or 2-7 in a fascicle, pale yellowish olivaceous to medium, brown, slightly paler and more narrow towards the tip, rearly geniculate, subtruncate at the apex, 15-150 x 4-6.5

2-5 in a divergent fascicle, pale brown to brown, straight to slightly bent or curved, geniculate, occasionally branched, multiseptate, obtuse to

subtruncate at the apex, conidial scars conspicuous, 60-250 x 3-6 µm

2-5 in a loose fascicle, pale to olivaceous brown,

septate, unbranched, straight, smooth walled, large conidial scars conspicuously thickened, 30-90 x

5-7.5 µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

11696 FR

<sup>11700</sup>FR

<sup>11711</sup>FR

hyaline, acicular to obclavate straight to curved, indistinctly multiseptate, subacute at the apex, truncate of subtruncate at the base, 25-130 x 3-5

hyaline, solitary,

acicular, substraight to mildly curved, multiseptate, acute to obtuse at the apex, truncate at the base, hilum thickened and darkened, 40-210

x 2-5 µm

hyaline, solitary, acicular, smooth walled, straight, base-truncate, apex-obtuse, hilum thickened and darkened, 40- 250 x 3-4.5 µm

µm

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

µm

lacking

lacking

lacking


**Ref. Coll. Accession No.** 

CALP

CALP <sup>11712</sup>FR

CALP

<sup>11713</sup>AR

<sup>11733</sup>AR

hyaline, solitary, acicular to obclavatecylindric, substraight to mildly curved, 2- 13 septate, nonconstricted,apexsubacute ,basetruncate, hilum conspicuously thickened, darkened, 35-150

x 3-4 µm

4.5µm,

hyaline, solitary, obclavate, smooth walled, straight, mildly curved, basetruncate, apexobtuse, 40-120 x2-

hilum thickened & darkened.

hyaline, acicular to obclavate, straight to mildly

curved, indistinctly multiseptate, truncate at the base, acute at the base, 30-150 x 2-3

µm

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

3-15 in a loose fascicle, brown to deep brown throughout, irregular in width,

straight to slightlybcurved, geniculate, not branched, 2-5 septate, obtuse to subtruncate at the apex, conidial scars small and conspicuous, 25- 210 x 4-5 µm

dense fascicle, pale olivaceous to medium brown, multiseptate, unbranched, straight, mildly geniculate towards the apex, smooth walled, large conidial scars conspicuously thickened, 40-150 x

5-7.5 µm

4-6.5 µm

2-5 in a fascicle or borne singly, olivaceous to medium brown, paler upward, rarely branched, mildly geniculate, scar conspicuously thickened, 25-180 x

present

lacking

lacking

*Cercospora hydrangeae* 

*Cercospora ipomoeae*

*Cercospora ipomoeae* 

*Hydrangea macrophylla* (Milflores)

*Ipomoea triloba*  (Little bell)

*Ipomoea batatas*  (Sweet potato)


Taxonomic Review of and Development

Cumagun, 2010)*.* 

*Cercospora adiantigena* 

*Cercospora basellaealbae* 

*Adiantum phillipense*  (Maiden hair fern)

*Basella alba*  (Vine spinachgreen)

of a Lucid Key for Philippine Cercosporoids and Related Fungi 11

olivaceous brown conidiophores with small conspicuously thickened and darkened conidiogenous loci, and obclavate-cylindrical conidia with obconically truncate base (Table 2). Teodoro (1937) had enumerated 65 species of *Cercospora* in the Philippines while 33 species were reported by Quimio and Abilay (1977). In the present study, 48 hosts exhibiting leaf spots were reported as caused by species of *Cercospora,* 32 were from medicinal plants. There were 30 first records of *Cercospora* leaf spots recorded in this study. All species of *Cercospora* associated with those hosts are known except for a species on *Basella albae*. It has not been described on this host; therefore, it warrants description on a new host record, with proposed species name of *Cercospora basellae-albae (*Begum and

> **Ref. Coll. Accession No.**

> > CALP

CALP

11735 FR

<sup>11715</sup>AR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

small to

fascicle, subhyaline, straight,

4-10 µm

µm

2-15 in a divergent fascicle, light brown, straight to rarely curved, unbranched, thick walled, septate, geniculate, with rounded apex, conidial scars distinct, 30-85 x 4-5

moderately long

hyaline, solitary, obclavatecylindrical, short conidia occasionally fusoid, septate, thin walled, smooth, apex obtuse, base short obconically truncate, hilam thickened and darkened, 40- 90 x 4-8 µm

hyaline, acicular to subcylindrical, straight to rarely curved, unbranched, smooth walled, septate, with truncate to obconically truncate base and obtuse apex, 20-80 x 2-

5 µm

subcylindrical to moderately geniculate to sinuous, unbranched, multiseptate, conidial scar thickened and darkened, 25-150 x

present

present


Reference: Chupp (1954); Ellis (1971, 1976); Guo & Hsieh (1995); Guo *et al.* (1998); Hsieh & Goh(1990); Saccardo (1886); Shin & Kim (2001); Vasudeva (1963).

\* AR= already reported, FR= first record.

Table 1. List and descriptions of formerly reported *Cercospora* species that were reclassified in this study as *Cercospora apii s.lat.* 

Some former species of *Cercospora* that are morphologically different from *C.apii s.lat.,* are now considered to *Cercospora s.str.* As far as known, *Cercospora s.str.* are host specific or with a host range confined to species of a single host genus or some allied host genera of a single family. This phenomenon is constantly being addressed via molecular studies (Crous et al., 2000, 2001). *Cercospora s.str.* is characterized by having stromata, with numerous, densely arranged rather short, solitary to fasciculate, subhyaline to light or

2-15 in a fascicle, pale olivaceous brown, straight, rarely septate and geniculate, unbranched, scars conspicuously thickened,10-50 x

4-5.5 µm

3-6.5 µm

4-6 µm

2-20 in a fascicle, pale to medium dark olivaceous brown, not branched, straight or geniculate, scars conspicuously thickened, 15-200 x

Reference: Chupp (1954); Ellis (1971, 1976); Guo & Hsieh (1995); Guo *et al.* (1998); Hsieh & Goh(1990);

Table 1. List and descriptions of formerly reported *Cercospora* species that were reclassified

Some former species of *Cercospora* that are morphologically different from *C.apii s.lat.,* are now considered to *Cercospora s.str.* As far as known, *Cercospora s.str.* are host specific or with a host range confined to species of a single host genus or some allied host genera of a single family. This phenomenon is constantly being addressed via molecular studies (Crous et al., 2000, 2001). *Cercospora s.str.* is characterized by having stromata, with numerous, densely arranged rather short, solitary to fasciculate, subhyaline to light or

2-5 in a fascicle, pale olivaceous to medium brown, paler toward the tip, not branched, multiseptate, mildly-geniculate, scars large and conspicuously thickened, 20-100 x

**Ref. Coll. Accession No.** 

> CALP <sup>11690</sup>FR

CALP

CALP

<sup>11704</sup>AR

<sup>11701</sup>AR

hyaline, solitary,

hyaline, solitary, acicular, straight to mildly curved, multiseptate, acute to subacute at the apex, truncate at the base, hilum thickened and darkened, 25-250 x 2-4.5 µm

hyaline, solitary, acicular/obclavat e, straight to mildly curved, apex-acute / subacute at basetruncate to subtruncate, hilum thickened and darkened, 20- 160 x 2-4 µm

acicular, multiseptate base-truncate, tip-acute hilum thickened and darkened, 40-150 x 2-3.5µm

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

*Cercospora moricola* 

*Cercospora nicotianae* 

*Cercospora zinniae* 

*Morus alba* 

*Nicotiana tabacum*  (Tobacco)

*Zinnia elegans*  (Zinnia) lacking

lacking

Saccardo (1886); Shin & Kim (2001); Vasudeva (1963).

\* AR= already reported, FR= first record.

in this study as *Cercospora apii s.lat.* 

(Mulberry) lacking

olivaceous brown conidiophores with small conspicuously thickened and darkened conidiogenous loci, and obclavate-cylindrical conidia with obconically truncate base (Table 2). Teodoro (1937) had enumerated 65 species of *Cercospora* in the Philippines while 33 species were reported by Quimio and Abilay (1977). In the present study, 48 hosts exhibiting leaf spots were reported as caused by species of *Cercospora,* 32 were from medicinal plants. There were 30 first records of *Cercospora* leaf spots recorded in this study. All species of *Cercospora* associated with those hosts are known except for a species on *Basella albae*. It has not been described on this host; therefore, it warrants description on a new host record, with proposed species name of *Cercospora basellae-albae (*Begum and Cumagun, 2010)*.* 


Taxonomic Review of and Development

*Dolichos lablab*  (Lab bean)

*Daucus carota*  (Carrote)

*Corchorus olitorius*  (Jute)

*Cercospora canescens* 

*Cercospora carotae* 

*Cercospora corchori* 

of a Lucid Key for Philippine Cercosporoids and Related Fungi 13

fasciculate, pale to medium dark brown, multiseptate, geniculate, rarely branched, apextruncate, conidial

hyaline, acicular, straight to curved, indistinctly multiseptate, apex-acute, base-truncate, thickened hilum, 25-200 x 2.5-5.5 µm

hyaline, filiform to cylindric, solitary, straight to slightly curved, 1-5 septa, rounded base, obtuse apex, hilum

thickened and darkened, 25- 95 x 3.5-5.5 µm

 hyaline, acicular to obclavate, straight to curved, indistinctly multiseptate, obtuse at the apex, baseobconically truncate , thickened hilum, 25-165 x 2.5-5 µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

11694 FR

<sup>11730</sup>AR

<sup>11732</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

densely

scars

6.5 µm

2.5-4 µm

2-7 in a fascicle or borne singly, pale to medium olivaceous brown, paler at the apex, septate, not branched, geniculate, subtruncate at tip, with thickened conidial scars, 40- 230 x 2-5.5 µm

conspicuously thickened, 2-4 µm wide, 20-200 x 3-

3-15 in a fascicle or borne singly, pale olivaceous brown, paler tips, upper portion slightly geniculate, straight, scars conspicuous thickened, 20-40 x

present

present

present


1-10 in a fascicle, pale olivaceous brown, fairly uniform in color and width, not branched, straight

2-15 in a divergent fascicle, emerging through stomata, pale olivaceous to medium brown, not branched, multiseptate, mildly geniculate,

hyaline, acicular, obclavate, straight to slightly curved, in distinctly multiseptate, rounded apex, truncate at the base with a thickened hilum, 15-70 x 1-4 µm.

hyaline, solitary, acicular to cylindrical, straight to mildly curved, multiseptate, acute to rounded at the apex, truncate at the base, hilum

thickened and darkened, 25- 250 x 2-4.5 µm

hyaline, acicular, straight to curved, indistinctly multiseptate, subacute to acute at the apex, truncate at the base, 25- 200 x 2-5 µm

or mildly geniculate with thickened conidial scars, sparingly septate, 30-75 x 4-6

but rarely geniculate in the upper portion, scars large and conspicuously thickened, 20-200 x

3-5.5 µm

µm

2-15 in a fascicle, pale olivaceous to medium brown, uniform in colour and width but paler the attenuated tips, rarely branched, multiseptate, mildly geniculate, conidial scars at the subtruncate tip, 25-400 x 3.5-6

µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

<sup>11703</sup>FR

<sup>11705</sup>AR

<sup>11674</sup>NHR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

present

present

present

*Cercospora basellaealbae* 

*Cercospora brassisicola*

*Cercospora brassicicola*

*Basella albae* (Vine spinachpurple)

*Brassica pekinensis*  (Pechay)

*Brassica campestris*  (Mustard)


Taxonomic Review of and Development

*Euphorbia*  sp.

(Euphorbia)

*Gendarussa vulgaris*  (Gendarussa)

*Ocimum sanctum*  (Basil)

present

present

lacking

*Cercospora euphorbiae* 

*Cercospora gendarussae* 

*Cercospora guatemalensis*

of a Lucid Key for Philippine Cercosporoids and Related Fungi 15

5-15 in a fascicle, pale olivaceous brown, uniform in colour and width, paler the tips, smooth wall, straight to mildly curved, not branched, not geniculate, multiseptate, medium conidial scar thickened and darkened, 15-65 x

hyaline, solitary, cylindroobclavate, subobtuse tip, obconically truncate base, straight to curved, multiseptate, hilum

thickened and darkened, 40- 100 x 3.5-5 µm

thickened and darkened, 45- 180 x 3-4 µm

hyaline, cylindric or acicular, straight to mildly curved, indistinctly multiseptate, base truncate to obconically truncate, tip rounded to conic, 45-120 x 2.5-4 µm

hyaline, solitary, acicular to cylidroobclavate, acute to rounded tip, obconically truncate base, straight to curved, multiseptate, hilum

4-6 µm

densely fasciculate, olivaceous brown, uniform in colour and width, paler the tips, smooth wall, straight to mildly curved, not branched, rarely geniculate,

multiseptate, large conidial scar thickened and darkened, 20-120 x

2-10 in a fascicle, pale to olivaceous brown, slightly paler and more narrow towards the tip, septate, not branched, straight to slightly curved, conidial scar conspicuous , 25- 100 x 4-5.5 µm

4-5.5 µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

11725 FR

<sup>11722</sup>FR

<sup>11721</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 


5-15 in a fascicle, pale to medium brown, slightly paler and more narrow towards the tip, springly septate, not branched, mildly geniculate, almost straight, large conidial scar at subtruncate tip, 30- 120 x 4-5.5 µm

2-15 in a fascicle, pale olivaceous brown, uniform in colour, usually straight, septate, not branched, conidial scars conspicuously thickened and darkened, 30-85 x

3.5-5.5 µm

2-5 in a small fascicle, pale to olivaceous brown, straight to mildly curved, not branched,

sometimes mildly geniculate,

multiseptate, large conidial scars conspicuous, 25-85

x 4.5-6 µm

hyaline, acicular to obclavate, straight to curved, indistinctly multiseptate, base truncate, tip acute, 40- 220 x 2.5-5 µm

hyaline, acicular to obclavatocylindrical, multiseptate, straight to mildly curved, tip acute to subacute and base truncate,

hilum

hyaline, cylindrical to obclavate, straight to mildly curved, multiseptate, obconically truncate base, rounded tip, hilum

thickened and darkened, 50- 150 x 3-4.5 µm

thickened and darkened, 40- 120 x 3-4.5 µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

<sup>11720</sup>FR

<sup>11729</sup>FR

<sup>11697</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

present

lacking

lacking

*Cercospora corchori* 

*Cercospora daturicola* 

*Cercospora eluesine* 

*Corchorus acutangulus* (saluyot)

*Datura metal*  (Datura)

*Eluesine indica*  (Dogs tail)


Taxonomic Review of and Development

*Lactuca sativa*  (Lettuce)

*Mentha arvensis*  (Apple mint plant)

*Mikania cordifolia*  (Climbing hemp weed)

*Cercospora lactucaesativae* 

*Cercospora menthicola* 

*Cercospora mikaniicola* 

of a Lucid Key for Philippine Cercosporoids and Related Fungi 17

2-10 in a fascicle or borne singly, pale olivaceous brown, slightly paler and narrower towards

hyaline, solitary, acicular to obclavate, straight to curved, indistinctly multiseptate, subacute at the

apex,

base-

subtruncate at the base, 20-200 x 3-5 µm.

hyaline,solitary , acicular, straight to mildly curved, multiseptate,

subtruncate to obconically truncate. Apexacute to rounded, hilum

thickened and darkened, 40- 120 x 2-4 µm

hyaline, solitary, obclavate, truncate at the base, acute the tip, hilum thickened and darkened, 40- 80 x 4-9 µm

the apex,

geniculate, conidial scar at subtruncate tip, 25- 100 x 4-5 µm

2-5 in a small fascicle, pale to olivaceous brown, multiseptate, not branched, straight to curved, mildly geniculate towards the apex, smooth walled, conidial

scars

4-6.5 µm

to mildly

scars

4-6.5 µm

conspicuously thickened, 35-120 x

2-5 in a fascicle, medium

olivaceous brown, closely septate, not branched, straight

geniculate, smooth walled, conidial

conspicuously thickened, 50-150 x

multiseptate, not branched, springly

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

11716 FR

<sup>11691</sup>FR

<sup>11699</sup>AR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

present

lacking

lacking


1-10 in a fascicle, pale to medium brown, paler and more narrow toward the tip, geniculate, rarely branched, large conidial scar at subtruncate tip, 25- 100 x 3-5 µm

2-10 in a fascicle, medium dark brown, uniform in

multiseptate, not branched, mildly geniculate, subtruncate at the apex, scars large and conspicuously thickened, 35-200 x

colour,

4-6 µm

5-8 in a small fascicle, pale olivacous brown, paler upwards, smooth wall, straight to mildly curved, not branched, geniculate, large conidial scar thickened and darkened, 45-300 x

5-5.5 µm

hyaline, acicular, sometimes curved, multiseptate, base truncate, tip acute, 40- 130 x 2-3 µm

hyaline, acicular, straight to curved, indistinctly multiseptate, hilum

thickened and darkened, 45- 300 x 2.5-5 µm

hyaline, solitary, acicularobclavate, subacute tip, truncate base, straight to curved, multiseptate, hilum

thickened and darkened, 45- 180 x 4.5-5.5

µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

<sup>11706</sup>FR

<sup>11698</sup>FR

<sup>11718</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

present

*Cercospora helianthicola*

*Cercospora kikuchii* 

*Cercospora labiatacearum* *Helianthus annuus*  (Sunflower)

*Glycine max*

*Pogostemon cablin*  (Patchouli)

lacking

(Soybean) present


Taxonomic Review of and Development

*Sesbania sesban*  (Sesbania)

*Cassia alata*  (Ring worm bush)

*Syndrella nodiflora*  (syndrella)

*Cercospora sesbaniae* 

*Cercospora simulate* 

*Cercospora syndrellae* 

of a Lucid Key for Philippine Cercosporoids and Related Fungi 19

2-15 in a fascicle, pale to very pale olivaceous brown, uniform in colour, 1-4 septate, width irregular, not branched, mildly geniculate, conidial scar conspicuously thickened, 20-65 x

hyaline, acicular, straight to curved, multiseptate, truncate base, tip obtuse, 25- 60 x 2.5-3.5 µm

hyaline, cylindroobclavate, straight to mildly curved, septate, base obconically truncate, tip obtuse, hilum thickened and darkened, 30- 100 x 2.5-4 µm

hyaline, cylindrical to obclavate, straight to curved, multiseptate, obconically truncate base, tip rounded, 45-100 x 2.5-4.5

µm

2.5-5.5 µm

2-15 in a fascicle, dark brown in colour, paler the tip, irregular width, not branched, upper portion mildly geniculate, multiseptate, medium conidial scar at subconic tip, 50-280 x 3.5-6

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

11727 FR

<sup>11726</sup>FR

<sup>11695</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

present

lacking

lacking

µm

5-10 in a fascicle, pale to olivaceous brown, straight to mildly curved, not branched, mildly geniculate,

multiseptate, large conidial scar thickened and darkened, 40-90 x

5-6.5 µm


3-12 in a fascicle, pale to medium olivaceous brown, multiseptate, rarely branched, straight, mildly geniculate, conidial scars conspicuously thickened, 20-110 x

hyaline, acicular, acute to subacute at the apex, truncate at the base with a thickened hilum, 30-130 x

3-4 µm

hyaline, acicular to obclavate, straight to mildly curved, indistinctly multiseptate, subacute to subobtuse at the apex, subtruncate to truncate at the base, hilum thickened and darkened, 20- 100 x 2-4 µm

hyaline, cylindric, straight to curved, indistinctly multiseptate, acute at the apex, truncate at the base, hilum

thickened and darkened, 30- 150 x 2-4.5 µm

4-6 µm

densely

fasciculate, pale olivaceous brown, fairly uniform in colour and width, sparingly septate, not branched, geniculate, large conidial scar present at

subtruncate tip, 20- 250 x 4-8 µm

2-5 in a small fascicle, olivaceous brown, slightly paler towards the apex, multiseptate, rarely branched, straight, mildly geniculate, large conidial scar present, 20-110 x 4-

5.5 µm

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

<sup>11723</sup>AR

<sup>11719</sup>AR

<sup>11702</sup>AR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

present

present

lacking

*Cercospora pulcherrimae*

*Cercospora ricinella* 

*Cercospora sesame* 

*Euphorbia pulcherrema* (Poinsettia)

*Ricinus communis*  (Castor bean)

*Sesamum orientale* (Sesame)


Taxonomic Review of and Development

*Pseudocerc ospora abelmoschi*

*Pseudocerc ospora alternanth eraenodiflorae*

*Alternanthera nodiflora*  (Alternenthera)

*Abelmoschus esculentus*  (Okra)

new records of *Pseudocercospora* diseases in the Philippines.

lacking or small

present

µm

10-25 in a divergent fascicle, emerging through the stromata, pale brown, straight to curved, not branched, sometimes geniculate, septate, scars inconspicuous, 10-55 x 4-5 µm

**Species Host Stromata Conidiophores Conidia** 

densely

fasciculate, pale to medium olevaceous brown, uniform in colour, 0-3 septate, sometimes constricted at the septa, irregular in width or slightly clavate, simple or branched, sparingly geniculate, sinuous, rounded or conically truncate at the apex, 10-50 x 3-5

of a Lucid Key for Philippine Cercosporoids and Related Fungi 21

*Pseudocercospora* was introduced by Spegazzini (1910). Deighton (1976) re-introduced this name and widened the concept of this genus considerably to include a wide range of cercosporoids with pigmented conidiophores and inconspicuous, unthickened, not darkened conidiogenous loci. It is the second largest Cercosporoid genus, with more than 300 published names (Kirk *et al.* 2001). In Taiwan, 198 species of *Pseudocercospora* have been recognized by Hsieh & Goh (1990). In the present study, 20 *Pseudocercospora* sp. were reported, of which 14 species caused diseases on medicinal plants (Table 3). There were 12

> **Ref. Coll. Accession No.**

> > CALP

CALP

11736 FR

<sup>11746</sup>AR

pale olivaceous to brown, obclavate to cylindric, straight to mildly curved, septate, acute to subobtuse at the apex, obconic to obconicaly truncate at the base, hilum unthickened and inconspicuous, 25- 80 x 2.5-4 µm

subhyaline to very pale olivaceous, obclavate with gradual attenuation, hyaline to pale brown, straight to mildly curved, tip subobtuse, base obconic, 3-12 septate, sometimes constricted at the septa, hilum unthickened and inconspicuous, 30- 110 x 2.5-4.5 µm

**Status of collection**


Reference: Chupp (1954); Ellis (1971, 1976); Guo & Hsieh (1995); Guo *et al.* (1998); Shin & Kim (2001); Vasudeva (1963).

\* AR= already reported, FR= first record, NHR= new host record.

Table 2. List and descriptions of formerly reported *Cercospora* species, that were reclassified in this study as of *Cercospora s. str.*

#### **3. Genus** *Pseudocercospora*

*P*s*eudocercospora* Speg (Crous & Braun, 2003).

*Stromata* lacking to well developed, usually pigmented; *conidiophores* are pigmented, pale olivaceous to medium dark brown with *conidiogenous loci* inconspicuous, unthickened and not darkened but somewhat refractive or rarely very slightly darkened, or only outer rim slightly darkened and refractive; *conidia* subhyaline to pigmented, solitary or catenulate, scolecosporous, hila unthickened and not darkened (Table 3).

**Type species:** *Pseudocercospora vitis* (Lev.) Speg.

hyaline, narrowly obclavate or filiform, straight, 3-10 septate, acute at the apex, obconic or long obconically truncate at the base; hilum thickened and darkened, 25- 90 x 2-3.5 µm

hyaline, cylindric to obclavate, straight to slightly curved, multiseptate, rounded apex and base obconically truncate, hilum conspicuously thickened, 25- 50 x 3.5-4 µm

**Ref. Coll. Accession No.** 

CALP

CALP

<sup>11692</sup>FR

<sup>11714</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

densely

x 2-3 µm

3-4.5 µm

Reference: Chupp (1954); Ellis (1971, 1976); Guo & Hsieh (1995); Guo *et al.* (1998); Shin & Kim (2001);

Table 2. List and descriptions of formerly reported *Cercospora* species, that were reclassified

*Stromata* lacking to well developed, usually pigmented; *conidiophores* are pigmented, pale olivaceous to medium dark brown with *conidiogenous loci* inconspicuous, unthickened and not darkened but somewhat refractive or rarely very slightly darkened, or only outer rim slightly darkened and refractive; *conidia* subhyaline to pigmented, solitary or catenulate,

2-8 in a fascicle, pale olivaceous brown, uniform in colour, straight to slightly curved, not branched, septate, mildly geniculate, conidial scars conspicuously thickened and darkened, 25-65 x

fasciculate, very pale olivaceous, cylindric, erect or sinuous, rarely septate or geniculate, truncate or rounded at the apex, conidial scars thickened conspicuous, 10-50

present

lacking

\* AR= already reported, FR= first record, NHR= new host record.

scolecosporous, hila unthickened and not darkened (Table 3).

*Cercospora tagetiserectae* 

*Cercospora tithoniae* 

Vasudeva (1963).

*Tagetes erecta*  (Marygold)

*Tithonia diversifolia*  (African sunflower)

in this study as of *Cercospora s. str.*

**3. Genus** *Pseudocercospora*

*P*s*eudocercospora* Speg (Crous & Braun, 2003).

**Type species:** *Pseudocercospora vitis* (Lev.) Speg.

*Pseudocercospora* was introduced by Spegazzini (1910). Deighton (1976) re-introduced this name and widened the concept of this genus considerably to include a wide range of cercosporoids with pigmented conidiophores and inconspicuous, unthickened, not darkened conidiogenous loci. It is the second largest Cercosporoid genus, with more than 300 published names (Kirk *et al.* 2001). In Taiwan, 198 species of *Pseudocercospora* have been recognized by Hsieh & Goh (1990). In the present study, 20 *Pseudocercospora* sp. were reported, of which 14 species caused diseases on medicinal plants (Table 3). There were 12 new records of *Pseudocercospora* diseases in the Philippines.


Taxonomic Review of and Development

*Pseudocerc ospora blumeae*

*Pseudocerc ospora bixicola*

*Pseudocerc ospora borreriae*

*Borreria micrantha*  (Borreria)

*Bixa orellanae*  (Bixa)

*Blumea balsamifera*  (Sambong)

of a Lucid Key for Philippine Cercosporoids and Related Fungi 23

pale olivaceous brown, mostly cylindrical, rarely obclavate, straight to slightly curved, septate, subobtuse

to broadly rounded at the apex, subtruncate to long obconically truncate at the base, hilum unthickened. 30- 110 x 3.5-5.5 µm

pale olivaceous, obclavate-

cylindric, straight to mildly curved, indistinctly 3-6 septate, subobtuse at the apex, obconic or obconically rounded at the base, hilum unthickened, , 30- 60 x 2-3 µm

subhyaline to pale or medium olevaceous, cylindric to obclavato-

cylindric, straight to mildly curved, 3-9 septate, base obconic to obconically truncate, hilum unthickened and inconspicuous, 30- 90 x 2.5-5 μm

5-25 in a fascicle, cylindric, pale to medium brown, uniform in colour and width, straight to curved, not branched, septate, mildly geniculate, rounded to truncate at the apex, conidial

**Ref. Coll. Accession No.**

CALP

CALP

CALP

11740 FR

<sup>11738</sup>FR

<sup>11739</sup>AR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

scars

densely

scars

unthickened, 15- 70 x 4-5 µm

fasciculate, pale olivaceous, cylindrical, septate,

branched, rarely geniculate, conically rounded at the apex, conidial

unthickened or inconspicuous, 15-40 x 2.5-4 µm

medium to dark brown, arise singly, uniform in colour, irregular in width, multiseptate, branched, slightly geniculate, curved to tortuous, small spore scars at bluntly rounded tip, 35-220 x 3-5.5

lacking

lacking

lacking or small

μm


pale olivaceous, cylindric to obclavato-

cylindric, straight to mildly curved, 2-8 septate, subobtuse to broadly rounded at the apex, sharply obconic or obconically truncate at the base, hilum unthickened and not darkened, 15- 90 x 2.5-5 µm

subhyaline, solitary, filiform to

narrowly

obclavate, straight to mildly curved, septate, subacute at the apex, truncate to obconically truncate at the base, hilum unthickened and not darkened; 30- 70 x 1.5-3 µm

**Ref. Coll. Accession No.**

CALP

CALP

<sup>11741</sup>AR

<sup>11679</sup>AR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

4-15 in a divergent fascicle, pale olivaceous to olivaceous brown, longer ones curved, sharply bent or undulate, branched, septate, sometimes slightly

constricted at the some septa, rarely geniculate, conic at the apex,

inconspicuous, 10-50 x 3-5 µm

10-40 in a divergent fascicle, pale olivaceous brown to brown , irregular in width, substraight to mildly curved, not geniculate, not branched, multiseptate, conidial scars inconspicuous, 15-50 x 2-3 µm

scars

*Pseudocerc ospora atromarginalis*

*Pseudocerc ospora balsaminicola*

*Impatiens balsamina*  (Balsam plant)

well develop ed

*Solanum nigrum*  (Black night shade)

lacking


Taxonomic Review of and Development

*Lantana camara*  (Lantana)

*Lycopersicon esculentum*  (Tomato)

well develop ed

lacking

*Pseudocerc ospora formosana*

*Pseudocerc ospora fuligena*

*Pseudocerc ospora gmelinae* 

*Gmelina arborea*  (Yemen)

of a Lucid Key for Philippine Cercosporoids and Related Fungi 25

narrowly obclavate, very pale olivaceous, straight to curved, indistinctly multiseptate, base long obconically truncate, tip subacute, hilum unthickened and inconspicuous, 30- 100 x 2.5-3.5 µm

subhyaline to pale olivaceous, cylindric to cylindro-obclavate, straight to mildly curved, rounded to obtuse at the apex, obconically

truncate, multiseptate, hilum

2-4 µm

unthickened, not darkened, 25-110 x

subhyaline to pale olivaceous brown, cylindro-obclavate, straight to mildly curved, base attenuated, tip subacute, hilum unthickened and inconspicuous, 30- 80 x 3-4.5 μm

**Ref. Coll. Accession No.**

CALP

CALP

CALP

11754 AR

<sup>11748</sup>AR

<sup>11747</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

small fascicle, very pale olivaceous to brown, sparingly septate, not geniculate, straight to undulate, scars inconspicuous, 25-40 x 3-4 µm

loosely fasciculate usually 5-15 per fascicle, pale olivaceous to very pale olivaceous brown, uniform in color, straight to sinuous, tip rounded or truncate, sometimes geniculate, not branched, septate, conidial

scars

unthickened, 15- 45 x 3-5 µm

2.8 in a small fascicle, pale olivaceous brown, straight to mildly geniculate, smooth, unbranched, septate, scars inconspicuous, 30-50 x 3-4.5 μm

lacking


pale olivaceous brown, obclavate or obclavatocylindric, straight to mildly curved, multiseptate, rounded at the apex, obconically truncate at the base, hilum unthickened and inconspicuous, 25- 100 x 3-5 µm

subhyaline to very pale olivaceous brown, cylindric to obclavate, straight to mildly curved, septate, apex rounded and base subtruncate, hilum unthickened, reflective, 20-80 x

3-5 µm

curved, multiseptate, subacute to obtuse at the apex, sharply obconic or obconically truncate at the base, hilum unthickened and inconspicuous, 25- 130 x 2-5 µm

subhyaline to very pale olivaceous brown, cylindric or cylindro-obclavate, straight to mildly

2-20 in a fascicle, emerging through stomata, olivaceous brown, cylindrical, septate, rarely geniculate, very rarely branched,

**Ref. Coll. Accession No.**

CALP

CALP

CALP

<sup>11737</sup>AR

<sup>11753</sup>FR

<sup>11742</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

scars

scars

inconspicuous, 18-55 x 3.5-5 µm

dense fascicle, subhyaline to pale olivaceous brown, straight to sinuous or mildly geniculate, occationally branched, septate, conic at the apex, scars inconspicuous, 15-60 x 3-5 µm

inconspicuous, 15-40 x 3-5 µm

olivaceous brown, in a dense fascicle, paler and narrower the tips, straight to mildly curved, not geniculate,

present

present

small

*Pseudocerc ospora chrysanthe micola*

*Pseudocerc ospora corchorica* 

*Pseudocerc ospora cruenta*

*Chrysanthe mum morifolium*  (Chrysanth emum)

*Corchorus capsularis*  (Jute)

*Phaseolus lunatus*  (Lima bean)


Taxonomic Review of and Development

*Sesbania sesban*  (Sesbania)

*Solanum melongena*  (Eggplant)

*Synedrella nodiflora*  (Syndrella)

*Pseudocerc ospora sesbanicola*

*Pseudocerc ospora solanimelongenicola*

*Pseudocerc ospora synedrellae*

of a Lucid Key for Philippine Cercosporoids and Related Fungi 27

pale olivaceous brown, cylindric, sometimes obclavato-

cylindric, straight or slightly curved, septate, rounded at the apex, truncate at the base, hilum unthickened, 18-30

olivaceous to pale brown, cylindric to cylindro-obclavate, straight to slightly curved, obtuse at

subhyaline to very pale olivaceous, narrowly obclavate or filiform, straight to slightly curved, indistinctly multiseptate, subacute at the apex, obconically truncate at the base, hilum unthickened and inconspicuous, 15- 90 x 2-3 µm

x 3-4.5 μm

the apex, obconically truncate at the base, hilum visible but not thickened, 30-100 x 3.5-5 µm

5-20 in a fascicle, emerging from stromata,

septate, rounded at the apex, scars inconspicuous, 15-65 x 3-4 μm

5-10 in a fascicle, pale olivaceous brown, paler towards the apex, straight or geniculate, sometimes branched, septate, conidial scars visible but not thickened, 30- 60 x 3.5-4.5 µm

2-15 in a fascicle , pale olivaceous brown, simple or branched, straight or slightly undulate, septate, sometimes constricted at the

septa, not geniculate, rounded or conical at the apex, 5-30 x 2-4

µm

**Ref. Coll. Accession No.**

CALP

CALP

CALP

11750 FR

<sup>11749</sup>FR

<sup>11752</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

lacking

present

lacking


pale olivaceous, subcylindric to slightly obclavate, straight or slightly curved, smooth, thin-walled, obtuse at the apex, shortly tapered at the base, hilum unthickened and inconspicuous, 18- 60 x 1.5-2.5 µm

subhyaline, cylindric to narrowly

x 3-4 µm

subhyaline, obclavate, straight to slightly curved, septate, rounded at

the apex, obconocally truncate base with unthickened hilum, 35-60 x 3-

5.5 µm

obclavate, straight to mildly curved, septate, subacute to subobtuse at the apex, truncate at the base, hilum unthickened 25-60

**Ref. Coll. Accession No.**

CALP

CALP

CALP

<sup>11743</sup>FR

<sup>11744</sup>FR

<sup>11745</sup>FR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

numerous, pale olivaceous, straight or slightly sinuous, sometimes slightly geniculate, smooth, simple,

septate, subtruncate at the apex, 5-30 x 2-4 µm, conidial

scars

densely

and

densely

unthickened

fasciculate, pale to very pale olivaceous brown, conidial scar unthickened

inconspicuous, 10-40 x 3-5 µm

fasciculate, pale olivaceous to yellowish brown, not branched, septate, mildly geniculate, conidial scar visible but not thickened, 10-35 x 2.5-4 µm

present

lacking

small

*Pseudocerc ospora jasminicola*

*Pseudocerc ospora ocimicola*

*Pseudocerc ospora pachyrrhizi* *Jasminum grandifloru*

(Jasmine)

*Ocimum basilicum*  (Sweet basil)

*Pachyrrhizus erosus*  (Turnip)

*m* 


Taxonomic Review of and Development

*Amaranthus viridis*  (Amaranth)

*Bougainvil lea spectabilis*  (Bougainvilla)

*Manihot esculenta*  (Cassava) well developed

*Passalora amaranthae*

*Passalora bougainvilleae*

*Passalora henningsii*

descriptions of the genus *Passalora* (Table 4).

well developed

**Species Host Stromata Conidiophores Conidia** 

brown, multiseptate, straight or slightly curved, not branched, moderately geniculate, conspicuously thickened small conidial scars,25- 100 x 3.5-6.5 µm

lacking 5-8 in a small

fascicle, pale to olivaceous brown, smooth, straight to

densely fasciculate, subhyaline to pale olivaceous brown, uniform in colour and width, 1-3 septate, straight or slightly curved, not branched, mildly geniculate, conidial scars conspicuously thickened, 15-50 x

3.5-5 µm

geniculate, aseptate, conidial scar conspicuous and darkened, 10- 75 x 5-7.5 µm

of a Lucid Key for Philippine Cercosporoids and Related Fungi 29

Bougainvilla, (Ponaya & Cumagun, 2008), *P. henningsii* on *Manihot esculenta,* formerly named as *Cercospora cassavae* Ellis & Everh. /*C. manihots* Henn*. / C. henningsii* Allesch (Crous and Braun, 2003) and *P. tinosporae* on *Tinospora reticulate.* The same host species and the associated fungi were reviewed in the study and were found to confirm with the

> densely fasciculate, pale to olivaceous

**Ref. Coll. Accession No.** 

CALP

CALP

CALP

11756 AR

<sup>11758</sup>AR

<sup>11757</sup>NS

pale olivaceous, cylindric or obclavatocylindric, straight to slightly curved, 3-7 septate, bluntly rounded at the apex, obconic at the base , small thickened hilum, 25-65 x 3.5-6.5 µm

pale to olivaceous brown, solitary, smooth, slightly

curved, cylindrical to obclavatocylindric, 3-6 septa, truncate at the base and rounded at the apex, hilum thickened and darkened, 30-65 x

5-10 µm

pale olivaceous, cylindric, straight to slightly curved, 3-6 septate, bluntly rounded at the apex, obconic at the base with a small thickened hilum, 25-60 x 4-6.5 µm

**Status of collection**


Reference: Chupp (1954); Ellis (1971); Guo & Hsieh (1995); Guo *et al.* (1998); Hsieh & Goh (1990); Saccardo (1886); Shin & Kim (2001); Vasudeva (1963); Yen & Lim (1980). \* AR= already reported, FR= first record.

Table 3. List and descriptions of *Pseudocercospora* species found in this study.

#### **4. Genus** *Passalora*

*Passalora* Fr. (Crous & Braun, 2003).

*Stromata* absent to well developed; *conidiophores* are solitary or loosely to densely fasciculate, unbranched or branched, continuous to pluriseptate, subhyaline to pigmented, conidiogenous loci conspicuous, scars thickened and darkened-refractive, c*onidia* solitary to catenate, simple or branched, amerosporous to scolecosporous, pale to distinctly pigmented (if subhyaline, conidia non-scolecosporous), smooth to finely verruculose, with few septa, hila thickened and darkened-refractive, more or less truncate (Crous & Braun, 2003).

**Type species:** *Passalora bacilligera* (Mont. & Fr.)

#### **4.1 Dichotomous Key to the Species** *Passalora*


Approximately 550 names of *Passalora* that have been published or amended in the world, compiled by Crous and Braun (2003). In this study, four already known and recorded *Passalora* species were found, namely *Passalora personata* on *Arachis hypogaea* (Quimio and Abilay, 1977), formerly named as *Cercospora arachidis*, *P. bougainvilleae* causing leaf spot of

subhyaline to pale olivaceous, cylindric to obclavate, straight to mildly curved, septate, acute to subacute at the apex and obconically truncate at the base, hilum unthickened 18-50 x 2.5-3.5 μm

**Ref. Coll. Accession No.**

CALP

<sup>11751</sup>AR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

Saccardo (1886); Shin & Kim (2001); Vasudeva (1963); Yen & Lim (1980).

pale olivaceous, borne singly or densely fasciculate, straight, not branched, not geniculate, 5-35 x 2-3.5 µm, conidial scar inconspicuous

Reference: Chupp (1954); Ellis (1971); Guo & Hsieh (1995); Guo *et al.* (1998); Hsieh & Goh (1990);

*Stromata* absent to well developed; *conidiophores* are solitary or loosely to densely fasciculate, unbranched or branched, continuous to pluriseptate, subhyaline to pigmented, conidiogenous loci conspicuous, scars thickened and darkened-refractive, c*onidia* solitary to catenate, simple or branched, amerosporous to scolecosporous, pale to distinctly pigmented (if subhyaline, conidia non-scolecosporous), smooth to finely verruculose, with few septa,

1. Stromata lacking---------------------------------------------------------------------------- *P. bougainvilleae*  1. Stromata present, sometimes well developed----------------------------------------------------------2 2. Conidiophores strongly geniculate--------------------------------------------- *P. personata* 2. Conidiophores straight to slightly geniculate-----------------------------------------------3 3. Conidiophores aseptate----------------------------------------------------------------------- *P. tinosporae* 3. Conidiophores septate----------------------------------------------------------------------------------------4 4. Conidiophores up to 50 um long------------------------------------------------ *P. henningsii* 

Approximately 550 names of *Passalora* that have been published or amended in the world, compiled by Crous and Braun (2003). In this study, four already known and recorded *Passalora* species were found, namely *Passalora personata* on *Arachis hypogaea* (Quimio and Abilay, 1977), formerly named as *Cercospora arachidis*, *P. bougainvilleae* causing leaf spot of

4. Conidiophores up to 100 um long--------------------------------------------- *P. amaranthae* 

hila thickened and darkened-refractive, more or less truncate (Crous & Braun, 2003).

Table 3. List and descriptions of *Pseudocercospora* species found in this study.

well develop ed

*Pseudocerc ospora viticis*

*Vitex nigundo*  (Lagundi)

\* AR= already reported, FR= first record.

*Passalora* Fr. (Crous & Braun, 2003).

**Type species:** *Passalora bacilligera* (Mont. & Fr.)

**4.1 Dichotomous Key to the Species** *Passalora*

**4. Genus** *Passalora*

Bougainvilla, (Ponaya & Cumagun, 2008), *P. henningsii* on *Manihot esculenta,* formerly named as *Cercospora cassavae* Ellis & Everh. /*C. manihots* Henn*. / C. henningsii* Allesch (Crous and Braun, 2003) and *P. tinosporae* on *Tinospora reticulate.* The same host species and the associated fungi were reviewed in the study and were found to confirm with the descriptions of the genus *Passalora* (Table 4).


Taxonomic Review of and Development

*Asperisporium* Maublanc. (Crous & Braun, 2003)

**Type species:** *Asperisporium caricae* (Speg.) Maubl.

described in the Philippines (Cumagun and Padilla, 2007).

well developed

Reference: Chupp (1954); Ellis (1971); Ellis & Holliday (1972).

**Species Host Stromata Conidiophores Conidia** 

**4.2 Genus** *Asperisporium*

septa (Ellis, 1971).

*Asperisporium moringae*

*oleifera.*

*Moringa oleifera (Moringa)*

\*AR= already reported. FR= first record.

*Periconiella* Saccardo (Ellis, 1971).

**4.3 Genus** *Periconiella*

of a Lucid Key for Philippine Cercosporoids and Related Fungi 31

*Sporodochia* punctiform, pulvinate, brown, olivaceous brown or black. *Mycelium* immersed. Stromata usually well-developed, erumpent. *Conidiophores* macronematous, mononematous, closely packed together forming sporodochia, usually rather short, unbranched or occasionally branched, straight or flexuous, hyaline to olivaceous brown, smooth, polyblastic, scars prominent. *Conidia* solitary, ellipsoidal, fusiform, obovoid, pyriform, clavate or obclavate, hyaline to brown or olivaceous brown, smooth or verrucose, with 0-3

The genus *Asperisporium,* introduced by Maublanc (1913) resembles *Passalora*, but differs in having verrucose conidia (Ellis 1971, 1976; von Arx, 1983). In the present study, only one species of *Asperisporium* was identified (Table 5). It was *Asperisporium moringae* (Thirum. & Govindu) Deighton on *Moringa oleifera*. This disease was reported in the Philippines by Quimio & Abilay (1977), with *Cercospora moringae* (Thirum. & Govindu) as the causal pathogen. The black spot of papaya caused by *Asperisporium caricae* was first recorded and

> pale olivaceous brown, straight, geniculate, 10-35 x 4-6 µm

Table 5. Characteristics of *Asperisporium moringae* associated with leaf spot of *Moringa* 

*Stromata* none; *conidiophores* macronematous, mononematous, each composed of an erect, straight or flexious, brown to dark blackish brown, smooth or verruculose; *conidiogenous cells*  polyblastic, integrated and terminal, sympodial, cylindrical, scars often numerous; *Conidia* solitary or occasionally in very short chains, simple, ellipsoidal, obclavate or obovoid,

**Ref. Coll. Accession No.** 

CALP

<sup>11761</sup>AR

pale olivaceous

brown, obclavate conico-truncate at the base, verruculose walled, mostly 1-2 septate, 20- 45 x 5.5-7.5 µm

**Status of collection**


Reference: Chupp (1954); Ellis (1971, 1976); Guo & Hsieh (1995); Guo *et al.* (1998); Hsieh & Goh(1990); Katsuki (1965); Saccardo (1886); Shin & Kim (2001); Vasudeva (1963). \* NS= new species, AR= already reported, FR= first record.

Table 4. List and descriptions of different species of *Passalora* that were formerly classified as *Cercospora* species in the Philippines.

A species associated with *Amaranthus viridis* was believed to be new. Its characteristics are close to *P.henningsii* in terms of having amphigenous colonies and with shorter conidiophores (15-50 µm). However, its characteristics are different from others, having darker septation on conidia, and its association with a new host that warrants a new species. The proposed new species was *Passalora amaranthae* on *Amaranthus viridis.*

#### **4.2 Genus** *Asperisporium*

30 Plant Pathology

solitary, subhyaline to pale olivaceous, filiform or obclavate or obclavato-

cylindric, usually very finely rough-walled, obtuse or

broadly rounded at the apex, truncate or obconically truncate at the base, hilum conspicuously thickened and darkened, 2-10 septate, 20-80 x 4-7.5 µm

subhyaline to very pale, cylindrical to obclavate, aseptate, straight to curved, obconically truncate base, subobtuse tip, 10-45 x 4-6 µm

densely fasciculate, pale olivacious, smooth, slightly or

geniculate, straight or slightly curved, not branched, 0-3 septate, conidial

**Ref. Coll. Accession No.** 

CALP

CALP

<sup>11760</sup>AR

<sup>11755</sup>AR

**Status of collection**

**Species Host Stromata Conidiophores Conidia** 

strongly

scars

conspicuously thickened, 20-90 x

3.5-6.5 µm

present densely fasciculate,

geniculate, multiseptate, conidial scars conspicuous and at bluntly rounded tip, 25-110 x 3.5-5.5

µm

The proposed new species was *Passalora amaranthae* on *Amaranthus viridis.*

Katsuki (1965); Saccardo (1886); Shin & Kim (2001); Vasudeva (1963).

\* NS= new species, AR= already reported, FR= first record.

*Cercospora* species in the Philippines.

subhyaline to very pale, not branched,

Reference: Chupp (1954); Ellis (1971, 1976); Guo & Hsieh (1995); Guo *et al.* (1998); Hsieh & Goh(1990);

Table 4. List and descriptions of different species of *Passalora* that were formerly classified as

A species associated with *Amaranthus viridis* was believed to be new. Its characteristics are close to *P.henningsii* in terms of having amphigenous colonies and with shorter conidiophores (15-50 µm). However, its characteristics are different from others, having darker septation on conidia, and its association with a new host that warrants a new species.

*Passalora personata*

*Passalora tinosporae* *Tinospora reticulate*  (Makabuhay)

*Arachis hypogea*  (Peanut) well developed

#### *Asperisporium* Maublanc. (Crous & Braun, 2003)

*Sporodochia* punctiform, pulvinate, brown, olivaceous brown or black. *Mycelium* immersed. Stromata usually well-developed, erumpent. *Conidiophores* macronematous, mononematous, closely packed together forming sporodochia, usually rather short, unbranched or occasionally branched, straight or flexuous, hyaline to olivaceous brown, smooth, polyblastic, scars prominent. *Conidia* solitary, ellipsoidal, fusiform, obovoid, pyriform, clavate or obclavate, hyaline to brown or olivaceous brown, smooth or verrucose, with 0-3 septa (Ellis, 1971).

#### **Type species:** *Asperisporium caricae* (Speg.) Maubl.

The genus *Asperisporium,* introduced by Maublanc (1913) resembles *Passalora*, but differs in having verrucose conidia (Ellis 1971, 1976; von Arx, 1983). In the present study, only one species of *Asperisporium* was identified (Table 5). It was *Asperisporium moringae* (Thirum. & Govindu) Deighton on *Moringa oleifera*. This disease was reported in the Philippines by Quimio & Abilay (1977), with *Cercospora moringae* (Thirum. & Govindu) as the causal pathogen. The black spot of papaya caused by *Asperisporium caricae* was first recorded and described in the Philippines (Cumagun and Padilla, 2007).


Reference: Chupp (1954); Ellis (1971); Ellis & Holliday (1972). \*AR= already reported. FR= first record.

Table 5. Characteristics of *Asperisporium moringae* associated with leaf spot of *Moringa oleifera.*

#### **4.3 Genus** *Periconiella*

*Periconiella* Saccardo (Ellis, 1971).

*Stromata* none; *conidiophores* macronematous, mononematous, each composed of an erect, straight or flexious, brown to dark blackish brown, smooth or verruculose; *conidiogenous cells*  polyblastic, integrated and terminal, sympodial, cylindrical, scars often numerous; *Conidia* solitary or occasionally in very short chains, simple, ellipsoidal, obclavate or obovoid,

Taxonomic Review of and Development

data were entered for developed Lucid key.

Fig. 1. Screen shot from the Lucid Builder.

Cercosporoid species on the right side.

**5.1 Lucid Builder** 

of a Lucid Key for Philippine Cercosporoids and Related Fungi 33

Lucid key consists of two programs: Lucid Builder and Lucid Player. The first program is a key development tool (Fig. 1) that allows taxonomists to input their knowledge base into a form that is readily accessible by other people. A Lucid Builder enables key developers to easily build their own keys. In the present study, information from 74 Cercosporoid fungi

In Lucid Builder, data were incorporated for example for *Cercospora adiantigena* on *Adiantum phillipense* (Fig. 2). The right side of the screen shows, all species that were inputted while left side provides the inputted characters for specific species. The information that were entered on the left side of the screen corresponds to the highlighted

hyaline or rather pale olive or olivaceous brown, without septa or with one or a few transverse septa.

**Type species:** *Periconiella velutina* (Wint.) Sacc.

In the present study, only one *Periconiella lygodii* on *Lygodium japonicum* was reported (Table 6). Four species of *Periconiella* have been reported to occur on ferns (Braun, 2004). He noted that *P.lygodii* is distinguished from all other species of *Periconiella* on ferns by having long, obclavate-cylindrical, pluriseptate, smooth conidia. This is the first record of *P. lygodii* on *Lygodium japonicum* in the Philippines, (Begum et al. 2009).


Reference: Chupp (1954); Ellis (1971); Braun (2004). \* FR =First Record.

Table 6. Characteristics of *Periconiella lygodii* associated with leaf spot of *Lygodium japonicum.*

#### **5. Lucid key for Philippine cercosporoid fungi**

Before the advent of computers, the traditional way in which scientists identify identify biological specimens was through the use of printed pathway (or dichotomous) keys. However, with the advent of database and multi-media-software, it is now possible to store large amounts of biological data and to access this information through easy-to-use matrixbased (or multi-access) keys. Lucid is one example of a multi-media matrix key.The Lucid Program from The Centre for Biological Information Technology CBIT, University of Queensland, which was licensed to the third author was used to develop the Lucid key. The activities involved in the process of designing, developing, producing and publishing a Lucid key on CD, DVD or the Internet are outlined as follows: (1) Establishing the scope of the key; (2) designing and scoring the key; (3) sourcing, developing and editing facts sheets, images and other multi-media associated with features and entities; (4) packaging up a prototype on CD or on the Internet; (5) beta testing and user testing of the key; (6) graphic design of CD and CD-insert or web; and (7) packing of key for release.

#### **5.1 Lucid Builder**

32 Plant Pathology

hyaline or rather pale olive or olivaceous brown, without septa or with one or a few

In the present study, only one *Periconiella lygodii* on *Lygodium japonicum* was reported (Table 6). Four species of *Periconiella* have been reported to occur on ferns (Braun, 2004). He noted that *P.lygodii* is distinguished from all other species of *Periconiella* on ferns by having long, obclavate-cylindrical, pluriseptate, smooth conidia. This is the first record of

> **Ref. Coll. Accession No.**

> > CALP 11680 and BRIP 52369

**Status of collection** 

FR

*P. lygodii* on *Lygodium japonicum* in the Philippines, (Begum et al. 2009).

Medium to medium –dark brown, straight, multiseptate, thick-walled, branched apical portion, two to four times

pale

olivaceous or olivaceous brown, obclavatecylindrical, smooth, conicotruncate at the base, apex obtuse or subobtuse, 1- 5 septate, 25- 75 x 3-5.5 µm

dichotomously or occasionally trichotomously branched, 90-350 x 2-5.5 µm

Table 6. Characteristics of *Periconiella lygodii* associated with leaf spot of *Lygodium japonicum.*

Before the advent of computers, the traditional way in which scientists identify identify biological specimens was through the use of printed pathway (or dichotomous) keys. However, with the advent of database and multi-media-software, it is now possible to store large amounts of biological data and to access this information through easy-to-use matrixbased (or multi-access) keys. Lucid is one example of a multi-media matrix key.The Lucid Program from The Centre for Biological Information Technology CBIT, University of Queensland, which was licensed to the third author was used to develop the Lucid key. The activities involved in the process of designing, developing, producing and publishing a Lucid key on CD, DVD or the Internet are outlined as follows: (1) Establishing the scope of the key; (2) designing and scoring the key; (3) sourcing, developing and editing facts sheets, images and other multi-media associated with features and entities; (4) packaging up a prototype on CD or on the Internet; (5) beta testing and user testing of the key; (6) graphic

**Species Host Stromata Conidiophores Conidia** 

lacking

Reference: Chupp (1954); Ellis (1971); Braun (2004). \* FR =First Record.

design of CD and CD-insert or web; and (7) packing of key for release.

**5. Lucid key for Philippine cercosporoid fungi** 

transverse septa.

*Periconiella lygodii*

*Lygodium japonicum*  (Japanese climbing fern)

**Type species:** *Periconiella velutina* (Wint.) Sacc.

Lucid key consists of two programs: Lucid Builder and Lucid Player. The first program is a key development tool (Fig. 1) that allows taxonomists to input their knowledge base into a form that is readily accessible by other people. A Lucid Builder enables key developers to easily build their own keys. In the present study, information from 74 Cercosporoid fungi data were entered for developed Lucid key.

Fig. 1. Screen shot from the Lucid Builder.

In Lucid Builder, data were incorporated for example for *Cercospora adiantigena* on *Adiantum phillipense* (Fig. 2). The right side of the screen shows, all species that were inputted while left side provides the inputted characters for specific species. The information that were entered on the left side of the screen corresponds to the highlighted Cercosporoid species on the right side.

Taxonomic Review of and Development

**5.2 Lucid Player** 

Fig. 3. Screen shot from the Lucid Player.

of a Lucid Key for Philippine Cercosporoids and Related Fungi 35

During an identification session, Lucid Player allows one to choose any question in its list at any time, but "stepping" through the key in a structured and sensible way will make one task of identification easier. The guidelines for making identification are as follows (1) familiarity with the specimen; (2) note and use of distinctive features; (3) answering easy

Familiarity with the characteristics of the specimen to be identified is essential. Briefly reviewing Lucid key and specimen's characteristics before one starts will facilitate the identification. In any key, some taxa may possess particularly distinctive features. Use of these may allow the taxon to be keyed out in a very few steps. At the very least, starting with particularly distinctive or striking features for the first character states selected may quickly reduce the list of entities remaining. One can select any features from any position in the list and start by browsing the list of features available for obvious features that one can quite quickly answer, as opposed to getting stuck on the first one. Lucid is designed to overcome problems associated with difficult and obscure features.Always choose multiple states if one is uncertain which state is the correct one to choose for a particular specimen. One can choose as many states as from any one feature. After a preliminary identification has been made, one can check the other information (notes or image) provided for the taxon.

The second program of the Lucid key is key interface or Lucid Player (Fig. 3) through which end-users interact with the Lucid key that has been developed and distributed either as a CD-Rom or via the Internet. The Lucid player enables users to view and interact with the key.

features first; 4) choosing multiple states; and (5) checking the result.

Fig. 2. Screen shot from the Lucid Builder- *Cercospora adiantigena* on *Adiantum phillipense. S*ymptoms of the disease and measurement of morphological characters (Left inset).

During an identification session, Lucid Player allows one to choose any question in its list at any time, but "stepping" through the key in a structured and sensible way will make one task of identification easier. The guidelines for making identification are as follows (1) familiarity with the specimen; (2) note and use of distinctive features; (3) answering easy features first; 4) choosing multiple states; and (5) checking the result.

Familiarity with the characteristics of the specimen to be identified is essential. Briefly reviewing Lucid key and specimen's characteristics before one starts will facilitate the identification. In any key, some taxa may possess particularly distinctive features. Use of these may allow the taxon to be keyed out in a very few steps. At the very least, starting with particularly distinctive or striking features for the first character states selected may quickly reduce the list of entities remaining. One can select any features from any position in the list and start by browsing the list of features available for obvious features that one can quite quickly answer, as opposed to getting stuck on the first one. Lucid is designed to overcome problems associated with difficult and obscure features.Always choose multiple states if one is uncertain which state is the correct one to choose for a particular specimen. One can choose as many states as from any one feature. After a preliminary identification has been made, one can check the other information (notes or image) provided for the taxon.

#### **5.2 Lucid Player**

34 Plant Pathology

Fig. 2. Screen shot from the Lucid Builder- *Cercospora adiantigena* on *Adiantum phillipense. S*ymptoms of the disease and measurement of morphological characters (Left inset).

The second program of the Lucid key is key interface or Lucid Player (Fig. 3) through which end-users interact with the Lucid key that has been developed and distributed either as a CD-Rom or via the Internet. The Lucid player enables users to view and interact with the key.

Fig. 3. Screen shot from the Lucid Player.

Taxonomic Review of and Development

**6. Summary and conclusions** 

*Passalora* were inputted into the Lucid key.

of the move towards providing taxonomic information on-line.

caused by *Asperisporium moringae* was reported on *Moringa oleifera.*

extension work in mycology and plant pathology.

*Biodiversity Series* 5:1 –231.

Press, St Paul, USA. 218.

Agrios, G.N. (2005). Plant Pathology. 5th ed. Academic Press. New York.

**7. References** 

4: 17-18.

of a Lucid Key for Philippine Cercosporoids and Related Fungi 37

present study only the data of the true Cercosporoids like, *Cercospora, Pseudocercospora* and

Identification of Cercosporoid fungi is a difficult task, and the Lucid key was created to help provide individuals with easily accessible tools to distinguish species. Recent experience suggests that computer-based identification keys will become an increasingly important part

The genus *Cercospora* is one of the largest genera of hyphomycetes. i.e., commonly associated with leaf spots and is responsible for great damages to beneficial plants, such as cereals, vegetables, ornamentals, forest trees, grasses. A total of 105 Cercosporoid diseases were identified, belonging to *Cercospora* (48), *Pseudocercospora* (20), *Passalora* (5), *Asperisporium* (1), *Cladosporium* (30,) and *Periconiella* (1). From the reported *Cercospora* species, 20 were *Cercospora apii s.lat* and 28 were *Cercospora s.str.* The first report of *Cercospora basellae-albae* in the Philippines was observed causing leaf spots on *Basella alba cv. Rubra (*Begum and Cumagun*,*  2010*).* Twenty eight first records of Cercospora leaf spots were reported. Among Pseudocercospora leaf spots, 12 first records were reported and all were host specific. A new species of *Passalora amaranthae* found on *Amaranthus viridis* was reported*.* Only one specimen

Lucid key is a powerful and highly flexible knowledge management software application designed to help users with identification and diagnostic tasks. Lucid is one of a multimedia matrix keys, that includes possible the storing of large amounts of information. Lucid key consists of two programs: Lucid Builder and Lucid Player. The first program is a key development tool, which allows developers to easily build their own keys. The second program of the Lucid key is the key interface or Lucid Player, through which end-users interacts with the Lucid key and enables users to view and interact with the key. In the present study, Lucid key was developed to identify Cercosporoid fungi, a total of 74 Cercosporoid fungi and their characters were entered into a program using Lucid Builder. Lucid has the advantage over printed dichotomous keys in that the user is able to skip an unanswerable couplet or question and still proceed with identification. Identification of Cercosporoid fungi is a difficult task, and the Lucid key was created to help provide individuals with easily accessible tools to distinguish species. The end product of the Lucid key of Philippine Cercosporoid fungi, in the future will be useful in teaching, research, and

Aptroot, A. (2006). *Mycosphaerella* and its anamorphs: 2. Conspectus of *Mycosphaerella*. *CBS* 

Barnett, H.L. and Hunter, B.B. (1998): Illustrated Genera of Imperfect Fungi 4th ed. APS

Begum, M.M., Shivas, R.G. and Cumagun, C.J.R. (2009). First record of *Periconiella lygodii*

occurring on *Lygodium japonicum* in the Philippines. *Australasian Plant Disease Notes*

Lucid Player allows one to input a list of character states that best describe the specimen to be identified. These character states can be selected (or de-selected) in any order, resulting in a shortening of the list of remaining taxa that best match the decribed specimen. The upper left side of the screen shows all characters for a given specimen while it's lower left side indicates the characters that were chosen. The upper right side of the screen provides the possible identity of the specimen while the lower right side shows the discarded taxa from the list (Fig. 4).

Fig. 4. Screen shot from the Lucid Player- *Passalora bougainvilleae.* 

In the present study, Lucid key was developed to identify Cercosporoid fungi, even though the dichotomous keys are the most common keys encountered. The use of dichotomous keys has a major disadvantage: if a couplet is difficult or impossible to answer, the identification session often ends there. Lucid has the advantage over printed dichotomous keys in that the user is able to skip an unanswerable couplet or question and still proceed with identification because Lucid key allows to start at any point and proceed in any order. Lucid guide for smut fungi of Australia has already been completed. Its accompanying CD, incorporating a Lucid Player, provides an easy- to-use, interactive key to smut species, with comprehensive fact-sheets, distribution maps, and over 1000 images (Vanky and Shivas, 2008). On the other hand, Lucid guide for smut fungi of Thailand is still underway in collaboration with Australian plant pathologists (Shivas, personal communication). Gerald (2005) reported that, "Diagnosing Postharvest Diseases of Cantaloupe" is the first Lucid key developed in the U.S. for a set of plant diseases and one of the first plant disease identification keys ever developed in Lucid. A Lucid key was developed for the identification of *Phytophthora species* in USA based on morphological and molecular characters (Ristaino *et al.* 2008). In the present study only the data of the true Cercosporoids like, *Cercospora, Pseudocercospora* and *Passalora* were inputted into the Lucid key.

Identification of Cercosporoid fungi is a difficult task, and the Lucid key was created to help provide individuals with easily accessible tools to distinguish species. Recent experience suggests that computer-based identification keys will become an increasingly important part of the move towards providing taxonomic information on-line.

### **6. Summary and conclusions**

36 Plant Pathology

Lucid Player allows one to input a list of character states that best describe the specimen to be identified. These character states can be selected (or de-selected) in any order, resulting in a shortening of the list of remaining taxa that best match the decribed specimen. The upper left side of the screen shows all characters for a given specimen while it's lower left side indicates the characters that were chosen. The upper right side of the screen provides the possible identity of the specimen while the lower right side shows the discarded taxa from

Fig. 4. Screen shot from the Lucid Player- *Passalora bougainvilleae.* 

In the present study, Lucid key was developed to identify Cercosporoid fungi, even though the dichotomous keys are the most common keys encountered. The use of dichotomous keys has a major disadvantage: if a couplet is difficult or impossible to answer, the identification session often ends there. Lucid has the advantage over printed dichotomous keys in that the user is able to skip an unanswerable couplet or question and still proceed with identification because Lucid key allows to start at any point and proceed in any order. Lucid guide for smut fungi of Australia has already been completed. Its accompanying CD, incorporating a Lucid Player, provides an easy- to-use, interactive key to smut species, with comprehensive fact-sheets, distribution maps, and over 1000 images (Vanky and Shivas, 2008). On the other hand, Lucid guide for smut fungi of Thailand is still underway in collaboration with Australian plant pathologists (Shivas, personal communication). Gerald (2005) reported that, "Diagnosing Postharvest Diseases of Cantaloupe" is the first Lucid key developed in the U.S. for a set of plant diseases and one of the first plant disease identification keys ever developed in Lucid. A Lucid key was developed for the identification of *Phytophthora species* in USA based on morphological and molecular characters (Ristaino *et al.* 2008). In the

the list (Fig. 4).

The genus *Cercospora* is one of the largest genera of hyphomycetes. i.e., commonly associated with leaf spots and is responsible for great damages to beneficial plants, such as cereals, vegetables, ornamentals, forest trees, grasses. A total of 105 Cercosporoid diseases were identified, belonging to *Cercospora* (48), *Pseudocercospora* (20), *Passalora* (5), *Asperisporium* (1), *Cladosporium* (30,) and *Periconiella* (1). From the reported *Cercospora* species, 20 were *Cercospora apii s.lat* and 28 were *Cercospora s.str.* The first report of *Cercospora basellae-albae* in the Philippines was observed causing leaf spots on *Basella alba cv. Rubra (*Begum and Cumagun*,*  2010*).* Twenty eight first records of Cercospora leaf spots were reported. Among Pseudocercospora leaf spots, 12 first records were reported and all were host specific. A new species of *Passalora amaranthae* found on *Amaranthus viridis* was reported*.* Only one specimen caused by *Asperisporium moringae* was reported on *Moringa oleifera.*

Lucid key is a powerful and highly flexible knowledge management software application designed to help users with identification and diagnostic tasks. Lucid is one of a multimedia matrix keys, that includes possible the storing of large amounts of information. Lucid key consists of two programs: Lucid Builder and Lucid Player. The first program is a key development tool, which allows developers to easily build their own keys. The second program of the Lucid key is the key interface or Lucid Player, through which end-users interacts with the Lucid key and enables users to view and interact with the key. In the present study, Lucid key was developed to identify Cercosporoid fungi, a total of 74 Cercosporoid fungi and their characters were entered into a program using Lucid Builder. Lucid has the advantage over printed dichotomous keys in that the user is able to skip an unanswerable couplet or question and still proceed with identification. Identification of Cercosporoid fungi is a difficult task, and the Lucid key was created to help provide individuals with easily accessible tools to distinguish species. The end product of the Lucid key of Philippine Cercosporoid fungi, in the future will be useful in teaching, research, and extension work in mycology and plant pathology.

#### **7. References**

Agrios, G.N. (2005). Plant Pathology. 5th ed. Academic Press. New York.


Taxonomic Review of and Development

*Bull. Acad. Sinica* 30: 117-132.

Book Co., Taipei. 376.

3).*Trans. Mycol. Soc.Japan* 16:1-15.

edn. CAB International, Oxon.

(APAN) and its Applications*:* 27-30.

University of the Philippines Los Banos. Philippines.

*Journal of Plant Pathology*, 90, S2.81-S2.465.

dispositorum. *Michelia* 2 :1-38.

from Korea 7; 1-302.

20: 309-467.

Waterloo, Canada. 42.

University.

Ottawa.

208-212.

810.

of a Lucid Key for Philippine Cercosporoids and Related Fungi 39

Gerald, J.H. (2005). Diagnosing Postharvest Diseases of Cantaloupe. North Carolina State

Goh, T. K. & W. H. Hsieh (1989). New species of *Cercospora* and allied genera of Taiwan. *Bot.* 

Guo, Y.L. and Hsieh, W.H. (1995). The genus *Pseudocercospora* in China. Mycosystema

Guo, Y.L., Liu, Y.J. and Hsieh, W.H. (1998). Flora Fungorum Sinicorum Vol. 9,

Katsuki, S. and Kobayashi. (1975). *Cercospora* of Japan and allied genera (Supplement

Kendrick, W.B. and Dicosmo, F. (1979). Teleomorph-anamorph connections in ascomycetes.

Kirk, P.M., Cannon, P.F., David, J.C. and Stalpers, L.A. (2001). Dictionary of the Fungi, 9th

Maublanc, A. (1913). Su rune maladie de feuilles du papayer "Carica papaya. *Lavoura* 16 :

Michaelides, J. Hunter, L. Kendrick, B. and Nag Raj, T.R. (1979). Synoptic Key to 200 Genera

Norton, G.A. (2000). Multi-media keys for identification and diagnostics: the Lucid

Pollack, F. G. (1987) An annotated compilation of *Cercospora* names. *Mycol. Mem*. 12:1-212. Ponaya, A.B. and Cumagun, C.J.R. (2008). First record of *Passalora bougainvilleae* causing leaf spot of bougainvillea in the Philippines. *Australasian Plant Disease Notes* 3: 3-4. Quimio T.H. and Abilay L.E. (1977). Cercospora Species and disease of Philippine Crops.

Ristaino, J.B., Haege, M.J. and Hu, C.H. (2008). Development of a *Phytophthora* Lucid key.

Saccardo, P.A. (1880). Conspectus generum fungorum Italie inferriorum, nempe as

Saccardo, P. A. (1886). Sylloge fungorum omnium hucusgue cognitorium. Vol. IV. Padova,

Shin, H.D. and Kim, J.D. (2001). *Cercospora* and allied genera from Korea. Plant Pathogens

Spegazzini, C.(1910). Myceters Argentinenses, Ser. V. *An. Mus. Nac. Hist. Nat. Buenos Aires*

Sutton, B.C. (1980). The coelomycetes: fungi imperfecti with pycnidia, acervuli, and stromata. Commonwealth Mycological Institute, Kew, Surrey, England, 696 p.

Stakman E.C. and Harrar J.G. (1957). 362. The Ronald Press Company. New York.

Sphaeropsidas, elanconieas et Hyphomycetes pertinentium. Systemate sporologico

In: The Whole Fungus, Vol.1:283-410. National Museum of Natural Sciences,

of Coelomycetes. University of Waterloo Biology Series 20. University of Waterloo,

experience. International Workshop of the Asia-Pacific Advanced Network

Monographieum Series No. 2. Int. Acad. Pub., Beijing, China. 388.

Hanlin, R.T. (1990). Illustrated Genera of Ascomycetes. 3 Vols. APS Press, St Paul, USA. Hsieh, W. H. and Goh, T.K. (1990). *Cercospora* and Similar Fungi from Taiwan. Maw Chang

Katsuki, S. (1965). *Cercospora* of Japan. *Trans. Mycol. Soc. Japan,* Extra Issue No. 1. 100.

*Pseudocercospora.* Science Press, Beijing, China.


Begum, M.M. and Cumagun, C.J.R. (2010). First record of *Cercospora basellae-albae* from the

Crous, P.W. and Braun, U. (2003). *Mycosphaerella* and its anamorphs. . Names published in

Crous, P.W. and Groenewald J.Z. (2006a). *Mycosphaerella alistairii*. *Fungal Planet* No. 4.

Crous, P.W. and Groenewald, J.Z. (2006b). *Mycosphaerella maxii*. *Fungal Planet* No. 6.

Crous, P.W. Aptroot, A. Kang, J.C. Braun, U., and Wingfield, M.J.(2000). The genus

Crous, P.W., Kang, J.C., and Braun, U. (2001). A phylogenetic redefinition of anamorph

Cumagun C.J.R. and Padilla, C.L. (2007). First record of *Asperisporium caricae* causing black spot of papaya in the Philippines. *Australasian Plant Disease Notes* 2:89-90. Deighton, F.C. (1967a). New names in *Mycosphaerella (M. arachidis and m. pruni-persicae)* and

Deighton, F.C. (1967b). Studies on *Cercospora* and allied genera. II. *Passalora, Cercosporidium* and some species of *Fusicladium* on *Euphorbia. Mycol. Papers* 112:1-80. Deighton, F.C. (1971). Studies on *Cercospora* and allied genera. III. *Cercospora. Mycol Papers*

Deighton, F.C. (1973). Studies on *Cercospora* and allied genera. IV. *Cercosporella* Sacc.,

Deighton, F.C. (1974). Studies on *Cercospora* and allied genera. V. *Mycovellosiella* Rangel. and

Deighton, F.C. (1976). Studies on *Cercospora* and allied genera. VI. *Pseudocercospora* Speg.,

Deighton, F.C.(1979). Studies on *Cercospora* and allied genera. VII. New Species and

Deighton, F.C. (1983). Studies on *Cercospora* and allied genera. VIII. Further notes on *Cercoseptoria* and some species and redispositions. *Mycol. Papers* 151: 1-13. Deighton, F.C. (1987). New species of *Pseudocercospora* and *Mycovellosiella*, and new

Ellis, M.B. (1971). Dematiaceous Hypomycetes. Kew, UK: Commonwealth Mycological

Ellis, M.B. (1976). More Dematiaceous Hypomycetes. Kew, UK Commonwealth Mycological

Ellis, M.B. and Holliday, P. (1972). *Asperisporium caricae*. CMI Descriptions of Pathogenic Fungi and Bacteria No. 347.Kew, UK: CAB International Mycological Institute

combinations into *Pseudocercospora* and *Mycovellosiella*. *Trans. Brit. Mycol.soc.* 88:

*Pseudocercosporella* gen. Nov. and *Pseudocercosporidium* gen.nov. *Mycol Papers*133: 1-

genera in *Mycosphaerella* based on ITS rDNA sequence and morphology. *Mycologia*

*Mycosphaerella* and its anamorphs. *Studies in Mycology* 45:107–121.

validation of *M. rosicola*. *Trans. Brit. Mycol. Soc.* 50:328-329.

a new species of *Ramulariopsis*. *Mycol. Papers* 137: 1-73.

redispositions. *Mycol. Papers* 144:1-56.

*Pantospora* Cif. And *Cercoseptoria* Petr. *Mycol. Papers* 140:1-168.

Braun, U. (2004). *Periconiella* species occurring on ferns. *Feddes Repertorium* 115: 50-55. Chupp, C. (1953). A monograph of the fungus genus *Cercospora*. Ithaca New York. 9-20. Chupp, C. (1954). A monograph of the fungus genus *Cercospora*. Ithaca. New York. 667. Crous, P.W. (1998). *Mycosphaerella* spp. and their anamorphs associated with leaf spot

Philippines. *Australasian Plant Disease Notes* 5: 115-116.

diseases of *Eucalyptus*. *Mycologia Memoir* 21: 1– 170.

(www.fungalplanet.org).

(www.fungalplanet.org).

93: 1081-1101.

124: 1-13.

365-391.

Institute.

Institute.

62.

*Cercospora* and *Passalora*. *CBS Biodiversity Series* 1:1 –571.


**2** 

*China* 

**General Description of** 

Genhua Yang and Chengyun Li

*Rhizoctonia* **Species Complex** 

*Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming, Yunnan* 

The genus concept of *Rhizoctonia* spp. was established by de Candolle (1815) (Sneh *et al*., 1998). However, the lack of specific characters led to the classification of a mixture of unrelated fungi as *Rhizoctonia* spp. (Parmeter and Whitney, 1970; Moore, 1987). Ogoshi (1975) enhanced the specificity of the genus concept for *Rhizoctonia* by elevating the following characteristics of *R. solani* to the genus level. Based on this revised genus concept, species of *Rhizoctonia* can be differentiated by mycelia color, number of nuclei per young vegetative hyphal cell and the morphology of their teleomorph. The teleomorph of

The anamorphs of *Rhizoctonia* are heterogeneous. Moore (1987) placed the anamorphs of *Thanatephorus* spp. in *Moniliopsis*. She reserved the genus *Rhizoctonia* for anamorph of ustomycetous fungi which have septa with simple pores. Moniliopsis species have smooth, broad hyphae with brown walls, multinucleate cells, dolipore septa with perforate parenthesomes and teleomorphs in the genera *Thanatephorus* and *Waitea*. Of the binucleate *Rhizoctonia* spp., the anamorphs of the *R*. *repens* group (teleomorph *Tulasnella*) were assigned to the new genus *Epulorhiza.* Anamorph of *Ceratobasidium* was assigned to the new genus *Ceratorhiza* (Moore, 1987). Moore's system is taxonomically correct and justified. At present, the concept of genus *Rhizoctonia* has become clear from these taxonomical studies at the molecular level (Gonzalez *et al*., 2001). However, many researchers (Sneh *et al*., 1998) in the world still retain the name *Rhizoctonia* for Moore's *Moniliopsis* spp., *Ceratorhiza* spp. and

Affinity for hyphal fusion (anastomosis) (Parmeter *et al*., 1969; Parmeter and Whitney, 1970; Ogoshi *et al*., 1983a; Burpee *et al*., 1980a) has been used to characterize isolates among *R*. *solani*, *R*. *zeae*, *R*. *oryzae*, *R*. *repens* and binucleate *Rhizoctoni*a spp. with *Ceratobasidium* teleomorphs. To date, isolates of *R. solani* have been assigned to 13 anastomosis groups (AG) and those of *R*. *zeae* and *R*. *oryzae* have each been assigned to their own one group (Sneh *et* 

Anastomosis reactions between hyphae of paired isolates of *R. solani* consist of several types; such as perfect fusion, imperfect fusion, contact fusion and no reaction (Matsumoto *et al*., 1932). At present, four categories of anastomosis (C3 to C0) defined by Carling *et al*. (1996)

*Rhizoctonia* spp. belongs to the sub-division Basidiomycota, class Hymenomycetes.

*Epulorhiza* spp.. Hence, I used the name of *Rhizoctonia* in this study.

*al*., 1998; Carling *et al*., 1999, 2002c).

**1. Introduction** 


### **General Description of**  *Rhizoctonia* **Species Complex**

Genhua Yang and Chengyun Li *Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming, Yunnan China* 

#### **1. Introduction**

40 Plant Pathology

Teodoro, N.T. (1937). Enumeration of Philippine fungi. Commonwealth of the Phill. Dept. of

Vanky, K. and Shivas, R.G. (2008). Fungi of Australia: The Smut Fungi. Australian Biological

Vasudeva, R.S. (1963). Indian Cercosporae. Indian Council of Agricultural Research, New

von Arx, J.A. (1983). *Mycosphaerella* and its anamorphs. Proc. K. Ned. Akad. Wet. Ser. C Biol.

Welles, C.G. (1924). Observation on taxonomic factors used in the genus *Cercospora*. *Science*

Welles, C.G. (1925). Taxonomic studies in the genus *Cercospora* in the Philippine

Wellman, F.L.(1972). Tropical American Plant Disease. 219 pp. The Scarecrow Press, Inc.

Yen JM, Lim G. (1980) *Cercospora* and allied genera of Singapore and the Malay peninsula.

Young, T. (2001) 101 Forest Fungi of Eastern Australia. Knowledge Books and

Agric. & Commerce. *Tech. Bull.* No. 4. 585.

The Gardens' Bulletin Singapore 33, 151–263.

Software,Brighton, Australia. CD ROM.

Resources Study. CSIRO Publishing.

Delhi. 245.

59:216-218.

New Jersey.

Med. Sci. 86, 1: 15-54.

Islands.*Am.J.Bot*.12:195-220.

The genus concept of *Rhizoctonia* spp. was established by de Candolle (1815) (Sneh *et al*., 1998). However, the lack of specific characters led to the classification of a mixture of unrelated fungi as *Rhizoctonia* spp. (Parmeter and Whitney, 1970; Moore, 1987). Ogoshi (1975) enhanced the specificity of the genus concept for *Rhizoctonia* by elevating the following characteristics of *R. solani* to the genus level. Based on this revised genus concept, species of *Rhizoctonia* can be differentiated by mycelia color, number of nuclei per young vegetative hyphal cell and the morphology of their teleomorph. The teleomorph of *Rhizoctonia* spp. belongs to the sub-division Basidiomycota, class Hymenomycetes.

The anamorphs of *Rhizoctonia* are heterogeneous. Moore (1987) placed the anamorphs of *Thanatephorus* spp. in *Moniliopsis*. She reserved the genus *Rhizoctonia* for anamorph of ustomycetous fungi which have septa with simple pores. Moniliopsis species have smooth, broad hyphae with brown walls, multinucleate cells, dolipore septa with perforate parenthesomes and teleomorphs in the genera *Thanatephorus* and *Waitea*. Of the binucleate *Rhizoctonia* spp., the anamorphs of the *R*. *repens* group (teleomorph *Tulasnella*) were assigned to the new genus *Epulorhiza.* Anamorph of *Ceratobasidium* was assigned to the new genus *Ceratorhiza* (Moore, 1987). Moore's system is taxonomically correct and justified. At present, the concept of genus *Rhizoctonia* has become clear from these taxonomical studies at the molecular level (Gonzalez *et al*., 2001). However, many researchers (Sneh *et al*., 1998) in the world still retain the name *Rhizoctonia* for Moore's *Moniliopsis* spp., *Ceratorhiza* spp. and *Epulorhiza* spp.. Hence, I used the name of *Rhizoctonia* in this study.

Affinity for hyphal fusion (anastomosis) (Parmeter *et al*., 1969; Parmeter and Whitney, 1970; Ogoshi *et al*., 1983a; Burpee *et al*., 1980a) has been used to characterize isolates among *R*. *solani*, *R*. *zeae*, *R*. *oryzae*, *R*. *repens* and binucleate *Rhizoctoni*a spp. with *Ceratobasidium* teleomorphs. To date, isolates of *R. solani* have been assigned to 13 anastomosis groups (AG) and those of *R*. *zeae* and *R*. *oryzae* have each been assigned to their own one group (Sneh *et al*., 1998; Carling *et al*., 1999, 2002c).

Anastomosis reactions between hyphae of paired isolates of *R. solani* consist of several types; such as perfect fusion, imperfect fusion, contact fusion and no reaction (Matsumoto *et al*., 1932). At present, four categories of anastomosis (C3 to C0) defined by Carling *et al*. (1996)

General Description of *Rhizoctonia* Species Complex 43

Isolates of *R. solani* that exhibits DNA base sequence homology and affinities for hyphal anastomosis may represent a diverging evolutionary unit (Kuninaga and Yokosawa, 1980). This hypothesis is supported by analysis of restriction fragment length polymorphisms (RFLPs) and the sequences with in ribosomal RNA genes (rDNA) among different anastomosis groups of *R. solani* (Vilgalys and Gonzalez, 1990; Gonzalez, *et al*., 2001; Carling

As mentioned above, many AGs and subgroups of *R*. *solani* and binucleate *Rhizoctonia* spp. have been reported as causal of agents Rhizoctonia diseases on a wide range of host species. However, little is known about the Rhizoctonia diseases and the anastomosis groups and subgroups of their causal fungi on vegetables, ornamentals and food crops in the Asian

**2. Characteristics of anastomosis groups and subgroups of** *Rhizoctonia* 

**AG-1 IA** (Li and Yan, 1990; Sneh *et al.*, 1998; Fenille *et al*., 2002; Naito, 2004).

Disease symptoms and host range of each AG and its subgroups are summarized as follows. In this review, the book by Sneh et al., 1998 entitled "Identification of *Rhizoctonia* Species"

Symptoms: sheath blight, foliar blight, leaf blight, web-blight, head rot, bottom rot, and

Host: rice (*Oryza sativa* L.), corn (*Zea mays* L.), barley (*Hordeum vulgare* L.), sorghum (*Sorghum vulgare* Pes.), potato (*Solanum tuberosum* L.), barnyard millet, common millet, soybean, peanut (*Arachis hypogaea* L.), lima bean, cabbage, leaf lettuce, Stevia, orchard grass, crimson clover, tall fescue (*Festuca arundiacea* Schreb), turfgrass, creeping bentgrass,

Note: This group has a tendency to attack aerial parts of the plants. Basidiospore infection of rice has been reported, but sclerotia are more important as an infection source. The optimum

Symptoms: sheath blight, leaf blight, foliar blight, web-blight, root rot, damping-off, head

Host: corn, sugar beet, gay feather (*Liatris* spp.), common bean, fig (*Ficus* L.), adzuki bean, soybean, cabbage, leaf lettuce, redtop, bentgrass, orchard grass, leaf lettuce, apple (*Malus pumila* Mill), Japanese pear, European pear, lion'ear (*Leonotis leonurus*), hortensia (*Hydrangea* spp.), *Larix* spp., gazania (*Gazania* spp.) *Cotoneaster* spp., Egyptian atar-cluster (*Pentas lanceolata*), Chinese lantern plant (*Physalis alkekeng* var. franchetii), *Hypericum patulum*, marigold, *Acacia* spp., rosemary, *Eucalyptus* spp., pine (*Pinus* L.), *Larix* spp., cypress

*et al*., 2002b).

tropics especially the southern parts of China.

*solani* **and binucleate** *Rhizoctonia* **spp.** 

**2.1 Multinucleate** *Rhizoctonia* **spp.** 

1. **AG-1: IA, IB, IC, ID** 

brown patch.

rot, and bottom rot.

provided a substitute for the reference before 1998.

perennial ryegrass, gentian (*Gentiana scabra*), and camphor.

growth temperature is higher than those of AG-1 IB.

**AG-1 IB** (Sneh *et al*., 1998; Naito, 2004;Yang *et al*., 2005b).

(*Cupressus* spp.), and elephant foot (*Amorphophallus Konjac*).

have been accepted by many researchers. These are useful for a better understanding of the genetic diversity of *R. solani* populations, because of the background genetically supported by vegetative or somatic compatibility (VC or SC) of confronted isolates (MacNish *et al*, 1997). Each of categories is as follows:

**C3:** walls fuse; membranes fuse, accompanied with protoplasm connection; anastomosis point frequently is not obvious; diameter of anastomosis point is equal or nearly equal hyphal diameter; anastomosing cells and adjacent cells may die, but generally do not. This category occurs for the same anastomosis group, same vegetative compatibility population (VCP) and the same isolate.

**C2:** wall connection is obvious, but membrane contact is uncertain; anastomosing and adjacent cells always die. This category occurs in same AG, but not between different VCPs.

**C1:** wall contact between hyphae is apparent, but both wall penetration and membranemembrane contact do not occur; occasionally one or both anastomosing cells and adjacent cells die. This category occurs between different AGs or in the same AG.

**C0:** no reaction. This category occurs between different AGs.

In general, hyphal fusion occurs at a high frequency (50%≥) within members of the same AG, with the exception of non-self-anastomosing isolates (Hyakumachi and Ui, 1988). On the other hand, hyphal fusion among members of different AGs occurs at either a low frequency (≤30%) or no fusion occurs. *Rhizoctonia* isolates giving C3 to C1 reactions in anastomosing test have been taken to be the same AG.

To date, isolates of multinucleate *R. solani* have been assigned to 13 anastomosis groups (AG-1 to AG-13), some of which include several subgroups and isolates of *R*. *zeae* and *R*. *oryzae* have been assigned to WAG-Z and WAG-O, respectively (Sneh *et al*., 1998; Carling *et al*., 1999, 2002c). Isolates of binucleate *Rhizoctonia* spp. with *Ceratobasidium* teleomorphs have been reported. A system developed in Japan (Ogoshi *et al*.,1979, 1983 a,b; Sneh *et al*., 1998; Hyakumachi *et al.*, 2005) includes 21 anastomosis groups designated AG-A to AG-U, in which at present AG-J and AG-M still are in question as members of binucleate *Rhizoctonia*. Another system developed in the USA (Burpee *et al*., 1980a) includes 7 anastomosis groups designed as CAG-1 to CAG -7. CAG-1 corresponds to AG-D, CAG-2 to AG-A, CAG-3 and CAG-6 to AG-E, CAG-4 to AG-F, CAG-5 to AG-R, and CAG-7 to AG-S (Sneh *et al*., 1998; Ogoshi *et al*., 1983a). At present, the anastomosis system based on AG-A through AG-U used in this review paper is widely accepted by many researchers.

Some homogenous groups of isolates of *R*. *solani* are well known as bridging isolates (AG-BI) that anastomose with members of different AGs (Carling, 1998). In general, there is no contradiction in the conventional anastomosis grouping system by taking anastomosis frequency into consideration. However, two exceptional cases where anastomosis frequency mismatched with morphological, physiological and pathogenic characteristics have been reported from tobacco (Nicoletti *et al*., 1999) and soybean (Naito and Kanematsu, 1994). These demonstrate the limitations of using hyphal anastomosis as the sole criteria for characterization and identification of closely related fungi. In addition, it is not easy to determine the subgroup of isolates within the same AG because no differences occur in their anastomosis reaction. Thus, in order to determine AGs or subgroups in *R. solani*, genetic analysis using molecular approaches that employ multiple genetic loci is needed.

have been accepted by many researchers. These are useful for a better understanding of the genetic diversity of *R. solani* populations, because of the background genetically supported by vegetative or somatic compatibility (VC or SC) of confronted isolates (MacNish *et al*,

**C3:** walls fuse; membranes fuse, accompanied with protoplasm connection; anastomosis point frequently is not obvious; diameter of anastomosis point is equal or nearly equal hyphal diameter; anastomosing cells and adjacent cells may die, but generally do not. This category occurs for the same anastomosis group, same vegetative compatibility population

**C2:** wall connection is obvious, but membrane contact is uncertain; anastomosing and adjacent cells always die. This category occurs in same AG, but not between different VCPs. **C1:** wall contact between hyphae is apparent, but both wall penetration and membranemembrane contact do not occur; occasionally one or both anastomosing cells and adjacent

In general, hyphal fusion occurs at a high frequency (50%≥) within members of the same AG, with the exception of non-self-anastomosing isolates (Hyakumachi and Ui, 1988). On the other hand, hyphal fusion among members of different AGs occurs at either a low frequency (≤30%) or no fusion occurs. *Rhizoctonia* isolates giving C3 to C1 reactions in

To date, isolates of multinucleate *R. solani* have been assigned to 13 anastomosis groups (AG-1 to AG-13), some of which include several subgroups and isolates of *R*. *zeae* and *R*. *oryzae* have been assigned to WAG-Z and WAG-O, respectively (Sneh *et al*., 1998; Carling *et al*., 1999, 2002c). Isolates of binucleate *Rhizoctonia* spp. with *Ceratobasidium* teleomorphs have been reported. A system developed in Japan (Ogoshi *et al*.,1979, 1983 a,b; Sneh *et al*., 1998; Hyakumachi *et al.*, 2005) includes 21 anastomosis groups designated AG-A to AG-U, in which at present AG-J and AG-M still are in question as members of binucleate *Rhizoctonia*. Another system developed in the USA (Burpee *et al*., 1980a) includes 7 anastomosis groups designed as CAG-1 to CAG -7. CAG-1 corresponds to AG-D, CAG-2 to AG-A, CAG-3 and CAG-6 to AG-E, CAG-4 to AG-F, CAG-5 to AG-R, and CAG-7 to AG-S (Sneh *et al*., 1998; Ogoshi *et al*., 1983a). At present, the anastomosis system based on AG-A through AG-U

Some homogenous groups of isolates of *R*. *solani* are well known as bridging isolates (AG-BI) that anastomose with members of different AGs (Carling, 1998). In general, there is no contradiction in the conventional anastomosis grouping system by taking anastomosis frequency into consideration. However, two exceptional cases where anastomosis frequency mismatched with morphological, physiological and pathogenic characteristics have been reported from tobacco (Nicoletti *et al*., 1999) and soybean (Naito and Kanematsu, 1994). These demonstrate the limitations of using hyphal anastomosis as the sole criteria for characterization and identification of closely related fungi. In addition, it is not easy to determine the subgroup of isolates within the same AG because no differences occur in their anastomosis reaction. Thus, in order to determine AGs or subgroups in *R. solani*, genetic

analysis using molecular approaches that employ multiple genetic loci is needed.

cells die. This category occurs between different AGs or in the same AG.

**C0:** no reaction. This category occurs between different AGs.

anastomosing test have been taken to be the same AG.

used in this review paper is widely accepted by many researchers.

1997). Each of categories is as follows:

(VCP) and the same isolate.

Isolates of *R. solani* that exhibits DNA base sequence homology and affinities for hyphal anastomosis may represent a diverging evolutionary unit (Kuninaga and Yokosawa, 1980). This hypothesis is supported by analysis of restriction fragment length polymorphisms (RFLPs) and the sequences with in ribosomal RNA genes (rDNA) among different anastomosis groups of *R. solani* (Vilgalys and Gonzalez, 1990; Gonzalez, *et al*., 2001; Carling *et al*., 2002b).

As mentioned above, many AGs and subgroups of *R*. *solani* and binucleate *Rhizoctonia* spp. have been reported as causal of agents Rhizoctonia diseases on a wide range of host species. However, little is known about the Rhizoctonia diseases and the anastomosis groups and subgroups of their causal fungi on vegetables, ornamentals and food crops in the Asian tropics especially the southern parts of China.

#### **2. Characteristics of anastomosis groups and subgroups of** *Rhizoctonia solani* **and binucleate** *Rhizoctonia* **spp.**

Disease symptoms and host range of each AG and its subgroups are summarized as follows. In this review, the book by Sneh et al., 1998 entitled "Identification of *Rhizoctonia* Species" provided a substitute for the reference before 1998.

#### **2.1 Multinucleate** *Rhizoctonia* **spp.**

#### 1. **AG-1: IA, IB, IC, ID**

**AG-1 IA** (Li and Yan, 1990; Sneh *et al.*, 1998; Fenille *et al*., 2002; Naito, 2004).

Symptoms: sheath blight, foliar blight, leaf blight, web-blight, head rot, bottom rot, and brown patch.

Host: rice (*Oryza sativa* L.), corn (*Zea mays* L.), barley (*Hordeum vulgare* L.), sorghum (*Sorghum vulgare* Pes.), potato (*Solanum tuberosum* L.), barnyard millet, common millet, soybean, peanut (*Arachis hypogaea* L.), lima bean, cabbage, leaf lettuce, Stevia, orchard grass, crimson clover, tall fescue (*Festuca arundiacea* Schreb), turfgrass, creeping bentgrass, perennial ryegrass, gentian (*Gentiana scabra*), and camphor.

Note: This group has a tendency to attack aerial parts of the plants. Basidiospore infection of rice has been reported, but sclerotia are more important as an infection source. The optimum growth temperature is higher than those of AG-1 IB.

**AG-1 IB** (Sneh *et al*., 1998; Naito, 2004;Yang *et al*., 2005b).

Symptoms: sheath blight, leaf blight, foliar blight, web-blight, root rot, damping-off, head rot, and bottom rot.

Host: corn, sugar beet, gay feather (*Liatris* spp.), common bean, fig (*Ficus* L.), adzuki bean, soybean, cabbage, leaf lettuce, redtop, bentgrass, orchard grass, leaf lettuce, apple (*Malus pumila* Mill), Japanese pear, European pear, lion'ear (*Leonotis leonurus*), hortensia (*Hydrangea* spp.), *Larix* spp., gazania (*Gazania* spp.) *Cotoneaster* spp., Egyptian atar-cluster (*Pentas lanceolata*), Chinese lantern plant (*Physalis alkekeng* var. franchetii), *Hypericum patulum*, marigold, *Acacia* spp., rosemary, *Eucalyptus* spp., pine (*Pinus* L.), *Larix* spp., cypress (*Cupressus* spp.), and elephant foot (*Amorphophallus Konjac*).

General Description of *Rhizoctonia* Species Complex 45

Undetermined subgroup: sesame (*Sesamum* Linn.), white mustard (*Sinapsis alba*), primrose *(Primula* spp.), white lace flower (*Ammi majus*), carnation, baby's-breath (*Gypsophila paniculata*),

Note: Undetermined subgroup: eggplant, sugar beet, tomato, and wheat. Their pathological

4. **AG-4: HG-I, HG-II, HG-III** (Baird, 1996; Holtz *et al*., 1996; Sneh *et al.*, 1998; Fenille *et al*., 2002; Ravanlou and Banihashemi, 2002; EI Hussieni, 2003; Kuramae *et al*., 2002, 2003;

Host: pea, sugar beet, melon, soybean, adzuki bean, common bean, snap bean, lima bean, carrot, spinach, taro, tomato (*Lycopersicon esculentum* Mill.), potato, alfalfa (*Medicago sativa* Linn.), elephant foot, arrowleaf clover, beans, barley, buckwheat, cabbage, canola, turnip, carnation, cauliflower, Chinese chive, chrysanthemum, corn, cotton (*Gossypium* Linn.), table beet, tobacco, turfgrass, wheat, white lupine, parsley (*Petroselinum* Hill), *Cineraria* Linn., stock, poinsettia, primrose, hybrid bouvardia, *Citrus* Linn., cauliflower, *Euphorbia* spp., geranium (*Pelargonium* spp.), Russel prairie gentian, statice (*Limonium* spp.), baby's-breath,

5. **AG-5** (Li, *et al*., 1998; Demirci, 1998; Sneh *et al*., 1998; Ravanlou and Banihashemi, 2002;

Symptoms: root rot, damping-off, black scurf, brown patch, and symbiosis (orchids).

rusell prairie gentian (*Eustoma grandiflorum*), snap bean, lima bean, and Chinese radish.

**AG 2-3:** (Naito and Kanematsu, 1994; Sumner *et al*., 2003).

Host: Zoysia grass.

Host: soybean.

**AG-2-4:** (Sumner, 1985).

Host: corn and carrot.

**AG-2-BI:** (Carling *et al*., 2002b). Symptoms;:nonpathogenic.

Note: former name is AG-BI.

PT: potato with black scurf symptoms.

Naito, 2004; Yang *et al*., 2005c).

and ecological information is less.

and *Astragalus membranaceus* 

Eken and Demirci, 2004; Naito, 2004).

TB: tobacco with target leaf spot symptoms.

Symptoms: leaf blight and root rot.

Note: basidiospores cause leaf spot of soybean.

Symptoms: crown rot, brace rot, and damping-off.

Host: isolates, obtained only from soils and plants in forests.

3. **AG 3: PT, TB** (Sneh *et al.*, 1998; Kuninaga *et al*., 2000).

Symptoms: black scurf, leaf spot, target leaf spot, and damping-off.

Symptoms: damping-off, root rot, stem canker, fruit rot, and stem rot.

**AG-1 IC** (Sneh *et al.,* 1998; Naito, 2004).

Symptoms: damping-off, summer blight, foot rot, crown rot canker, and root rot.

Host: sugar beet, carrot (*Daucus carota* L.), buckwheat (*Eriogonum* Michx), flax (*Linum usitatissimum* L.), soybean, bean (*Phaseolus* L.), cabbage, pineapple (*Ananas comosus* (Linn.) Merr.), panicum (*Panicum* spp.), spinach (*Spinacia oleracea* L.), and radish (*Raphanus sativus* Linn).

**AG-1 ID** (Priyatmojo *et al.*, 2001).

Symptom: leaf spot.

Host: coffee (*Coffea* Linn).

Note: this subgroup was recently reported in the Philippines (Priyatmojo *et al.*, 2001)

Undetermined subgroup: buckwheat, flax, spinach, radish, and durian (*Durio zibethinus* Murr.).

#### 2. **AG-2: 2-1, 2-2 IIIB, 2-2 IV, 2-2 Lp, 2-3, 2-4, 2-BI.**

**AG-2-1** (Satoh *et al*., 1997; Camporota and Perrin, 1998; Sneh *et al*., 1998; Rollins *et al*., 1999; Khan and Kolte, 2000; Naito, 2004)

Symptoms: damping-off, leaf rot, leaf blight, root rot, foot rot, bottom rot, and bud rot.

Host: sugar beet, wheat (*Triticum aestivum* Linn.), potato, cowpea (*Vigna unguiculata* (Linn.) Walp), canola, rape (*Brassica napus* Linn.), cauliflower (*Brassica oleracea* var. *botrytis* Linn.), mustard (*Sinapis* Linn.), turnip (*Brassica rapa* Linn.), pepper (Piper Linn.), *Silene armeria,*  spinach, leaf lettuce, strawberry (*Fragaria ananassa* Duchesne), tulip (*Tulipa gesneriana* Linn.), tobacco (*Nicotiana* Linn.), clover (*Medicago* Linn.), and table beet.

Note: This group includes the AG-2-1 tulip strain (former AG-2t) and the AG-2-1 tobacco strain (former homogenous Nt-isolates) (Kuninaga *et al.*, 2000).

**AG-2-2 III B** (Sneh *et al.*, 1998; Priyatmojo *et al.*, 2001; Naito, 2004).

Symptoms: brown sheath blight, dry root rot, root rot, brown patch, large patch, black scurf, stem rot, stem blight, *Rhizoctonia* rot, damping-off, stem rot, collar rot, and crown brace rot.

Host: rice, soybean, corn, sugar beet, edible burdock (*Arctium lappa*), taro (*Colocasia esculenta*), *Dryopteris* spp., elephant foot, crocus, saffron (*Crocus sativus* Linn.), redtop, bentgrass, St. Augustine grass, turf, balloon flower (*Platycodon grandiflorum*), Christmas-bells (*Sandersonia aurantiaca*), *Hedera rhombea*, mat rash, *gladiolus*, ginger, and *Iris* Linn..

**AG-2-2 IV:** (Sneh *et al*., 1998; Naito, 2004).

Symptoms: leaf blight, foliage rot, root rot, and stem rot.

Host: sugar beet, carrot, eggplant (*Solanum* Linn), pepper, spinach, stevenia (*Stevenia* Adams et Fisch), and turfgrass.

**AG-2-2 LP:** (Aoyagi *et al*., 1998).

Symptoms: large patch.

Host: Zoysia grass.

44 Plant Pathology

Host: sugar beet, carrot (*Daucus carota* L.), buckwheat (*Eriogonum* Michx), flax (*Linum usitatissimum* L.), soybean, bean (*Phaseolus* L.), cabbage, pineapple (*Ananas comosus* (Linn.) Merr.), panicum (*Panicum* spp.), spinach (*Spinacia oleracea* L.), and radish (*Raphanus sativus*

Symptoms: damping-off, summer blight, foot rot, crown rot canker, and root rot.

Note: this subgroup was recently reported in the Philippines (Priyatmojo *et al.*, 2001)

Undetermined subgroup: buckwheat, flax, spinach, radish, and durian (*Durio zibethinus*

**AG-2-1** (Satoh *et al*., 1997; Camporota and Perrin, 1998; Sneh *et al*., 1998; Rollins *et al*., 1999;

Host: sugar beet, wheat (*Triticum aestivum* Linn.), potato, cowpea (*Vigna unguiculata* (Linn.) Walp), canola, rape (*Brassica napus* Linn.), cauliflower (*Brassica oleracea* var. *botrytis* Linn.), mustard (*Sinapis* Linn.), turnip (*Brassica rapa* Linn.), pepper (Piper Linn.), *Silene armeria,*  spinach, leaf lettuce, strawberry (*Fragaria ananassa* Duchesne), tulip (*Tulipa gesneriana* Linn.),

Note: This group includes the AG-2-1 tulip strain (former AG-2t) and the AG-2-1 tobacco

Symptoms: brown sheath blight, dry root rot, root rot, brown patch, large patch, black scurf, stem rot, stem blight, *Rhizoctonia* rot, damping-off, stem rot, collar rot, and crown brace rot. Host: rice, soybean, corn, sugar beet, edible burdock (*Arctium lappa*), taro (*Colocasia esculenta*), *Dryopteris* spp., elephant foot, crocus, saffron (*Crocus sativus* Linn.), redtop, bentgrass, St. Augustine grass, turf, balloon flower (*Platycodon grandiflorum*), Christmas-bells

Host: sugar beet, carrot, eggplant (*Solanum* Linn), pepper, spinach, stevenia (*Stevenia* Adams

(*Sandersonia aurantiaca*), *Hedera rhombea*, mat rash, *gladiolus*, ginger, and *Iris* Linn..

Symptoms: damping-off, leaf rot, leaf blight, root rot, foot rot, bottom rot, and bud rot.

**AG-1 IC** (Sneh *et al.,* 1998; Naito, 2004).

**AG-1 ID** (Priyatmojo *et al.*, 2001).

Khan and Kolte, 2000; Naito, 2004)

**AG-2-2 IV:** (Sneh *et al*., 1998; Naito, 2004).

et Fisch), and turfgrass.

Symptoms: large patch.

**AG-2-2 LP:** (Aoyagi *et al*., 1998).

Symptoms: leaf blight, foliage rot, root rot, and stem rot.

2. **AG-2: 2-1, 2-2 IIIB, 2-2 IV, 2-2 Lp, 2-3, 2-4, 2-BI.** 

tobacco (*Nicotiana* Linn.), clover (*Medicago* Linn.), and table beet.

strain (former homogenous Nt-isolates) (Kuninaga *et al.*, 2000).

**AG-2-2 III B** (Sneh *et al.*, 1998; Priyatmojo *et al.*, 2001; Naito, 2004).

Symptom: leaf spot.

Host: coffee (*Coffea* Linn).

Linn).

Murr.).

**AG 2-3:** (Naito and Kanematsu, 1994; Sumner *et al*., 2003).

Symptoms: leaf blight and root rot.

Host: soybean.

Note: basidiospores cause leaf spot of soybean.

**AG-2-4:** (Sumner, 1985).

Symptoms: crown rot, brace rot, and damping-off.

Host: corn and carrot.

**AG-2-BI:** (Carling *et al*., 2002b).

Symptoms;:nonpathogenic.

Host: isolates, obtained only from soils and plants in forests.

Note: former name is AG-BI.

Undetermined subgroup: sesame (*Sesamum* Linn.), white mustard (*Sinapsis alba*), primrose *(Primula* spp.), white lace flower (*Ammi majus*), carnation, baby's-breath (*Gypsophila paniculata*), rusell prairie gentian (*Eustoma grandiflorum*), snap bean, lima bean, and Chinese radish.

3. **AG 3: PT, TB** (Sneh *et al.*, 1998; Kuninaga *et al*., 2000).

Symptoms: black scurf, leaf spot, target leaf spot, and damping-off.

PT: potato with black scurf symptoms.

TB: tobacco with target leaf spot symptoms.

Note: Undetermined subgroup: eggplant, sugar beet, tomato, and wheat. Their pathological and ecological information is less.

4. **AG-4: HG-I, HG-II, HG-III** (Baird, 1996; Holtz *et al*., 1996; Sneh *et al.*, 1998; Fenille *et al*., 2002; Ravanlou and Banihashemi, 2002; EI Hussieni, 2003; Kuramae *et al*., 2002, 2003; Naito, 2004; Yang *et al*., 2005c).

Symptoms: damping-off, root rot, stem canker, fruit rot, and stem rot.

Host: pea, sugar beet, melon, soybean, adzuki bean, common bean, snap bean, lima bean, carrot, spinach, taro, tomato (*Lycopersicon esculentum* Mill.), potato, alfalfa (*Medicago sativa* Linn.), elephant foot, arrowleaf clover, beans, barley, buckwheat, cabbage, canola, turnip, carnation, cauliflower, Chinese chive, chrysanthemum, corn, cotton (*Gossypium* Linn.), table beet, tobacco, turfgrass, wheat, white lupine, parsley (*Petroselinum* Hill), *Cineraria* Linn., stock, poinsettia, primrose, hybrid bouvardia, *Citrus* Linn., cauliflower, *Euphorbia* spp., geranium (*Pelargonium* spp.), Russel prairie gentian, statice (*Limonium* spp.), baby's-breath, and *Astragalus membranaceus* 

5. **AG-5** (Li, *et al*., 1998; Demirci, 1998; Sneh *et al*., 1998; Ravanlou and Banihashemi, 2002; Eken and Demirci, 2004; Naito, 2004).

Symptoms: root rot, damping-off, black scurf, brown patch, and symbiosis (orchids).

General Description of *Rhizoctonia* Species Complex 47

Host: strawberry, sugar beet, bean, pea, sunflower (*Helianthus annuus* Linn.), tomato, melon, cucumbear (*Cucumis sativas* Linn.), leaf lettuce, spinach, peanut, potato, *Solanum tuberosum*,

Note: Some isolates in this group form mycorrhizal associations with orchids.

Symptoms: grey sclerotium disease, sclerotium disease, gray southern blight. Host: rice, *Echinochloa crugalli* subsp. *submitica* var. typica, and foxtail millet.

Symptoms: brown sclerotium disease, grey sclerotium disease, and sheath spot.

Symptoms: sharp eye spot, yellow patch, foot rot, Sclerotium disease, snow mold, root rot,

Host: cereals, turf grass, wheat, barley, sugar beet, clove, pea, onions (*Allium cepa* Linn.),

Note: Recently this group is classified into subgroup AG-D (I) that causes Rhizoctonia patch

Host: bean, pea, radish, onion, leaf lettuce, tomato lima bean, snap bean, soybean, peanut, cowpea (*Vigna Savi*), flax, sugar beet, *Rhododendron* Linn., long leaf pine (*Pinus palustris* 

Host: bean, pea, radish, onion, peanut, leaf lettuce, tomato, subterranean clover radish,

potato, cotton, bean, soybean, mat rush, foxtail millet, and subterranean clover.

and winter patch diseases. AG-D (II) causes elephant footprint disease.

Symptoms: web-blight, damping-off, seedlings, and symbiosis (orchids).

Mill.), slash, lobolly pine (*Pinus taeda* Linn.), and rye (*Secale cereale* Linn.).

tomato, cotton, taro, strawberry (source: DDJB), and *Fragaria x ananassa*.

Host: orchids, sugar beet seedlings, subterranean clover, and wheat.

**2.2 Binucleate** *Rhizoctonia* **spp.** 

and apple.

2. **AG-B: a and b.** 

**AG-Ba** (Sneh *et al*., 1998).

**AG-Bb** (Sneh *et al*., 1998).

Host: fox tail, millet, and rice.

Symptoms: symbiosis (orchids).

5. **AG-E** (Sneh *et al*., 1998).

Symptoms: none.

3. **AG-C** (Sneh *et al*., 1998; Hayakawa *et al*., 1999).

Note: No important pathogens have been reported. 4. **AG-D: I, II** (Sneh *et al*., 1998; Toda *et al*.,1999).

damping-off, lesions on stems, and winter stem rot.

6. **AG-F** (Sneh *et al*., 1998; Eken and Demirci, 2004).

1. **AG-A:** (Mazzola, 1997; Sneh *et al*., 1998).

Symptoms: root rot, damping-off, browning, and tortoise shell.

Host: soybean, adzuki bean, apple, barley, chickpea, common bean, lima bean, potato, strawberry, sugar beet, table beet, tobacco, turfgrass, wheat, and white lupine.

6. **AG-6: HG-I, GV** (Mazzola, 1997; Meyer *et al*., 1998; Sneh *et al*., 1998; Carling *et al*., 1999; Pope and Carter, 2001; Naito, 2004)

Symptom: root rot, crater rot, and symbiosis (orchids).

Host: apple, wheat, carrot, and carnation.

Note: all isolates from forests are nonpathogenic.

7. **AG-7:** (Naito, *et al.*, 1993; Baird and Carling, 1995; Carling, 1997, 2000; Carling *et al.*, 1998)

Symptoms: damping-off, root rot, and black scurf.

Host: carnation, cotton, soybean, watermelon (*Citrullus lanatus* (Thunb.) Mansfeld), *Raphanus* Linn., and potato.

8. **AG-8:** (Sneh *et al.*, 1998; Naito, 2004).

Symptoms: bare patch.

Host: barley, cereals, green pepper, potato, and wheat.

9. **AG-9:** (Sneh *et al*., 1998; Naito, 2004).

Symptoms: black scurf.

Host: potato, crucifers, wheat, and barley.

10. **AG-10:** (Sneh *et al*., 1998.)

Symptoms: weak pathogenic.

Host: barley and wheat.

11. **AG-11:** (Kumar *et al*., 2002).

Symptoms: damping-off and hypocotyls rot.

Host: barley, lupine, soybean, and wheat.

Note: this group is considered as bridging isolates (anastomose with each members of AG-2- 1, AG-2 BI, AG-8) (Carling *et al*., 1996).

12. **AG-12:** (Kumar *et al.*, 2002).

Symptoms: symbiosis (orchids).

Host: *Dactylorhiza aristata* (Orchidaceae).

13. **AG-13:** (Carling *et al*., 2002a).

Symptoms: none.

Host: cotton.

#### **2.2 Binucleate** *Rhizoctonia* **spp.**

1. **AG-A:** (Mazzola, 1997; Sneh *et al*., 1998).

Symptoms: root rot, damping-off, browning, and tortoise shell.

Host: strawberry, sugar beet, bean, pea, sunflower (*Helianthus annuus* Linn.), tomato, melon, cucumbear (*Cucumis sativas* Linn.), leaf lettuce, spinach, peanut, potato, *Solanum tuberosum*, and apple.

Note: Some isolates in this group form mycorrhizal associations with orchids.

#### 2. **AG-B: a and b.**

46 Plant Pathology

Host: soybean, adzuki bean, apple, barley, chickpea, common bean, lima bean, potato,

6. **AG-6: HG-I, GV** (Mazzola, 1997; Meyer *et al*., 1998; Sneh *et al*., 1998; Carling *et al*., 1999;

7. **AG-7:** (Naito, *et al.*, 1993; Baird and Carling, 1995; Carling, 1997, 2000; Carling *et al.*,

Host: carnation, cotton, soybean, watermelon (*Citrullus lanatus* (Thunb.) Mansfeld),

Note: this group is considered as bridging isolates (anastomose with each members of AG-2-

strawberry, sugar beet, table beet, tobacco, turfgrass, wheat, and white lupine.

Pope and Carter, 2001; Naito, 2004)

Host: apple, wheat, carrot, and carnation.

8. **AG-8:** (Sneh *et al.*, 1998; Naito, 2004).

9. **AG-9:** (Sneh *et al*., 1998; Naito, 2004).

Host: potato, crucifers, wheat, and barley.

Symptoms: damping-off and hypocotyls rot.

Host: barley, lupine, soybean, and wheat.

1, AG-2 BI, AG-8) (Carling *et al*., 1996).

Host: *Dactylorhiza aristata* (Orchidaceae).

12. **AG-12:** (Kumar *et al.*, 2002). Symptoms: symbiosis (orchids).

13. **AG-13:** (Carling *et al*., 2002a).

Symptoms: none.

Host: cotton.

1998)

*Raphanus* Linn., and potato.

Symptoms: bare patch.

Symptoms: black scurf.

Host: barley and wheat.

10. **AG-10:** (Sneh *et al*., 1998.) Symptoms: weak pathogenic.

11. **AG-11:** (Kumar *et al*., 2002).

Symptom: root rot, crater rot, and symbiosis (orchids).

Note: all isolates from forests are nonpathogenic.

Symptoms: damping-off, root rot, and black scurf.

Host: barley, cereals, green pepper, potato, and wheat.

**AG-Ba** (Sneh *et al*., 1998).

Symptoms: grey sclerotium disease, sclerotium disease, gray southern blight.

Host: rice, *Echinochloa crugalli* subsp. *submitica* var. typica, and foxtail millet.

**AG-Bb** (Sneh *et al*., 1998).

Symptoms: brown sclerotium disease, grey sclerotium disease, and sheath spot.

Host: fox tail, millet, and rice.

3. **AG-C** (Sneh *et al*., 1998; Hayakawa *et al*., 1999).

Symptoms: symbiosis (orchids).

Host: orchids, sugar beet seedlings, subterranean clover, and wheat.

Note: No important pathogens have been reported.

4. **AG-D: I, II** (Sneh *et al*., 1998; Toda *et al*.,1999).

Symptoms: sharp eye spot, yellow patch, foot rot, Sclerotium disease, snow mold, root rot, damping-off, lesions on stems, and winter stem rot.

Host: cereals, turf grass, wheat, barley, sugar beet, clove, pea, onions (*Allium cepa* Linn.), potato, cotton, bean, soybean, mat rush, foxtail millet, and subterranean clover.

Note: Recently this group is classified into subgroup AG-D (I) that causes Rhizoctonia patch and winter patch diseases. AG-D (II) causes elephant footprint disease.

5. **AG-E** (Sneh *et al*., 1998).

Symptoms: web-blight, damping-off, seedlings, and symbiosis (orchids).

Host: bean, pea, radish, onion, leaf lettuce, tomato lima bean, snap bean, soybean, peanut, cowpea (*Vigna Savi*), flax, sugar beet, *Rhododendron* Linn., long leaf pine (*Pinus palustris*  Mill.), slash, lobolly pine (*Pinus taeda* Linn.), and rye (*Secale cereale* Linn.).

6. **AG-F** (Sneh *et al*., 1998; Eken and Demirci, 2004).

Symptoms: none.

Host: bean, pea, radish, onion, peanut, leaf lettuce, tomato, subterranean clover radish, tomato, cotton, taro, strawberry (source: DDJB), and *Fragaria x ananassa*.

General Description of *Rhizoctonia* Species Complex 49

In this chapter, we described the classification of *Rhizoctonia* spp. complex. Mutinucleate *Rhizoctonia* spp. included 13 anastomosis, of which AG 1-4 were strong pathogenic on many plants and AG 6-10 were orchid mycorrhizae. Binucleate *Rhizoctonia* spp. included 18 anastomosis groups, but AG-U belonged to AG-P and AG-T belonged to AG-A (Sharon et al., 2008), which were weak or nonpathogenic to plants and some AGs were orchid mycorrhizae.

This work was supported by the National Basic Research Program (No. 2011CB100400) from The Ministry of Science and Technology of China and the National Natural Science Funds,

Aoyagi, T., Kageyama, K. & Hyakumachi, M. (1998) Characterization and survival of

Baird, R. E. (1996) First report of *Rhizoctonia solani* AG-4 on canola in Georgia. Plant Dis.

Baird, R. E. & Carling, D. E. (1995) First report of *Rhizoctonia solani* AG-7 in Indiana. Plant

Botha, A., Denman, S., Lamprecht, S. C., Mazzola, M. & Crous, P. W. (2003) Characterization

Burpee, L. L., Sanders, P. L., Cole, H. Jr. & Sherwood, R. T. (1980a) Anastomosis groups

Camporota, P. & Perrin, R. (1998) Characterization of *Rhizoctonia* species involved in tree seedling damping-off in French forest nurseries. Appl. Soil Ecol. 10(1/2), 65-71. Carling, D. E. (1997) First report of *Rhizoctonia solani* AG-7 in Georgia. Plant Dis. 82(1), 127.

*Rhizoctonia solani* AG2-2 LP associated with large patch disease of zoysia grass.

and pathogenicity of *Rhizoctonia* isolates associated with black root rot of strawberries in the Western Cape Province, South Africa. Australasian Plant Pathol.

among isolates of *Ceratobasidium cornigerum* and related fungi. Mycologia 72, 689-

18. **AG-S** (Demirci, 1998; Sneh *et al*., 1998).

Host: azalea, wheat, barley, and azalea. 19. **AG-T:** (Hyakumachi *et al.,* 2005).

Symptoms: no specific diseases.

Symptoms: stem rot and root rot.

Symptoms: stem rot and root rot.

20. **AG-U:** (Hyakumachi *et al*., 2005).

Host: miniature roses (*Rosa rugosa* Thunb.).

China (30660006, and 31160352), respectively.

Plant Dis. 82, 857-863.

80(1), 104.

Dis. 79(3), 321.

32(2), 195-201.

701.

Host: miniature roses.

**3. Summary** 

**4. Acknowledgments** 

**5. References** 

7. **AG-G** (Mazzola, 1997; Sneh *et al*., 1998; Leclerc *et al*., 1999; Martin, 2000; Botha *et al*., 2003; Fenille *et al*., 2005).

Symptoms: damping-off, root rot, and browning.

Host: strawberry, sugar beet, bean, pea, tomato, melon, sunflower, peanut, yacoon, apple, *Rhododendron* Linn., and *Fragaria x ananassa*.

Note: Non-pathogenic binucleate *Rhizoctonia* spp. provide effective protection to young bean seedlings against root rot caused by *R. solani* AG-4 (Leclerc *et al*., 1999).

8. **AG-H** (Hayakawa *et al*., 1999).

Symptoms: symbiosis (orchids).

Host: *Dactylorhiza aristata* (Orchidaceae).

9. **AG-I** (Mazzola, 1997; Sneh *et al*., 1998; Ravanlou and Banihashemi, 2002)

Symptoms: root rot and symbiosis (orchids).

Host: strawberry, sugar beet, wheat, apple, orchids, and *Fragaria x ananassa.*

10. **AG-J**: (Sneh *et al*.*,* 1998).

Symptoms: none.

Host: apple.

11. **AG-K** (Demirci, 1998; Li *et al*., 1998; Sneh *et al*., 1998; Ravanlou and Banihashemi, 2002).

Symptoms: none.

Host: sugar beet, radish, tomato, carrot, onion, wheat, maize, *Allium cepa* (source: DDJB),

*Pyrus communis* (pear) (source: DDJB), and Fragaria x ananassa.


Host: apple.

15. **AG-P:** (Sneh *et al*., 1998; Yang *et al*., 2006).

Symptoms: black rot and wirestem.

Host: tea (*Camellia* Linn.), red birch.

16. **AG-Q:** (Sneh *et al.*, 1998).

Symptoms: none.

Host: (Bentgrass).

17. **AG-R:** (Sneh *et al*., 1998;Yang *et al*., 2006).

Symptoms: wirestem

Host: bean, pea, radish, onion, leaf lettuce, tomato, lima bean, snap bean, soybean, cowpea, peanuts, red birch, and azalea.

18. **AG-S** (Demirci, 1998; Sneh *et al*., 1998).

Symptoms: no specific diseases.

Host: azalea, wheat, barley, and azalea.

19. **AG-T:** (Hyakumachi *et al.,* 2005).

Symptoms: stem rot and root rot.

Host: miniature roses.

48 Plant Pathology

7. **AG-G** (Mazzola, 1997; Sneh *et al*., 1998; Leclerc *et al*., 1999; Martin, 2000; Botha *et al*.,

Host: strawberry, sugar beet, bean, pea, tomato, melon, sunflower, peanut, yacoon, apple,

Note: Non-pathogenic binucleate *Rhizoctonia* spp. provide effective protection to young bean

11. **AG-K** (Demirci, 1998; Li *et al*., 1998; Sneh *et al*., 1998; Ravanlou and Banihashemi, 2002).

Host: sugar beet, radish, tomato, carrot, onion, wheat, maize, *Allium cepa* (source: DDJB),

14. **AG-O:** No special diseases have been reported (Mazzola, 1997; Sneh *et al*., 1998).

Host: bean, pea, radish, onion, leaf lettuce, tomato, lima bean, snap bean, soybean, cowpea,

seedlings against root rot caused by *R. solani* AG-4 (Leclerc *et al*., 1999).

9. **AG-I** (Mazzola, 1997; Sneh *et al*., 1998; Ravanlou and Banihashemi, 2002)

Host: strawberry, sugar beet, wheat, apple, orchids, and *Fragaria x ananassa.*

*Pyrus communis* (pear) (source: DDJB), and Fragaria x ananassa.

12. AG-L: No special diseases have been reported (Sneh *et al*., 1991). 13. **AG-N:** No special diseases have been reported (Sneh *et al*.*,* 1991).

2003; Fenille *et al*., 2005).

8. **AG-H** (Hayakawa *et al*., 1999). Symptoms: symbiosis (orchids).

10. **AG-J**: (Sneh *et al*.*,* 1998).

Symptoms: none.

Symptoms: none.

Host: apple.

Host: apple.

Host: *Dactylorhiza aristata* (Orchidaceae).

Symptoms: root rot and symbiosis (orchids).

15. **AG-P:** (Sneh *et al*., 1998; Yang *et al*., 2006).

17. **AG-R:** (Sneh *et al*., 1998;Yang *et al*., 2006).

Symptoms: black rot and wirestem. Host: tea (*Camellia* Linn.), red birch.

16. **AG-Q:** (Sneh *et al.*, 1998).

peanuts, red birch, and azalea.

Symptoms: none. Host: (Bentgrass).

Symptoms: wirestem

Symptoms: damping-off, root rot, and browning.

*Rhododendron* Linn., and *Fragaria x ananassa*.

20. **AG-U:** (Hyakumachi *et al*., 2005).

Symptoms: stem rot and root rot.

Host: miniature roses (*Rosa rugosa* Thunb.).

#### **3. Summary**

In this chapter, we described the classification of *Rhizoctonia* spp. complex. Mutinucleate *Rhizoctonia* spp. included 13 anastomosis, of which AG 1-4 were strong pathogenic on many plants and AG 6-10 were orchid mycorrhizae. Binucleate *Rhizoctonia* spp. included 18 anastomosis groups, but AG-U belonged to AG-P and AG-T belonged to AG-A (Sharon et al., 2008), which were weak or nonpathogenic to plants and some AGs were orchid mycorrhizae.

#### **4. Acknowledgments**

This work was supported by the National Basic Research Program (No. 2011CB100400) from The Ministry of Science and Technology of China and the National Natural Science Funds, China (30660006, and 31160352), respectively.

#### **5. References**


General Description of *Rhizoctonia* Species Complex 51

Kumar, S., Sivasithamparam, K. & Sweetingham, M. W. (2002) Prolific production of

Kuninaga, S. & Yokosawa, R. (1980) A comparison of DNA compositions among anastomosis groups in *Rhizoctonia solani* Kühn. Ann. Phytopathol. Soc. Japan 46,: 150-158. Kuninaga, S., Nicoletti, R., Lahoz, E. & Naito, S. (2000). Ascription of Nt-isolates of

Leclerc, P. C., Balmas, V., Charest, P. M. & Jabaji,H. S. (1999) Development of reliable

Li, H.R. & Yan, S.Q. (1990) Studies on the strains of pathogens of sheath blight of rice in the

Li, H.R., Wu, B.C. & Yan, S. Q. (1998) Aetiology of *Rhizoctonia* in sheath blight of maize in

MacNish, G. C., Carling, D. E. & Brainard, K. A. (1997) Relationship of microscopic

Martin, F. N. (2000) *Rhizoctonia* spp. recovered from strawberry roots in central coastal

Mazzola, M. (1997) Identification and pathogenicity of *Rhizoctonia* spp. isolated from apple

Meyer, L., Wehner, F. C., Nel, L. H. & Carling, D. E. (1998) Characterization of the crater

Moore, R. T. (1987) The genera of *Rhizoctonia*-like fungi: *Asorhizoctonia, Ceratorhiza* gen. nov., *Epulorhiza* gen. nov., *Moniliopsis* and *Rhizoctonia*. Mycotaxon 29, 91-99. Naito, S. (2004) Rhizoctonia diseases: Taxonomy and population biology. Proceeding of the

Naito, S. & Kanematsu, S. (1994) Characterization and pathogenicity of a new anastomosis

Naito, S., Mohamad, D., Nasution, A &. Purwanti, H. (1993) Soilborne diseases and ecology

Nicoletti, R., Lahoz, E., Kanematsu, S., Naito, S. & Contillo, R. (1999) Characterization of

Ogoshi, A. (1975) Grouping of *Rhizoctonia solani* Kühn and their perfect stages. Rev. Plant.

Ogoshi, A. & Ui, T. (1979) Specificity in vitamin requirement among anastomosis groups of

Ogoshi, A., Oniki, M., Araki, T. & Ui, T. (1983a) Anastomosis groups of binucleate

International Seminar on Biological Control of Soilborne Plant Diseases, Japan-

subgroup AG-2-3 of *Rhizoctonia solani* Kühn isolated from leaves of soybean. Ann.

*Rhizoctonia solani* isolates from tobacco fields related to anastomosis groups 2–1 and

*Rhizoctonia* in Japan and North America and their perfect states. Mycol. Soc. Japan

compatible populations (VCP) in AG-8. Mycol. Res. 100, 61-68.

disease strain of *Rhizoctonia solani*. Phytopathology 88, 366-371.

Argentina Joint Study, Buenos Aires, Argentina, p.18-31.

BI (AG 2–1 and AG BI). J. Phytopathology 147 (2), 71-77.

*Rhizoctonia solani* Kühn. Ann. Phytopathol.Soc. Jpn. 45, 47-53.

of pathogens on soybean roots in Indonesia. JARQ 26, 247-253.

roots and orchard soils. Phytopathology 87, 582-587.

lupin. Annal. Appl. Biol. 141(1), 11-18.

sequence similarity. J. Plant Pathol. 82, 61-64.

using PCR. Mycol. Res. 103(9), 1165-1172.

Sichuan. Plant Pathol. 47 (1), 16-21.

California. Phytopathology 90, 345-353.

Phytopath. Soc. Japan 60(6), 681-690.

Protect. Res. 8, 93-103.

24, 79-87.

English abstract).

sclerotia in soil by *Rhizoctonia solani* anastomosis group (AG) 11 pathogenic on

*Rhizoctonia solani* to anastomosis group 2-1 (AG-2-1) on account of rDNA-ITS

molecular markers to detect non-pathogenic binucleate *Rhizoctonia* isolates (AG-G)

east and south of Sichuan Province. Acta Mycologica Sinica 9: 41-9. (Chinese with

vegetative reactions in *Rhizoctonia solani* and the occurrence of vegetatively


Carling, D. E. (2000) Anastomosis groups and subsets of anastomosis groups of *Rhizoctonia* 

Carling, D. E., Brainard, K.A., Virgen-Calleros, G. & Olalde-Portugal, V.F. (1998) First report of *Rhizoctonia solani* AG-7 on potato in Mexico. Plant Dis. 82(1), 127. Carling, D. E., Kuninaga, S. & Brainard, K. A. (2002b) Hyphal anastomosis reactions, rDNA-

Carling, D. E. (1996) Grouping in *Rhizoctonia solani* by hyphal anastomosis reaction. In: Sneh,

Carling, D. E., Baird, R. E., Gitaitis, R. D., Brainard, K. A. & Kuninaga, S. (2002c)

de Candolle, A. 1815. Uredo rouille des cereals In Forafran caise, famille des champigons p.83. Demirci, E. (1998) *Rhizoctonia* species and anastomosis groups isolated from barley and

Eken, C. & Demirci, E. (2004) Anastomosis groups and pathogenicity of *Rhizoctonia solani* 

Fenille, R.C., Ciampi, M.B., Souza, N.L., Nakataniand, A.K. and Kuramae, E.E.(2005)

Fenille, R. C., Luizde S. N. & Kuramae, E. E. (2002) Characterization of *Rhizoctonia solani* associated with soybean in Brazil. Eur. J. of Plant Pathol. 108(8), 783-792. Gonzalez, D., Carling, D. E., Kuninaga, S., Vilgalys, R. & Cubeta, M. A. (2001) Ribosomal

Hayakawa, S., Uetake, Y. & Ogoshi, A.(1999) Identification of symbiotic rhizoctonia from

Holtz, B. A., Michailides, T. J., Feguson, L., Hancock, J. G. & Weinhold, A. R. (1996) First report of *Rhizoctonia solani* (AG-4) on pistachio rootstock seedlings. Plant Dis. 80(11), 1303. Hyakumachi, M., Priyatmojo, A., Kubota, M. & Fukui, H. (2005) New anastomosis groups,

Hyyakumachi, M. and Ui,T. (1988)Development of the teleomorph of non-self-anastomosing isolates of Rhizoctonia solani by a buried-slide method plant pathol. 37(3):438-440 Khan, R. U. & Kolte, S. J. (2000) Some seedling diseases of rapeseed-mustard and their

the Faculty of Agriculture Hokkaido University 69(2), 129-141.

flower and miniature roses. Phytopathology 95,784-792.

control. Indian Phytopathol. 55(1), 102-103.

*Rhizoctonia*. National Chung Hsing University, Taichung, Taiwan .14 pp. Carling, D. E., Baird, R. E., Gitaitis, R. D., Brainard, K. A. & Kuninaga, S. (2002a)

*solani*. Phytopathology 92(8), 893-899.

anastomosis group. Phytopathology 89(10), 942-946.

Publishers, Dordecht, The Netherlands, pp 37-47.

wheat in Erzurum, Turkey. Plant Pathol. 47(1), 10-15.

sonchifolius) in Brazail. Plant patho. 54,325-330.

*solani*. Phytopathology 92,893-899.

Mycologia 93 (6), 1138-1150.

86(1), 49-52.

*solani*. Abstract in Proceedings of the Third International Symposium on

Characterization of AG-13, a newly reported anastomosis group of *Rhizoctonia* 

internal transcribed spacer sequences, and virulence levels among subsets of *Rhizoctonia solani* anastomosis group-2 (AG-2) and AG-BI. Phytopathology 92, 43-50. Carling, D. E., Pope, E. J., Brainard, K. A. & Carter, D. A. (1999) Characterization of

mycorrhizal isolates of *Rhizoctonia solani* from an orchid, including AG-12, a new

B., Jabaji-Hare, S., Neat, S. and Dijst, G. *et al*., (eds). *Rhizoctonia* species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic

Characterization of AG-13, a newly reported anastomosis group of *Rhizoctonia* 

and binucleate *Rhizoctonia* isolates from bean in Erzurum, Turkey J. Plant Pathol.

Binucleate Rhizoctonia sp. AG-G causing rot rot in yacon (Smallanyhus

DNA systematics of *Ceratobasidium* and *Thanatephorus* with *Rhizoctonia* anamorphs.

naturally occurring protocorms and roots of *Dactylorhiza aristata* (Orchidaceae). J. of

AG-Tand AG-U, of binucleate *Rhizoctonia* spp. causing root and stem rot of cut-


**3** 

**Phytobacterial Type VI Secretion System –** 

Panagiotis F. Sarris1,2, Emmanouil A. Trantas2, Nicholas Skandalis3,

Microbes, and their distant relatives, plants, are thought to have co-evolved during the last 2 billion years. Most of the plant-associated prokaryotes are commensals, found primarily on leaf surfaces or roots, and have no discernible or known effects on plant growth or physiology; others evolved more or less intimate relationships with plants such as N-fixing symbioses, endophytic existence or plant growth-promoting (rhizobacterial) associations; yet others, the minority, wage outright hostility with plants, inciting various diseases.

Although some phytopathogenic bacteria internalize themselves in the plant vascular system, most of them colonize plant tissues extracellularly and target plant cell wall and membrane or internal cellular structures, signaling systems and metabolic machinery from the outside. For targeting they deploy phytotoxic metabolites, hormones, polysaccharides, enzymes for the hydrolysis of cell walls and other catalytic macromolecular effectors (and, exceptionally, DNA) as "ballistic missiles". To accomplish efficient transport of macromolecules across the bacterial and/or the plant cell envelop (plant cell wall and membrane), Gram-negative bacteria possess a suite of specialized transport systems, dedicated to the transport of selected sets of proteins from the bacterial cytoplasm to the external environment or into other living cells. Type I to type VI secretion systems (abbreviated T1SS to T6SS) form channels by assembling oligomeric macromolecular complexes of varying composition and sophistication. These assemblies function as molecular machines, are broadly conserved across Gram-negative bacteria and

**1. Introduction** 

**Gene Distribution, Phylogeny, Structure** 

**and Biological Functions** 

Anastasia P. Tampakaki4, Maria Kapanidou1,

*1Department of Biology, University of Crete, Heraklion* 

*Technological Educational Institute of Crete, Heraklion 3Benaki Phytopathological Institute, Kifisia, Athens* 

> *4Department of Agricultural Biotechnology, Agricultural University of Athens, Athens*

*6University of California, Berkeley, CA,* 

*1,2,3,4,5Greece 6USA* 

*5Institute of Molecular Biology and Biotechnology,* 

*Foundation for Research and Technology-Hellas, Heraklion* 

Michael Kokkinidis1,5 and Nickolas J. Panopoulos1,6

*2Department of Plant Sciences, School of Agricultural Technology,* 


### **Phytobacterial Type VI Secretion System – Gene Distribution, Phylogeny, Structure and Biological Functions**

Panagiotis F. Sarris1,2, Emmanouil A. Trantas2, Nicholas Skandalis3, Anastasia P. Tampakaki4, Maria Kapanidou1, Michael Kokkinidis1,5 and Nickolas J. Panopoulos1,6 *1Department of Biology, University of Crete, Heraklion 2Department of Plant Sciences, School of Agricultural Technology, Technological Educational Institute of Crete, Heraklion 3Benaki Phytopathological Institute, Kifisia, Athens 4Department of Agricultural Biotechnology, Agricultural University of Athens, Athens 5Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion 6University of California, Berkeley, CA, 1,2,3,4,5Greece 6USA* 

#### **1. Introduction**

52 Plant Pathology

Ogoshi, A., Oniki, M., Araki, T. & Ui, T. (1983b) Studies on the anastomosis groups of

Parmeter, J. R. J., Sherwood, R. T. & Platt, W. D. (1969) Anastomosis grouping among

Parmeter, J. R. Jr. & Whitney, H. S. (1970) Taxonomy and nomenclature of the imperfect

Pope, E. J. & Carter, D. A. (2001) Phylogenetic placement and host specificity of mycorrhizal

Priyatmojo, A., Escopalao, V. E., Tangonan, N. G., Pascual, C. B., Suga, H., Kageyama, K. &

Ravanlou, A. & Banihashemi, Z. (2002) Isolation of some anastomosis groups of *Rhizoctonia*

Rollins, P. A., Keinath, A. P. & Farnham, M. W. (1999) Effect of inoculum type and

Satoh, Y., Kanehira, T. & Shinohara, M. (1997) Occurrence of seedling damping-off of Jew's

Sharon, M., Kuninaga, S., Hyakumachi, M., Naito, S., & Sneh B. (2008) Classification of

Sneh, B., Burpee, L. and Ogoshi, A. (1998) Identification of *Rhizoctonia* species. The APS, St.

Sumner, D. R. (1985) First report of *Rhizoctonia solani* AG-2-4 on carrot in Georgia. Plant Dis.

Sumner, D. R., Phatak, S. C. & Carling, D. E. (2003) Characterization and pathogenicity of a

Toda, T., Hyakumachi, M., Suga, H., Kageyama, K., Tanaka, A. & Tani, T. (1999)

Yang G. H., Chen H. R., Naito, S., Wu, J. Y., He, X. H. & Duan, C. F. (2005b) Occurrence of

Yang, G. H., Naito, S., Ogoshi, A. & Dong, W. H. (2006) Identification, isolation frequency

based on rDNA and RAPD analyses. Eur. J. Plant Pathol. 105, 835-846. Vilgalys, R. & Gonzalez, D. (1990) Ribosomal DNA restriction fragment length

polymorphism in *Rhizoctonia solani*. Phytopathology 80, 151-158.

controlled environment. Can. J. Plant Pathol. 21(2), 119-124.

Kaiho 38(2): 87-91 (Japanese with English abstract).

classical anastomosis grouping. *Mycoscience* 49, 93–114.

isolates of *Thanatephorus cucumeris*. Phytopathology 59, 1270-1278.

*solani*. University of California Press, Berkeley. 255 pp.

Mycologia 93(4), 712-719.

Phytopathology 91,1054-1061.

38(3-4), 67-69.

Paul, Minesota.

of soybean. Plant Dis. 87(10), 1264.

China. J. Gen. Plant Pathol.71, 377–379.

Phytopathology 154(2), 80-83.

69, 25-27.

binucleate *Rhizoctonia* and their perfect states. J. Fac. Agr. Hokkaido Univ. 61, 244-260.

state. Pages 7-19 in: J. R. Parmeter Jr., (ed.) Biology and Pathology of *Rhizoctonia* 

isolates belonging to AG-6 and AG-12 in the *Rhizoctonia solani* species complex.

Hyakumachi, M. (2001) Characterization of a new subgroup of *Rhizoctonia solani*  anastomosis group 1 (AG-1 ID), causal agent of a necrotic leaf spot on coffee.

associated with wheat root and crown in Fars province. Iranian J. Plant Pathol.

anastomosis group of *Rhizoctonia solani* causing wirestem of cabbage seedlings in a

mallow, *Corchorus olitorius* caused by *Rhizoctonia solani* AG-2-1. Nippon Kingakukai

*Rhizoctonia* spp. using rDNA-ITS sequence analysis supports the genetic basis of the

new anastomosis subgroup AG-2-3 of *Rhizoctonia solani* Kuün isolated from leaves

Differentiation of *Rhizoctonia* AG-D isolates from turfgrass into subgroups I and II

foliar rot of pak choy and Chinese mustard caused by *Rhizoctonia solani* AG-1 IB in

and pathogenicity of *Rhizoctonia* spp. causing the wirestem of red birch in China. J.

Microbes, and their distant relatives, plants, are thought to have co-evolved during the last 2 billion years. Most of the plant-associated prokaryotes are commensals, found primarily on leaf surfaces or roots, and have no discernible or known effects on plant growth or physiology; others evolved more or less intimate relationships with plants such as N-fixing symbioses, endophytic existence or plant growth-promoting (rhizobacterial) associations; yet others, the minority, wage outright hostility with plants, inciting various diseases.

Although some phytopathogenic bacteria internalize themselves in the plant vascular system, most of them colonize plant tissues extracellularly and target plant cell wall and membrane or internal cellular structures, signaling systems and metabolic machinery from the outside. For targeting they deploy phytotoxic metabolites, hormones, polysaccharides, enzymes for the hydrolysis of cell walls and other catalytic macromolecular effectors (and, exceptionally, DNA) as "ballistic missiles". To accomplish efficient transport of macromolecules across the bacterial and/or the plant cell envelop (plant cell wall and membrane), Gram-negative bacteria possess a suite of specialized transport systems, dedicated to the transport of selected sets of proteins from the bacterial cytoplasm to the external environment or into other living cells. Type I to type VI secretion systems (abbreviated T1SS to T6SS) form channels by assembling oligomeric macromolecular complexes of varying composition and sophistication. These assemblies function as molecular machines, are broadly conserved across Gram-negative bacteria and

Phytobacterial Type VI Secretion System –

but others may merely await identification.

mechanisms of these bacterial nanomachines.

surface (Pukatzki et al., 2007).

Gene Distribution, Phylogeny, Structure and Biological Functions 55

studies in a few bacteria further suggest that each T6SSs assumes a different role in the interactions of the harbouring organism with others. However, it is not known if there are T6SSs that can target both prokaryotes and eukaryotes. Unlike the Type III secretion systems (T3SSs), only few T6SS substrates have been identified and experimentally verified to date,

It is now becoming increasingly clear that T6SS probably represents an evolutionary adaptation of a transmembrane protein translocation mechanism and at least some of its core components may share a common ancestor with bacteriophages. Common evolutionary ancestry and similar design are features ostensibly shared between other macromolecular transport systems and other bacterial devices that have evolved to serve entirely different biological functions (e.g. between T2SS and type IV pili, T3SS and flagella, or T4SS and conjugative pili). Indeed, the study of the macromolecular assembly process in these systems cross-feeds our understanding about structure, function and molecular

The T6SS appears to be an injectisome, with some of its core component proteins structurally related to the cell-puncturing devices of tailed bacteriophages, and at least in some well-studied cases, have been shown capable of translocating effector proteins into the host cell cytoplasm (Bingle et al., 2008; Cascales, 2008; Filloux et al., 2008, 2011a; 2011b; Shrivastava & Mande 2008; Russell et al., 2011; Zheng et al., 2011), as is the case with the T3SS and T4SS. In the human pathogenic species *V. cholerae* and *P. aeruginosa* T6SS exports haemolysin-coregulated proteins (Hcp) and Valine-glycine repeat (Vgr) proteins; for these proteins the role of effectors associated with cytotoxicity in some *in vitro* models has been proposed (Pukatzki et al., 2006; Mougous et al., 2006). However, VgrG and Hcp display mutual dependence for secretion in *V. cholerae*, *Edwardsiella tarda* and enteroaggregative *E. coli* (Pukatzki et al., 2007; Zheng & Leung, 2007; Dudley et al., 2006), suggesting that these proteins might be not only passengers but also components of the secretion machine, a fact also supported by recent structural studies (see section 3.3). Such "dual function" could be related to distinct protein domains. In particular, the N-terminal domains of Vgr proteins show strong homology with the T4 bacteriophage base plate components gp27 and gp5 (Pukatzki et al., 2007) and a conserved core followed by a highly polymorphic C-terminal domain. The *V. cholerae* VgrG1 protein has a C-terminal domain homologous to the actin cross-linking domain (ACD) of the RtxA toxin, while VgrG1 from *Aeromonas hydrophila* possesses actin ADP-ribosylating activity (Pukatzki et al., 2007; Sheahan et al., 2004; Suarez et al., 2010). Some VgrGs ("evolved" VgrGs) from various bacterial species possess various effector-like C-terminal domains: a) a tropomyosin-like domain, which is thought to manipulate actin filaments during *Yersinia* infections, b) a pertactin-like, YadA-like, mannose-binding-like, or fibronectin-like domains or share similarities with peptidoglycan- or fibronectin-binding sequences and c) homologs of the eukaryotic lysosomal cathepsin D protein (Cascales, 2008). On the other hand, it was suggested that some VgrG orthologs may not be injected but may remain attached to the bacterial cell

Similarly to T3S systems, Hcp-secreted proteins lack N-terminal hydrophobic signal sequences, indicating secretion in a Sec- or Tat-independent manner, and a probable crossing of the bacterial cell envelope in a single step (Bingle et al., 2008; Pallen et al., 2003). Furthermore, Hcp-secreted proteins seem to have intracellular targets in eukaryotic hosts.

play important roles in the virulence of pathogens. In general, most of these systems require a component providing energy to the secretion process (usually an ATPase), an outer-membrane protein, and various components involved either in scaffolding the macromolecular complex into the cell envelope or in the specific recognition of secreted substrates. It is noteworthy that certain components of these secretion machines are thought to be derived from other membrane-bound multiprotein structures serving a different purpose (see below).

#### **2. Historical highlights**

The T6SS is a relatively recent discovery, first identified as a protein secretion apparatus involved in virulence of *Vibrio cholerae* in the *Dictyostelium* (Mougous et al., 2006) and *Pseudomonas aeruginosa* in mouse models (Pukatzki et al., 2006). In several cases it has been shown to be important for bacterial virulence (host-pathogen interaction) and has attracted strong interest because it has been found via *in silico* analysis in the genomes of a large number of Gram-negative bacteria associated with human and animal diseases. While initially considered an atypical type IV secretion system (T4SS), various lines of evidence have established its identity as a distinct protein transport system (Bladergroen et al., 2003; Roest et al., 1997; Pukatski et al. 2006, Boyer et al., 2009).

An interesting twist to the T6SS story is the discovery of multiple copies of gene clusters coding for T6SS homologs in a large number of sequenced eubacterial genomes, including those of several plant-associated species (KEGG gene database; http://www.genome.jp/ kegg-bin/get\_htext?ko02044.keg). These species are mostly within the class of *Proteobacteria*, but also within the *Planctomycetes* and *Acidobacteria* (Tseng et al., 2009; Boyer et al., 2009). Several T6SS gene clusters are within "pathogenicity islands", for example, *P. aeruginosa-*HSI (Hcp-secretion island), enteroaggregative *Escherichia coli* (EAEC-*pheU*), *Salmonella typhimurium*-SCI (*Salmonella* Centrisome Island), *Francisella tularensis-*FPI (*Francisella* Pathogenicity Island), *Agrobacterium tumefaciens* (Wu et al., 2008)*, Pectobacterium atrosepticum* (Liu et al., 2008) and *Xanthomonas oryzae* (Tseng et al., 2009), which indicates relationship to virulence or survival in the host. Bioinformatic analysis revealed that most of the "avirulent" bacterial species studied (i.e. bacteria that have no known host) lack T6SS orthologs but active protein secretion or the ability to invade hosts await experimental testing (Shrivastava & Mande 2008; Bingle et al., 2008). Many interesting highlights are best expressed by the imaginative titles of many publications cited in this chapter.

#### **3. T6SS genes, proteins, injectisomes**

#### **3.1 Gene content and proteins of T6SS clusters**

T6SSs are typically encoded by clusters of 12 to over 20 genes, with 13 genes thought to constitute the minimal number needed to produce a functional apparatus (Boyer et al., 2009). They are found mostly in α-, β-, and γ-proteobacteria (about 25% of the sequenced genomes; Bingle et al., 2008). Recently, Chow and Mazmanian (2010) characterized a T6SS in *Helicobacter hepaticus*, which belongs to the ε-(epsilon) subgroup of proteobacteria. These clusters (frequently referred to as T6SS loci in the literature) often occur in multiple, nonorthologous copies/genome (i.e. are not the result of simple duplication), indicating that they have probably been acquired by horizontal gene transfer (Sarris at al., 2011). Detailed

play important roles in the virulence of pathogens. In general, most of these systems require a component providing energy to the secretion process (usually an ATPase), an outer-membrane protein, and various components involved either in scaffolding the macromolecular complex into the cell envelope or in the specific recognition of secreted substrates. It is noteworthy that certain components of these secretion machines are thought to be derived from other

The T6SS is a relatively recent discovery, first identified as a protein secretion apparatus involved in virulence of *Vibrio cholerae* in the *Dictyostelium* (Mougous et al., 2006) and *Pseudomonas aeruginosa* in mouse models (Pukatzki et al., 2006). In several cases it has been shown to be important for bacterial virulence (host-pathogen interaction) and has attracted strong interest because it has been found via *in silico* analysis in the genomes of a large number of Gram-negative bacteria associated with human and animal diseases. While initially considered an atypical type IV secretion system (T4SS), various lines of evidence have established its identity as a distinct protein transport system (Bladergroen et al., 2003;

An interesting twist to the T6SS story is the discovery of multiple copies of gene clusters coding for T6SS homologs in a large number of sequenced eubacterial genomes, including those of several plant-associated species (KEGG gene database; http://www.genome.jp/ kegg-bin/get\_htext?ko02044.keg). These species are mostly within the class of *Proteobacteria*, but also within the *Planctomycetes* and *Acidobacteria* (Tseng et al., 2009; Boyer et al., 2009). Several T6SS gene clusters are within "pathogenicity islands", for example, *P. aeruginosa-*HSI (Hcp-secretion island), enteroaggregative *Escherichia coli* (EAEC-*pheU*), *Salmonella typhimurium*-SCI (*Salmonella* Centrisome Island), *Francisella tularensis-*FPI (*Francisella* Pathogenicity Island), *Agrobacterium tumefaciens* (Wu et al., 2008)*, Pectobacterium atrosepticum* (Liu et al., 2008) and *Xanthomonas oryzae* (Tseng et al., 2009), which indicates relationship to virulence or survival in the host. Bioinformatic analysis revealed that most of the "avirulent" bacterial species studied (i.e. bacteria that have no known host) lack T6SS orthologs but active protein secretion or the ability to invade hosts await experimental testing (Shrivastava & Mande 2008; Bingle et al., 2008). Many interesting highlights are best expressed by the imaginative titles of many publications

T6SSs are typically encoded by clusters of 12 to over 20 genes, with 13 genes thought to constitute the minimal number needed to produce a functional apparatus (Boyer et al., 2009). They are found mostly in α-, β-, and γ-proteobacteria (about 25% of the sequenced genomes; Bingle et al., 2008). Recently, Chow and Mazmanian (2010) characterized a T6SS in *Helicobacter hepaticus*, which belongs to the ε-(epsilon) subgroup of proteobacteria. These clusters (frequently referred to as T6SS loci in the literature) often occur in multiple, nonorthologous copies/genome (i.e. are not the result of simple duplication), indicating that they have probably been acquired by horizontal gene transfer (Sarris at al., 2011). Detailed

membrane-bound multiprotein structures serving a different purpose (see below).

Roest et al., 1997; Pukatski et al. 2006, Boyer et al., 2009).

**2. Historical highlights** 

cited in this chapter.

**3. T6SS genes, proteins, injectisomes** 

**3.1 Gene content and proteins of T6SS clusters** 

studies in a few bacteria further suggest that each T6SSs assumes a different role in the interactions of the harbouring organism with others. However, it is not known if there are T6SSs that can target both prokaryotes and eukaryotes. Unlike the Type III secretion systems (T3SSs), only few T6SS substrates have been identified and experimentally verified to date, but others may merely await identification.

It is now becoming increasingly clear that T6SS probably represents an evolutionary adaptation of a transmembrane protein translocation mechanism and at least some of its core components may share a common ancestor with bacteriophages. Common evolutionary ancestry and similar design are features ostensibly shared between other macromolecular transport systems and other bacterial devices that have evolved to serve entirely different biological functions (e.g. between T2SS and type IV pili, T3SS and flagella, or T4SS and conjugative pili). Indeed, the study of the macromolecular assembly process in these systems cross-feeds our understanding about structure, function and molecular mechanisms of these bacterial nanomachines.

The T6SS appears to be an injectisome, with some of its core component proteins structurally related to the cell-puncturing devices of tailed bacteriophages, and at least in some well-studied cases, have been shown capable of translocating effector proteins into the host cell cytoplasm (Bingle et al., 2008; Cascales, 2008; Filloux et al., 2008, 2011a; 2011b; Shrivastava & Mande 2008; Russell et al., 2011; Zheng et al., 2011), as is the case with the T3SS and T4SS. In the human pathogenic species *V. cholerae* and *P. aeruginosa* T6SS exports haemolysin-coregulated proteins (Hcp) and Valine-glycine repeat (Vgr) proteins; for these proteins the role of effectors associated with cytotoxicity in some *in vitro* models has been proposed (Pukatzki et al., 2006; Mougous et al., 2006). However, VgrG and Hcp display mutual dependence for secretion in *V. cholerae*, *Edwardsiella tarda* and enteroaggregative *E. coli* (Pukatzki et al., 2007; Zheng & Leung, 2007; Dudley et al., 2006), suggesting that these proteins might be not only passengers but also components of the secretion machine, a fact also supported by recent structural studies (see section 3.3). Such "dual function" could be related to distinct protein domains. In particular, the N-terminal domains of Vgr proteins show strong homology with the T4 bacteriophage base plate components gp27 and gp5 (Pukatzki et al., 2007) and a conserved core followed by a highly polymorphic C-terminal domain. The *V. cholerae* VgrG1 protein has a C-terminal domain homologous to the actin cross-linking domain (ACD) of the RtxA toxin, while VgrG1 from *Aeromonas hydrophila* possesses actin ADP-ribosylating activity (Pukatzki et al., 2007; Sheahan et al., 2004; Suarez et al., 2010). Some VgrGs ("evolved" VgrGs) from various bacterial species possess various effector-like C-terminal domains: a) a tropomyosin-like domain, which is thought to manipulate actin filaments during *Yersinia* infections, b) a pertactin-like, YadA-like, mannose-binding-like, or fibronectin-like domains or share similarities with peptidoglycan- or fibronectin-binding sequences and c) homologs of the eukaryotic lysosomal cathepsin D protein (Cascales, 2008). On the other hand, it was suggested that some VgrG orthologs may not be injected but may remain attached to the bacterial cell surface (Pukatzki et al., 2007).

Similarly to T3S systems, Hcp-secreted proteins lack N-terminal hydrophobic signal sequences, indicating secretion in a Sec- or Tat-independent manner, and a probable crossing of the bacterial cell envelope in a single step (Bingle et al., 2008; Pallen et al., 2003). Furthermore, Hcp-secreted proteins seem to have intracellular targets in eukaryotic hosts.

Phytobacterial Type VI Secretion System –

**3.2 Regulation** 

available.

*hydrophila* homologs.

(Bernard et al., 2011).

Gene Distribution, Phylogeny, Structure and Biological Functions 57

Although T6SS gene expression inside macrophages has been demonstrated for several animal and human pathogens (e. g. *Burkholderia pseudomallei, S. enterica, V. cholerae* and *Francisella* (Shalom et al., 2007; Parsons & Heffron 2005; de Bruin et al., 2007), and to a small extent in plant pathogens, the signals triggering T6SS expression are largely unknown. For example, in *V. cholerae,* upon phagocytosis, expression of the T6SS induces cytoskeleton rearrangements through the secretion of the actin cross-linking domain of VgrG (Ma et al., 2009). Likewise, the Hcp1 of *P. aeruginosa* is induced during the infection of cystic fibrosis patients (Mougous et al., 2006), while Hcp3 is expressed upon addition of epithelial cell extracts (Chugani & Greenberg, 2007). No information concerning the expression of Hcp2 is

The T6SS regulation involves various transcriptional activators such as AraC, TetR-, and MarR-like proteins, σ54-like factors and heat-stable nucleoid-structural (H-NS) proteins (Bernard et al., 2010). Likewise, two-component systems, like the ferric uptake regulator Fur and the Quorum sensing (QS) related regulators like LuxI, LuxR and acyl homoserine lactones (AHL) are reported to be involved in regulation of T6SS expression (Bernard et al., 2010). It has been also reported (Mougous et al., 2007) that the regulation of the HSI-1 in *P. aeruginosa* PAO1 is influenced by the sensor kinases RedS and the LadS, resulting in opposite patterns of regulation for the type III and type VI secretion systems in this bacterium, as in *S. enterica* (Parsons & Heffron 2005). T6SS gene expression in *P. syringae pv. syringae* B278a is also regulated by the two sensor kinases RetS (negatively) and LadS (positively). Two more proteins, PpkA and PppA, seem to play an important role in T6SS gene regulation (Mougous et al., 2007). PpkA is a kinase, which becomes activated by autophosphorylation under certain environmental conditions whereas PppA is a phosphatase, which counteracts with the action of PpkA. Both proteins act on a common protein substrate, Fha1 (*f*ork *h*ead-*a*ssociated domain protein; Mougous et al., 2007). Gene expression in HSI-2 and HSI-3 is proposed to be regulated by two σ54 factors which are encoded in the respective T6SS clusters of *P. aeruginosa*, as well as in their *V. cholerae* and *A.* 

Recent work reported the identification of Fur as the main regulator of the enteroaggregative *E. coli sci1* T6SS gene cluster. A detailed analysis of the promoter region showed the presence of conserved motifs, which are target of the DNA adenine methylase Dam (Brunet et al., 2011). The authors showed that the *sci1* gene cluster expression is under the control of an epigenetic methylation-dependent switch: Fur binding prevents methylation of a conserved motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. In other work (Bernard et al., 2011), several clusters were identified (including those of *V. cholerae*, *A. hydrophila*, *P. atrosepticum*, *P. aeruginosa*, *Pseudomonas syringae* pv. *tomato*, and a *Marinomonas* sp.) as having typical –24/–12 sequences, enhancer binding motifs recognized by the alternate sigma factor σ54 which directs the RNA polymerase to cognate promoters and requires the action of a bacterial enhancer binding protein (bEBP), which binds to *cis*-acting upstream activating sequences. The authors further showed that putative bEBPs are encoded within the T6SS gene clusters possessing σ54 boxes and, through *in vitro* binding and *in vivo* reporter fusion assays, they demonstrated that the expression of these clusters is dependent on both σ54 and bEBPs

Thus, the Hcp protein of *A. hydrophila* was found in culture supernatants, as well as in the cytosol and the membrane of human epithelial cells after infection. Hcp secretion was independent of the T3SS and the flagellar system and the secreted protein was capable of binding to the murine macrophages from the outside, in addition to being translocated into mammalian model host cells; heterologous expression of this protein in HeLa cells increased the rate of apoptosis mediated by caspase 3 activation (Suarez et al., 2008). These findings are consistent with Hcp being secreted/translocated by T6SS, along with other yet unidentified effectors. The Hcp1 protein from pathogenic *P. aeruginosa* was also shown to be actively secreted in cystic fibrosis patients resulting in Hcp specific antibody production. Likewise, a novel T6SS protein, VasX, which is required for pathogenicity against the amoeboid host model *Dictyostelium discoideum* has recently been described. VasX is unique because it contains a putative pleckstrin homology domain which is typically only found in eukaryotic and not in bacterial proteins. VasX can bind to mammalian membrane lipids, an interaction mediated by the putative pleckstrin homology domain. It has been proposed that this domain may direct VasX to specific targets within the host cell resulting in disruption of host cell signaling (Miyata et al., 2011).

Another hallmark of T6SSs clusters is a gene coding for an AAA+ Clp-like ATPase , named 17 ClpV, belonging to a sub class of ClpB ATPases which comprise hexameric enzymes involved in protein quality control. A possible role of T6SS Clp-ATPase members might be the unfolding of substrates to be secreted, as demonstrated for the T4SS and the T3SS ATPases (Cascales, 2008). However, the *Salmonella enterica* T6SS Clp protein forms oligomeric complexes with ATP hydrolytic activity but fails to unfold aggregated proteins (Schlieker et al., 2005). A study by Bonemann et al. (2009) revealed the involvement of the ClpV protein of *V. cholerae* in remodelling supramolecular assemblies formed by two core components VipA/VipB (synonyms: ImpB/ImpC) which are crucial for T6SS secretion and virulence (see section 3.3). However, some bacterial species (e.g. *Rhizobium leguminosarum*  and *Francisella tularensis*) may not contain functional Clp homologs within their T6SS clusters (Filloux et al., 2008).

Another T6SS-linked gene, *icmF* (intracellular multiplication in macrophages)*,* has been previously studied in the context of T4SS secretion and shown to be necessary for efficient secretion*.* This protein carries three transmembrane domains (Sexton et al., 2004) and is partially required for *Legionella pneumophila* replication in macrophages (Purcell & Shuman 1998). Furthermore, the lack of IcmF resulted in a reduced level of another core protein, DotU, suggesting that the two proteins interact or are co-regulated. It was also shown that the lack of DotU and/or IcmF affected the stability of other core components, which suggests that DotU and IcmF assist in assembly and stability of a functional T6SS (Filloux et al., 2008). Furthermore, IcmF has been proposed to function as a further energizing component (Bonemann et al., 2009). Amino acid sequence analysis predicts that IcmF is located in the inner membrane and consists of a cytosolic and a periplasmic domain. The cytosolic domain has a conserved Walker-A motif, indicating a function as an ATPase during secretion, consistent with the finding that IcmF mutations prevent Hcp secretion (Pukatzki et al., 2006; Zheng & Leung 2007). An *icmF* mutant of avian pathogenic *E. coli* had decreased adherence to and invasion of epithelial cells, as well as decreased intra-macrophage survival and was also defective for biofilm formation on abiotic surfaces (Pace et al., 2011).

#### **3.2 Regulation**

56 Plant Pathology

Thus, the Hcp protein of *A. hydrophila* was found in culture supernatants, as well as in the cytosol and the membrane of human epithelial cells after infection. Hcp secretion was independent of the T3SS and the flagellar system and the secreted protein was capable of binding to the murine macrophages from the outside, in addition to being translocated into mammalian model host cells; heterologous expression of this protein in HeLa cells increased the rate of apoptosis mediated by caspase 3 activation (Suarez et al., 2008). These findings are consistent with Hcp being secreted/translocated by T6SS, along with other yet unidentified effectors. The Hcp1 protein from pathogenic *P. aeruginosa* was also shown to be actively secreted in cystic fibrosis patients resulting in Hcp specific antibody production. Likewise, a novel T6SS protein, VasX, which is required for pathogenicity against the amoeboid host model *Dictyostelium discoideum* has recently been described. VasX is unique because it contains a putative pleckstrin homology domain which is typically only found in eukaryotic and not in bacterial proteins. VasX can bind to mammalian membrane lipids, an interaction mediated by the putative pleckstrin homology domain. It has been proposed that this domain may direct VasX to specific targets within the host cell resulting in disruption of

Another hallmark of T6SSs clusters is a gene coding for an AAA+ Clp-like ATPase , named 17 ClpV, belonging to a sub class of ClpB ATPases which comprise hexameric enzymes involved in protein quality control. A possible role of T6SS Clp-ATPase members might be the unfolding of substrates to be secreted, as demonstrated for the T4SS and the T3SS ATPases (Cascales, 2008). However, the *Salmonella enterica* T6SS Clp protein forms oligomeric complexes with ATP hydrolytic activity but fails to unfold aggregated proteins (Schlieker et al., 2005). A study by Bonemann et al. (2009) revealed the involvement of the ClpV protein of *V. cholerae* in remodelling supramolecular assemblies formed by two core components VipA/VipB (synonyms: ImpB/ImpC) which are crucial for T6SS secretion and virulence (see section 3.3). However, some bacterial species (e.g. *Rhizobium leguminosarum*  and *Francisella tularensis*) may not contain functional Clp homologs within their T6SS

Another T6SS-linked gene, *icmF* (intracellular multiplication in macrophages)*,* has been previously studied in the context of T4SS secretion and shown to be necessary for efficient secretion*.* This protein carries three transmembrane domains (Sexton et al., 2004) and is partially required for *Legionella pneumophila* replication in macrophages (Purcell & Shuman 1998). Furthermore, the lack of IcmF resulted in a reduced level of another core protein, DotU, suggesting that the two proteins interact or are co-regulated. It was also shown that the lack of DotU and/or IcmF affected the stability of other core components, which suggests that DotU and IcmF assist in assembly and stability of a functional T6SS (Filloux et al., 2008). Furthermore, IcmF has been proposed to function as a further energizing component (Bonemann et al., 2009). Amino acid sequence analysis predicts that IcmF is located in the inner membrane and consists of a cytosolic and a periplasmic domain. The cytosolic domain has a conserved Walker-A motif, indicating a function as an ATPase during secretion, consistent with the finding that IcmF mutations prevent Hcp secretion (Pukatzki et al., 2006; Zheng & Leung 2007). An *icmF* mutant of avian pathogenic *E. coli* had decreased adherence to and invasion of epithelial cells, as well as decreased intra-macrophage survival and was also defective for biofilm formation on

host cell signaling (Miyata et al., 2011).

clusters (Filloux et al., 2008).

abiotic surfaces (Pace et al., 2011).

Although T6SS gene expression inside macrophages has been demonstrated for several animal and human pathogens (e. g. *Burkholderia pseudomallei, S. enterica, V. cholerae* and *Francisella* (Shalom et al., 2007; Parsons & Heffron 2005; de Bruin et al., 2007), and to a small extent in plant pathogens, the signals triggering T6SS expression are largely unknown. For example, in *V. cholerae,* upon phagocytosis, expression of the T6SS induces cytoskeleton rearrangements through the secretion of the actin cross-linking domain of VgrG (Ma et al., 2009). Likewise, the Hcp1 of *P. aeruginosa* is induced during the infection of cystic fibrosis patients (Mougous et al., 2006), while Hcp3 is expressed upon addition of epithelial cell extracts (Chugani & Greenberg, 2007). No information concerning the expression of Hcp2 is available.

The T6SS regulation involves various transcriptional activators such as AraC, TetR-, and MarR-like proteins, σ54-like factors and heat-stable nucleoid-structural (H-NS) proteins (Bernard et al., 2010). Likewise, two-component systems, like the ferric uptake regulator Fur and the Quorum sensing (QS) related regulators like LuxI, LuxR and acyl homoserine lactones (AHL) are reported to be involved in regulation of T6SS expression (Bernard et al., 2010). It has been also reported (Mougous et al., 2007) that the regulation of the HSI-1 in *P. aeruginosa* PAO1 is influenced by the sensor kinases RedS and the LadS, resulting in opposite patterns of regulation for the type III and type VI secretion systems in this bacterium, as in *S. enterica* (Parsons & Heffron 2005). T6SS gene expression in *P. syringae pv. syringae* B278a is also regulated by the two sensor kinases RetS (negatively) and LadS (positively). Two more proteins, PpkA and PppA, seem to play an important role in T6SS gene regulation (Mougous et al., 2007). PpkA is a kinase, which becomes activated by autophosphorylation under certain environmental conditions whereas PppA is a phosphatase, which counteracts with the action of PpkA. Both proteins act on a common protein substrate, Fha1 (*f*ork *h*ead-*a*ssociated domain protein; Mougous et al., 2007). Gene expression in HSI-2 and HSI-3 is proposed to be regulated by two σ54 factors which are encoded in the respective T6SS clusters of *P. aeruginosa*, as well as in their *V. cholerae* and *A. hydrophila* homologs.

Recent work reported the identification of Fur as the main regulator of the enteroaggregative *E. coli sci1* T6SS gene cluster. A detailed analysis of the promoter region showed the presence of conserved motifs, which are target of the DNA adenine methylase Dam (Brunet et al., 2011). The authors showed that the *sci1* gene cluster expression is under the control of an epigenetic methylation-dependent switch: Fur binding prevents methylation of a conserved motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. In other work (Bernard et al., 2011), several clusters were identified (including those of *V. cholerae*, *A. hydrophila*, *P. atrosepticum*, *P. aeruginosa*, *Pseudomonas syringae* pv. *tomato*, and a *Marinomonas* sp.) as having typical –24/–12 sequences, enhancer binding motifs recognized by the alternate sigma factor σ54 which directs the RNA polymerase to cognate promoters and requires the action of a bacterial enhancer binding protein (bEBP), which binds to *cis*-acting upstream activating sequences. The authors further showed that putative bEBPs are encoded within the T6SS gene clusters possessing σ54 boxes and, through *in vitro* binding and *in vivo* reporter fusion assays, they demonstrated that the expression of these clusters is dependent on both σ54 and bEBPs (Bernard et al., 2011).

Phytobacterial Type VI Secretion System –

separate sets of effector proteins.

Gene Distribution, Phylogeny, Structure and Biological Functions 59

The crystal structure of the *E. tarda* EvpC protein, an Hcp1 homolog from the virulent protein gene cluster EVP which contains a conserved T6SS, has been recently determined at 2.8 Å resolution (PDB accession code 3EAA) and revealed a high structural similarity with Hcp1 (Jobichen et al., 2010). In solution, EvpC exists as a dimer at low concentrations and as a hexamer at high concentrations. In the crystals symmetry-related EvpC molecules form hexameric rings which stack to form tubes similar to Hcp1. Structure-based mutagenesis has revealed a critical role for EvpC secretion for three negatively charged N-terminal residues, and three positively charged C-terminal ones (Jobichen et al., 2010). This secretion impairment of EvpC decreases the virulence of the T6SS-containing pathogenic bacteria.

The structure of the N-terminal fragment (residues 1-483 out of 824) of the VgrG protein encoded by the *E. coli* CFT073 gene c3393 was determined at a resolution of 2.6 Å (PDB ID 2P5Z). The protein shows striking structural similarities (Leiman et al., 2009) to the structure of the complex (gp5)3-(gp27)3 of the T4 bacteriophage cell-puncturing device (PDB ID 1K28). VgrG can be described as a fusion of T4 gp27 and gp5; at the level of equivalent domains VgrG shares the highest structural homology with gp27 and exhibits only minor modifications relative to gp5. The VgrG structure comprises a domain (residues 380-470) of unknown function which is conserved in all VgrGs. This domain (DUF586) is the equivalent of the oligosaccharide/oligonucleotide-binding (OB)-fold domain of T4 gp5. The secondary structure prediction of the C-terminal part of VgrG (residues 490-820) which follows the OBfold domain, shows repetitive β-strands (5-10 residues each) flanked by glycines. It is likely, that these strands form a β-helix that is equivalent to the triple stranded β-helix in trimeric gp5 which is involved in membrane penetration. The trimeric structure of the N-terminal VgrG fragment in the crystal, probably indicates that the complete VgrG protein may also adopt a trimeric structure that is equivalent to the (gp5)3-(gp27)3 complex. Sequence analyses suggest that effector domains are fused to the C-termini of many VgrG proteins ('evolved' VgrGs). The gp5 C-terminal β-helix has a 23 KDa extension of unknown function corresponding to a VgrG effector domain. Since many VgrG proteins do not contain an additional C-terminal domain, it may be concluded that different T6SS injectisomes service

The structural similarities of Hcp and VgrG to components of the injection apparatus of tailed bacteriophages are highly suggestive that the two proteins might be structural components of T6SS, rather than effector proteins. The absence of 'evolved' VgrGs in many T6SSs suggests also that Hcp/VgrG might act as a conduit for T6SS effectors. The inner diameter of the Hcp tubule (40 Å) would allow the passage of proteins in an unfolded form; delivery of effectors to target cells might involve the cell-puncturing activity of VgrG.

The energy for the translocation of secreted proteins to the extracellular environment is provided by two components of the T6SS that have been introduced in section 3.1: IcmF and ClpV (Bonemann et al., 2009). IcmF is a membrane-embedded component that forms a structure spanning the inner and outer membranes. ClpV does not interact with the exoproteins Hcp and VgrG, but binds specifically with its N-domain to two cytosolic proteins, VipA (COG3516) and VipB (COG3517), that are conserved and essential components of T6SS (Bonemann et al., 2009, 2010). VipA and VipB interact with each other forming a tubular, cogweel-like structure larger than 200 kDa, with a diameter of approximately 300Å and a central channel of 100Å in diameter, and a length ranging from 25 to 500nm. Electron microscopy studies of VipA/VipB suggest that there is an overall

A study by Zheng et al. (2011) provides new insights into the functional requirements of secretion as well as killing of bacterial and eukaryotic phagocytic cells by *V. cholerae* by analyzing non-polar mutations (in-frame deletions) in each gene predicted to code for *V. cholera* T6SS components. They grouped 17 proteins into four categories: twelve proteins (VipA, VipB, VCA0109–VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two proteins (VasH and VCA0122) were thought to be regulators that are required for T6SS gene expression and virulence; another two (VCA0121 and VgrG-3) were not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; one protein (VCA0118) was not required for Hcp expression or secretion but still played a role in both amoebae and bacterial killing and may therefore be an effector protein. ClpV was required for *Dictyostelium* virulence but was less important for killing *E. coli*. In addition, VgrG-2 which is encoded outside of the T6SS cluster was required for bacterial killing but VgrG-1 was not and several genes in the same putative operon as *vgrG*-1 and *vgrG*-2 also contributed to *Dictyostelium* virulence but had a smaller effect on *E. coli* killing.

#### **3.3 Structure and functions of T6SS proteins and injectisomes**

In contrast to other bacterial secretion systems (e.g. T3SS) there is only a small number of experimentally determined structures of T6SS core proteins or of their macromolecular assemblies. These structural studies have allowed within a relatively short time to understand important aspects of T6SS function to a considerable detail. Additional insights into structure-function relationships of T6SS have been deduced from structural/sequence similarities observed between T6SS proteins and a) components of cell puncturing devices utilized by tailed bacteriophages for DNA delivery or b) proteins from other types of bacterial secretion systems; experimental verification of the relationships derived by analogies to other systems remains largely to be delivered.

Medium to high resolution crystal structures exist for the *P. aeruginosa* Hcp1 protein, the Nterminal fragment of VgrG from the uropathogenic *E. coli* CFT073, and EvpC from *E. tarda*. Electron microscopy has been used in the study of the assemblies of various T6SS proteins including *P. aeruginosa* Hcp1, Hcp2 and Hcp3, the *E. coli* CFT073 Hcp, and *V. cholerae* VipA/VipB complex.

The structures of Hcp and VgrG provide the strongest evidence about the evolutionary relatedness between proteins of T6SS and phage tails with extended homologies existing at the levels of structure, assembly and function. The structure of Hcp1 (Mougous et al., 2006) was determined at a resolution of 1.95 Å (PDB ID 1Y12) and was found to be strikingly similar to that of the gpVN tail tube protein of phage lambda, with only minor deviations between the two structures (Pell et al., 2009). In the Hcp1 crystal, symmetry-related molecules assemble into hexameric ring which can be superimposed onto the trimeric pseudohexamer formed by the tube domains of the T4 bacteriophage gp27 trimer. Sequence analyses suggest that Hcp is evolutionary related to a further viral protein, the T4 tail tube protein gp19. Strikingly, the packing of Hcp1 hexamers in the crystals studied produces tubelike structures which are geometrically nearly identical to the T4 tail tube which is composed of stacked gp19 hexamer. The Hcp hexameric rings can also be induced to polymerize *in vitro* into stable nanotubes through the introduction of cysteine mutations capable of engaging in disulfide bridges formation across the hexamers (Ballister et al., 2008).

A study by Zheng et al. (2011) provides new insights into the functional requirements of secretion as well as killing of bacterial and eukaryotic phagocytic cells by *V. cholerae* by analyzing non-polar mutations (in-frame deletions) in each gene predicted to code for *V. cholera* T6SS components. They grouped 17 proteins into four categories: twelve proteins (VipA, VipB, VCA0109–VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two proteins (VasH and VCA0122) were thought to be regulators that are required for T6SS gene expression and virulence; another two (VCA0121 and VgrG-3) were not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; one protein (VCA0118) was not required for Hcp expression or secretion but still played a role in both amoebae and bacterial killing and may therefore be an effector protein. ClpV was required for *Dictyostelium* virulence but was less important for killing *E. coli*. In addition, VgrG-2 which is encoded outside of the T6SS cluster was required for bacterial killing but VgrG-1 was not and several genes in the same putative operon as *vgrG*-1 and *vgrG*-2 also contributed to *Dictyostelium* virulence but had a smaller effect on *E. coli* killing.

In contrast to other bacterial secretion systems (e.g. T3SS) there is only a small number of experimentally determined structures of T6SS core proteins or of their macromolecular assemblies. These structural studies have allowed within a relatively short time to understand important aspects of T6SS function to a considerable detail. Additional insights into structure-function relationships of T6SS have been deduced from structural/sequence similarities observed between T6SS proteins and a) components of cell puncturing devices utilized by tailed bacteriophages for DNA delivery or b) proteins from other types of bacterial secretion systems; experimental verification of the relationships derived by

Medium to high resolution crystal structures exist for the *P. aeruginosa* Hcp1 protein, the Nterminal fragment of VgrG from the uropathogenic *E. coli* CFT073, and EvpC from *E. tarda*. Electron microscopy has been used in the study of the assemblies of various T6SS proteins including *P. aeruginosa* Hcp1, Hcp2 and Hcp3, the *E. coli* CFT073 Hcp, and *V. cholerae*

The structures of Hcp and VgrG provide the strongest evidence about the evolutionary relatedness between proteins of T6SS and phage tails with extended homologies existing at the levels of structure, assembly and function. The structure of Hcp1 (Mougous et al., 2006) was determined at a resolution of 1.95 Å (PDB ID 1Y12) and was found to be strikingly similar to that of the gpVN tail tube protein of phage lambda, with only minor deviations between the two structures (Pell et al., 2009). In the Hcp1 crystal, symmetry-related molecules assemble into hexameric ring which can be superimposed onto the trimeric pseudohexamer formed by the tube domains of the T4 bacteriophage gp27 trimer. Sequence analyses suggest that Hcp is evolutionary related to a further viral protein, the T4 tail tube protein gp19. Strikingly, the packing of Hcp1 hexamers in the crystals studied produces tubelike structures which are geometrically nearly identical to the T4 tail tube which is composed of stacked gp19 hexamer. The Hcp hexameric rings can also be induced to polymerize *in vitro* into stable nanotubes through the introduction of cysteine mutations capable of engaging in

**3.3 Structure and functions of T6SS proteins and injectisomes** 

analogies to other systems remains largely to be delivered.

disulfide bridges formation across the hexamers (Ballister et al., 2008).

VipA/VipB complex.

The crystal structure of the *E. tarda* EvpC protein, an Hcp1 homolog from the virulent protein gene cluster EVP which contains a conserved T6SS, has been recently determined at 2.8 Å resolution (PDB accession code 3EAA) and revealed a high structural similarity with Hcp1 (Jobichen et al., 2010). In solution, EvpC exists as a dimer at low concentrations and as a hexamer at high concentrations. In the crystals symmetry-related EvpC molecules form hexameric rings which stack to form tubes similar to Hcp1. Structure-based mutagenesis has revealed a critical role for EvpC secretion for three negatively charged N-terminal residues, and three positively charged C-terminal ones (Jobichen et al., 2010). This secretion impairment of EvpC decreases the virulence of the T6SS-containing pathogenic bacteria.

The structure of the N-terminal fragment (residues 1-483 out of 824) of the VgrG protein encoded by the *E. coli* CFT073 gene c3393 was determined at a resolution of 2.6 Å (PDB ID 2P5Z). The protein shows striking structural similarities (Leiman et al., 2009) to the structure of the complex (gp5)3-(gp27)3 of the T4 bacteriophage cell-puncturing device (PDB ID 1K28). VgrG can be described as a fusion of T4 gp27 and gp5; at the level of equivalent domains VgrG shares the highest structural homology with gp27 and exhibits only minor modifications relative to gp5. The VgrG structure comprises a domain (residues 380-470) of unknown function which is conserved in all VgrGs. This domain (DUF586) is the equivalent of the oligosaccharide/oligonucleotide-binding (OB)-fold domain of T4 gp5. The secondary structure prediction of the C-terminal part of VgrG (residues 490-820) which follows the OBfold domain, shows repetitive β-strands (5-10 residues each) flanked by glycines. It is likely, that these strands form a β-helix that is equivalent to the triple stranded β-helix in trimeric gp5 which is involved in membrane penetration. The trimeric structure of the N-terminal VgrG fragment in the crystal, probably indicates that the complete VgrG protein may also adopt a trimeric structure that is equivalent to the (gp5)3-(gp27)3 complex. Sequence analyses suggest that effector domains are fused to the C-termini of many VgrG proteins ('evolved' VgrGs). The gp5 C-terminal β-helix has a 23 KDa extension of unknown function corresponding to a VgrG effector domain. Since many VgrG proteins do not contain an additional C-terminal domain, it may be concluded that different T6SS injectisomes service separate sets of effector proteins.

The structural similarities of Hcp and VgrG to components of the injection apparatus of tailed bacteriophages are highly suggestive that the two proteins might be structural components of T6SS, rather than effector proteins. The absence of 'evolved' VgrGs in many T6SSs suggests also that Hcp/VgrG might act as a conduit for T6SS effectors. The inner diameter of the Hcp tubule (40 Å) would allow the passage of proteins in an unfolded form; delivery of effectors to target cells might involve the cell-puncturing activity of VgrG.

The energy for the translocation of secreted proteins to the extracellular environment is provided by two components of the T6SS that have been introduced in section 3.1: IcmF and ClpV (Bonemann et al., 2009). IcmF is a membrane-embedded component that forms a structure spanning the inner and outer membranes. ClpV does not interact with the exoproteins Hcp and VgrG, but binds specifically with its N-domain to two cytosolic proteins, VipA (COG3516) and VipB (COG3517), that are conserved and essential components of T6SS (Bonemann et al., 2009, 2010). VipA and VipB interact with each other forming a tubular, cogweel-like structure larger than 200 kDa, with a diameter of approximately 300Å and a central channel of 100Å in diameter, and a length ranging from 25 to 500nm. Electron microscopy studies of VipA/VipB suggest that there is an overall

Phytobacterial Type VI Secretion System –

Fig. 1. *Part 1*

Gene Distribution, Phylogeny, Structure and Biological Functions 61

resemblance between the VirA/VirB tubules and the T4 tail sheath structure which accommodates the viral tail tube proteins (Aksyuk et al., 2009); the diameter of the inner channel of VirA/VirB tubules is sufficient to encase Hcp tubes. In-frame deletion mutants of *vipA* and *vipB* genes could no longer secrete Hcp and VgrG proteins, although the total levels of the proteins were not affected (Bonemann et al., 2009), thus suggesting a crucial role of VirA and VirB for T6SS function. Importantly, there is no evidence for an interaction of VipA and VipB with the cytosolic proteins Hcp and VgrG. VipA/VipB cogwheel-like tubules are disassembled by ClpV; this ClpV-mediated remodelling of VipA/VipB tubules into smaller complexes (100kDa), has been suggested as an essential step in T6SS secretion, revealing an unexpected role for this ATPase component in a bacterial protein secretion system. The recent characterization of the *P. syringae* pv. *syringae* T6SS proteins ImpB (177aa) and ImpC (500aa) (homologs of VipA and VipB respectively) suggest that the two proteins form supramolecular structures of comparable size to the assemblies of VipA/VipB (M. Kokkinidis, unpublished results). These ongoing studies represent the first structural analysis of the T6SS of a plant pathogen.

#### **4. T6SS role in host colonization and interbacterial interactions**

The T6SS was initially thought to play a role primarily in bacterial pathogenicity and host colonization. Roest et al. (1997) and Bladergroen et al. (2003) characterized *Rhizobium* loci (*imp*) that hinder effective nodulation on certain plants. Imp mutants (impaired in nodulation) were deficient in the secretion of an effector protein (RbsB-like) and were mapped in a cluster of 16 genes (from pRL120462 [impN-like] to pRL120480 [vgrG-like], Fig. 1). The later authors predicted that these genes encoded a new protein secretion system, which was later named T6SS by Pukatzki et al. (2006). However, it is also found in environmental isolates and recent findings suggest that they may also function in a broader biological context: to mediate cooperative or competitive interactions between bacteria, including bacterial biofilm formation, or to promote the establishment of commensal or mutualistic relationships between bacteria and eukaryotes (Aschtgen et al., 2008; Hood et al., 2010; Jani & Cotter, 2010; Schwarz et al., 2010a; Russell et al., 2011; Zheng et al., 2011). For example, *P. aeruginosa* is capable of secreting the antibacterial factors Tse (*T*ype VI *s*ecretion *e*xported) (Hood et al., 2010). Tse2 is a toxic protein from *P. aeruginosa* (PA2702) and arrests growth of both prokaryotic and eukaryotic cells when expressed intracelularly. It is proposed to be an export substrate of the *P. aeruginosa* HSI-I (Hood et al., 2010). Tse2 expressing bacterial cells produce also an immunity protein, Tsi2 (PA2703), preventing cell death when co-expressed with Tse2. It is noteworthy that *tse2*/*tsi2* are not found in other, phylogenetically close *Pseudomonas* species (*P. entomophila* and *P. mendocina*), suggesting that it is a species-restricted regulon that responds to specific needs (Sarris & Scoulica, 2011). That the T6SS can target bacteria has also been demonstrated experimentally for *Burkholderia thailandensis* (Hood et al., 2010; Schwarz et al., 2010b) and *V. cholerae* (MacIntyre et al., 2010; Zheng et al., 2011). In *B. thailandensis* two of the five T6SSs assume specialized functions: either in the survival of the organism in a murine host (T6SS-5), or against other bacteria (T6SS-1), since strains lacking the bacterial-targeting T6SS-1 could not persist in a mixed biofilm with competing bacteria (Schwarz et al., 2010b). Miyata et al. (2010) speculate that *V. cholerae* uses its T6SS to outcompete bacterial neighbours as well as eukaryotic predators

resemblance between the VirA/VirB tubules and the T4 tail sheath structure which accommodates the viral tail tube proteins (Aksyuk et al., 2009); the diameter of the inner channel of VirA/VirB tubules is sufficient to encase Hcp tubes. In-frame deletion mutants of *vipA* and *vipB* genes could no longer secrete Hcp and VgrG proteins, although the total levels of the proteins were not affected (Bonemann et al., 2009), thus suggesting a crucial role of VirA and VirB for T6SS function. Importantly, there is no evidence for an interaction of VipA and VipB with the cytosolic proteins Hcp and VgrG. VipA/VipB cogwheel-like tubules are disassembled by ClpV; this ClpV-mediated remodelling of VipA/VipB tubules into smaller complexes (100kDa), has been suggested as an essential step in T6SS secretion, revealing an unexpected role for this ATPase component in a bacterial protein secretion system. The recent characterization of the *P. syringae* pv. *syringae* T6SS proteins ImpB (177aa) and ImpC (500aa) (homologs of VipA and VipB respectively) suggest that the two proteins form supramolecular structures of comparable size to the assemblies of VipA/VipB (M. Kokkinidis, unpublished results). These ongoing studies represent the first structural

**4. T6SS role in host colonization and interbacterial interactions** 

outcompete bacterial neighbours as well as eukaryotic predators

The T6SS was initially thought to play a role primarily in bacterial pathogenicity and host colonization. Roest et al. (1997) and Bladergroen et al. (2003) characterized *Rhizobium* loci (*imp*) that hinder effective nodulation on certain plants. Imp mutants (impaired in nodulation) were deficient in the secretion of an effector protein (RbsB-like) and were mapped in a cluster of 16 genes (from pRL120462 [impN-like] to pRL120480 [vgrG-like], Fig. 1). The later authors predicted that these genes encoded a new protein secretion system, which was later named T6SS by Pukatzki et al. (2006). However, it is also found in environmental isolates and recent findings suggest that they may also function in a broader biological context: to mediate cooperative or competitive interactions between bacteria, including bacterial biofilm formation, or to promote the establishment of commensal or mutualistic relationships between bacteria and eukaryotes (Aschtgen et al., 2008; Hood et al., 2010; Jani & Cotter, 2010; Schwarz et al., 2010a; Russell et al., 2011; Zheng et al., 2011). For example, *P. aeruginosa* is capable of secreting the antibacterial factors Tse (*T*ype VI *s*ecretion *e*xported) (Hood et al., 2010). Tse2 is a toxic protein from *P. aeruginosa* (PA2702) and arrests growth of both prokaryotic and eukaryotic cells when expressed intracelularly. It is proposed to be an export substrate of the *P. aeruginosa* HSI-I (Hood et al., 2010). Tse2 expressing bacterial cells produce also an immunity protein, Tsi2 (PA2703), preventing cell death when co-expressed with Tse2. It is noteworthy that *tse2*/*tsi2* are not found in other, phylogenetically close *Pseudomonas* species (*P. entomophila* and *P. mendocina*), suggesting that it is a species-restricted regulon that responds to specific needs (Sarris & Scoulica, 2011). That the T6SS can target bacteria has also been demonstrated experimentally for *Burkholderia thailandensis* (Hood et al., 2010; Schwarz et al., 2010b) and *V. cholerae* (MacIntyre et al., 2010; Zheng et al., 2011). In *B. thailandensis* two of the five T6SSs assume specialized functions: either in the survival of the organism in a murine host (T6SS-5), or against other bacteria (T6SS-1), since strains lacking the bacterial-targeting T6SS-1 could not persist in a mixed biofilm with competing bacteria (Schwarz et al., 2010b). Miyata et al. (2010) speculate that *V. cholerae* uses its T6SS to

analysis of the T6SS of a plant pathogen.

Fig. 1. *Part 1*

Phytobacterial Type VI Secretion System –

Gene Distribution, Phylogeny, Structure and Biological Functions 63

like amoebae and mammalian immune cells. Chow & Mazmanian (2010) further propose that pathobionts of the human gastrointestinal tract, such as *H. hepaticus,* may have evolved a T6SS as a mechanism to actively maintain a non-pathogenic, symbiotic relationship in the GI tract by regulating bacterial colonization and host inflammation; they hypothesize that alteration in the composition of the microbiota, known as dysbiosis, may be a critical factor in various

Knowledge on the functionalities and biological roles of T6SS in plant-associated bacteria is poor and limited to relatively few systems (reviewed in Records, 2011). With regard to phytopathogens, functionality has been demonstrated for *A. tumefaciens, P. atrosepticum*, and two pathovars of *Pseudomonas syringae*. A study with *P. s.* pv. *tomato* DC3000 (Wang et al., 2008) has shed some light on the possible role of the T6SSs in pathogenicity. Deletions of the entire T6SS clusters (T6SS-II or T6SS-III; grouping is based on our phylogenetic analysis, see sections 5, and 6) or both copies of *icmF (icmF1* and *icmF2*) caused reduction of bacterial population in *Nicotiana benthamiana* and milder symptoms on tomato leaves. When either the T6SS-II or T6SS-III cluster was deleted, both symptoms severity and bacterial populations were reduced. However, *vgrG1* or *vgrG2* deletions had no effect on disease development on tomato or on *N. benthamiana*. However, an insertional mutant of the *clpV/B* gene of the T6SS maintained the ability for *in planta* multiplication and produced disease symptoms similar to those caused by wild-type strain (Records & Gross 2010). RNA transcripts of the *icmF* homologs of the *P. syringae* pv. *tomato* DC3001 and *P. syringae* pv. *phaseolicola* 1448a were detected by RT-PCR in both rich and minimal media, indicating that the gene is probably expressed in both strains (Sarris et al., 2010). Microarray analysis showed that the *A. tumefaciens* T6SS is induced by mildly acidic conditions, such as encountered in plant tissues and in the rhizosphere (Yuan et al., 2008) and deletion of *hcp* resulted in reduced tumorigenesis on potato tuber slices (Wu et al., 2008). The *P. atrosepticum* T6SS is induced by potato tuber extracts (Mattinen et al., 2007). Transcriptome profiling (Liu et al., 2008) also indicated regulation of the T6SS of *P. atrosepticum* by quorum sensing, as deletion of *expI*, a gene responsible for N-(3-oxohexanoyl)-L-homoserine lactone synthesis. Furthermore, deletions in either ECA3438 (*impJ*) or ECA3444 (*vipB*) resulted in slightly reduced virulence in potato stems and tubers. However, mutation of the ECA3432 (*icmF*) gene resulted in increased potato tuber maceration, indicating that the T6SS may be involved in antipathogenesis activity (Yuan et al., 2008). Whether T6SS mechanisms engage other aspects of *P. syringae-*host/vector biology, antagonism and predation in the plant or other micro-environments remain open questions. Among the symbiotic N-fixing bacteria, extended symbiosis phenotypes of certain rhizobia have been linked to a T6SS. The presence of T6SS homologs in the sequenced genomes of many rhizobia presents opportunities to

human inflammatory disorders such as inflammatory bowel disease and colon cancer.

further investigate it's role in bacteria-plant symbiosis (Fauvart & Michiels, 2008).

Further to our recently published study (Sarris et al., 2010), a genome-wide *in silico* analysis was carried out for 13 phytobacterial species and more than 30 strains from different genera, to identify conserved gene clusters encoding for T6SSs by BLASTP and reverse BLAST analysis of sequences deposited in various genome databases (e.g. KEGG, NCBI, RizoBase), both complete annotated and draft sequenced phytobacterial genomes. The baits consisted of protein sequences encoded by the *P. syringae* Hcp secretion islands I, II and III (HSI-I, II,

**5. Mining phytobacterial for T6SS homologs** 

Fig. 1. Maps of T6SS clusters of plant-associated bacteria. Orthologs are indicated by the same color. The genes adjacent to or encoded by the T6SS gene clusters but not recognized as orthologs are indicated by light beige arrows. Arrows indicate the transcriptional direction. The gene locus numbers are referred in the text and the published or annotated gene designations are indicated above the genes of each cluster.

Fig. 1. Maps of T6SS clusters of plant-associated bacteria. Orthologs are indicated by the same color. The genes adjacent to or encoded by the T6SS gene clusters but not recognized as orthologs are indicated by light beige arrows. Arrows indicate the transcriptional direction. The gene locus numbers are referred in the text and the published or annotated

gene designations are indicated above the genes of each cluster.

Fig. 1. *Part 2*

like amoebae and mammalian immune cells. Chow & Mazmanian (2010) further propose that pathobionts of the human gastrointestinal tract, such as *H. hepaticus,* may have evolved a T6SS as a mechanism to actively maintain a non-pathogenic, symbiotic relationship in the GI tract by regulating bacterial colonization and host inflammation; they hypothesize that alteration in the composition of the microbiota, known as dysbiosis, may be a critical factor in various human inflammatory disorders such as inflammatory bowel disease and colon cancer.

Knowledge on the functionalities and biological roles of T6SS in plant-associated bacteria is poor and limited to relatively few systems (reviewed in Records, 2011). With regard to phytopathogens, functionality has been demonstrated for *A. tumefaciens, P. atrosepticum*, and two pathovars of *Pseudomonas syringae*. A study with *P. s.* pv. *tomato* DC3000 (Wang et al., 2008) has shed some light on the possible role of the T6SSs in pathogenicity. Deletions of the entire T6SS clusters (T6SS-II or T6SS-III; grouping is based on our phylogenetic analysis, see sections 5, and 6) or both copies of *icmF (icmF1* and *icmF2*) caused reduction of bacterial population in *Nicotiana benthamiana* and milder symptoms on tomato leaves. When either the T6SS-II or T6SS-III cluster was deleted, both symptoms severity and bacterial populations were reduced. However, *vgrG1* or *vgrG2* deletions had no effect on disease development on tomato or on *N. benthamiana*. However, an insertional mutant of the *clpV/B* gene of the T6SS maintained the ability for *in planta* multiplication and produced disease symptoms similar to those caused by wild-type strain (Records & Gross 2010). RNA transcripts of the *icmF* homologs of the *P. syringae* pv. *tomato* DC3001 and *P. syringae* pv. *phaseolicola* 1448a were detected by RT-PCR in both rich and minimal media, indicating that the gene is probably expressed in both strains (Sarris et al., 2010). Microarray analysis showed that the *A. tumefaciens* T6SS is induced by mildly acidic conditions, such as encountered in plant tissues and in the rhizosphere (Yuan et al., 2008) and deletion of *hcp* resulted in reduced tumorigenesis on potato tuber slices (Wu et al., 2008). The *P. atrosepticum* T6SS is induced by potato tuber extracts (Mattinen et al., 2007). Transcriptome profiling (Liu et al., 2008) also indicated regulation of the T6SS of *P. atrosepticum* by quorum sensing, as deletion of *expI*, a gene responsible for N-(3-oxohexanoyl)-L-homoserine lactone synthesis. Furthermore, deletions in either ECA3438 (*impJ*) or ECA3444 (*vipB*) resulted in slightly reduced virulence in potato stems and tubers. However, mutation of the ECA3432 (*icmF*) gene resulted in increased potato tuber maceration, indicating that the T6SS may be involved in antipathogenesis activity (Yuan et al., 2008). Whether T6SS mechanisms engage other aspects of *P. syringae-*host/vector biology, antagonism and predation in the plant or other micro-environments remain open questions. Among the symbiotic N-fixing bacteria, extended symbiosis phenotypes of certain rhizobia have been linked to a T6SS. The presence of T6SS homologs in the sequenced genomes of many rhizobia presents opportunities to further investigate it's role in bacteria-plant symbiosis (Fauvart & Michiels, 2008).

#### **5. Mining phytobacterial for T6SS homologs**

Further to our recently published study (Sarris et al., 2010), a genome-wide *in silico* analysis was carried out for 13 phytobacterial species and more than 30 strains from different genera, to identify conserved gene clusters encoding for T6SSs by BLASTP and reverse BLAST analysis of sequences deposited in various genome databases (e.g. KEGG, NCBI, RizoBase), both complete annotated and draft sequenced phytobacterial genomes. The baits consisted of protein sequences encoded by the *P. syringae* Hcp secretion islands I, II and III (HSI-I, II,

Phytobacterial Type VI Secretion System –

Gene Distribution, Phylogeny, Structure and Biological Functions 65

Table 1. Gene content of the putative T6SS genes of phytobacterial species, indicating locus names and COG numbers (not available for OmpA). (+): present, (++): present in a second copy, (-): missing. \*Species abbreviations: AAsC: *Acidovorax avenae* subsp. *citrulli*; AT: *Agrobacterium tumefaciens*; AV: *Agrobacterium vitis*; BJ: *Bradyrhizobium japonicum*; PA: *Pectobacterium atrocepticum*; EA: *Erwinia amylovora*; EP: *Erwinia pyrifoliae*; ML: *Mesorhizobium loti*; PS: *Pseudomonas syringae*; RS: *Ralstonia solanacearum*; RE: *Rhizobium etli*; RL: *Rhizobium leguminosarum*; Xsp: *Xanthomonas* spp.; CT: *Cupriavidus taiwanensis*; DZ: *Dickeya zeae*; DD: *Dickeya dadantii*; PA: *Pantoea ananatis*. \*\*The numbers in the second row of the table denote the percentage of cases where the gene/protein is present among the species/strains examined. \*\*\*Latin numerals I, II, III denote T6SS-I, T6SS-II and T6SS-III, respectively. A table of the locus

numbers from the KEGG database is available upon request from P.F. Sarris.

III, here referred to interchangeably as T6SS-I, T6SS-II and T6SS-III, respectively) (Sarris et al., 2010), as well as their homologs from other known T6SS clusters (Boyer et al., 2009). Clusters containing at least five genes encoding proteins with similarity to known T6SS core proteins were considered as part of a putative T6SS locus. The genomic regions thus identified were then extended by examining four kilo-bases up- and down-stream for putative conserved genes associated with T6SS by "orthologue" and "paralogue finder" analysis against all the KEGG deposited genomes. Maps of the genomic islands were constructed manually in PowerPoint Microsoft office software. For sequence alignment and phylogenetic tree construction, the conserved proteins ImpL, ImpG, IpmC and ImpH from all phytobacterial species deposited at the NCBI and KEGG databases were edited with the DNAman computer package (Lynnon Co) and were included for sequence alignment and tree construction. Phylogenetic relations were inferred using the neighbour-joining method (Saitou & Nei 1987) offered in MEGA4 software (Tamura et al., 2007). In Table 1 and Fig. 1 the clusters are identified by the organsim's initials, and in the phylogenetic trees the organism's name/strain number and the T6SS groupings used in the text and figure legends are given.

#### **6. Phylogenetic analysis of phytobacterial T6SS**

#### **6.1 Phylogenies based on overall gene content**

Table 1 shows the presence/absence of a T6SS protein homolog (indicated with the plus sign [+] and minus [-] sign, respectively). Homologs of each T6SS-related bait protein exist in members of all phytobacterial species/strains, but with substantial differences among strains in gene content and copy number. Nine core genes, *impK*, *impB*, *impC*, *impG impH*, *impJ*, *hcp*, *impL* and *clpB*, are found in 100% of the species examined, with *clpB* seemingly present as a pseudo-gene in *Rhizobium leguminosarum)*. On the other hand, homologs of genes such as *ompA* and *impD* are present only in 14% of the species/strains examined, a finding of unclear significance. Several instances of multiple T6SS clusters were identified in the same strain, mostly in distant locations with respect to each other. These clusters are depicted in Fig. 1, based on the phylogenetic analysis described in the sections below.

To analyse the evolutionary history of phytobacterial T6SS, a distance tree was initially constructed, depicting the phylogenetic relationships and gene composition among the T6SSs of all species examined based on the data in Table 1. Initially, the evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei 1987) by scoring each locus as present (+) or absent (-), including all the data (Fig. 2). The constructed tree essentially gives a graphic representation of the data in Table 1 and reveals two distinct T6SS clusters, in terms of the presence or absence of the genetic elements. The tree shows extensive intermixing among the T6SSs of various bacterial species/groups, with several deep and shallow branches, many with low bootstrap values. Noteworthy in this tree is the close relationship between: *a*. the *Xanthomonas* T6SS-I and II and rhizobial T6SSs (except *Rhizobium leguminosarum*), *b*. the two phytopathogenic enterobacteria T6SS-II (*Erwinia amylovora*, and *Erwinia pyrifoliae*) and the *Acidovorax* T6SS, *c*. the sole T6SS of the *Agrobacteria* spp., *Rh. leguminosarum* and *Xanthomonas* spp. T6SS-II *d*. the *Ralstonia solanacearum* and *Xanthomonas*  spp. T6SS-III and *e*. between other phypathogenic enterobacterial T6SSs (*E. amylovora* and *P. ananatis* T6SS-III, the *P. atrosepticum* T6SS-I and the T6SS of the two *Dickeya* species).

III, here referred to interchangeably as T6SS-I, T6SS-II and T6SS-III, respectively) (Sarris et al., 2010), as well as their homologs from other known T6SS clusters (Boyer et al., 2009). Clusters containing at least five genes encoding proteins with similarity to known T6SS core proteins were considered as part of a putative T6SS locus. The genomic regions thus identified were then extended by examining four kilo-bases up- and down-stream for putative conserved genes associated with T6SS by "orthologue" and "paralogue finder" analysis against all the KEGG deposited genomes. Maps of the genomic islands were constructed manually in PowerPoint Microsoft office software. For sequence alignment and phylogenetic tree construction, the conserved proteins ImpL, ImpG, IpmC and ImpH from all phytobacterial species deposited at the NCBI and KEGG databases were edited with the DNAman computer package (Lynnon Co) and were included for sequence alignment and tree construction. Phylogenetic relations were inferred using the neighbour-joining method (Saitou & Nei 1987) offered in MEGA4 software (Tamura et al., 2007). In Table 1 and Fig. 1 the clusters are identified by the organsim's initials, and in the phylogenetic trees the organism's name/strain number and the T6SS groupings used in the text and

Table 1 shows the presence/absence of a T6SS protein homolog (indicated with the plus sign [+] and minus [-] sign, respectively). Homologs of each T6SS-related bait protein exist in members of all phytobacterial species/strains, but with substantial differences among strains in gene content and copy number. Nine core genes, *impK*, *impB*, *impC*, *impG impH*, *impJ*, *hcp*, *impL* and *clpB*, are found in 100% of the species examined, with *clpB* seemingly present as a pseudo-gene in *Rhizobium leguminosarum)*. On the other hand, homologs of genes such as *ompA* and *impD* are present only in 14% of the species/strains examined, a finding of unclear significance. Several instances of multiple T6SS clusters were identified in the same strain, mostly in distant locations with respect to each other. These clusters are depicted in Fig. 1, based on the phylogenetic analysis described in the sections below.

To analyse the evolutionary history of phytobacterial T6SS, a distance tree was initially constructed, depicting the phylogenetic relationships and gene composition among the T6SSs of all species examined based on the data in Table 1. Initially, the evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei 1987) by scoring each locus as present (+) or absent (-), including all the data (Fig. 2). The constructed tree essentially gives a graphic representation of the data in Table 1 and reveals two distinct T6SS clusters, in terms of the presence or absence of the genetic elements. The tree shows extensive intermixing among the T6SSs of various bacterial species/groups, with several deep and shallow branches, many with low bootstrap values. Noteworthy in this tree is the close relationship between: *a*. the *Xanthomonas* T6SS-I and II and rhizobial T6SSs (except *Rhizobium leguminosarum*), *b*. the two phytopathogenic enterobacteria T6SS-II (*Erwinia amylovora*, and *Erwinia pyrifoliae*) and the *Acidovorax* T6SS, *c*. the sole T6SS of the *Agrobacteria* spp., *Rh. leguminosarum* and *Xanthomonas* spp. T6SS-II *d*. the *Ralstonia solanacearum* and *Xanthomonas*  spp. T6SS-III and *e*. between other phypathogenic enterobacterial T6SSs (*E. amylovora* and *P.* 

*ananatis* T6SS-III, the *P. atrosepticum* T6SS-I and the T6SS of the two *Dickeya* species).

figure legends are given.

**6. Phylogenetic analysis of phytobacterial T6SS** 

**6.1 Phylogenies based on overall gene content** 


Table 1. Gene content of the putative T6SS genes of phytobacterial species, indicating locus names and COG numbers (not available for OmpA). (+): present, (++): present in a second copy, (-): missing. \*Species abbreviations: AAsC: *Acidovorax avenae* subsp. *citrulli*; AT: *Agrobacterium tumefaciens*; AV: *Agrobacterium vitis*; BJ: *Bradyrhizobium japonicum*; PA: *Pectobacterium atrocepticum*; EA: *Erwinia amylovora*; EP: *Erwinia pyrifoliae*; ML: *Mesorhizobium loti*; PS: *Pseudomonas syringae*; RS: *Ralstonia solanacearum*; RE: *Rhizobium etli*; RL: *Rhizobium leguminosarum*; Xsp: *Xanthomonas* spp.; CT: *Cupriavidus taiwanensis*; DZ: *Dickeya zeae*; DD: *Dickeya dadantii*; PA: *Pantoea ananatis*. \*\*The numbers in the second row of the table denote the percentage of cases where the gene/protein is present among the species/strains examined. \*\*\*Latin numerals I, II, III denote T6SS-I, T6SS-II and T6SS-III, respectively. A table of the locus numbers from the KEGG database is available upon request from P.F. Sarris.

Phytobacterial Type VI Secretion System –

73 100

87 74

83

100

100

100

**0.1**

indicates the different bacterial species.

57

100

96

100

Gene Distribution, Phylogeny, Structure and Biological Functions 67

100 100

100

100

100

100

100

100

100

100

99 100

50 100

 *Xanthomonas oryzae HSI-I*

 *Acidovorax aven.citrulli Pantoea ananatis HSI-II Erwinia pyrifoliae Erwinia amylovora HSI-II Bradyrhizobium japonicum Mesorhizobium loti Rhizobium etli HSI-I Rhizobium leguminosarum Agrobacterium tumefaciens Agrobacterium vitis Xanthomonas oryzae HSI-III Xanthomonas oryz.oryzicola HSI-III*

 *Ralstonia solanacearum Cupriavidus taiwanensis HSI-II Pseudomonas syr.tomatoDC HSI-II Pseudomonas syr.tomatoT1 HSI-II Pseudomonas sav.savastanoi HSI-II Pseudomonas syr.aesculi HSI-II Pseudomonas syr.oryzae HSI-II Pseudomonas syr.tomatoDC HSI-III*

 *Pseudomonas sav.savastanoi HSI-I Pseudomonas syr.tabaci HSI-I Pseudomonas syr.aesculi HSI-I Pseudomonas syr.syringae HSI-I Pseudomonas syr.phaseolicola HSI-I Pseudomonas syr.tomatoT1 HSI-I Pseudomonas syr.oryzae HSI-I*

 *Pectobacterium atrosepticum*

 *Erwinia amylovora HSI-III Pantoea ananatis HSI-III*

 *Dickeya dadantii Dickeya zeae*

 *Pseudomonas syr.tabaci HSI-II*

100

100 100

Fig. 3. Phylogenetic clustering of the plant-associated bacterial species based on the sequence of four T6SS core proteins (ImpC ImpG, ImpH and ImpL). The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei 1987). The bootstrap consensus tree inferred from 5000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the proteins analysed replicates (Felsenstein 1985). For other details see Fig. 2 legend. There were a total of 1729 positions in the final dataset. Phylogenetic analyses were conducted with MEGA4 (Tamura et al., 2007). Difference in tree branch colors

The consensus phylogenetic tree obtained shows four deep branches. One branch hosts the majority of the phytobacterial T6SSs, except for those of *P. syringae* pathovars (T6SSs-I, II and III), the *Dickeya* spp. T6SS and *P. carotovorum* T6SS. In this branch several T6SS groups are evident. One group includes the *Xanthomonas* spp. T6SS-I and T6SS-II together with the T6SS of *Rhizobium etli* T6SS-II. A second group includes the *C. taiwanensis* T6SS-I, *A. avenae* subsp. *citrulli*, *P. ananatis* T6SS-II and the *Erwinia* spp. T6SS-II. The *B. japonicum* T6SS

100

99

99

100

100

100

100

100

 *Xanthomonas oryz.oryzicola HSI-I Xanthomonas cam.vesicatoria HSI-I Xanthomonas cam.vesicatoria HSI-II Xanthomonas axonopodis HSI-II Rhizobium etli HSI-II*

 *Cupriavidus taiwanensis HSI-I*

Fig. 2. Distance tree of T6SS of various plant pathogenic bacteria; constructed with data of Table 1, through a matrix where each gene locus was scored as (+) when present or as (-) when not present. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987). The bootstrap consensus tree inferred from 5000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the species analysed (Felsenstein 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl & Pauling 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 29 positions in the final dataset. Phylogenetic analyses were conducted with MEGA4 (Tamura et al., 2007).

#### **6.2 Phylogenetic analysis of four core proteins**

Subsequently, we carried out a phylogenetic cluster analysis of four highly conserved T6SS core proteins (ImpC, ImpG, IpmH and ImpL-like) by combining the protein sequences and forcing the software to reckon phylogenetic analysis of all four proteins. Data clustering was accomplished by the Neighbor-Joining method (Saitou and Nei, 1987) and are presented as a tree for the evaluation of similarity/distances among each T6SS in each species (Fig, 3) and is used as a basis to infer the T6SS phylogenies discussed below.

 *Rhizobium etli*

 *Pantoea ananatis (T6SS II) Acidovorax avenae subsp citrulli*

 *Erwinia amylovora (T6SS II) Erwinia pyrifoliae (T6SS II)*

41 92

 *Pseudomonas syringae (T6SS II)*

64

 *Pseudomonas syringae (T6SS III)*

90

Fig. 2. Distance tree of T6SS of various plant pathogenic bacteria; constructed with data of Table 1, through a matrix where each gene locus was scored as (+) when present or as (-) when not present. The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987). The bootstrap consensus tree inferred from 5000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the species analysed (Felsenstein 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (5000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl & Pauling 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 29 positions in the final dataset. Phylogenetic analyses were conducted with MEGA4

Subsequently, we carried out a phylogenetic cluster analysis of four highly conserved T6SS core proteins (ImpC, ImpG, IpmH and ImpL-like) by combining the protein sequences and forcing the software to reckon phylogenetic analysis of all four proteins. Data clustering was accomplished by the Neighbor-Joining method (Saitou and Nei, 1987) and are presented as a tree for the evaluation of similarity/distances among each T6SS in each species (Fig, 3) and

 *Dickeya zeae Dickeya dadantii*

 *Cupriavidus taiwanensis (T6SS I) Cupriavidus taiwanensis (T6SS II) Ralstonia solanacearum Xanthomonas spp (T6SS III) Pseudomonas syringae (T6SS I)*

54

30

24

26

4

25

38

43 35

45

49

**0.05**

(Tamura et al., 2007).

**6.2 Phylogenetic analysis of four core proteins** 

is used as a basis to infer the T6SS phylogenies discussed below.

39

44

24

65

53 79

 *Bradyrhizobium japonicum Xanthomonas spp (T6SS I) Mesorhizobium loti*

 *Xanthomonas spp (T6SS II) Agrobacterium vitis*

 *Rhizobium leguminosarum*

 *Erwinia amylovora (T6SS III) Pantoea ananatis (T6SS III) Pectobacterium atrocepticum (T6SS I)*

 *Agrobacterium tumefaciens*

Fig. 3. Phylogenetic clustering of the plant-associated bacterial species based on the sequence of four T6SS core proteins (ImpC ImpG, ImpH and ImpL). The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei 1987). The bootstrap consensus tree inferred from 5000 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the proteins analysed replicates (Felsenstein 1985). For other details see Fig. 2 legend. There were a total of 1729 positions in the final dataset. Phylogenetic analyses were conducted with MEGA4 (Tamura et al., 2007). Difference in tree branch colors indicates the different bacterial species.

The consensus phylogenetic tree obtained shows four deep branches. One branch hosts the majority of the phytobacterial T6SSs, except for those of *P. syringae* pathovars (T6SSs-I, II and III), the *Dickeya* spp. T6SS and *P. carotovorum* T6SS. In this branch several T6SS groups are evident. One group includes the *Xanthomonas* spp. T6SS-I and T6SS-II together with the T6SS of *Rhizobium etli* T6SS-II. A second group includes the *C. taiwanensis* T6SS-I, *A. avenae* subsp. *citrulli*, *P. ananatis* T6SS-II and the *Erwinia* spp. T6SS-II. The *B. japonicum* T6SS

Phytobacterial Type VI Secretion System –

distant from the others (Fig. 4).

Marchi, 1992).

**7. T6SS in phytopathogenic bacterial species 7.1** *Pseudomonas* **spp. (***P. syringae* **and** *P. savastanoi***)**

spots on Indian horse chestnut in India in 1969 (Green et al., 2010).

order and protein sequence similarity to the T6SS-II (Figs. 3 and 4).

Gene Distribution, Phylogeny, Structure and Biological Functions 69

and T6SS-III, the *P. aeruginosa* HSI-II, *P. fluorescens* T6SS-II and the *P. entomophila* four T6SS core proteins. In this group, the *P. syringae* T6SS-III is phylogenetically very close to the *P. aeruginosa* HSI-II and *P. fluorescens* T6SS-II. The *P. syringe* T6SS-III is present only in *P. syringae* pv. *tomato* strain DC3000, and has the same gene order and high protein homology with the T6SS-II of the *P. aeruginosa* PA14 HS-II (data not shown), which reinforces the view of Yan and colleagues (Yan et al., 2008) that the model plant pathogen *P. s.* pv. *tomato* DC3000 is a very atypical tomato strain. Thus, it appears that the *P. syringae* T6SS-III may have been horizontally acquired and maintained through vertical transfer and is remarkably well conserved in *P. s.* pv. *tomato* DC3000, *P. aeruginosa* and *P. fluorescens* species with distant genetic relatedness and very distinct, opportunistic relationships with plants. A second distinct group in the same branch is formed by the T6SSs –I and –III of *P. putida*. The second branch in the four-protein tree is formed by the *P. fluorescens* T6SS-I and the *P. aeruginosa* HSI-I and –III. Finally, the third branch of the four protein tree includes the *P. syringae* T6SS-I grouped with the *P. putida* T6SS-II. Remarkably, in the two *P. fluorescens* strains analysed the sole T6SS in Pf-5 and the T6SS-I and T6SS-II in Pf0-1 are quite distant. Two of the three T6SSs found in the *P. putida* strains studied (T6SS-I in Pf-5 and PKT2440, and T6SS-III in the later strain) are very close relatives while the third one (T6SS-II) is very

The species *P. syringae* (*P.s.; γ-proteobacteria*) comprises phytopathogenic members that are placed in infra-sub-specific groupings (pathovars); collectively, they infect a wide range of plant species and can also live as epiphytes in the plant phyllosphere, until conditions are favourable for disease development (Hirano & Upper, 2000). Some non-pathogenic strains of *P. syringae* have been used as biocontrol agents against post-harvest rots (Janisiewicz &

Bioinformatics analysis (Sarris et al., 2010) revealed the presence of multiple T6SS clusters and putative effectors in six fully or partially sequenced genomes of *P.s.* pathovars. In a subsequent study, two more draft genomes of the bleeding cankers pathogen of the horse chestnut trees (*Aesculus hippocastanum*), *P. syringae* pv. *aesculi* (*Pae*), became available in the NCBI gene database (Green et al., 2010). The two strains show genomic differences implicated in host association and fitness: one strain was isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-*Pae*), and the second (I-*Pae*) from leaf

Genome comparisons between the sequence assemblies of E-*Pae* and I-*Pae* revealed differences in a number of genomic regions including two T6SS clusters and a large number of putative effectors that are present in I-*Pae* but absent from E-*Pae*. In the phylogenetic trees of Figs 3 and 4, the two *Pae* T6SS clusters group together with the *P. syringae* T6SS-I & II respectively. Analysis of the draft genome sequence of a close relative, *P. savastanoi* pv. *savastanoi* strain NCPPB 3335 (Rodriguez-Palenzuela et al., 2010) indicated also the presence of two putative T6SS gene clusters:, one (AER-0002618 to AER-0002633) highly similar to the *P. syringae* T6SS-I and another (AER-0002971 to AER-0002983) that is more similar in gene

appears distinct (with bootstrap value 83) but very close to a subgroup formed by the *M. loti* and *R. etli* T6SS-II. Another group in this phylogenetic branch includes the *R. leguminosarum* and *Agrobacterium* spp. T6SSs. Finally, in the same branch are the *Xanthomonas* spp. T6SS-III, grouped together with the *R. solanacearum* and *C. taiwanensis* T6SS-II. In the second phylogenetic branch are grouped only the *P. syringae* pathovars. As previously reported (Sarris et al., 2010), in this group there are two distinct sub-groups. The first one includes the *P. syringae* T6SS-II and T6SS-III, while the second carries only the *P. syringae* T6SS-I. Finally, two more branches were formed but without bootstrap value. The first one includes the T6SSs of *Dickeya* spp. and *P. atrosepticum*, while the second consists of the *P. ananatis* T6SS-II and *Erwinia* spp. T6SS-III.The phylogenetic relationships of the *P. syringae* T6SSs were further examined by constructing an additional phylogenetic tree of the four core proteins (ImpC, ImpG, IpmH and ImpL-like) by including representatives of fully sequenced nonphytopathogenic fluorescent *Pseudomonas* and the *R. solanacearum T6SS, C. taiwanensis* T6SS-II, *P. ananatis* T6SS-II and *Erwinia* spp. T6SS-III, as these appear as reference species based on their distant relationships in the tree of Fig. 3. The *Pseudomonas* tree shows three deep branches (not including out-group species), each including species with high boostrap values (Fig. 4). The first branch includes a group which is formed by the *P. syringae* T6SS-II

Fig. 4. T6SS evolutionary relationships of 30 fluorescent *Pseudomonas* T6SSs. The evolutionary history was inferred using four (4) T6SS core proteins (ImpC, ImpG, ImpH, ImpL), by the Neighbor-Joining method (Saitou & Nei, 1987). The optimal tree with the sum of branch length = 6.49662029 is shown. For other details see Fig. 2 and Fig. 3 legends). Difference in tree branch colors indicates the different *Pseudomonas* species, while the outgroup species are presented with black.

appears distinct (with bootstrap value 83) but very close to a subgroup formed by the *M. loti* and *R. etli* T6SS-II. Another group in this phylogenetic branch includes the *R. leguminosarum* and *Agrobacterium* spp. T6SSs. Finally, in the same branch are the *Xanthomonas* spp. T6SS-III, grouped together with the *R. solanacearum* and *C. taiwanensis* T6SS-II. In the second phylogenetic branch are grouped only the *P. syringae* pathovars. As previously reported (Sarris et al., 2010), in this group there are two distinct sub-groups. The first one includes the *P. syringae* T6SS-II and T6SS-III, while the second carries only the *P. syringae* T6SS-I. Finally, two more branches were formed but without bootstrap value. The first one includes the T6SSs of *Dickeya* spp. and *P. atrosepticum*, while the second consists of the *P. ananatis* T6SS-II and *Erwinia* spp. T6SS-III.The phylogenetic relationships of the *P. syringae* T6SSs were further examined by constructing an additional phylogenetic tree of the four core proteins (ImpC, ImpG, IpmH and ImpL-like) by including representatives of fully sequenced nonphytopathogenic fluorescent *Pseudomonas* and the *R. solanacearum T6SS, C. taiwanensis* T6SS-II, *P. ananatis* T6SS-II and *Erwinia* spp. T6SS-III, as these appear as reference species based on their distant relationships in the tree of Fig. 3. The *Pseudomonas* tree shows three deep branches (not including out-group species), each including species with high boostrap values (Fig. 4). The first branch includes a group which is formed by the *P. syringae* T6SS-II

> *Pantoea ananatis T6SS-III Erwinia amylovora T6SS-II*

 *Pseudomonas putidaKT2440 T6SS-III Pseudomonas putidaKT2440 T6SS-I Pseudomonas putidaF1 T6SS-I Pseudomonas fluorescensPf-5 T6SS Pseudomonas aeruginosaPA14 HSI-I*

 *Pseudomonas fluorescensPf0-1 T6SS-I Pseudomonas aeruginosaPA14 HSI-III*

 *Pseudomonas syr.tabaci T6SS-I*

 *Pseudomonas sav.savastanoi T6SS-II Pseudomonas syr.aesculi T6SS-II Pseudomonas syr.oryzae T6SS-II Pseudomonas syr.tomatoDC T6SS-II Pseudomonas syr.tomatoT1 T6SS-II Pseudomonas entomophila T6SS Pseudomonas aeruginosaPA14 HSI-II Pseudomonas syr.tomatoDC T6SS-III Pseudomonas fluorescensPf0-1 T6SS-II*

 *Pseudomonas syr.tabaci T6SS-II Pseudomonas putidaKT2440 T6SS-II Pseudomonas putidaF1 T6SS-II Pseudomonas syr.tomatoT1 T6SS-I Pseudomonas syr.oryzae T6SS-I Pseudomonas syr.syringae T6SS-I Pseudomonas syr.phaseolicola T6SS-I Pseudomonas syr.aesculi T6SS-I Pseudomonas sav.savastanoi T6SS-I*

100

100

100

58

100

58

100

100

98 100

100

100

40 100

100

58 100

100

group species are presented with black.

100

58 41 42

58

**0.1**

53

35

58

100

 *Ralstonia solanacearum T6SS Cupriavidus taiwanensis T6SS-II*

Fig. 4. T6SS evolutionary relationships of 30 fluorescent *Pseudomonas* T6SSs. The

evolutionary history was inferred using four (4) T6SS core proteins (ImpC, ImpG, ImpH, ImpL), by the Neighbor-Joining method (Saitou & Nei, 1987). The optimal tree with the sum of branch length = 6.49662029 is shown. For other details see Fig. 2 and Fig. 3 legends). Difference in tree branch colors indicates the different *Pseudomonas* species, while the outand T6SS-III, the *P. aeruginosa* HSI-II, *P. fluorescens* T6SS-II and the *P. entomophila* four T6SS core proteins. In this group, the *P. syringae* T6SS-III is phylogenetically very close to the *P. aeruginosa* HSI-II and *P. fluorescens* T6SS-II. The *P. syringe* T6SS-III is present only in *P. syringae* pv. *tomato* strain DC3000, and has the same gene order and high protein homology with the T6SS-II of the *P. aeruginosa* PA14 HS-II (data not shown), which reinforces the view of Yan and colleagues (Yan et al., 2008) that the model plant pathogen *P. s.* pv. *tomato* DC3000 is a very atypical tomato strain. Thus, it appears that the *P. syringae* T6SS-III may have been horizontally acquired and maintained through vertical transfer and is remarkably well conserved in *P. s.* pv. *tomato* DC3000, *P. aeruginosa* and *P. fluorescens* species with distant genetic relatedness and very distinct, opportunistic relationships with plants. A second distinct group in the same branch is formed by the T6SSs –I and –III of *P. putida*. The second branch in the four-protein tree is formed by the *P. fluorescens* T6SS-I and the *P. aeruginosa* HSI-I and –III. Finally, the third branch of the four protein tree includes the *P. syringae* T6SS-I grouped with the *P. putida* T6SS-II. Remarkably, in the two *P. fluorescens* strains analysed the sole T6SS in Pf-5 and the T6SS-I and T6SS-II in Pf0-1 are quite distant. Two of the three T6SSs found in the *P. putida* strains studied (T6SS-I in Pf-5 and PKT2440, and T6SS-III in the later strain) are very close relatives while the third one (T6SS-II) is very distant from the others (Fig. 4).

#### **7. T6SS in phytopathogenic bacterial species**

#### **7.1** *Pseudomonas* **spp. (***P. syringae* **and** *P. savastanoi***)**

The species *P. syringae* (*P.s.; γ-proteobacteria*) comprises phytopathogenic members that are placed in infra-sub-specific groupings (pathovars); collectively, they infect a wide range of plant species and can also live as epiphytes in the plant phyllosphere, until conditions are favourable for disease development (Hirano & Upper, 2000). Some non-pathogenic strains of *P. syringae* have been used as biocontrol agents against post-harvest rots (Janisiewicz & Marchi, 1992).

Bioinformatics analysis (Sarris et al., 2010) revealed the presence of multiple T6SS clusters and putative effectors in six fully or partially sequenced genomes of *P.s.* pathovars. In a subsequent study, two more draft genomes of the bleeding cankers pathogen of the horse chestnut trees (*Aesculus hippocastanum*), *P. syringae* pv. *aesculi* (*Pae*), became available in the NCBI gene database (Green et al., 2010). The two strains show genomic differences implicated in host association and fitness: one strain was isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-*Pae*), and the second (I-*Pae*) from leaf spots on Indian horse chestnut in India in 1969 (Green et al., 2010).

Genome comparisons between the sequence assemblies of E-*Pae* and I-*Pae* revealed differences in a number of genomic regions including two T6SS clusters and a large number of putative effectors that are present in I-*Pae* but absent from E-*Pae*. In the phylogenetic trees of Figs 3 and 4, the two *Pae* T6SS clusters group together with the *P. syringae* T6SS-I & II respectively. Analysis of the draft genome sequence of a close relative, *P. savastanoi* pv. *savastanoi* strain NCPPB 3335 (Rodriguez-Palenzuela et al., 2010) indicated also the presence of two putative T6SS gene clusters:, one (AER-0002618 to AER-0002633) highly similar to the *P. syringae* T6SS-I and another (AER-0002971 to AER-0002983) that is more similar in gene order and protein sequence similarity to the T6SS-II (Figs. 3 and 4).

Phytobacterial Type VI Secretion System –

the *C. taiwanensis* T6SS-II.

**7.4** *Erwinia* **spp. (***E. pyrifoliae* **and** *E. amylovora***)** 

than mere passive dispersal (Smits et al., 2010).

Gene Distribution, Phylogeny, Structure and Biological Functions 71

*campesrtis* pv. *vesicatoria* XCV2120 (*X.c.v.*), one in *X. axonopodis* 4122 (*X.a.c.*) and three in each, *X.o* and *X.o.o.* One T6SS cluster in XCV2120 extends from XCV2120 (*impB*-like) to XCV2143 (*impA*-like) and is referred to here as T6SS-I, while a second cluster is located from XCV4202 (*impA*-like) to XCV4243 (*impB*-like) and referred as T6SS-II (Fig. 1). This cluster contains two interruptions by some apparently T6SS-unrelated putative ORFs, one, between XCV4214 (*impI*-like) and XCV4236 (*clpB*-like), and another between XCV4202 (*impA*-like) and XCV4208 (*impM*-like). Two T6SS clusters are found in *X.c.v.*: one of them is phylogenetically closer to the T6SS-I found in *X.o.* and *X.o.o* while the second seems to more closely related to the sole T6SS locus of *X.a.c* (referred to as T6SS-II in Fig. 3) which spans from XAC4112 (*impA*-like) to XAC4147 (*impB*-like) (Fig. 1). The *X.a.c.* T6SS locus is almost collinear for the T6SS related genes and for the two T6SS-unrelated putative ORF insertions with the T6SS of *X.c.v*. Whole genome comparisons (Potnis et al., 2011) recently enabled an extensive analysis of the presence and distribution of T6SS among *Xanthomonas* strains representing 15 pathovars. *X.o.* and *X.o.o.* carry the T6SS-I, located from XOO3034 (*impA*-like) to XOO3052 (*impB*-like) and Xoryp\_12330 (*impA*-like) to Xoryp\_12445 (*impB*-like), and a second T6SS which is referred to as T6SS-III in Fig. 1 and is located between XOO3517 (*impL*-like) and XOO3474 (*vgrG*-like) in *X.o.* and Xoryp\_06365 (*impL*-like) and Xoryp\_06645 (*vgrG*-like) in *X.o.o*. This locus contains a large number of T6SS unrelated ORF insertions, and is phylogenetically distant from the *Xanthomonas* T6SS-I and T6SS-II, while is phylogenetically close to the *R. solanacearum* and

Bacteria of the genus *Erwinia* (γ-proteobacteria) are plant-associated as pathogens, saprophytes and epiphytes and exhibit considerable heterogeneity, forming four phylogenetic clades that are intermixed with members of other genera, such as *E. coli*, *Klebsiella pneumoniae*, and *Serratia marcescens* (Kwon et al., 1997)*. E. amylovora* (*E.a*) originated in North America and has spread to other continents, threatening the native apple germplasm in Central Asia. *E. pyrifoliae* (*E.p.*) is a newly described necrotrophic pathogen initially isolated in the middle 1990's from Japanese pear (*Pyrus pyrifolia*), but could not be found in later years in the previously affected orchards (Kim et al., 2001). *E.p.* causes fire blight symptoms essentially indistinguishable from those of *E.a.* infection (Kim et al., 2001) but has more limited host range. Both pathogens share many common virulence factors, including two distinct type III secretion systems (T3SS) and genes for desferrioxamine

The genome sequence of the highly virulent strain Ea273 (ATCC 49946) isolated from diseased apple (*Malus pumila* cv. Rhode Island Greening) in New York State, is over 99.99% identical to that of the European isolate CFBP 1430 (ERWAC), indicating minimal divergence since the global dispersion of *E.a.* However, a large-scale rearrangement of the genome resulting in repositioning of two large portions of the chromosome has taken place. Additionally, ERWAC has only the smaller plasmid (pEA29) found in *E.a.* and its two T3SSs on genomic islands PAI-2 and PAI-3 have high homology to the insect endosymbiont *Sodalis glossidinius* str. *morsitans* (SODGM) and to the mammalian pathogens *Salmonella* and *Yersinia* spp., indirectly suggesting a closer insect association

biosynthesis. However, *E.p* lacks a third T3SS cluster found in *E. amylovora*.

#### **7.2** *Ralstonia solanacearum*

*R. solanacearum* (*R.s.*, previously known as *Pseudomonas solanacearum; β-proteobacteria*), the causative agent of bacterial wilt of solanaceous plants, is a soil-resident bacterium with wide geographical distribution in all warm and tropical areas and the causal agent of vascular wilt disease in over 200 annual and perennial plant species, representing over 50 botanical families, both monocots and dicots. It also causes disease on the model plant *Arabidopsis thaliana*, and along with *P. syringe* pv. *tomato* DC3000, is one of the leading models in the study of plant-microbe interactions.

The genome of *R.s.* strain GMI1000, as was annotated in KEGG gene data base has one T6SS cluster comprising 16 genes, lying between and *impL*-like (RS01945; also designated as RSp0763) and a *vgrG*-like RS01970, also designated as RSp0738). This cluster seems to be interrupted by some apparently T6SS-unrelated putative open reading frames (11 ORFs) indicating possible gene rearrangements. The gene clusters RS01945-46-47 (also designated as RSp0763-62-61) and RS01967-68-69-70 (also designated as RSp0741-40-39-38) have been annotated as putative *impL-impM-ompA* and *vasD-impJ-impK-vgr* respectively, and are found in reverse orientation compared to the middle section of the cluster, which include genes annotated as *vgr-clpB-impH-impG-impF-hcp-impC-impB* [from RS01949 to RS01966 (also designated as RSp0743 to RSp0749)]. Thus far, there is no experimental evidence for a role of the T6SS in the *R.s.* pathogenicity.

The consensus phylogenetic analysis tree of four *R.s.* T6SS core proteins revealed the closest phylogenetic proximity of the *R.s.* T6SS core components to those of the T6SS-II of *Cupriavidus taiwanensis* which belongs to the phylogenetically distant β-Rhizobium group (Fig. 3), with the same gene order, and the next closest relatives being those of the *Xanthomonas* Τ6SS-ΙΙΙ and the *Ralstonia* T6SS studied (also β-proteobacteria), indicating very recent common ancestry.

#### **7.3** *Xanthomonas* **spp.**

The genus *Xanthomonas* (γ-*Proteobacteria*) consists of 27 plant-associated species, each with many pathovars, which collectively, cause disease on at least 124 monocot species and 268 dicot species, including fruit and nut trees, solanaceous and brassicaceous plants, and cereals (Hayward, 1993; Leyns et al., 1984). *X. campestris* pv. *vesicatoria* (Doidge; syn. *Xanthomonas vesicatoria*) has tomato and pepper as principal hosts. However, various other *Solanaceae*, mainly weeds, have been recorded as incidental hosts. *X. axonopodis* pv. *citri* (*X.a.c.*) is the causal agent of "citrus canker", which affects most commercial citrus cultivars, resulting in significant losses worldwide. (Stall & Seymour, 1983). *X. oryzae* includes the two non-European rice pathogens pvs. *oryzae* and *oryzicola* (*X.o.* & *X.o.o.*). The principal host of both pathovars is rice and high-yielding cultivars are often highly susceptible. *Oryza sativa*  subsp. *japonica* is usually more resistant than subsp. *indica* to pv. *oryzicola*. *X.o.* invades the vascular tissue, while *X.o.o.* proliferates in the parenchyma (Nino-Liu et al., 2006).

Experimental evidence for a role of T6SS in *Xanthomonas* pathogenicity or other aspects of its life cycle is limited. Our data base search revealed a variable situation vis-à-vis T6SS homologs among several sequenced strains of *Xanthomonas*; All three *X. campestris* pv. *campestris* (*X.c.c.*) strains (ATCC33913, Xcc8004 and B100) and the avirulent *X. populi* and *X. codiaii* seem to lack T6SS-related genes, while two distinct T6SS loci are found in *X.* 

*R. solanacearum* (*R.s.*, previously known as *Pseudomonas solanacearum; β-proteobacteria*), the causative agent of bacterial wilt of solanaceous plants, is a soil-resident bacterium with wide geographical distribution in all warm and tropical areas and the causal agent of vascular wilt disease in over 200 annual and perennial plant species, representing over 50 botanical families, both monocots and dicots. It also causes disease on the model plant *Arabidopsis thaliana*, and along with *P. syringe* pv. *tomato* DC3000, is one of the leading models in the

The genome of *R.s.* strain GMI1000, as was annotated in KEGG gene data base has one T6SS cluster comprising 16 genes, lying between and *impL*-like (RS01945; also designated as RSp0763) and a *vgrG*-like RS01970, also designated as RSp0738). This cluster seems to be interrupted by some apparently T6SS-unrelated putative open reading frames (11 ORFs) indicating possible gene rearrangements. The gene clusters RS01945-46-47 (also designated as RSp0763-62-61) and RS01967-68-69-70 (also designated as RSp0741-40-39-38) have been annotated as putative *impL-impM-ompA* and *vasD-impJ-impK-vgr* respectively, and are found in reverse orientation compared to the middle section of the cluster, which include genes annotated as *vgr-clpB-impH-impG-impF-hcp-impC-impB* [from RS01949 to RS01966 (also designated as RSp0743 to RSp0749)]. Thus far, there is no experimental evidence for a role of

The consensus phylogenetic analysis tree of four *R.s.* T6SS core proteins revealed the closest phylogenetic proximity of the *R.s.* T6SS core components to those of the T6SS-II of *Cupriavidus taiwanensis* which belongs to the phylogenetically distant β-Rhizobium group (Fig. 3), with the same gene order, and the next closest relatives being those of the *Xanthomonas* Τ6SS-ΙΙΙ and the *Ralstonia* T6SS studied (also β-proteobacteria), indicating very

The genus *Xanthomonas* (γ-*Proteobacteria*) consists of 27 plant-associated species, each with many pathovars, which collectively, cause disease on at least 124 monocot species and 268 dicot species, including fruit and nut trees, solanaceous and brassicaceous plants, and cereals (Hayward, 1993; Leyns et al., 1984). *X. campestris* pv. *vesicatoria* (Doidge; syn. *Xanthomonas vesicatoria*) has tomato and pepper as principal hosts. However, various other *Solanaceae*, mainly weeds, have been recorded as incidental hosts. *X. axonopodis* pv. *citri* (*X.a.c.*) is the causal agent of "citrus canker", which affects most commercial citrus cultivars, resulting in significant losses worldwide. (Stall & Seymour, 1983). *X. oryzae* includes the two non-European rice pathogens pvs. *oryzae* and *oryzicola* (*X.o.* & *X.o.o.*). The principal host of both pathovars is rice and high-yielding cultivars are often highly susceptible. *Oryza sativa*  subsp. *japonica* is usually more resistant than subsp. *indica* to pv. *oryzicola*. *X.o.* invades the

vascular tissue, while *X.o.o.* proliferates in the parenchyma (Nino-Liu et al., 2006).

Experimental evidence for a role of T6SS in *Xanthomonas* pathogenicity or other aspects of its life cycle is limited. Our data base search revealed a variable situation vis-à-vis T6SS homologs among several sequenced strains of *Xanthomonas*; All three *X. campestris* pv. *campestris* (*X.c.c.*) strains (ATCC33913, Xcc8004 and B100) and the avirulent *X. populi* and *X. codiaii* seem to lack T6SS-related genes, while two distinct T6SS loci are found in *X.* 

**7.2** *Ralstonia solanacearum* 

study of plant-microbe interactions.

the T6SS in the *R.s.* pathogenicity.

recent common ancestry.

**7.3** *Xanthomonas* **spp.** 

*campesrtis* pv. *vesicatoria* XCV2120 (*X.c.v.*), one in *X. axonopodis* 4122 (*X.a.c.*) and three in each, *X.o* and *X.o.o.* One T6SS cluster in XCV2120 extends from XCV2120 (*impB*-like) to XCV2143 (*impA*-like) and is referred to here as T6SS-I, while a second cluster is located from XCV4202 (*impA*-like) to XCV4243 (*impB*-like) and referred as T6SS-II (Fig. 1). This cluster contains two interruptions by some apparently T6SS-unrelated putative ORFs, one, between XCV4214 (*impI*-like) and XCV4236 (*clpB*-like), and another between XCV4202 (*impA*-like) and XCV4208 (*impM*-like). Two T6SS clusters are found in *X.c.v.*: one of them is phylogenetically closer to the T6SS-I found in *X.o.* and *X.o.o* while the second seems to more closely related to the sole T6SS locus of *X.a.c* (referred to as T6SS-II in Fig. 3) which spans from XAC4112 (*impA*-like) to XAC4147 (*impB*-like) (Fig. 1). The *X.a.c.* T6SS locus is almost collinear for the T6SS related genes and for the two T6SS-unrelated putative ORF insertions with the T6SS of *X.c.v*. Whole genome comparisons (Potnis et al., 2011) recently enabled an extensive analysis of the presence and distribution of T6SS among *Xanthomonas* strains representing 15 pathovars. *X.o.* and *X.o.o.* carry the T6SS-I, located from XOO3034 (*impA*-like) to XOO3052 (*impB*-like) and Xoryp\_12330 (*impA*-like) to Xoryp\_12445 (*impB*-like), and a second T6SS which is referred to as T6SS-III in Fig. 1 and is located between XOO3517 (*impL*-like) and XOO3474 (*vgrG*-like) in *X.o.* and Xoryp\_06365 (*impL*-like) and Xoryp\_06645 (*vgrG*-like) in *X.o.o*. This locus contains a large number of T6SS unrelated ORF insertions, and is phylogenetically distant from the *Xanthomonas* T6SS-I and T6SS-II, while is phylogenetically close to the *R. solanacearum* and the *C. taiwanensis* T6SS-II.

#### **7.4** *Erwinia* **spp. (***E. pyrifoliae* **and** *E. amylovora***)**

Bacteria of the genus *Erwinia* (γ-proteobacteria) are plant-associated as pathogens, saprophytes and epiphytes and exhibit considerable heterogeneity, forming four phylogenetic clades that are intermixed with members of other genera, such as *E. coli*, *Klebsiella pneumoniae*, and *Serratia marcescens* (Kwon et al., 1997)*. E. amylovora* (*E.a*) originated in North America and has spread to other continents, threatening the native apple germplasm in Central Asia. *E. pyrifoliae* (*E.p.*) is a newly described necrotrophic pathogen initially isolated in the middle 1990's from Japanese pear (*Pyrus pyrifolia*), but could not be found in later years in the previously affected orchards (Kim et al., 2001). *E.p.* causes fire blight symptoms essentially indistinguishable from those of *E.a.* infection (Kim et al., 2001) but has more limited host range. Both pathogens share many common virulence factors, including two distinct type III secretion systems (T3SS) and genes for desferrioxamine biosynthesis. However, *E.p* lacks a third T3SS cluster found in *E. amylovora*.

The genome sequence of the highly virulent strain Ea273 (ATCC 49946) isolated from diseased apple (*Malus pumila* cv. Rhode Island Greening) in New York State, is over 99.99% identical to that of the European isolate CFBP 1430 (ERWAC), indicating minimal divergence since the global dispersion of *E.a.* However, a large-scale rearrangement of the genome resulting in repositioning of two large portions of the chromosome has taken place. Additionally, ERWAC has only the smaller plasmid (pEA29) found in *E.a.* and its two T3SSs on genomic islands PAI-2 and PAI-3 have high homology to the insect endosymbiont *Sodalis glossidinius* str. *morsitans* (SODGM) and to the mammalian pathogens *Salmonella* and *Yersinia* spp., indirectly suggesting a closer insect association than mere passive dispersal (Smits et al., 2010).

Phytobacterial Type VI Secretion System –

**7.7** *Dickeya* **spp. (***D. dadantii* **and** *D. zeae***)** 

Gene Distribution, Phylogeny, Structure and Biological Functions 73

genes (ECA4275, ECA2866, ECA0456 and ECA3672), are also present. Interestingly, virulence assays, performed with mutants in ECA3438 and ECA3444, in potato stems and tubers, showed significantly reduced virulence compared with the wild type strain in both cases (Liu et al., 2008). In our phylogenetic analysis the *P.a.* T6SS is presented as a member of a distinct

*Dickeya dadantii* (*D.d.*; formerly *Erwinia chrysanthemi;* γ-proteobacteria) is an opportunistic plant pathogen causing soft-rot, wilt, and blights on a wide range of plant species, such as maize, pineapple, banana, rice, tobacco, tomato, *Brachiaria ruziziensis* and *Chrysanthemum morifolium*. It possesses two O-serogroups, O: 1 and O: 6. *D.d.* is also highly virulent on the pea aphid *Acyrthosiphon pisum*, and possesses four genes encoding homologs of the Cyt family of insecticidal toxins from *Bacillus thuringiensis* (Grenier et al., 2006). *Dickeya zeae*  (*D.z.*; formerly *Erwinia chrysanthemi*) was isolated from soft rot and wilt of a various range of plants, such as *Zea mays*, *Ananas comosus*, *Brachiaria ruziziensis*, *Chrysanthemum morifolium*, *Musa* spp., *Nicotiana tabacum*, *Oryza sativa* and *Solanum tuberosum*, as well as from water

Two strains, *D.d.* Ech586 and *D.z.* Ech1591 (Lucas et al., 2009) that have been examined contain identical T6SS loci consisting of 17 genes lying from Dd586\_1304 (*vasL*-like) to Dd586\_1272 (*hcp*-like) for *D.d.*, with a disruption of several apparently T6SS-unrelated putative ORF insertions between Dd586\_1290 (*impB*-like) and Dd586\_1273 (*vgrG*-like) genes (Fig. 1). The *D.z.* T6SS locus spans from Dd1591\_2793 (*vasL*-like) to Dd1591\_2826 (*hcp*-like) with a disruption of several apparently T6SS-unrelated ORF insertions between Dd1591\_2807 (*impB*-like) and Dd1591\_2825 (*vgrG*-like) genes (Fig. 1). The two clusters are almost identical and the four core T6SS proteins examined form a distinct phylogenetic

*A. avenae* subsp. *citrulli* (*A.c.;* β-proteobacteria) is formerly known as *Pseudomonas pseudoalcaligenes* subsp. *citrulli* and is the causal agent of bacterial fruit blotch. It spreads by infested seeds, infected transplants, and occurs naturally in wild hosts. It can be asymptomatic on older plants, which can lead to high numbers of infected young plants early in the planting season. A T6SS cluster is found in *A.c.* strain AAC00-1 consisting of 16 genes between Aave\_1482 (*clp*-like ATPase) and Aave\_1465 (*hcp*-like) as annotated in Fig. 1. The T6SS locus is contiguous, except of two putative ORF insertions between the Aave\_1468 (*fha*-like) and Aave\_1465 genes that are apparently T6SS-unrelated. The *A.c.* T6SS cluster lacks a *vgrG* homolog, which potentially raises questions about the system's functionality. To date, there is no experimental evidence concerning a role of this system in *A.c.*-host interactions. Phylogenetically the *A.c.* T6SS cluster forms a sub-group with the *C. taiwanensis* 

*Agrobacterium* strains (α-proteobacteria) invade the crown, roots and stems of a great variety of plants via wounds causing overgrowths (crown gall, hairy root, and cane gall). *A.*

phylogenetic branch comprising the *P.a.* and *Dickeya* spp. T6SSs (Fig. 3).

samples. *D.z.* in contrast to *D.d.* possesses more than nine O-serogroups.

branch which includes the *P. atrosepticum* T6SS (Fig. 3).

T6SS-I, *P. ananatis* T6SS-II, *Erwinia* spp. T6SS-II (Fig. 3).

**7.9** *Agrobacterium* **spp. (***A. tumefaciens* **and** *A. vitis***)** 

**7.8** *Acidovorax avenae* **subsp.** *citrulli* 

*E.p.* strain Ep1/96 harbors one T6SS cluster which spans the region from EpC\_06150 (*vasD*like) to EpC\_06440 (*vgrG2*-like) and shares sequence and gene order homology with the one of the *E.a* T6SS clusters [designated T6SS-II in Fig. 1, 2 and 3), starting from EAMY\_3027 (*vasD*like) to EAMY\_3000 (*vgrG*-like)]. These clusters are very close phylogenetic relatives and have as next closest relative the *Pantoea ananatis* T6SS-II (Fig. 3). Furthermore, a small T6SS cluster of four ORFs (not presented in Fig. 1) is present in both *E.p.* and *E.a.* (*E.p*.:EpC\_19520-EpC\_19550 and *E.a.*: EAMY\_1620-EAMY\_1623). *E.a.* also harbors a second T6SS cluster (designated T6SS-III in Figs. 1, 2 and 3) which spans from EAMY\_3228 (*impB*-like) to EAMY\_3201 (*vasL*-like). This cluster exhibits gene order and sequence relatedness to *P. ananatis* T6SS-III (Fig. 1). Similar results were reported for *E.p.* DSM 12163T and *E.a* CFBP 1430 based on whole genome sequence analysis (Smits et al., 2010). There are no experimental data concerning the biological role of the T6SS in *Erwinia* spp. and most genes within the T6SS clusters are uncharacterized.

#### **7.5** *Pantoea ananatis*

The genus *Pantoea* (γ-proteobacteria) consists of both important plant pathogens and clinically relevant species. Clinical isolates have been reported to cause bacteraemia in humans. *P. ananatis* (*P.a.*) is considered an unconventional plant associated species, being associated with plants as an epiphyte, endophyte, pathogen, or symbiont, but can also occupy unusual ecological niches (e.g. contaminating aviation jet fuel tanks). It's ice nucleation activity has been exploited in the food industry and in the biological control of insects (Coutinho & Venter, 2009).

The exact role of T6SS in the adaptive capacity of *P.a*. is not known, but such a role might be considered likely as there has been no evidence of T2SS or T3SS in any of the strains sequenced so far (De Maayer et al., 2010). Our database search in the sequenced *P.a.* strain LMG 20103 revealed one truncated and two entire T6SS clusters named T6SS-I, II and III respectively in Figs 1 and 3. The truncated T6SS-I is located from PANA\_1650 (a possible T6SS related protein kinase A; PknA=ImpN) to PANA\_1656 (*impK*-like). The obvious absence of some basic T6SS core genes raises questions about the functionality of this T6SS locus. The T6SS-II locus is embedded between PANA\_2352 (*vgrG*-like) to PANA\_2372 (*vasD*-like) while the T6SS-III locus is divided in two sub-loci the first one is located from PANA\_4130 (*vasL*-like) to PANA\_4138 (*impL*-like), the second one, being located a few apparently T6SS-unrelated putative ORFs away, from PANA\_4144 (*vgrG*-like) to PANA\_4151 (*impB*-like). Phylogenetically, the *P.a.* T6SS-I seems to be related to some random distributed T6SS related ORFs in the genome of *E. amylovora* (data not shown). The *P.a.* T6SS-II is phylogenetically closer in protein sequence and the gene order to the *Erwinia* spp. T6SS-II (Figs 1 and 3). Interestingly, the *P.a.* T6SS-III forms a distant phylogenetic branch with the *E.a.* T6SS-III and appears as an out-group in our analysis (Fig. 3).

#### **7.6** *Pectobacterium atrosepticum*

*P. atrosepticum* (*P.a.*; formerly *Erwinia carotovora* subsp*. atroseptica;* γ-proteobacteria) is a member of the pectolytic *Erwinia* responsible for the soft rot and blackleg of potato stems and tubers. A T6SS locus is found in *P.a.* strain SCRI1043 extending from ECA3427 (*impB*-like) to ECA3445 (*vgrG*-like) and is here referred as *Erwinia* spp*.* T6SS-I (Fig. 1). One additional solitary locus, designated as *vgrG*-like gene (ECA2104), as well as four other loci, designated as *hcp*-like genes (ECA4275, ECA2866, ECA0456 and ECA3672), are also present. Interestingly, virulence assays, performed with mutants in ECA3438 and ECA3444, in potato stems and tubers, showed significantly reduced virulence compared with the wild type strain in both cases (Liu et al., 2008). In our phylogenetic analysis the *P.a.* T6SS is presented as a member of a distinct phylogenetic branch comprising the *P.a.* and *Dickeya* spp. T6SSs (Fig. 3).

#### **7.7** *Dickeya* **spp. (***D. dadantii* **and** *D. zeae***)**

72 Plant Pathology

*E.p.* strain Ep1/96 harbors one T6SS cluster which spans the region from EpC\_06150 (*vasD*like) to EpC\_06440 (*vgrG2*-like) and shares sequence and gene order homology with the one of the *E.a* T6SS clusters [designated T6SS-II in Fig. 1, 2 and 3), starting from EAMY\_3027 (*vasD*like) to EAMY\_3000 (*vgrG*-like)]. These clusters are very close phylogenetic relatives and have as next closest relative the *Pantoea ananatis* T6SS-II (Fig. 3). Furthermore, a small T6SS cluster of four ORFs (not presented in Fig. 1) is present in both *E.p.* and *E.a.* (*E.p*.:EpC\_19520-EpC\_19550 and *E.a.*: EAMY\_1620-EAMY\_1623). *E.a.* also harbors a second T6SS cluster (designated T6SS-III in Figs. 1, 2 and 3) which spans from EAMY\_3228 (*impB*-like) to EAMY\_3201 (*vasL*-like). This cluster exhibits gene order and sequence relatedness to *P. ananatis* T6SS-III (Fig. 1). Similar results were reported for *E.p.* DSM 12163T and *E.a* CFBP 1430 based on whole genome sequence analysis (Smits et al., 2010). There are no experimental data concerning the biological role of the T6SS in *Erwinia* spp. and most genes within the T6SS clusters are uncharacterized.

The genus *Pantoea* (γ-proteobacteria) consists of both important plant pathogens and clinically relevant species. Clinical isolates have been reported to cause bacteraemia in humans. *P. ananatis* (*P.a.*) is considered an unconventional plant associated species, being associated with plants as an epiphyte, endophyte, pathogen, or symbiont, but can also occupy unusual ecological niches (e.g. contaminating aviation jet fuel tanks). It's ice nucleation activity has been exploited in the food industry and in the biological control of

The exact role of T6SS in the adaptive capacity of *P.a*. is not known, but such a role might be considered likely as there has been no evidence of T2SS or T3SS in any of the strains sequenced so far (De Maayer et al., 2010). Our database search in the sequenced *P.a.* strain LMG 20103 revealed one truncated and two entire T6SS clusters named T6SS-I, II and III respectively in Figs 1 and 3. The truncated T6SS-I is located from PANA\_1650 (a possible T6SS related protein kinase A; PknA=ImpN) to PANA\_1656 (*impK*-like). The obvious absence of some basic T6SS core genes raises questions about the functionality of this T6SS locus. The T6SS-II locus is embedded between PANA\_2352 (*vgrG*-like) to PANA\_2372 (*vasD*-like) while the T6SS-III locus is divided in two sub-loci the first one is located from PANA\_4130 (*vasL*-like) to PANA\_4138 (*impL*-like), the second one, being located a few apparently T6SS-unrelated putative ORFs away, from PANA\_4144 (*vgrG*-like) to PANA\_4151 (*impB*-like). Phylogenetically, the *P.a.* T6SS-I seems to be related to some random distributed T6SS related ORFs in the genome of *E. amylovora* (data not shown). The *P.a.* T6SS-II is phylogenetically closer in protein sequence and the gene order to the *Erwinia* spp. T6SS-II (Figs 1 and 3). Interestingly, the *P.a.* T6SS-III forms a distant phylogenetic

branch with the *E.a.* T6SS-III and appears as an out-group in our analysis (Fig. 3).

*P. atrosepticum* (*P.a.*; formerly *Erwinia carotovora* subsp*. atroseptica;* γ-proteobacteria) is a member of the pectolytic *Erwinia* responsible for the soft rot and blackleg of potato stems and tubers. A T6SS locus is found in *P.a.* strain SCRI1043 extending from ECA3427 (*impB*-like) to ECA3445 (*vgrG*-like) and is here referred as *Erwinia* spp*.* T6SS-I (Fig. 1). One additional solitary locus, designated as *vgrG*-like gene (ECA2104), as well as four other loci, designated as *hcp*-like

**7.5** *Pantoea ananatis* 

insects (Coutinho & Venter, 2009).

**7.6** *Pectobacterium atrosepticum* 

*Dickeya dadantii* (*D.d.*; formerly *Erwinia chrysanthemi;* γ-proteobacteria) is an opportunistic plant pathogen causing soft-rot, wilt, and blights on a wide range of plant species, such as maize, pineapple, banana, rice, tobacco, tomato, *Brachiaria ruziziensis* and *Chrysanthemum morifolium*. It possesses two O-serogroups, O: 1 and O: 6. *D.d.* is also highly virulent on the pea aphid *Acyrthosiphon pisum*, and possesses four genes encoding homologs of the Cyt family of insecticidal toxins from *Bacillus thuringiensis* (Grenier et al., 2006). *Dickeya zeae*  (*D.z.*; formerly *Erwinia chrysanthemi*) was isolated from soft rot and wilt of a various range of plants, such as *Zea mays*, *Ananas comosus*, *Brachiaria ruziziensis*, *Chrysanthemum morifolium*, *Musa* spp., *Nicotiana tabacum*, *Oryza sativa* and *Solanum tuberosum*, as well as from water samples. *D.z.* in contrast to *D.d.* possesses more than nine O-serogroups.

Two strains, *D.d.* Ech586 and *D.z.* Ech1591 (Lucas et al., 2009) that have been examined contain identical T6SS loci consisting of 17 genes lying from Dd586\_1304 (*vasL*-like) to Dd586\_1272 (*hcp*-like) for *D.d.*, with a disruption of several apparently T6SS-unrelated putative ORF insertions between Dd586\_1290 (*impB*-like) and Dd586\_1273 (*vgrG*-like) genes (Fig. 1). The *D.z.* T6SS locus spans from Dd1591\_2793 (*vasL*-like) to Dd1591\_2826 (*hcp*-like) with a disruption of several apparently T6SS-unrelated ORF insertions between Dd1591\_2807 (*impB*-like) and Dd1591\_2825 (*vgrG*-like) genes (Fig. 1). The two clusters are almost identical and the four core T6SS proteins examined form a distinct phylogenetic branch which includes the *P. atrosepticum* T6SS (Fig. 3).

#### **7.8** *Acidovorax avenae* **subsp.** *citrulli*

*A. avenae* subsp. *citrulli* (*A.c.;* β-proteobacteria) is formerly known as *Pseudomonas pseudoalcaligenes* subsp. *citrulli* and is the causal agent of bacterial fruit blotch. It spreads by infested seeds, infected transplants, and occurs naturally in wild hosts. It can be asymptomatic on older plants, which can lead to high numbers of infected young plants early in the planting season. A T6SS cluster is found in *A.c.* strain AAC00-1 consisting of 16 genes between Aave\_1482 (*clp*-like ATPase) and Aave\_1465 (*hcp*-like) as annotated in Fig. 1. The T6SS locus is contiguous, except of two putative ORF insertions between the Aave\_1468 (*fha*-like) and Aave\_1465 genes that are apparently T6SS-unrelated. The *A.c.* T6SS cluster lacks a *vgrG* homolog, which potentially raises questions about the system's functionality. To date, there is no experimental evidence concerning a role of this system in *A.c.*-host interactions. Phylogenetically the *A.c.* T6SS cluster forms a sub-group with the *C. taiwanensis*  T6SS-I, *P. ananatis* T6SS-II, *Erwinia* spp. T6SS-II (Fig. 3).

#### **7.9** *Agrobacterium* **spp. (***A. tumefaciens* **and** *A. vitis***)**

*Agrobacterium* strains (α-proteobacteria) invade the crown, roots and stems of a great variety of plants via wounds causing overgrowths (crown gall, hairy root, and cane gall). *A.*

Phytobacterial Type VI Secretion System –

**8.3** *Bradyrhizobium japonicum* 

*Xanthomonas* T6SS-I and III (Fig. 3).

**8.5** *Cupriavidus taiwanensis* 

these T6SS clusters in host colonization.

**8.4** *Mesorhizobium loti* **(***Rhizobium loti***)**

Gene Distribution, Phylogeny, Structure and Biological Functions 75

genomic islands (Fig. 1). The T6SS-I spans from RHECIAT\_PC0000958 (*vgrG*-like) to RHECIAT\_PC0000933 (*impL*-like). While the T6SS-II seems to be divided in two segments with opposite gene orientations, located from RHECIAT\_PB0000227 (*impL*-like) to RHECIAT\_PB0000224 (*impJ*-like) and from RHECIAT\_PB0000217 (*impA*-like) to RHECIAT\_PB0000210 (*vgrG*-like). Furthermore, the *Clp*-like ATPase is absent in T6SS-II, which raises questions about the functionality of the cluster (Fig. 1). Phylogenetically, the four core proteins of the *R.e.* CIAT 652 T6SS-I are more closely related to those of the *Mesorhizobium loti* T6SS*,* forming a distinct sub-group, while those of the T6SS-II branch with

*B. japonicum* (*B.j*. α-proteobacteria; *Rhizobium japonicum* in earlier references) utilizes similar mechanisms, as the other symbiotic bacteria, to establish a symbiotic relationship. The *B.j.* strain USDA110 genome consists of a single circular chromosome of about 9 Mbp and has no plasmids. Our i*n silico* genome mining results were in agreement to Boyer et al. (2009) and Records et al. (2011) revealing the presence of one T6SS gene cluster consisting of 17 ORFs located between blr3604 (*ImpN*-like) and bll3587 (*clpB*-like) (Fig. 1). Phylogenetically, the *B.j.* T6SS forms a distinct branch from other *Rhizobium* T6SS and clusters closest to the

*M. loti* (*M.l.* α-proteobacteria), a symbiont of *Lotus japonicus* contains a chromosomal symbiosis island, similar to what is observed with other rhizobacteria. *M.l.* strain MAFF303099 contains one T6SS which is located between mlr2363 (*impN*-like) and mll2335 (*clpB*-like) gene loci (Fig.

*C. taiwanensis* (*C.t.* β-proteobacteria; originally called *Ralstonia taiwanensis* or *Wautersia taiwanensis*), belongs to the β-rhizobia group. It was first isolated from a nodule from the legume *Mimosa pudica* in Taiwan. The type strain LMG19424 has a three-replicon genome made up of two chromosomes of 3.5 Mb, and 2.4 Mb, and a 0.5 kb symbiotic plasmid which carries the genes essential for nodulation and nitrogen fixation (Amadou et al., 2008). In our *in silico* genome mining, two T6SS gene clusters were found in the LMG19424, one (T6SS-I) consisting of 15 genes located between RALTA\_A0602 (*impM*-like) and RALTA\_A0622 (*impA*-like), with six apparently T6SS-unrelated putative ORF insertions between RALTA\_A0611 (*vgrG*-like) and RALTA\_A0618 (*vasD*-like) genes (Fig. 1). Phylogenetically, the *C.t.* T6SS-I groups together with the T6SSs of *A. avenae* pv. *citrulli*, *P. ananatis* T6SS-II, *E. pyrifoliae* and *E. amylovora* T6SS-II (Fig. 3). The second cluster (T6SS-II) consists of 18 genes located between RALTA\_B1008 (*vgrG*-like) and RALTA\_B1029 (impL-like) and containing four putative ORF insertions apparently unrelated to T6SS, between RALTA\_B1019 (*clpB*like ATPase) and RALTA\_B1025 (*impA*-like) genes (Fig. 1). Based on gene order and sequence similarities, the C. t. T6SS-II and *R. solanacearum* T6SS appear phylogenetically close (Fig. 3). There are no studies concerning the functionality and/or the contribution of

1). In contrast to *R.l.*, *M.l.* possesses *vasD* while it apparently lacks *impE* (Figs. 1 and 3).

the *Xanthomonas* spp. T6SS-I and T6SS-II, forming a separate sub-group (Fig. 3).

*tumefaciens (A.t.*) have a wide host range among dicotyledonous plants, whereas other possess a very limited host range [*A. rubi* (*A.r.*) and *A. vitis* (*A.v*.) form galls on raspberries and grapes, respectively]. *A. rhizogenes* causes hairy roots on many plants. The ability to cause disease is associated with transmissible plasmids which may move from one strain to another. *A.t.* strain C58 is a representative of biovar I, which has also been extensively modified for biotechnological uses. *A.v.* strain S4 is a virulent biovar III isolated from *Vitis vinifera* (grape) cv. Izsaki Sarfeher crown galls in Kecskemet, Hungary in 1981 (Szegedi et al., 1988; 1996). *A.v.* strains not only cause galls on grapevines but also necrosis on grapevine roots and a hypersensitive response on non-host plants.

The two sequenced species, *A.t.* strain C58 and *A.v.* strain S4 seem to harbor almost identical T6SS gene cluster. According to our database search, those loci are lying between Atu4330 (*impN*-like) and Atu4348 (*vgrG*-like) in *A.t.* C58 and between Avi\_6039 (*impN*-like) and Avi\_6054 (*hcp*-like) in *A.v*. S4 (Fig. 1). *A.v*. seems to lack the *vgrG*-like gene upstream of the *clpV*-like gene (Fig. 1), while *A.t.* carries a *vgrG*-like gene in the solitary locus Atu\_3642 outside the T6SS cluster. However, the *A.v.* has five additional *vgrG*-like genes at the solitary loci Avi\_1646, Avi\_2758, Avi\_3254, Avi\_7056, Avi\_7557, which also are located outside the T6SS cluster. Phylogenetically the *Agrobacterium* spp. T6SSs branch more closely to the *Rhizobium leguminosarum* T6SS phylogenetic sub-group (Fig. 3).

#### **8. T6SS in plant symbiotic bacteria**

#### **8.1** *Rhizobium leguminosarum*

*R. leguminosarum* bv. *viciae* 3841 (*R.l.;* α-proteobacteria) has a genomic portfolio consisting of seven circular DNA modules (totalling about 7,8 Mb): one circular chromosome of about 5 Mb and six plasmids varying in size from 147 kb to 870 kb. A T6SS is one of the many protein secretion systems identified in *R.l.*, (Krehenbrink & Downie, 2008). The T6SS gene cluster was initially reported to contain 14 genes (*impA*-*impN*); later an *hcp* and a *vgrG* gene homolog (pRL120477 and pRL120480), coding for secreted proteins, were added. Our data mining in the genome of *R.l.,* 3841 leave open the presence of a functional Clp-like ATPase because of multiple sequence insertions and point mutations in the *clpV/B* gene (locus No: pRL120476) (Fig. 1), which is annotated as pseudogene in the KEGG, RhizoBase and NCBI databases. Phylogenetically, the *R.l.* T6SS clusters with the *Agrobacterium* spp. T6SSs and form a distinct phylogenetic branch (Fig. 3).

#### **8.2** *Rhizobium etli*

*R. etli* (*R.e.* a-proteobacteria) contributes to a significant proportion of nitrogen coming to the earth through microorganisms and is the prime species found associated with cultivated beans in the Americas. Although there is no experimental evidence for a functional T6SS in *R.e.*, bioinformatic analysis revealed two putative T6SSs, one of which (T6SS-I) is similar in organization to that of *R.l.* (Bladergroen et al., 2003) (Fig. 1), and in our phylogenetic distance tree (Fig. 3) it branches with the *Mesorhizobium loti* T6SS.

Our data mining revealed two interesting features. First, of the two strains of *R.e.* sequenced to date, *R.e.* CFN 42 and *R.e.* CIAT 652, only the latter seems to have T6SS core genes. Second, in this strain there are two T6SS gene clusters (T6SS-I and –II) located in distinct genomic islands (Fig. 1). The T6SS-I spans from RHECIAT\_PC0000958 (*vgrG*-like) to RHECIAT\_PC0000933 (*impL*-like). While the T6SS-II seems to be divided in two segments with opposite gene orientations, located from RHECIAT\_PB0000227 (*impL*-like) to RHECIAT\_PB0000224 (*impJ*-like) and from RHECIAT\_PB0000217 (*impA*-like) to RHECIAT\_PB0000210 (*vgrG*-like). Furthermore, the *Clp*-like ATPase is absent in T6SS-II, which raises questions about the functionality of the cluster (Fig. 1). Phylogenetically, the four core proteins of the *R.e.* CIAT 652 T6SS-I are more closely related to those of the *Mesorhizobium loti* T6SS*,* forming a distinct sub-group, while those of the T6SS-II branch with the *Xanthomonas* spp. T6SS-I and T6SS-II, forming a separate sub-group (Fig. 3).

#### **8.3** *Bradyrhizobium japonicum*

74 Plant Pathology

*tumefaciens (A.t.*) have a wide host range among dicotyledonous plants, whereas other possess a very limited host range [*A. rubi* (*A.r.*) and *A. vitis* (*A.v*.) form galls on raspberries and grapes, respectively]. *A. rhizogenes* causes hairy roots on many plants. The ability to cause disease is associated with transmissible plasmids which may move from one strain to another. *A.t.* strain C58 is a representative of biovar I, which has also been extensively modified for biotechnological uses. *A.v.* strain S4 is a virulent biovar III isolated from *Vitis vinifera* (grape) cv. Izsaki Sarfeher crown galls in Kecskemet, Hungary in 1981 (Szegedi et al., 1988; 1996). *A.v.* strains not only cause galls on grapevines but also necrosis on grapevine

The two sequenced species, *A.t.* strain C58 and *A.v.* strain S4 seem to harbor almost identical T6SS gene cluster. According to our database search, those loci are lying between Atu4330 (*impN*-like) and Atu4348 (*vgrG*-like) in *A.t.* C58 and between Avi\_6039 (*impN*-like) and Avi\_6054 (*hcp*-like) in *A.v*. S4 (Fig. 1). *A.v*. seems to lack the *vgrG*-like gene upstream of the *clpV*-like gene (Fig. 1), while *A.t.* carries a *vgrG*-like gene in the solitary locus Atu\_3642 outside the T6SS cluster. However, the *A.v.* has five additional *vgrG*-like genes at the solitary loci Avi\_1646, Avi\_2758, Avi\_3254, Avi\_7056, Avi\_7557, which also are located outside the T6SS cluster. Phylogenetically the *Agrobacterium* spp. T6SSs branch more closely to the

*R. leguminosarum* bv. *viciae* 3841 (*R.l.;* α-proteobacteria) has a genomic portfolio consisting of seven circular DNA modules (totalling about 7,8 Mb): one circular chromosome of about 5 Mb and six plasmids varying in size from 147 kb to 870 kb. A T6SS is one of the many protein secretion systems identified in *R.l.*, (Krehenbrink & Downie, 2008). The T6SS gene cluster was initially reported to contain 14 genes (*impA*-*impN*); later an *hcp* and a *vgrG* gene homolog (pRL120477 and pRL120480), coding for secreted proteins, were added. Our data mining in the genome of *R.l.,* 3841 leave open the presence of a functional Clp-like ATPase because of multiple sequence insertions and point mutations in the *clpV/B* gene (locus No: pRL120476) (Fig. 1), which is annotated as pseudogene in the KEGG, RhizoBase and NCBI databases. Phylogenetically, the *R.l.* T6SS clusters with the *Agrobacterium* spp. T6SSs and

*R. etli* (*R.e.* a-proteobacteria) contributes to a significant proportion of nitrogen coming to the earth through microorganisms and is the prime species found associated with cultivated beans in the Americas. Although there is no experimental evidence for a functional T6SS in *R.e.*, bioinformatic analysis revealed two putative T6SSs, one of which (T6SS-I) is similar in organization to that of *R.l.* (Bladergroen et al., 2003) (Fig. 1), and in our phylogenetic

Our data mining revealed two interesting features. First, of the two strains of *R.e.* sequenced to date, *R.e.* CFN 42 and *R.e.* CIAT 652, only the latter seems to have T6SS core genes. Second, in this strain there are two T6SS gene clusters (T6SS-I and –II) located in distinct

roots and a hypersensitive response on non-host plants.

*Rhizobium leguminosarum* T6SS phylogenetic sub-group (Fig. 3).

distance tree (Fig. 3) it branches with the *Mesorhizobium loti* T6SS.

**8. T6SS in plant symbiotic bacteria** 

form a distinct phylogenetic branch (Fig. 3).

**8.2** *Rhizobium etli* 

**8.1** *Rhizobium leguminosarum* 

*B. japonicum* (*B.j*. α-proteobacteria; *Rhizobium japonicum* in earlier references) utilizes similar mechanisms, as the other symbiotic bacteria, to establish a symbiotic relationship. The *B.j.* strain USDA110 genome consists of a single circular chromosome of about 9 Mbp and has no plasmids. Our i*n silico* genome mining results were in agreement to Boyer et al. (2009) and Records et al. (2011) revealing the presence of one T6SS gene cluster consisting of 17 ORFs located between blr3604 (*ImpN*-like) and bll3587 (*clpB*-like) (Fig. 1). Phylogenetically, the *B.j.* T6SS forms a distinct branch from other *Rhizobium* T6SS and clusters closest to the *Xanthomonas* T6SS-I and III (Fig. 3).

#### **8.4** *Mesorhizobium loti* **(***Rhizobium loti***)**

*M. loti* (*M.l.* α-proteobacteria), a symbiont of *Lotus japonicus* contains a chromosomal symbiosis island, similar to what is observed with other rhizobacteria. *M.l.* strain MAFF303099 contains one T6SS which is located between mlr2363 (*impN*-like) and mll2335 (*clpB*-like) gene loci (Fig. 1). In contrast to *R.l.*, *M.l.* possesses *vasD* while it apparently lacks *impE* (Figs. 1 and 3).

#### **8.5** *Cupriavidus taiwanensis*

*C. taiwanensis* (*C.t.* β-proteobacteria; originally called *Ralstonia taiwanensis* or *Wautersia taiwanensis*), belongs to the β-rhizobia group. It was first isolated from a nodule from the legume *Mimosa pudica* in Taiwan. The type strain LMG19424 has a three-replicon genome made up of two chromosomes of 3.5 Mb, and 2.4 Mb, and a 0.5 kb symbiotic plasmid which carries the genes essential for nodulation and nitrogen fixation (Amadou et al., 2008). In our *in silico* genome mining, two T6SS gene clusters were found in the LMG19424, one (T6SS-I) consisting of 15 genes located between RALTA\_A0602 (*impM*-like) and RALTA\_A0622 (*impA*-like), with six apparently T6SS-unrelated putative ORF insertions between RALTA\_A0611 (*vgrG*-like) and RALTA\_A0618 (*vasD*-like) genes (Fig. 1). Phylogenetically, the *C.t.* T6SS-I groups together with the T6SSs of *A. avenae* pv. *citrulli*, *P. ananatis* T6SS-II, *E. pyrifoliae* and *E. amylovora* T6SS-II (Fig. 3). The second cluster (T6SS-II) consists of 18 genes located between RALTA\_B1008 (*vgrG*-like) and RALTA\_B1029 (impL-like) and containing four putative ORF insertions apparently unrelated to T6SS, between RALTA\_B1019 (*clpB*like ATPase) and RALTA\_B1025 (*impA*-like) genes (Fig. 1). Based on gene order and sequence similarities, the C. t. T6SS-II and *R. solanacearum* T6SS appear phylogenetically close (Fig. 3). There are no studies concerning the functionality and/or the contribution of these T6SS clusters in host colonization.

Phytobacterial Type VI Secretion System –

and/or other macromolecules as well.

yet unknown elements" (Filloux, 2011b).

**10. Acknowledgments** 

Learning and Religious Affairs.

28, No 7, pp 821-829.

**11. References** 

Gene Distribution, Phylogeny, Structure and Biological Functions 77

Future studies are needed and expected to further advance the T6SS field. It is important to remember that formal evidence of the translocation of effector proteins into plant cells through T6SS is presently lacking, as is also the case for the molecular targets of these effectors. Paraphrasing Schwarz et al. (2010a), outstanding questions for future research on T6SS include the following: What are the physiological role(s) and adaptive significance of T6S-mediated plant cell targeting in disease, symbiosis, and interbacterial interactions in the environment? What is the significance of additional *vgrG* and *hcp* genes Are host- and bacterial cell-targeting T6SSs discernible by sequence or gene content? Are there T6SSs that can target both eukaryotic and prokaryotic cells? Are there other T6S substrates that await identification? Given the resemblance T6SS components to bacteriophage proteins it is also tempting to ask if T6SS transports only protein substrates

It is instructive to point out that the genes coding for several secretion systems, including T6SS, were first identified in phenotypic screens of mutants altered in their interaction with higher eukaryotes. It is conceivable that new secretion systems may be identified in other appropriately designed screens in multi-organism settings. Bioinformatic sourcing of genome, transcriptome and proteome data may point to new potential candidates, as occurred historically with the T6SS. Finally, the striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps invite speculation that new systems may even be predicted, "the way Mendeleïev had anticipated characteristics of

We apologize to any researchers whose work was not included in this review due to space limitations. This work was supported by PYTHAGORAS, and PENED 03ED375 grants of the Greek General Secretariat for Research and Development implemented within the framework of the "Reinforcement Programme of Human Research Manpower" (PENED) and co-financed by National and Community Funds (25% from the Greek Ministry of Development General Secretariat of Research and Technology and 75% from EU-European Social Fund) and by the EPEAEK graduate programs in Plant Molecular Biology and Biotechnology and Protein Biotechnology of the Greek Ministry of Education, Lifelong

Aksyuk, A. A., Leiman, P. G., Kurochkina, L. P., Shneider, M. M., Kostyuchenko, V. A.,

Amadou, C., Pascal, G., Mangenot, S., Glew, M., Bontemps, C., Capela, D., Carrere, S.,

Mesyanzhinov, V. V. & Rossmann, M. G. (2009). The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. *EMBO Journal*, Vol.

Cruveiller, S., Dossat, C., Lajus, A., Marchetti, M., Poinsot, V., Rouy, Z., Servin, B., Saad, M., Schenowitz, C., Barbe, V., Batut, J., Medigue, C. & Masson-Boivin, C.

#### **9. Prospects**

Protein secretion is fundamental to bacterial virulence and several systems mediate pathogenesis and other types of bacteria-host interaction. Beyond the other recognized secretion systems of Gram-negative bacteria with established role in host-pathogen interactions, the T6SS is of particular interest in this respect and has been shown to be important for bacterial virulence and for interaction with the host in various ways, often leading to "anti-pathogenesis" (Jani & Cotter, 2010). Nevertheless, its role and function in most bacteria is not clearly established and formal evidence for protein secretion/translocation into plant cells is scant. At present, we do not fully understand how the T6SS works and are only beginning to understand the biological role/s of the T6SS in plant-associated bacterial life. Multiple copies of T6SS in a single bacterial strain appear to be a frequent phenomenon, and this holds true for many plant associated species. Recent studies (Boyer et al., 2009; Filloux et al., 2008) have established the presence of multiple copies of apparently complete and/or degenerate T6SS loci in about one quarter of the proteobacterial genomes examined, that they generally display different phylogenetic origins and are not a result of recent duplication events, suggesting sustained and constrained mechanisms that favour this trend. Based on our own analysis (Sarris & Scoulica, 2011; Sarris et al., 2011), most strains of *Pseudomonas syringae,* the insect pathogenic *Pseudomonas entomophila* strain L48, the human opportunistic pathogen *Pseudomonas mendocina* strain ymp, and most of the *Pseudomonas* strains studied by Barret et al. (2011) typically carry T6SS from more than one phylogenetic clade and/or additional *vgrG* and *hcp* genes.

Although the majority of the recent studies concern the contribution of T6SS in bacterial pathogenicity (positively), many bacteria with genomes encoding putative T6SS are not known to be pathogens or symbionts, and T6SS may also function in non-pathogenic bacteria-host interactions and/or in interactions not involving eukaryotic partners. In *R. leguminosarum* the T6SS limits host-range and in *S. typhimurium* and *H. hepaticus* the evidence suggests a possible role of T6SS in limiting of bacterial virulence and, therefore, contribution to host colonization (Bladergroen et al., 2003; Parsons & Heffron, 2005; Jani & Cotter, 2010; Chow & Mazmanian, 2010). A relatively new twist in the system's repertoire of biological functions in a broader context is the finding that bacteria engage each other in a T6SS-dependent manner and can provide protection for a bacterium against cell contactinduced growth inhibition caused by other species of bacteria (Hood et al., 2010; Schwarz et al., 2010a). This leads to speculation that this pathway is of general significance to interbacterial interactions in polymicrobial diseases and the environment.

T6SS clusters occur with high frequency, have divergent phylogenies and individual strains or species often possess non-orthologous clusters with distinct or overlapping functions in bacterial interactions with multiple hosts, antagonists or predators unsuspected at present. In a recent study of the ocean metagenome (Persson et al., 2009) the T6SS was more abundant among γ-proteobacteria than other protein transport systems. The weight of present evidence suggests, at least indirectly, an apparently rampant lateral transfer of T6SS clusters/genes in the microbial world, which could be a significant driver for newly emerging pathogens, as proposed for the gastroenteritis agent *E. tarda* (Leung et al., 2011).

Future studies are needed and expected to further advance the T6SS field. It is important to remember that formal evidence of the translocation of effector proteins into plant cells through T6SS is presently lacking, as is also the case for the molecular targets of these effectors. Paraphrasing Schwarz et al. (2010a), outstanding questions for future research on T6SS include the following: What are the physiological role(s) and adaptive significance of T6S-mediated plant cell targeting in disease, symbiosis, and interbacterial interactions in the environment? What is the significance of additional *vgrG* and *hcp* genes Are host- and bacterial cell-targeting T6SSs discernible by sequence or gene content? Are there T6SSs that can target both eukaryotic and prokaryotic cells? Are there other T6S substrates that await identification? Given the resemblance T6SS components to bacteriophage proteins it is also tempting to ask if T6SS transports only protein substrates and/or other macromolecules as well.

It is instructive to point out that the genes coding for several secretion systems, including T6SS, were first identified in phenotypic screens of mutants altered in their interaction with higher eukaryotes. It is conceivable that new secretion systems may be identified in other appropriately designed screens in multi-organism settings. Bioinformatic sourcing of genome, transcriptome and proteome data may point to new potential candidates, as occurred historically with the T6SS. Finally, the striking similarities between secretion systems and type IV pili, flagella, bacteriophage tail, or efflux pumps invite speculation that new systems may even be predicted, "the way Mendeleïev had anticipated characteristics of yet unknown elements" (Filloux, 2011b).

#### **10. Acknowledgments**

76 Plant Pathology

Protein secretion is fundamental to bacterial virulence and several systems mediate pathogenesis and other types of bacteria-host interaction. Beyond the other recognized secretion systems of Gram-negative bacteria with established role in host-pathogen interactions, the T6SS is of particular interest in this respect and has been shown to be important for bacterial virulence and for interaction with the host in various ways, often leading to "anti-pathogenesis" (Jani & Cotter, 2010). Nevertheless, its role and function in most bacteria is not clearly established and formal evidence for protein secretion/translocation into plant cells is scant. At present, we do not fully understand how the T6SS works and are only beginning to understand the biological role/s of the T6SS in plant-associated bacterial life. Multiple copies of T6SS in a single bacterial strain appear to be a frequent phenomenon, and this holds true for many plant associated species. Recent studies (Boyer et al., 2009; Filloux et al., 2008) have established the presence of multiple copies of apparently complete and/or degenerate T6SS loci in about one quarter of the proteobacterial genomes examined, that they generally display different phylogenetic origins and are not a result of recent duplication events, suggesting sustained and constrained mechanisms that favour this trend. Based on our own analysis (Sarris & Scoulica, 2011; Sarris et al., 2011), most strains of *Pseudomonas syringae,* the insect pathogenic *Pseudomonas entomophila* strain L48, the human opportunistic pathogen *Pseudomonas mendocina* strain ymp, and most of the *Pseudomonas* strains studied by Barret et al. (2011) typically carry T6SS from more than one phylogenetic clade and/or

Although the majority of the recent studies concern the contribution of T6SS in bacterial pathogenicity (positively), many bacteria with genomes encoding putative T6SS are not known to be pathogens or symbionts, and T6SS may also function in non-pathogenic bacteria-host interactions and/or in interactions not involving eukaryotic partners. In *R. leguminosarum* the T6SS limits host-range and in *S. typhimurium* and *H. hepaticus* the evidence suggests a possible role of T6SS in limiting of bacterial virulence and, therefore, contribution to host colonization (Bladergroen et al., 2003; Parsons & Heffron, 2005; Jani & Cotter, 2010; Chow & Mazmanian, 2010). A relatively new twist in the system's repertoire of biological functions in a broader context is the finding that bacteria engage each other in a T6SS-dependent manner and can provide protection for a bacterium against cell contactinduced growth inhibition caused by other species of bacteria (Hood et al., 2010; Schwarz et al., 2010a). This leads to speculation that this pathway is of general significance to

T6SS clusters occur with high frequency, have divergent phylogenies and individual strains or species often possess non-orthologous clusters with distinct or overlapping functions in bacterial interactions with multiple hosts, antagonists or predators unsuspected at present. In a recent study of the ocean metagenome (Persson et al., 2009) the T6SS was more abundant among γ-proteobacteria than other protein transport systems. The weight of present evidence suggests, at least indirectly, an apparently rampant lateral transfer of T6SS clusters/genes in the microbial world, which could be a significant driver for newly emerging pathogens, as proposed for the gastroenteritis agent *E. tarda* (Leung et al., 2011).

interbacterial interactions in polymicrobial diseases and the environment.

**9. Prospects** 

additional *vgrG* and *hcp* genes.

We apologize to any researchers whose work was not included in this review due to space limitations. This work was supported by PYTHAGORAS, and PENED 03ED375 grants of the Greek General Secretariat for Research and Development implemented within the framework of the "Reinforcement Programme of Human Research Manpower" (PENED) and co-financed by National and Community Funds (25% from the Greek Ministry of Development General Secretariat of Research and Technology and 75% from EU-European Social Fund) and by the EPEAEK graduate programs in Plant Molecular Biology and Biotechnology and Protein Biotechnology of the Greek Ministry of Education, Lifelong Learning and Religious Affairs.

#### **11. References**


Phytobacterial Type VI Secretion System –

2937.

1267-1282.

No 1, pp 1-9.

6, pp 1570-1583.

pp 25-37.

*Evolution*, Vol. 39, No 4, pp 783-791.

Microbiology Today, May 2011,, pp 96-101.

*hippocastanum*. *PLoS ONE*, Vol. 5, No 4, pp e10224.

*Molecular Biology Reviews*, Vol. 64, No 3, pp 624-653.

*Host & Microbe*, Vol. 8, No 1, pp 2-6.

*and Environmental Microbiology*, Vol. 72, No 3, pp 1956-1965.

L., eds, pp. 51-54, Chapman & Hall, London, United Kingdom

Gene Distribution, Phylogeny, Structure and Biological Functions 79

De Maayer, P., Chan, W. Y., Venter, S. N., Toth, I. K., Birch, P. R. J., Joubert, F. & Coutinho,

Dudley, E. G., Thomson, N. R., Parkhill, J., Morin, N. P. & Nataro, J. P. (2006). Proteomic and

Fauvart, M. & Michiels, J. (2008). Rhizobial secreted proteins as determinants of host

Felsenstein, J. (1985). Confidence-Limits on Phylogenies - an Approach Using the Bootstrap.

Filloux, A. (2011a). The bacterial type VI secretion system: on the bacteeriophage trail.

Filloux, A. (2011b). Protein secretion systems in *Pseudomonas aeruginosa*: An Essay on diversity, evolution, and function. *Frontiers in Microbiology*, Vol. 2, No, 155 pp1-21. Filloux, A., Hachani, A. & Bleves, S. (2008). The bacterial type VI secretion machine: yet

Green, S., Studholme, D. J., Laue, B. E., Dorati, F., Lovell, H., Arnold, D., Cottrell, J. E.,

Grenier, A. M., Duport, G., Pages, S., Condemine, G. & Rahbe, Y. (2006). The phytopathogen

Hayward, A. C. (1993). The host of *Xanthomonas*, In: *Xanthomonas*, Swings, J. G., Civerolo, E.

Hirano, S. S. & Upper, C. D. (2000). Bacteria in the leaf ecosystem with emphasis on

Hood, R. D., Singh, P., Hsu, F., Guvener, T., Carl, M. A., Trinidad, R. R., Silverman, J. M.,

Jani, A. J. & Cotter, P. A. (2010). Type VI Secretion: Not Just for pathogenesis anymore. *Cell* 

Janisiewicz, W. J. & Marchi, A. (1992). Control of storage rots on various pear cultivars with a saprophytic strain of *Pseudomonas syringae*. *Plant Disease*, Vol. 76, No, pp 555-560. Jobichen, C., Chakraborty, S., Li, M., Zheng, J., Joseph, L., Mok, Y. K., Leung, K. Y. &

T. A. (2010). Genome sequence of *Pantoea ananatis* LMG20103, the causative agent of Eucalyptus Blight and Dieback. *Journal of Bacteriology*, Vol. 192, No 11, pp 2936-

microarray characterization of the AggR regulon identifies a *pheU* pathogenicity island in enteroaggregative *Escherichia coli*. *Molecular Microbiology*, Vol. 61, No 5, pp

specificity in the rhizobium-legume symbiosis. *FEMS Microbiology Letters*, Vol. 285,

another player for protein transport across membranes. *Microbiology*, Vol. 154, No

Bridgett, S., Blaxter, M., Huitema, E., Thwaites, R., Sharp, P. M., Jackson, R. W. & Kamoun, S. (2010). Comparative genome analysis provides insights into the evolution and adaptation of *Pseudomonas syringae* pv. *aesculi* on *Aesculus* 

*Dickeya dadantii* (*Erwinia chrysanthemi* 3937) is a pathogen of the pea aphid. *Applied* 

*Pseudomonas syringae*-a pathogen, ice nucleus, and epiphyte. *Microbiology and* 

Ohlson, B. B., Hicks, K. G., Plemel, R. L., Li, M., Schwarz, S., Wang, W. Y., Merz, A. J., Goodlett, D. R. & Mougous, J. D. (2010). A type VI secretion system of *Pseudomonas aeruginosa* targets a toxin to bacteria. *Cell Host & Microbe*, Vol. 7, No 1,

Sivaraman, J. (2010). Structural basis for the secretion of EvpC: a key type VI secretion system protein from *Edwardsiella tarda*. *PLoS ONE*, Vol. 5, No 9, pp e12910.

(2008). Genome sequence of the beta-rhizobium *Cupriavidus taiwanensis* and comparative genomics of rhizobia. *Genome Research*, Vol. 18, No 9, pp 1472-1483.


Ballister, E. R., Lai, A. H., Zuckermann, R. N., Cheng, Y. & Mougous, J. D. (2008). In vitro

*Proceedings of the National Academy of Sciences*, Vol. 105, No 10, pp 3733-3738. Barret, M., Egan, F., Fargier E., Morrissey, J. P. & O'Gara, F. (2011). Genomic analysis of the

Bernard, C. S., Brunet, Y. R., Gueguen, E. & Cascales, E. (2010). Nooks and Crannies in type VI secretion regulation. *Journal of Bacteriology*, Vol. 192, No 15, pp 3850-3860. Bernard, C. S., Brunet, Y. R., Gavioli, M., Lloubes, R. & Cascales, E. (2011). Regulation of

Bingle, L. E., Bailey, C. M. & Pallen, M. J. (2008). Type VI secretion: a beginner's guide.

Bladergroen, M. R., Badelt, K. & Spaink, H. P. (2003). Infection-blocking genes of a symbiotic

Bonemann, G., Pietrosiuk, A. & Mogk, A. (2010). Tubules and donuts: a type VI secretion

Boyer, F., Fichant, G., Berthod, J., Vandenbrouck, Y. & Attree, I. (2009). Dissecting the

Brunet, Y. R., Bernard, C. S., Gavioli, M., Lloubes, R. & Cascales, E. (2011). An epigenetic

Chugani, S. & Greenberg, E. P. (2007). The influence of human respiratory epithelia on

Coutinho, T. A. & Venter, S. N. (2009). *Pantoea ananatis*: an unconventional plant pathogen.

De Bruin, O., Ludu, J. & Nano, F. (2007). The *Francisella* pathogenicity island protein IglA

Type VI secretion gene custer. *PLoS Genetics*, Vol. 7, No 7, pp e1002205. Cascales, E. (2008). The type VI secretion toolkit. *EMBO Reports*, Vol. 9, No 8, pp 735-741. Chow, J. & Mazmanian, S. K. (2010). A pathobiont of the microbiota balances host

secretion. *Molecular Plant-Microbe Interactions*, Vol. 16, No 1, pp 53-64. Bonemann, G., Pietrosiuk, A., Diemand, A., Zentgraf, H. & Mogk, A. (2009). Remodelling of

*Escherichia coli*. *Journal of Bacteriology*, Vol. 190, No 22, pp 7523-7531.

effectors uncovered. Microbiology, vpl.157, No 6, p.1726-1740.

*Journal of Bacteriology*, Vol. 193, No 9, pp 2158-2167.

secretion. *EMBO Journal*, Vol. 28, No 4, pp 315-325.

*Molecular Plant Pathoogyl*, Vol. 10, No 3, pp 325-335.

*Microbiology*, Vol. 7, No 1, pp 1.

pp 104.

276.

35.

story. *Molecular Microbiology*, Vol. 76, No 4, pp 815-821.

*Current Opinions in Microbiology*, Vol. 11, No 1, pp 3-8.

(2008). Genome sequence of the beta-rhizobium *Cupriavidus taiwanensis* and comparative genomics of rhizobia. *Genome Research*, Vol. 18, No 9, pp 1472-1483. Aschtgen, M.-S., Bernard, C. S., De Bentzmann, S., Lloubes, R. & Cascales, E. (2008). SciN is

an outer membrane lipoprotein required for Type VI secretion in enteroaggregative

self-assembly of tailorable nanotubes from a simple protein building block.

type VI secretion systems in Pseudomonas spp: novel 3 clusters and putative

type VI secretion gene clusters by σ54 and cognate enhancer binding proteins.

*Rhizobium leguminosarum* strain that are involved in temperature-dependent protein

VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein

bacterial type VI secretion system by a genome wide *in silico* analysis: what can be learned from available microbial genomic resources? *BMC Genomics*, Vol. 10, No,

switch involving overlapping fur and DNA methylation optimizes expression of a

colonization and intestinal inflammation. *Cell Host & Microbe*, Vol. 7, No 4, pp 265-

*Pseudomonas aeruginosa* gene expression. *Microbial Pathogenesis*, Vol. 42, No 1, pp 29-

localizes to the bacterial cytoplasm and is needed for intracellular growth. *BMC* 


Phytobacterial Type VI Secretion System –

324.

2962.

Gene Distribution, Phylogeny, Structure and Biological Functions 81

Mougous, J. D., Gifford, C. A., Ramsdell, T. L. & Mekalanos, J. J. (2007). Threonine

Nino-Liu, D. O., Ronald, P. C. & Bogdanove, A. J. (2006). *Xanthomonas oryzae* pathovars:

Pace, F., Boldrin, J., Nakazato, G., Lancellotti, M., Sircili, M., Guedes, E., Silveira, W. &

Pallen, M. J., Chaudhuri, R. R. & Henderson, I. R. (2003). Genomic analysis of secretion

Parsons, D. A. & Heffron, F. (2005). sciS, an icmF homolog in *Salmonella enterica* serovar

Pell, L. G., Kanelis, V., Donaldson, L. W., Howell, P. L. & Davidson, A. R. (2009). The phage

Persson, O. P., Pinhassi, J., Riemann, L., Marklund, B. I., Rhen, M., Normark, S., Gonzalez, J.

Pukatzki, S., Ma, A. T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W. C.,

Pukatzki, S., Ma, A. T., Revel, A. T., Sturtevant, D. & Mekalanos, J. J. (2007). Type VI

Purcell, M. & Shuman, H. A. (1998). The *Legionella pneumophila* icmGCDJBF genes are

Records, A. R. & Gross, D. C. (2010). Sensor kinases RetS and LadS regulate *Pseudomonas* 

Records, A. R. (2011). The type VI secretion system: a multipurpose delivery system with a

marine bacteria. *Environmental Microbiology*, Vol. 11, No 6, pp 1348-1357. Potnis, N., Krasileva, Chow, K. V., Almeida, N. F., Patil, P., Ryan, R., Sharlach, M., Behlau,

systems. *Current Opinions in Microbiology*, Vol. 6, No 5, pp 519-527.

*aeruginosa*. *Nature Cell Biology*, Vol. 9, No 7, pp 797-803.

*Academy of Sciences USA*, Vol. 106, No 11, pp 4160-4165.

*Immunity*, Vol. 73, No 7, pp 4338-4345.

12, No 146, pp 2-23.

39, pp 15508-15513.

pp 2245-2255.

14, pp 3584-3596.

757.

1528-1533.

phosphorylation post-translationally regulates protein secretion in *Pseudomonas* 

model pathogens of a model crop. *Molecular Plant Pathology*, Vol. 7, No 5, pp 303-

Sperandio, V. (2011). Characterization of IcmF of the type VI secretion system in an avian pathogenic *Escherichia coli* (APEC) strain. *Microbiology*, vol 157, Pt 10, pp 2954-

*typhimurium*, limits intracellular replication and decreases virulence. *Infection and* 

lambda major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system. *Proceedings of the National* 

M. & Hagstrom, A. (2009). High abundance of virulence gene homologues in

F., Dow, J. M., White, F., Preston, J., Vinatzer, B., Koebnik, R., Setubal, J. C., Norman, D. J., Staskawicz B. & Jones J. B. (2011). Comparative genomics reveals diversity among xanthomonads infecting tomato and pepper. *BMC Genomics*, Vol.

Heidelberg, J. F. & Mekalanos, J. J. (2006). Identification of a conserved bacterial protein secretion system in *Vibrio cholerae* using the *Dictyostelium* host model system. *Proceedings of the National Academy of Sciences USA*, Vol. 103, No 5, pp

secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. *Proceedings of the National Academy of Sciences USA*, Vol. 104, No

required for killing of human macrophages. *Infection and Immunity*, Vol. 66, No 5,

*syringae* type VI secretion and virulence factors. *Journal of Bacteriology*, Vol. 192, No

phage-like machinery. *Molecular Plant-Microbe Interactions*, Vol. 24, No 7, pp 751-


Kim, W. S., Jock, S., Paulin, J.-P., Rhim, S.-L. & Geider, K. (2001). Molecular detection and

Krehenbrink, M. & Downie, J. A. (2008). Identification of protein secretion systems and

Kwon, S. W., Go, S. J., Kang, H. W., Ryu, J. C. & Jo, J. K. (1997). Phylogenetic analysis of

Leiman, P. G., Basler, M., Ramagopal, U. A., Bonanno, J. B., Sauder, J. M., Pukatzki, S.,

Leyns, F., De Cleene, M., Swings, J. & De Ley, J. (1984). The host range of the genus

Liu, H., Coulthurst, S. J., Pritchard, L., Hedley, P. E., Ravensdale, M., Humphris, S., Burr, T.,

Ma, A. T., Mcauley, S., Pukatzki, S. & Mekalanos, J. J. (2009). Translocation of a *Vibrio* 

Macintyre, D. L., Miyata, S. T., Kitaoka, M. & Pukatzki, S. (2010). The *Vibrio cholerae* type VI

Mattinen, L., Nissinen, R., Riipi, T., Kalkkinen, N. & Pirhonen, M. (2007). Host-extract

Miyata, S. T., Kitaoka, M., Wieteska, L., Frech, C., Chen, N. & Pukatzki, S. (2010). The *Vibrio* 

Miyata, S. T., Kitaoka, M., Brooks, T. M., Mcauley, S. B. & Pukatzki, S. (2011). *Vibrio cholerae*

Mougous, J. D., Cuff, M. E., Raunser, S., Shen, A., Zhou, M., Gifford, C. A., Goodman, A. L.,

*discoideum*. *Infection and Immunity*, Vol. 79, No 7, pp 2941-2949.

*Academy of Sciences USA*, Vol. 107, No 45, pp 19520-19524.

*atrosepticum*. *Proteomics*, Vol. 7, No 19, pp 3527-3537.

apparatus. *Science*, Vol. 312, No 5779, pp 1526-1530.

*Frontiers in Microbiology*, Vol. 1, No 117, pp 1-7.

pathogen. *Plant Disease*, Vol. 85, No 11, pp 1183-1188.

*Systematic Bacteriology*, Vol. 47, No 4, pp 1061-1067.

*Infection*, Vol. *in press*, doi:10.1016/j.micinf.2011.08.005.

*Host & Microbe*, Vol. 5, No 3, pp 234-243.

*Xanthomonas*. *The Botanical Review*, Vol. 50, No 3, pp 308-356.

9, No 9, pp 55.

*databases*.

differentiation of *Erwinia pyrifoliae* and host range analysis of the asian pear

novel secreted proteins in *Rhizobium leguminosarum* bv. *viciae*. *BMC Genomics*, Vol.

*Erwinia* species based on 16S rRNA gene sequences. *International Journal of* 

Burley, S. K., Almo, S. C. & Mekalanos, J. J. (2009). Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. *Proceedings of the National Academy of Sciences USA*, Vol. 106, No 11, pp 4154-4159. Leung, K. Y., Siame, B. A., Tenkink, B. J., Noort, R. J. & Mok, Y. K. (2011). *Edwardsiella tarda* -

Virulence mechanisms of an emerging gastroenteritis pathogen. *Microbes and* 

Takle, G., Brurberg, M. B., Birch, P. R., Salmond, G. P. & Toth, I. K. (2008). Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen *Pectobacterium atrosepticum*. *PLoS Pathogens*, Vol. 4, No 6, pp e1000093. Lucas, S., Copeland, A., Lapidus, A., Glavina Del Rio, T., Tice, H., Bruce, D., Goodwin, L.,

Pitluck, S., Chertkov, O., Brettin, T., Detter, J. C., Han, C., Larimer, F., Land, M., Hauser, L., Kyrpides, N., Ovchinnicova, G., Balakrishnan, V., Glasner, J. & Perna, N. T. (2009). Complete sequence of *Dickeya zeae* Ech1591, In: *EMBL/GenBank/DDBJ* 

*cholerae* type VI secretion effector requires bacterial endocytosis by host cells. *Cell* 

secretion system displays antimicrobial properties. *Proceedings of the National* 

induced changes in the secretome of the plant pathogenic bacterium *Pectobacterium* 

*cholerae* Type VI Secretion System: Evaluating its role in the human disease cholera.

requires the type VI secretion system virulence factor VasX to kill *Dictyostelium* 

Joachimiak, G., Ordonez, C. L., Lory, S., Walz, T., Joachimiak, A. & Mekalanos, J. J. (2006). A virulence locus of *Pseudomonas aeruginosa* encodes a protein secretion


Phytobacterial Type VI Secretion System –

No 8, pp e2955.

23, No 4, pp 384-393.

*Disease*, Vol. 67, No 5, pp 581-585.

*Pathogenesis*, Vol. 44, No 4, pp 344-361.

*Interactions*, Vol. 9, No 2, pp 139-143.

Vol. 24, No 8, pp 1596-1599.

Suppl 1, No, pp S2.

pp 494-507.

cells. *Microbiology*, Vol. 156, No 12, pp 3678-3688.

*Molecular Plant Pathology*, Vol. 32, No 2, pp 237-247.

Gene Distribution, Phylogeny, Structure and Biological Functions 83

Shrivastava, S. & Mande, S. S. (2008). Identification and functional characterization of gene

Smits, T. H., Rezzonico, F., Kamber, T., Blom, J., Goesmann, A., Frey, J. E. & Duffy, B. (2010).

Stall, R. E. & Seymour, C. P. (1983). Canker, a threat to citrus in the gulf-coast states. *Plant* 

Suarez, G., Sierra, J. C., Sha, J., Wang, S., Erova, T. E., Fadl, A. A., Foltz, S. M., Horneman, A.

Suarez, G., Sierra, J. C., Kirtley, M. L. & Chopra, A. K. (2010). Role of Hcp, a type 6 secretion

Szegedi, E., Czako, M., Otten, L. & Koncz, C. S. (1988). Opines in crown gall Tumors

Szegedi, E., Czako, M. & Otten, L. (1996). Further evidence that the vitopine-type pTi's of

Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evolutionary

Tseng, T. T., Tyler, B. M. & Setubal, J. C. (2009). Protein secretion systems in bacterial-host

Wang, Y.-Y. (2008). Characterization of type six secretion systems in *Pseudomonas syringae* 

Wu, H. Y., Chung, P. C., Shih, H. W., Wen, S. R. & Lai, E. M. (2008). Secretome analysis

*Applied and Environmental Microbiology*, Vol. 74, No 10, pp 3171-3181. Yuan, Z. C., Liu, P., Saenkham, P., Kerr, K. & Nester, E. W. (2008). Transcriptome profiling

pv. *tomato* DC3000: National University of Taiwan, MSc thesis.

components of Type VI Secretion system in bacterial genomes. *PLoS ONE*, Vol. 3,

Complete genome sequence of the fire blight pathogen *Erwinia amylovora* CFBP 1430 and comparison to other *Erwinia* spp. *Molecular Plant-Microbe Interactions*, Vol.

J. & Chopra, A. K. (2008). Molecular characterization of a functional type VI secretion system from a clinical isolate of *Aeromonas hydrophila*. *Microbial* 

system effector, of *Aeromonas hydrophila* in modulating activation of host immune

induced by biotype 3 isolates of *Agrobacterium tumefaciens*. *Physiological and* 

*Agrobacterium vitis* represent a novel group of Ti plasmids. *Molecular Plant-Microbe* 

Genetics Analysis (MEGA) software version 4.0. *Molecular Biology and Evolution*,

associations, and their description in the Gene Ontology. *BMC Microbiology*, Vol. 9

uncovers an Hcp-family protein secreted via a type VI secretion system in *Agrobacterium tumefaciens*. *Journal of Bacteriology*, Vol. 190, No 8, pp 2841-2850. Yan, S., Liu, H., Mohr, T. J., Jenrette, J., Chiodini, R., Zaccardelli, M., Setubal, J. C. &

Vinatzer, B. A. (2008). Role of recombination in the evolution of the model plant pathogen *Pseudomonas syringae* pv. *tomato* DC3000, a very atypical tomato strain.

and functional analysis of *Agrobacterium tumefaciens* reveals a general conserved response to acidic conditions (pH 5.5) and a complex acid-mediated signaling involved in *Agrobacterium*-plant interactions. *Journal of Bacteriology*, Vol. 190, No 2,

Zheng, J. & Leung, K. Y. (2007). Dissection of a type VI secretion system in *Edwardsiella tarda*.

*Molecular Microbiology*, Vol. 66, No 5, pp 1192-1206.


Rodriguez-Palenzuela, P., Matas, I. M., Murillo, J., Lopez-Solanilla, E., Bardaji, L., Perez-

Roest, H. P., Mulders, I. H. M., Spaink, H. P., Wijffelman, C. A. & Lugtenberg, B. J. J. (1997).

Russell, A. B., Hood, R. D., Bui, N. K., Leroux, M., Vollmer, W. & Mougous, J. D. (2011).

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. *Molecular Biology and Evolution*, Vol. 4, No 4, pp 406-425. Sarris, P. F., Skandalis, N., Kokkinidis, M. & Panopoulos, N. J. (2010). *In silico* analysis

Sarris, P. F. & Scoulica, E. V. (2011). *Pseudomonas entomophila* and *Pseudomonas mendocina*:

Sarris, P. F., Zoumadakis, C., Panopoulos, N. J. & Scoulica, E. (2011). Distribution of the

Schlieker, C., Zentgraf, H., Dersch, P. & Mogk, A. (2005). ClpV, a unique Hsp100/Clp

Schwarz, S., Hood, R. D. & Mougous, J. D. (2010a). What is type VI secretion doing in all

Schwarz, S., West, T. E., Boyer, F., Chiang, W.-C., Carl, M. A., Hood, R. D., Rohmer, L.,

*PLoS Pathogen,* Vol. 6, No 8, e1001068. doi:10.1371/journal.ppat.1001068. Sexton, J. A., Miller, J. L., Yoneda, A., Kehl-Fie, T. E. & Vogel, J. P. (2004). *Legionella* 

Shalom, G., Shaw, J. G. & Thomas, M. S. (2007). In vivo expression technology identifies a

invasion of macrophages. *Microbiology*, Vol. 153, No Pt 8, pp 2689-2699. Sheahan, K.-L., Cordero, C. L. & Fullner Satchell, K. J. (2004). Identification of a domain

those bugs? *Trends in Microbiology*, Vol. 18, No 12, pp 531-537.

*Plant-Microbe Interactions*, Vol. 10, No 7, pp 938-941.

*Genetics and Evolution*, Vol. 11, No 6, pp 1352-1360.

*Evolution*, Vol. 11, No 1, pp 157–166.

*Infect Immun*, Vol. 72, No 10, pp 5983-5992.

6, pp 1604-1620.

7356, pp 343-347.

1127.

9803.

Martinez, I., Rodriguez-Moskera, M. E., Penyalver, R., Lopez, M. M., Quesada, J. M., Biehl, B. S., Perna, N. T., Glasner, J. D., Cabot, E. L., Neeno-Eckwall, E. & Ramos, C. (2010). Annotation and overview of the *Pseudomonas savastanoi* pv. *savastanoi* NCPPB 3335 draft genome reveals the virulence gene complement of a tumour-inducing pathogen of woody hosts. *Environmental Microbiology*, Vol. 12, No

A *Rhizobium leguminosarum* Biovar *trifolii* locus not localized on the Sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group. *Molecular* 

Type VI secretion delivers bacteriolytic effectors to target cells. *Nature*, Vol. 475, No

reveals multiple putative type VI secretion systems and effector proteins in *Pseudomonas syringae* pathovars. *Molecular Plant Pathology*, Vol. 11, No 6, pp 795-804.

Potential models for studying the bacterial type VI secretion system. *Infection,* 

putative type VI secretion system core genes in *Klebsiella* spp. *Infection Genetics and* 

member of pathogenic proteobacteria. *Biological Chemistry*, Vol. 386, No 11, pp 1115-

Tolker-Nielsen, T., Skerrett, S. J. & Mougous, J. D. (2010b). *Burkholderia* Type VI Secretion Systems have distinct roles in eukaryotic and bacterial cell interactions.

*pneumophila* DotU and IcmF are required for stability of the Dot/Icm complex.

type VI secretion system locus in *Burkholderia pseudomallei* that is induced upon

within the multifunctional *Vibrio cholerae* RTX toxin that covalently cross-links actin. *Proceedings of the National Academy of Sciences USA*, Vol. 101, No 26, pp 9798-


**1. Introduction** 

increasing product rejections*.* 

*B. cinerea*, but to a lesser extend.

**4** 

*Greece* 

**Novel Elicitors Induce Defense** 

*Department of Greenhouse Cultivation and Floriculture,* 

Cut flower production and trade in the E.U. and the rest of the world holds the main share within the ornamental horticulture industry. Despite the global economic crisis started in 2008, changes in cut flower trade, such as the merge of the 2 major auctions in Holland (i.e. VBA and FloraHolland), resulted in stabilization or even small increases in stem number sales for the years 2008-2010 (Evans & Van der Ploeg, 2008; Anonymous, 2011). In other words, the importance of cut flower industry in global economy is undisputed, but also

Product quality of horticultural crops has been the main area of research the past decades. Growers and sellers have been seeking for best possible product quality and highest possible profits. However, problems in quality after pathogen infections at some point of production, or during storage or transportation eventually result in economic losses (van Meeteren, 2009). *Botrytis cinerea* is the single-most important pathogen infecting ornamental plants and cut flowers postharvest and substantially reduce growers' and sellers' income by

*B. cinerea* Pers. is a common fungal pathogen that infects glasshouse-grown ornamental crops under cool and humid conditions with latent symptoms, which develop during storage or transportation (Elad, 1988). Growers and sellers around the world are deeply concerned by such infection problems. In Europe, for instance, large quantities of *B. cinerea*infected cut freesias from Τhe Netherlands are rejected in the UK by wholesalers and retailers at certain times of the year (Darras et al., 2004). These rejections result in immediate economic losses and make cooperation between growers and importers problematical. The problem is equally substantial for roses (Elad, 1988; Elad et al., 1993), gerberas (Salinas & Verhoeff, 1995) and Geraldton waxflowers (Joyce, 1993), although species such as chrysanthemum (Dirkse, 1982), narcissus (O'Neill et al., 2004), lisianthus (Wegulo & Vilchez, 2007), dianthus, ranunculus and cyclamen (Seglie et al., 2009) eventually suffer infections by

Infections by *B. cinerea* are usually managed by conventional fungicides applied protectively at certain times of the year and especially during autumn and spring when most infections occur. However, extensive use of fungicides such as dicarboximides, has led to the

reflects the human need for ornamental plant consumption as part of a better life.

**Responses in Cut Flowers** 

*Technological Educational Institute of Kalamata* 

Anastasios I. Darras


### **Novel Elicitors Induce Defense Responses in Cut Flowers**

Anastasios I. Darras

*Department of Greenhouse Cultivation and Floriculture, Technological Educational Institute of Kalamata Greece* 

#### **1. Introduction**

84 Plant Pathology

Zheng, J., Ho, B. & Mekalanos, J. J. (2011). Genetic analysis of anti-amoebae and anti-

Zuckerkandl, E. & Pauling, L. (1965). Evolutionary divergence and convergence in proteins,

6, No 8, pp e23876.

Press, New York.

bacterial activities of the Type VI secretion system in *Vibrio cholerae*. *PLoS ONE*, Vol.

In: *Evolving Genes and Proteins*, Bryson, V., Vogel, H. J., eds, pp. 97-166, Academic

Cut flower production and trade in the E.U. and the rest of the world holds the main share within the ornamental horticulture industry. Despite the global economic crisis started in 2008, changes in cut flower trade, such as the merge of the 2 major auctions in Holland (i.e. VBA and FloraHolland), resulted in stabilization or even small increases in stem number sales for the years 2008-2010 (Evans & Van der Ploeg, 2008; Anonymous, 2011). In other words, the importance of cut flower industry in global economy is undisputed, but also reflects the human need for ornamental plant consumption as part of a better life.

Product quality of horticultural crops has been the main area of research the past decades. Growers and sellers have been seeking for best possible product quality and highest possible profits. However, problems in quality after pathogen infections at some point of production, or during storage or transportation eventually result in economic losses (van Meeteren, 2009). *Botrytis cinerea* is the single-most important pathogen infecting ornamental plants and cut flowers postharvest and substantially reduce growers' and sellers' income by increasing product rejections*.* 

*B. cinerea* Pers. is a common fungal pathogen that infects glasshouse-grown ornamental crops under cool and humid conditions with latent symptoms, which develop during storage or transportation (Elad, 1988). Growers and sellers around the world are deeply concerned by such infection problems. In Europe, for instance, large quantities of *B. cinerea*infected cut freesias from Τhe Netherlands are rejected in the UK by wholesalers and retailers at certain times of the year (Darras et al., 2004). These rejections result in immediate economic losses and make cooperation between growers and importers problematical. The problem is equally substantial for roses (Elad, 1988; Elad et al., 1993), gerberas (Salinas & Verhoeff, 1995) and Geraldton waxflowers (Joyce, 1993), although species such as chrysanthemum (Dirkse, 1982), narcissus (O'Neill et al., 2004), lisianthus (Wegulo & Vilchez, 2007), dianthus, ranunculus and cyclamen (Seglie et al., 2009) eventually suffer infections by *B. cinerea*, but to a lesser extend.

Infections by *B. cinerea* are usually managed by conventional fungicides applied protectively at certain times of the year and especially during autumn and spring when most infections occur. However, extensive use of fungicides such as dicarboximides, has led to the

Novel Elicitors Induce Defense Responses in Cut Flowers 87

In regards to pot plants, *B. cinerea* disease symptoms on geranium (*Pelargonium zonale*) flowers has been described by Strider (1985) as flower blight, leaf blight and stem rot. Martinez et al. (2008) published a detailed report on infection of *Pelargonium x hortorum, Euphorbia pulcherrima, Lantana camara, Lonicera japonica, Hydrangea macrophylla, and Cyclamen persicum* by *B. cinerea.* They reported that growth of *B. cinerea* isolates in-vitro from the above mentioned ornamentals varied significantly. *B. cinerea* showed a high degree of phenotypical variability among the isolates, not only as regards to visual aspects of the colonies but also to some morphological structures such as conidium length, conidiophores, sclerotia production, and hyphae (Martinez et al., 2008). Increased susceptibility to grey mold from 10% to 80% in stems and from 3% to 14% in leaves was observed after using elevated levels of N supply (i.e. from 7.15 to 57.1 mM) for begonia plant (*Begonia x* 

Host-pathogen specificity involves factors that determine the virulence of the pathogen and also factors that confer resistance on the host (Lucas, 1997). Many theories have been proposed concerning mechanisms by which pathogens either achieve or fail to infect host tissue. A model concerning specific gene-for-gene interactions determining the host range of pathogens in wild plant species was first proposed by Flor (1971). In a gene-for-gene system, recognition of the pathogen by the host occurs when a resistance (R) gene of the host

**Pathogen genes Resistant or susceptible genes in the plant** 

a (-) indicate incompatible interaction and, therefore, no infection. (+) indicate compatible interaction

Table 1. Quadratic check of gene combinations and disease reaction types in host-pathogen

According to this model, avr gene products secreted by hyphae or located on the surface of the pathogen bind to a receptor located on the cell membranes of host's epidermal cells. Binding triggers a cascade of defence responses by the host. Every other possible match in the system could lead to infection (Table 1). Thus, a combination of a resistant host gene and a virulent pathogen leads to a compatible host-pathogen interaction. In both cases, when an avr race of the pathogen matches with a susceptible host and a virulent pathogen matches with a susceptible host, the host fails to recognize the pathogen and infection

Culture filtrates or extracts from microbial cells can act as potent inducers of plant defence responses (Chappell & Hahlbrock, 1984; Kombrink & Hahlbrock, 1986; Fritzemeier et al.,

systems in which the gene-for-gene concept for one gene operates (Lucas, 1997).

A (avirulent) dominant AR (-)a Ar (+) a (virulent) recessive aR (+) ar (+)

R (resistant) dominant

r (susceptible) recessive

*tuberhybrida* Voss) cultivation (Pitchay et al., 2007).

**Virulent or avirulent** 

and, therefore, infection.

occurs (Flor, 1971).

**1.2 Review on host-pathogen interactions and on defence responses** 

interacts with an avirulence (avr) gene of the pathogen (Table 1).

appearance of resistant *B. cinerea* strains (Pappas, 1997). Alternative methods to control *B. cinerea* disease (i.e. grey mold) within the concept of integrated disease management (IDM) programs are sought by growers and help overcome resistance by the pathogen.

Elicitor-based disease management constitutes an attractive socio-environmentally sound strategy (Joyce & Johnson, 1999). Known activators of plant defence reactions, such as 2,6 dichloroisoniciotinic acid (INA), salicylic acid (SA), 3-aminobutyric acid (BABA), Acibenzolar-S-methyl [ASM; benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester; benzothiadiazole or BTH; CGA 245704] and methyl jasmonate (MeJA), have been shown to enhance natural defence mechanisms or induce systemic defence responses such as SAR or ISR in plants, thereby providing prospects for IDM (Terry & Joyce, 2004a).

#### **1.1** *Botrytis cinerea* **infecting cut flowers and ornamental pot plants**

*Botrytis cinerea* Pers. belongs to the Class Deuteromycetes and the Phylum Ascomycota. The disease caused by *B. cinerea* is called grey mold. The fungus is pathogenic to most of the cultivated ornamental pot plants and cut flowers. For example, infection of gerbera (*Gerbera jamesonii*) flowers occurs inside the glasshouse during crop cultivation, but symptoms develop after a latent period at storage or transportation following fluctuations in temperature (Salinas & Verhoeff, 1995). Favourable temperature and relative humidity (RH) for the pathogen after harvest results in rapid disease development (Salinas et al., 1989). Grey mold on gerbera and freesia flowers is observed as small necrotic, dark-brown fleck lesions 'spots'. Similar symptoms developed in the laboratory under controlled conditions following artificial inoculation of gerbera or freesia inflorescences at temperatures ranging from 4 to 25°C (Salinas & Verhoeff, 1995; Darras et al., 2006a). Infection of freesia (*Freesia hybrida*) inflorescences after artificial inoculation occurred in less than 24 h at 12°C and 80- 90% RH. Even at the low temperature of 5°C, disease symptoms were evident in a saturated atmosphere (ca. 100% RH) within the first 24 h of incubation.

*B. cinerea* is also pathogenic to Geraldton waxflower (*Chamelaucium uncinatum*), the Australian native plant which holds a high ornamental and commercial value (Joyce, 1993; Tomas et al., 1995). Geraldton waxflower sprigs artificially inoculated with *B. cinerea* showed increased abscission of flowers from their pedicels.

*B. cinerea* infects rose (*Rosa hybrida*) flowers and produces necrotic spots or blister-like patches on petal surfaces (Pie & De Leeuw, 1991; Williamson et al., 1995). Infection has been described by Elad (1988) as restricted, brown, volcano-like shaped lesions. *B. cinerea* damages phylloclades of ruscus (*Ruscus aculeatus*) by causing small, dark water soaked necrotic lesions encircled by a faint halo. These lesions later become brown without growing in size (Elad et al., 1993).

Infection of lisianthus (*Eustoma russellianum*) flowers has been recently reported by Wegulo & Vilchez (2007). Significant (*P* ≤ 0.03) positive correlations between stem lesion length of naturally infected plants in the glasshouse (*R* = 0.74) and stem lesion length of artificially inoculated ones (*R* = 0.62) with the disease incidence score, and with the percent of necrosis (*R* = 0.71) of detached leaves were reported (Wegulo & Vilchez, 2007). From all the 12 lisianthus cultivars tested, 'Magic Champagne' was suggested as the most resistant and proposed as ideal for commercial cultivation.

appearance of resistant *B. cinerea* strains (Pappas, 1997). Alternative methods to control *B. cinerea* disease (i.e. grey mold) within the concept of integrated disease management (IDM)

Elicitor-based disease management constitutes an attractive socio-environmentally sound strategy (Joyce & Johnson, 1999). Known activators of plant defence reactions, such as 2,6 dichloroisoniciotinic acid (INA), salicylic acid (SA), 3-aminobutyric acid (BABA), Acibenzolar-S-methyl [ASM; benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester; benzothiadiazole or BTH; CGA 245704] and methyl jasmonate (MeJA), have been shown to enhance natural defence mechanisms or induce systemic defence responses such as SAR or

*Botrytis cinerea* Pers. belongs to the Class Deuteromycetes and the Phylum Ascomycota. The disease caused by *B. cinerea* is called grey mold. The fungus is pathogenic to most of the cultivated ornamental pot plants and cut flowers. For example, infection of gerbera (*Gerbera jamesonii*) flowers occurs inside the glasshouse during crop cultivation, but symptoms develop after a latent period at storage or transportation following fluctuations in temperature (Salinas & Verhoeff, 1995). Favourable temperature and relative humidity (RH) for the pathogen after harvest results in rapid disease development (Salinas et al., 1989). Grey mold on gerbera and freesia flowers is observed as small necrotic, dark-brown fleck lesions 'spots'. Similar symptoms developed in the laboratory under controlled conditions following artificial inoculation of gerbera or freesia inflorescences at temperatures ranging from 4 to 25°C (Salinas & Verhoeff, 1995; Darras et al., 2006a). Infection of freesia (*Freesia hybrida*) inflorescences after artificial inoculation occurred in less than 24 h at 12°C and 80- 90% RH. Even at the low temperature of 5°C, disease symptoms were evident in a saturated

*B. cinerea* is also pathogenic to Geraldton waxflower (*Chamelaucium uncinatum*), the Australian native plant which holds a high ornamental and commercial value (Joyce, 1993; Tomas et al., 1995). Geraldton waxflower sprigs artificially inoculated with *B. cinerea*

*B. cinerea* infects rose (*Rosa hybrida*) flowers and produces necrotic spots or blister-like patches on petal surfaces (Pie & De Leeuw, 1991; Williamson et al., 1995). Infection has been described by Elad (1988) as restricted, brown, volcano-like shaped lesions. *B. cinerea* damages phylloclades of ruscus (*Ruscus aculeatus*) by causing small, dark water soaked necrotic lesions encircled by a faint halo. These lesions later become brown without growing

Infection of lisianthus (*Eustoma russellianum*) flowers has been recently reported by Wegulo & Vilchez (2007). Significant (*P* ≤ 0.03) positive correlations between stem lesion length of naturally infected plants in the glasshouse (*R* = 0.74) and stem lesion length of artificially inoculated ones (*R* = 0.62) with the disease incidence score, and with the percent of necrosis (*R* = 0.71) of detached leaves were reported (Wegulo & Vilchez, 2007). From all the 12 lisianthus cultivars tested, 'Magic Champagne' was suggested as the most resistant and

programs are sought by growers and help overcome resistance by the pathogen.

ISR in plants, thereby providing prospects for IDM (Terry & Joyce, 2004a).

**1.1** *Botrytis cinerea* **infecting cut flowers and ornamental pot plants** 

atmosphere (ca. 100% RH) within the first 24 h of incubation.

showed increased abscission of flowers from their pedicels.

in size (Elad et al., 1993).

proposed as ideal for commercial cultivation.

In regards to pot plants, *B. cinerea* disease symptoms on geranium (*Pelargonium zonale*) flowers has been described by Strider (1985) as flower blight, leaf blight and stem rot. Martinez et al. (2008) published a detailed report on infection of *Pelargonium x hortorum, Euphorbia pulcherrima, Lantana camara, Lonicera japonica, Hydrangea macrophylla, and Cyclamen persicum* by *B. cinerea.* They reported that growth of *B. cinerea* isolates in-vitro from the above mentioned ornamentals varied significantly. *B. cinerea* showed a high degree of phenotypical variability among the isolates, not only as regards to visual aspects of the colonies but also to some morphological structures such as conidium length, conidiophores, sclerotia production, and hyphae (Martinez et al., 2008). Increased susceptibility to grey mold from 10% to 80% in stems and from 3% to 14% in leaves was observed after using elevated levels of N supply (i.e. from 7.15 to 57.1 mM) for begonia plant (*Begonia x tuberhybrida* Voss) cultivation (Pitchay et al., 2007).

#### **1.2 Review on host-pathogen interactions and on defence responses**

Host-pathogen specificity involves factors that determine the virulence of the pathogen and also factors that confer resistance on the host (Lucas, 1997). Many theories have been proposed concerning mechanisms by which pathogens either achieve or fail to infect host tissue. A model concerning specific gene-for-gene interactions determining the host range of pathogens in wild plant species was first proposed by Flor (1971). In a gene-for-gene system, recognition of the pathogen by the host occurs when a resistance (R) gene of the host interacts with an avirulence (avr) gene of the pathogen (Table 1).


a (-) indicate incompatible interaction and, therefore, no infection. (+) indicate compatible interaction and, therefore, infection.

Table 1. Quadratic check of gene combinations and disease reaction types in host-pathogen systems in which the gene-for-gene concept for one gene operates (Lucas, 1997).

According to this model, avr gene products secreted by hyphae or located on the surface of the pathogen bind to a receptor located on the cell membranes of host's epidermal cells. Binding triggers a cascade of defence responses by the host. Every other possible match in the system could lead to infection (Table 1). Thus, a combination of a resistant host gene and a virulent pathogen leads to a compatible host-pathogen interaction. In both cases, when an avr race of the pathogen matches with a susceptible host and a virulent pathogen matches with a susceptible host, the host fails to recognize the pathogen and infection occurs (Flor, 1971).

Culture filtrates or extracts from microbial cells can act as potent inducers of plant defence responses (Chappell & Hahlbrock, 1984; Kombrink & Hahlbrock, 1986; Fritzemeier et al.,

Novel Elicitors Induce Defense Responses in Cut Flowers 89

interactions does the pathogen infect and colonize the host. Accumulation of phytoalexins can occur as part of the HR (Dixon et al., 1994). However, it is not clear whether the HR triggers the production of phytoalexins and other antimicrobial compounds or if their

Phytoalexins are low molecular weight antimicrobial compounds produced de-novo by some plants. They accumulate during infection by pathogens or after injury or stress (Ebel, 1986; Isaac, 1992; Kuć, 1995). Accumulation of phytoalexins is mainly observed when fungi, rather than bacteria, viruses or nematodes, try to infect the plant. Accumulation is a result of specific elicitors released either by the fungal cell walls or by the plant cell walls (Ebel, 1986). Elicitors of phytoalexins include a large number of compounds including inorganic salts (Perrin & Cruickshank, 1965), oligoglucans (Sharp et al., 1984), ethylene (Chalutz & Stahmann, 1969), fatty acids (Bostock et al., 1981), and chitosan oligomers (Kendra & Hadwiger, 1984). Over 200 compounds, microorganisms and physiological stresses have been reported to elicit pisatin in pea, phaseollin and kievitone in green bean and glyceollin

Most phytoalexins have been isolated from dicot plants, but they are also present in monocots including rice, corn, sorghum, wheat, barley and onions (Kuć, 1995). There is no published work on phytoalexins in cut flower species. Phytoalexins have been found in almost every part of the plant including roots, stems, leaves and fruits (Kuć, 1995). Such plant species produces a characteristic set of phytoalexins derived from secondary metabolism, in most cases via the phenylpropanoid pathway (Ebel, 1986; Kombrink & Somssich, 1995; Kuć, 1995). Phytoalexins belong to a number of key chemical groups including phenolics (e.g. flavonoids and coumarins), polyacetylenes, isoprenes, terpenoids and steroids (Ebel, 1986). They are produced by both resistant and susceptible tissues and resistance appears to be related with the total phytoalexin concentration (Kuć, 1995). Phytoalexins can affect fungal growth by inhibiting germ tube elongation and colony growth (Elad, 1997). The main effect of phytoalexins on fungi is via their cell membranes. Direct contact of phytoalexins with fungal cell walls resulted in fungal plasma membrane disruption and loss of the ultrastructural integrity (Elad, 1997). In compatible interactions, the pathogen apparently tolerates accumulated phytoalexins, detoxifies them, suppresses phytoalexin accumulation and/or avoids eliciting phytoalexin production (Kuć, 1995). Overcoming phytoalexin accumulation is attributed to either suppressor molecules released by the pathogen (i.e. low molecular weight polysaccharides or glycopeptides) or suppression of the intensity and timing of signal genes that could trigger phytoalexin

Pathogenesis related proteins (PR-proteins) accumulate either in extracellular space or the vacuole after various types of plant stress, including pathogen infection (Stermer, 1995; Sticher et al., 1997). PR-proteins accumulate at the site of infection as well as in uninfected tissues (Van Loon & Gerritsen, 1989; Ryals et al., 1996). Although healthy plants may contain traces of PR-proteins, the transcription of genes encoding PR-proteins is up-regulated following pathogen attack, elicitor treatment, wounding or stress (Stermer, 1995; Sticher et al., 1997; Van Loon, 1997). Signal compounds responsible for initiating PR-protein production include salicylic acid, ethylene, the enzyme xylanase, the polypeptide systemin

accumulation is a direct result of elicitation (Kombrink & Somssich, 1995).

**1.2.2 Local acquired resistance** 

in soybean (Kuć, 1991).

accumulation (Kuć, 1995).

1987; Keller et al., 1999). For instance, extracts from fungal cell walls when applied to plant tissue induced the synthesis and accumulation of phytoalexins (Yoshikawa et al., 1993). Active components in such chemical, biological and physical extracts are referred to as elicitors. This term is now generally used to denote agents, which induce plant defence responses, including accumulation of PR-proteins, cell wall structural (strengthening) changes, and hypersensitive cell death (Kombrink & Hahlbrock, 1986).

#### **1.2.1 Rapid defence responses**

The first step in the rapid defence responses by plants is recognition of the infection attempt by the pathogen. Pathogen recognition results in a signalling cascade to neighbouring cells and in initial molecular defence responses (Kombrink & Somssich, 1995). Examples of elicitor-active components produced by pathogenic fungi include the -glucan elicitor and the 42 kDa glycoprotein derived from the fungus *Phytophthora megasperma*, the oligo-1,4- galacturonides from *Cladosporium fulvum* and *Rhynchosporium secalis*, and the harpin protein from *Erwinia amylovora*. These compounds activate defence responses when they bind to host receptors during incompatible host-pathogen interactions (Ebel & Cosio, 1994; Kombrink & Somssich, 1995).

In parsley cells, the existence of a receptor was proposed by Ebel & Cosio (1994). The intracellular changes were subsequent signals activated by the receptor and transported to host plasma membrane. Changes in H+, K+, Cl – and Ca2+ fluxes across the plasma membrane and H2O2 increase within 2-5 min can occur (Nurnberger et al., 1994; Nurnberger & Scheel, 2001).

The activity of active oxygen species (e.g. O- , H2O2) and the rapidity of their production after invasion characterize the rapid defence response of the host (Dixon et al., 1994; Ebel & Cosio, 1994; Bolwell, 1999). These toxic active oxygen species cause host cell death at the infection site (Kombrink & Somssich, 1995). It has been suggested that reactive oxygen species (ROS) could have a dual function in disease resistance (Kombrink & Schmelzer, 2001). Firstly, ROS participate directly in cell death during HR and, thereby, results in direct pathogen inhibition. Secondly, ROS have a role in signal diffusion for cellular protectant induction and associated defence responses in neighbouring cells (Kombrink & Schmelzer, 2001).

The HR is part of the initial plant defence responses and involves localized cell death at the infection site (Kombrink & Schmelzer, 2001). Thus, the HR is a result of host recognition of infection attempts made by a pathogenic bacterium or fungus. Specific elicitor-molecules comprise signals, which induce these initial defence responses. When pathogenic bacteria are injected inside a non-host plant under artificial conditions they are killed by the HR as a result of being surrounded by dead cells. The HR may occur when either virulent strains of bacteria are injected inside a resistant host or avirulent strains of bacteria are injected inside a susceptible host (Agrios, 1997). HR associated isolation of the pathogen inside necrotic cells causes the pathogen loses its ability to take-up nutrients and grow into adjacent healthy cells (Kombrink & Schmelzer, 2001).

Elicitors which do not cause an HR can also activate defence-related compounds (Schroder et al., 1992; Atkinson, 1993; Kuć 1995; Kombrink & Schmelzer, 2001). Activation of these compounds can be similar for both compatible and incompatible host-pathogen interactions (Schroder et al., 1992; Kombrink and Schmelzer, 2001). However, only with compatible interactions does the pathogen infect and colonize the host. Accumulation of phytoalexins can occur as part of the HR (Dixon et al., 1994). However, it is not clear whether the HR triggers the production of phytoalexins and other antimicrobial compounds or if their accumulation is a direct result of elicitation (Kombrink & Somssich, 1995).

#### **1.2.2 Local acquired resistance**

88 Plant Pathology

1987; Keller et al., 1999). For instance, extracts from fungal cell walls when applied to plant tissue induced the synthesis and accumulation of phytoalexins (Yoshikawa et al., 1993). Active components in such chemical, biological and physical extracts are referred to as elicitors. This term is now generally used to denote agents, which induce plant defence responses, including accumulation of PR-proteins, cell wall structural (strengthening)

The first step in the rapid defence responses by plants is recognition of the infection attempt by the pathogen. Pathogen recognition results in a signalling cascade to neighbouring cells and in initial molecular defence responses (Kombrink & Somssich, 1995). Examples of elicitor-active components produced by pathogenic fungi include the -glucan elicitor and the 42 kDa glycoprotein derived from the fungus *Phytophthora megasperma*, the oligo-1,4- galacturonides from *Cladosporium fulvum* and *Rhynchosporium secalis*, and the harpin protein from *Erwinia amylovora*. These compounds activate defence responses when they bind to host receptors during incompatible host-pathogen interactions (Ebel & Cosio, 1994;

In parsley cells, the existence of a receptor was proposed by Ebel & Cosio (1994). The intracellular changes were subsequent signals activated by the receptor and transported to host plasma membrane. Changes in H+, K+, Cl – and Ca2+ fluxes across the plasma membrane and H2O2 increase within 2-5 min can occur (Nurnberger et al., 1994; Nurnberger

invasion characterize the rapid defence response of the host (Dixon et al., 1994; Ebel & Cosio, 1994; Bolwell, 1999). These toxic active oxygen species cause host cell death at the infection site (Kombrink & Somssich, 1995). It has been suggested that reactive oxygen species (ROS) could have a dual function in disease resistance (Kombrink & Schmelzer, 2001). Firstly, ROS participate directly in cell death during HR and, thereby, results in direct pathogen inhibition. Secondly, ROS have a role in signal diffusion for cellular protectant induction and associated

The HR is part of the initial plant defence responses and involves localized cell death at the infection site (Kombrink & Schmelzer, 2001). Thus, the HR is a result of host recognition of infection attempts made by a pathogenic bacterium or fungus. Specific elicitor-molecules comprise signals, which induce these initial defence responses. When pathogenic bacteria are injected inside a non-host plant under artificial conditions they are killed by the HR as a result of being surrounded by dead cells. The HR may occur when either virulent strains of bacteria are injected inside a resistant host or avirulent strains of bacteria are injected inside a susceptible host (Agrios, 1997). HR associated isolation of the pathogen inside necrotic cells causes the pathogen loses its ability to take-up nutrients and grow into adjacent healthy

Elicitors which do not cause an HR can also activate defence-related compounds (Schroder et al., 1992; Atkinson, 1993; Kuć 1995; Kombrink & Schmelzer, 2001). Activation of these compounds can be similar for both compatible and incompatible host-pathogen interactions (Schroder et al., 1992; Kombrink and Schmelzer, 2001). However, only with compatible

defence responses in neighbouring cells (Kombrink & Schmelzer, 2001).

, H2O2) and the rapidity of their production after

changes, and hypersensitive cell death (Kombrink & Hahlbrock, 1986).

**1.2.1 Rapid defence responses** 

Kombrink & Somssich, 1995).

The activity of active oxygen species (e.g. O-

cells (Kombrink & Schmelzer, 2001).

& Scheel, 2001).

Phytoalexins are low molecular weight antimicrobial compounds produced de-novo by some plants. They accumulate during infection by pathogens or after injury or stress (Ebel, 1986; Isaac, 1992; Kuć, 1995). Accumulation of phytoalexins is mainly observed when fungi, rather than bacteria, viruses or nematodes, try to infect the plant. Accumulation is a result of specific elicitors released either by the fungal cell walls or by the plant cell walls (Ebel, 1986). Elicitors of phytoalexins include a large number of compounds including inorganic salts (Perrin & Cruickshank, 1965), oligoglucans (Sharp et al., 1984), ethylene (Chalutz & Stahmann, 1969), fatty acids (Bostock et al., 1981), and chitosan oligomers (Kendra & Hadwiger, 1984). Over 200 compounds, microorganisms and physiological stresses have been reported to elicit pisatin in pea, phaseollin and kievitone in green bean and glyceollin in soybean (Kuć, 1991).

Most phytoalexins have been isolated from dicot plants, but they are also present in monocots including rice, corn, sorghum, wheat, barley and onions (Kuć, 1995). There is no published work on phytoalexins in cut flower species. Phytoalexins have been found in almost every part of the plant including roots, stems, leaves and fruits (Kuć, 1995). Such plant species produces a characteristic set of phytoalexins derived from secondary metabolism, in most cases via the phenylpropanoid pathway (Ebel, 1986; Kombrink & Somssich, 1995; Kuć, 1995). Phytoalexins belong to a number of key chemical groups including phenolics (e.g. flavonoids and coumarins), polyacetylenes, isoprenes, terpenoids and steroids (Ebel, 1986). They are produced by both resistant and susceptible tissues and resistance appears to be related with the total phytoalexin concentration (Kuć, 1995). Phytoalexins can affect fungal growth by inhibiting germ tube elongation and colony growth (Elad, 1997). The main effect of phytoalexins on fungi is via their cell membranes. Direct contact of phytoalexins with fungal cell walls resulted in fungal plasma membrane disruption and loss of the ultrastructural integrity (Elad, 1997). In compatible interactions, the pathogen apparently tolerates accumulated phytoalexins, detoxifies them, suppresses phytoalexin accumulation and/or avoids eliciting phytoalexin production (Kuć, 1995). Overcoming phytoalexin accumulation is attributed to either suppressor molecules released by the pathogen (i.e. low molecular weight polysaccharides or glycopeptides) or suppression of the intensity and timing of signal genes that could trigger phytoalexin accumulation (Kuć, 1995).

Pathogenesis related proteins (PR-proteins) accumulate either in extracellular space or the vacuole after various types of plant stress, including pathogen infection (Stermer, 1995; Sticher et al., 1997). PR-proteins accumulate at the site of infection as well as in uninfected tissues (Van Loon & Gerritsen, 1989; Ryals et al., 1996). Although healthy plants may contain traces of PR-proteins, the transcription of genes encoding PR-proteins is up-regulated following pathogen attack, elicitor treatment, wounding or stress (Stermer, 1995; Sticher et al., 1997; Van Loon, 1997). Signal compounds responsible for initiating PR-protein production include salicylic acid, ethylene, the enzyme xylanase, the polypeptide systemin

Novel Elicitors Induce Defense Responses in Cut Flowers 91

SA is still required for SAR expression (Van Loon, 1997). The importance of SA in the onset of SAR was determined using transgenic tobacco and Arabidopsis plants engineered to over-express SA-hydroxylase. Transformed plants with the naphthalene hydroxylase G

SA is produced from phenylalanine via coumaric and benzoic acid (Mauch-Mani and Slusarenko, 1996; Ryals et al., 1996; Sticher et al., 1997). Biosynthesis of SA starts with the conversion of phenylalanine to trans-cinnamic acid (Sticher et al., 1997). From transcinnamic acid, either benzoic acid (BA) or ortho-coumaric acid are produced. Both compounds are SA precursors (Sticher et al., 1997). Pallas et al. (1996) showed that tobacco plants epigenetically suppressed in PAL expression produced a much lower concentration of SA and other phenylpropanoid derivatives when artificially inoculated with tobacco mosaic virus (TMV). This was seen, firstly, due to the lack of resistance to TMV upon secondary infection, and, secondly, to the absence of PR protein induction in systemic leaves

Jasmonic acid (JA) and its methyl ester (MeJA) are derived from linolenic acid. They are cyclopentanine-based compounds that occur naturally in many plant species (Sembdner & Parthier, 1993; Creelman & Mullet, 1997). Linolenic acid levels or its availability could determine JA biosynthetic rate (Farmer & Ryan, 1992; Conconi et al., 1996). The level of JA in plants varies as a function of tissue and cell type, developmental stage, and in response to various environmental stimuli (Creelman & Mullet, 1997). For example, in soybean seedlings, JA levels are higher in the hypocotyls hook (a zone of cell division) and young plumules as compared to the zone of cell elongation and more mature regions of the stem, older leaves and roots (Creelman & Mullet, 1997). High JA levels are also found in flowers and pericarp tissues of developing reproductive structures (Creelman & Mullet, 1997). Jasmonates are wide spread in Angiosperms, Gymnosperms and algae (Parthier, 1991). They can mediate gene expression in response to various environmental and developmental processes (Wasternack & Parthier, 1997). These processes include wounding (Schaller & Ryan, 1995), pathogen attack (Epple et al., 1997), fungal elicitation (Nojiri et al., 1996), touch (Sharkey, 1996), nitrogen storage (Staswick, 1990), and cell wall strengthening (Creelman et al., 1992). Wounding of tomato leaves produced an 18-amino acid polypeptide called systemin, the first polypeptide hormone discovered in plants so far (Pearce et al., 1991). Systemin was released from damaged cells into the apoplast and transported out of the

Upon herbivore wounding, a systemic signal is delivered from systemin and results in an ABA-dependent rise of linoleic acid. Systemin was believed to bind to the plasma membrane of target cells and thereby initiate JA biosynthesis (Schaller & Ryan, 1995). JA accumulation can also be induced by oligosaccharides derived from plant cell walls and by elicitors, such as chitosans derived from fungal cell walls (Gundlach et al., 1992; Doares et al., 1995; Nojiri et al., 1996). JA also activates gene expression encoding proteinase inhibitors (Creelman & Mullet, 1997). Proteinase inhibitors are known antidigestive proteins that block the action of herbivore proteolytic enzymes (Creelman & Mullet, 1997). Thereby, proteinase inhibitors help the host to avoid consumption by herbivores. Proteinase inhibitors were accumulated in tomato plants after wounding (O'Donnell et al., 1996) and after irradiation with UV-C (Conconi et al. 1996). In response to wounding, ethylene and JA act together to regulate gene expression of proteinase inhibitors (O'Donnell et al., 1996). Exposing tomato

(NahG) gene produced low levels of SA and SAR expression was blocked.

wounded leaf via the phloem (Schaller & Ryan, 1995) (Fig. 1).

(Pallas et al., 1996).

and jasmonic acid (Agrios, 1997). The importance of PR-proteins lies in their range of defence activities (Van Loon et al., 1994). A number of PR-proteins release molecules that may act as elicitors (Keen & Yoshikawa, 1983). PR-proteins accumulation has been observed in monocots as well as in dicots (Redolfi, 1983). However, there is no published work on PRproteins induced in flower species. Eleven families of PR-proteins have been recognized so far (Van Loon et al., 1994). Some inhibit pathogen development during microbial infection by inhibiting fungal spore production and germination. Others are associated with strengthening of the host cell wall via its outgrowths and papillae (Agrios, 1997). Both -1,3 glucanases and chitinases, PR-2 and PR-3, respectively, are known to have direct antifungal activity (Mauch et al., 1988; Van Loon, 1997). However, many pathogens have evolved mechanisms to reduce the antifungal impact of PR-proteins (Van Loon, 1997). For example, many chitin-containing fungi are not inhibited by host-produced chitinases.

Plant secondary metabolites are divided into the three main categories of terpenes, phenolic compounds and nitrogen containing secondary metabolites (i.e. alkaloids) (Taiz & Zeiger, 1998). All secondary metabolites are produced through one of the major mevalonic, malonic or shikimic pathways (Taiz & Zeiger, 1998). Phenylalanine is a common amino acid produced via the shikimic pathway (Hahlbrock & Scheel, 1989). The most abundant classes of secondary phenolic compounds in plants are derived from phenylalanine via elimination of an ammonia molecule to form cinnamic acid. This reaction is catalyzed by phenylalanine ammonia lyase (PAL), the key enzyme of phenylpropanoid metabolism (Hahlbrock & Scheel, 1989). Derivatives of phenylpropanoid pathway include lowmolecular-weight flower pigments, antibiotic phytoalexins, UV-protectants, insect repellents, and signal molecules in plant-microbe interactions (Hahlbrock & Scheel, 1989; Kombrink & Somssich, 1995).

The main phenylpropanoid pathway branches leading to formation of flavonoids, isoflavonoids, coumarins, soluble esters, wall bound phenolics, lignin and suberin. This diverse spectrum of compounds has a wide range of properties (Hahlbrock & Scheel, 1989). For example, the lignin pathway is an important phenylpropanoid pathway branch that produces precursors for lignin deposition (Grisebach, 1981). Various enzymes implicated in the biosynthesis of lignin appeared to be induced in plants in response to infection or elicitor treatment (Matern & Kneusel, 1988). However, not all studies show a role of lignin and cell lignification in disease inhibition (Garrod et al., 1982). Furanocoumarins derived from the furanocoumarin pathway in parsley are considered potent phytoalexins (Beier & Oertli, 1983). Flavonoid and furanocoumarin production as a response to UV light or fungal elicitor treatment respectively was associated with up-regulation of PAL, 4-coumarate: CoA-ligase (4CL) and chalcone synthase (CHS). Up-regulation was based on rapid changes in amounts and activities of the corresponding mRNAs (Chappell & Hahlbrock, 1984).

After pathogen recognition by the host, a cascade of early responses is induced including ion fluxes, phosphorylation events, and generation of active oxygen species (Kombrink & Somssich, 1995). SA acts as a secondary signal molecule and its levels increase during the defence induction process. Thus, SA is required for initiation of synthesis of various defence-related proteins such as the PR-proteins (Van Loon, 1997; Metraux, 2001). SA accumulation endogenously in tobacco and cucumber plants lead to the HR and the SAR responses. However, SA is not necessarily the translocated signal (elicitor) for the onset of SAR. Rather, SA exerts an effect locally (Vernooij et al., 1994; Ryals et al., 1996). Nonetheless,

and jasmonic acid (Agrios, 1997). The importance of PR-proteins lies in their range of defence activities (Van Loon et al., 1994). A number of PR-proteins release molecules that may act as elicitors (Keen & Yoshikawa, 1983). PR-proteins accumulation has been observed in monocots as well as in dicots (Redolfi, 1983). However, there is no published work on PRproteins induced in flower species. Eleven families of PR-proteins have been recognized so far (Van Loon et al., 1994). Some inhibit pathogen development during microbial infection by inhibiting fungal spore production and germination. Others are associated with strengthening of the host cell wall via its outgrowths and papillae (Agrios, 1997). Both -1,3 glucanases and chitinases, PR-2 and PR-3, respectively, are known to have direct antifungal activity (Mauch et al., 1988; Van Loon, 1997). However, many pathogens have evolved mechanisms to reduce the antifungal impact of PR-proteins (Van Loon, 1997). For example,

Plant secondary metabolites are divided into the three main categories of terpenes, phenolic compounds and nitrogen containing secondary metabolites (i.e. alkaloids) (Taiz & Zeiger, 1998). All secondary metabolites are produced through one of the major mevalonic, malonic or shikimic pathways (Taiz & Zeiger, 1998). Phenylalanine is a common amino acid produced via the shikimic pathway (Hahlbrock & Scheel, 1989). The most abundant classes of secondary phenolic compounds in plants are derived from phenylalanine via elimination of an ammonia molecule to form cinnamic acid. This reaction is catalyzed by phenylalanine ammonia lyase (PAL), the key enzyme of phenylpropanoid metabolism (Hahlbrock & Scheel, 1989). Derivatives of phenylpropanoid pathway include lowmolecular-weight flower pigments, antibiotic phytoalexins, UV-protectants, insect repellents, and signal molecules in plant-microbe interactions (Hahlbrock & Scheel, 1989;

The main phenylpropanoid pathway branches leading to formation of flavonoids, isoflavonoids, coumarins, soluble esters, wall bound phenolics, lignin and suberin. This diverse spectrum of compounds has a wide range of properties (Hahlbrock & Scheel, 1989). For example, the lignin pathway is an important phenylpropanoid pathway branch that produces precursors for lignin deposition (Grisebach, 1981). Various enzymes implicated in the biosynthesis of lignin appeared to be induced in plants in response to infection or elicitor treatment (Matern & Kneusel, 1988). However, not all studies show a role of lignin and cell lignification in disease inhibition (Garrod et al., 1982). Furanocoumarins derived from the furanocoumarin pathway in parsley are considered potent phytoalexins (Beier & Oertli, 1983). Flavonoid and furanocoumarin production as a response to UV light or fungal elicitor treatment respectively was associated with up-regulation of PAL, 4-coumarate: CoA-ligase (4CL) and chalcone synthase (CHS). Up-regulation was based on rapid changes in amounts

After pathogen recognition by the host, a cascade of early responses is induced including ion fluxes, phosphorylation events, and generation of active oxygen species (Kombrink & Somssich, 1995). SA acts as a secondary signal molecule and its levels increase during the defence induction process. Thus, SA is required for initiation of synthesis of various defence-related proteins such as the PR-proteins (Van Loon, 1997; Metraux, 2001). SA accumulation endogenously in tobacco and cucumber plants lead to the HR and the SAR responses. However, SA is not necessarily the translocated signal (elicitor) for the onset of SAR. Rather, SA exerts an effect locally (Vernooij et al., 1994; Ryals et al., 1996). Nonetheless,

many chitin-containing fungi are not inhibited by host-produced chitinases.

and activities of the corresponding mRNAs (Chappell & Hahlbrock, 1984).

Kombrink & Somssich, 1995).

SA is still required for SAR expression (Van Loon, 1997). The importance of SA in the onset of SAR was determined using transgenic tobacco and Arabidopsis plants engineered to over-express SA-hydroxylase. Transformed plants with the naphthalene hydroxylase G (NahG) gene produced low levels of SA and SAR expression was blocked.

SA is produced from phenylalanine via coumaric and benzoic acid (Mauch-Mani and Slusarenko, 1996; Ryals et al., 1996; Sticher et al., 1997). Biosynthesis of SA starts with the conversion of phenylalanine to trans-cinnamic acid (Sticher et al., 1997). From transcinnamic acid, either benzoic acid (BA) or ortho-coumaric acid are produced. Both compounds are SA precursors (Sticher et al., 1997). Pallas et al. (1996) showed that tobacco plants epigenetically suppressed in PAL expression produced a much lower concentration of SA and other phenylpropanoid derivatives when artificially inoculated with tobacco mosaic virus (TMV). This was seen, firstly, due to the lack of resistance to TMV upon secondary infection, and, secondly, to the absence of PR protein induction in systemic leaves (Pallas et al., 1996).

Jasmonic acid (JA) and its methyl ester (MeJA) are derived from linolenic acid. They are cyclopentanine-based compounds that occur naturally in many plant species (Sembdner & Parthier, 1993; Creelman & Mullet, 1997). Linolenic acid levels or its availability could determine JA biosynthetic rate (Farmer & Ryan, 1992; Conconi et al., 1996). The level of JA in plants varies as a function of tissue and cell type, developmental stage, and in response to various environmental stimuli (Creelman & Mullet, 1997). For example, in soybean seedlings, JA levels are higher in the hypocotyls hook (a zone of cell division) and young plumules as compared to the zone of cell elongation and more mature regions of the stem, older leaves and roots (Creelman & Mullet, 1997). High JA levels are also found in flowers and pericarp tissues of developing reproductive structures (Creelman & Mullet, 1997). Jasmonates are wide spread in Angiosperms, Gymnosperms and algae (Parthier, 1991). They can mediate gene expression in response to various environmental and developmental processes (Wasternack & Parthier, 1997). These processes include wounding (Schaller & Ryan, 1995), pathogen attack (Epple et al., 1997), fungal elicitation (Nojiri et al., 1996), touch (Sharkey, 1996), nitrogen storage (Staswick, 1990), and cell wall strengthening (Creelman et al., 1992). Wounding of tomato leaves produced an 18-amino acid polypeptide called systemin, the first polypeptide hormone discovered in plants so far (Pearce et al., 1991). Systemin was released from damaged cells into the apoplast and transported out of the wounded leaf via the phloem (Schaller & Ryan, 1995) (Fig. 1).

Upon herbivore wounding, a systemic signal is delivered from systemin and results in an ABA-dependent rise of linoleic acid. Systemin was believed to bind to the plasma membrane of target cells and thereby initiate JA biosynthesis (Schaller & Ryan, 1995). JA accumulation can also be induced by oligosaccharides derived from plant cell walls and by elicitors, such as chitosans derived from fungal cell walls (Gundlach et al., 1992; Doares et al., 1995; Nojiri et al., 1996). JA also activates gene expression encoding proteinase inhibitors (Creelman & Mullet, 1997). Proteinase inhibitors are known antidigestive proteins that block the action of herbivore proteolytic enzymes (Creelman & Mullet, 1997). Thereby, proteinase inhibitors help the host to avoid consumption by herbivores. Proteinase inhibitors were accumulated in tomato plants after wounding (O'Donnell et al., 1996) and after irradiation with UV-C (Conconi et al. 1996). In response to wounding, ethylene and JA act together to regulate gene expression of proteinase inhibitors (O'Donnell et al., 1996). Exposing tomato

Novel Elicitors Induce Defense Responses in Cut Flowers 93

SAR is activated following induction of local acquired resistance (LAR). SAR is potentially induced after the HR and after challenge with virulent strains of a pathogen or elicitor treatment. It develops systemically in distant parts of the infected plant (Lawton et al., 1996; Ryals et al., 1996; Metraux, 2001). SAR protects plants from a broad range of potential

Specific genes induced in different plant species during SAR have been called SAR-genes (Kessmann et al., 1994; Stermer, 1995; Ryals et al., 1996; Sticher et al., 1997). Most of SARgenes encode PR-proteins such as those accumulated after inoculation of tobacco with TMV (Ward et al., 1991). These include PR-1 (PR-1a, PR-1b, PR-1c), -1,3-glucanases (PR-2a, PR-2b, PR-2c), chitinases (PR-3a, PR-3b), hevein-like proteins (PR-4a, PR-4b), thaumatin like proteins (PR-5a, PR-5b), acidic and basic isoforms of class III chitinase, an extracellular -1,3 glucanase and the basic isoform of PR-1 (Ward et al., 1991). SAR and SAR-gene activation has been observed in various dicots (Kessmann et al., 1994; Ryals et al., 1996). SAR activation involves species specificity (Ryals et al., 1992). For example, acidic PR-1 is only weakly expressed in cucumber. In contrast, acidic PR-1 is the main protein accumulating in tobacco and Arabidopsis. A number of homologous SAR-genes have been identified in monocots. Homologs of the PR-1 family were found in maize and barley and other PRproteins in maize (Nasser et al., 1988). Gorlach et al. (1996) isolated a group of wheat genes (WCI or wheat chemically induced) induced after chemical treatment with potent SAR inducers. WCI genes seemed to act in a similar manner to SAR-genes in dicots after chemical

Recent research has revealed that JA and ethylene play key roles in signal transduction pathways associated with plant defence responses (Pieterse and van Loon, 1999; Thomma et al., 2000). Inoculation with a necrotizing pathogen resulted predominantly in activation of the SA-dependent SAR response. This response leads to the accumulation of salicylic acid inducible PR-proteins and the expression of SAR (Ryals et al., 1996; Pieterse & van Loon, 1999) (Fig. 2, pathway 2). JA and ethylene inducible defence responses are induced by nonnecrotizing rhizobacteria and lead to the ISR phenomenon (Pieterse et al., 1996; Pieterse et al., 1998) (Fig. 2, pathway 1). Both pathways 1 and 2 are regulated in Arabidopsis plants

Depending on the invading pathogen, the composition of defence compounds produced after infection can vary between SA- and JA/ethylene-inducible pathways (Fig. 2, pathways

Wounding can also result in JA and ethylene inducible defence response activation (Fig. 2, pathway 4) (O'Donnell et al., 1996; Wasternack & Parthier, 1997). However, resultant products of the wounding pathway differ from those induced upon pathogen infection (O'Donnell et al., 1996; Rojo et al., 1999). A second distinct wound-signalling pathway leading to wound responsive (WR) gene expression has been found in Arabidopsis plants (Titarenko et al., 1997; Rojo et al., 1998) (Fig. 2, pathway 6). Upon wounding, Arabidopsis plants carrying the coi1 (JA-insensitive) mutant gene expressed the wound responsive genes choline kinase (CK) and wound responsive (WR3) indicating that the induced pathway was totally independent of JA. UV irradiation of tomato leaves also resulted in induction of the same defensive genes normally activated through the octadecanoid pathway after wounding (Conconi et al., 1996). This response is blocked after SA treatment, confirming the

**1.2.3 Systemic defence responses (i.e. SAR, ISR) and signalling pathways** 

pathogens (Kessmann et al, 1994).

treatment with plant activators (Gorlach et al., 1996).

2 and 3) (Ryals et al., 1996; Epple et al., 1997; Dong, 1998).

carrying the NPR1 gene.

leaves to increasing doses of 254 nm UV-C resulted in increased proteinase inhibitors gene expression. Expression of proteinase inhibitors in wounded (Doares et al., 1995; O'Donnell et al., 1996) or UV-C treated (Conconi et al., 1996) tomato leaves was markedly reduced upon treatment with SA. From linoleic acid, jasmonic acid is produced. Ethylene is required in the jasmonic-signalling cascade (O'Donnell et al., 1995).

Fig. 1. The octadecanoid-signalling pathway for defence gene expression in tomato (Schaller and Ryan, 1995).

leaves to increasing doses of 254 nm UV-C resulted in increased proteinase inhibitors gene expression. Expression of proteinase inhibitors in wounded (Doares et al., 1995; O'Donnell et al., 1996) or UV-C treated (Conconi et al., 1996) tomato leaves was markedly reduced upon treatment with SA. From linoleic acid, jasmonic acid is produced. Ethylene is required

> Oligo galacturonide

Oligogalacturo nide

**Local signals**

Chitosan Burning

Ultraviolet radiation

Gene expression

Fig. 1. The octadecanoid-signalling pathway for defence gene expression in tomato (Schaller

COOH

Jasmonic acid

COOH

COOH

Lipoxygenase

Allene oxide synthase

12-oxo-PDA

Linoleic acis

in the jasmonic-signalling cascade (O'Donnell et al., 1995).

Systemic signal i l

Membrane

O

O

Herbivore wounding

ABA

and Ryan, 1995).

Systemin translocat ed in the phloem

#### **1.2.3 Systemic defence responses (i.e. SAR, ISR) and signalling pathways**

SAR is activated following induction of local acquired resistance (LAR). SAR is potentially induced after the HR and after challenge with virulent strains of a pathogen or elicitor treatment. It develops systemically in distant parts of the infected plant (Lawton et al., 1996; Ryals et al., 1996; Metraux, 2001). SAR protects plants from a broad range of potential pathogens (Kessmann et al, 1994).

Specific genes induced in different plant species during SAR have been called SAR-genes (Kessmann et al., 1994; Stermer, 1995; Ryals et al., 1996; Sticher et al., 1997). Most of SARgenes encode PR-proteins such as those accumulated after inoculation of tobacco with TMV (Ward et al., 1991). These include PR-1 (PR-1a, PR-1b, PR-1c), -1,3-glucanases (PR-2a, PR-2b, PR-2c), chitinases (PR-3a, PR-3b), hevein-like proteins (PR-4a, PR-4b), thaumatin like proteins (PR-5a, PR-5b), acidic and basic isoforms of class III chitinase, an extracellular -1,3 glucanase and the basic isoform of PR-1 (Ward et al., 1991). SAR and SAR-gene activation has been observed in various dicots (Kessmann et al., 1994; Ryals et al., 1996). SAR activation involves species specificity (Ryals et al., 1992). For example, acidic PR-1 is only weakly expressed in cucumber. In contrast, acidic PR-1 is the main protein accumulating in tobacco and Arabidopsis. A number of homologous SAR-genes have been identified in monocots. Homologs of the PR-1 family were found in maize and barley and other PRproteins in maize (Nasser et al., 1988). Gorlach et al. (1996) isolated a group of wheat genes (WCI or wheat chemically induced) induced after chemical treatment with potent SAR inducers. WCI genes seemed to act in a similar manner to SAR-genes in dicots after chemical treatment with plant activators (Gorlach et al., 1996).

Recent research has revealed that JA and ethylene play key roles in signal transduction pathways associated with plant defence responses (Pieterse and van Loon, 1999; Thomma et al., 2000). Inoculation with a necrotizing pathogen resulted predominantly in activation of the SA-dependent SAR response. This response leads to the accumulation of salicylic acid inducible PR-proteins and the expression of SAR (Ryals et al., 1996; Pieterse & van Loon, 1999) (Fig. 2, pathway 2). JA and ethylene inducible defence responses are induced by nonnecrotizing rhizobacteria and lead to the ISR phenomenon (Pieterse et al., 1996; Pieterse et al., 1998) (Fig. 2, pathway 1). Both pathways 1 and 2 are regulated in Arabidopsis plants carrying the NPR1 gene.

Depending on the invading pathogen, the composition of defence compounds produced after infection can vary between SA- and JA/ethylene-inducible pathways (Fig. 2, pathways 2 and 3) (Ryals et al., 1996; Epple et al., 1997; Dong, 1998).

Wounding can also result in JA and ethylene inducible defence response activation (Fig. 2, pathway 4) (O'Donnell et al., 1996; Wasternack & Parthier, 1997). However, resultant products of the wounding pathway differ from those induced upon pathogen infection (O'Donnell et al., 1996; Rojo et al., 1999). A second distinct wound-signalling pathway leading to wound responsive (WR) gene expression has been found in Arabidopsis plants (Titarenko et al., 1997; Rojo et al., 1998) (Fig. 2, pathway 6). Upon wounding, Arabidopsis plants carrying the coi1 (JA-insensitive) mutant gene expressed the wound responsive genes choline kinase (CK) and wound responsive (WR3) indicating that the induced pathway was totally independent of JA. UV irradiation of tomato leaves also resulted in induction of the same defensive genes normally activated through the octadecanoid pathway after wounding (Conconi et al., 1996). This response is blocked after SA treatment, confirming the

Novel Elicitors Induce Defense Responses in Cut Flowers 95

Disease management in the past has been achieved by various methods including resistant cultivars, biological control, crop rotation, tillage, and chemical pesticides (Kessmann et al., 1994). Recently, the use of abiotic and/or biotic agents, as well as, synthetic compounds that

A chemical is generally characterized as a plant activator when it induces natural and/or systemic defence responses, activate gene expression and provide protection on the same spectrum of diseases exerted by a wild type host (Kessmann et al., 1996; Ruess et al., 1996). Plant activators, normally, do not exert direct antimicrobial activity against pathogens when used for disease control, but rather work through their mutagenic elicitation effect and help

**1.3.1 Acibenzolar-S-methyl (ASM; benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl** 

**response** 


Acidic peroxidase, class III chitinase and - 1,3-glucanase

Merlot *B. cinerea* na 0.3 mM Iriti et al., 2004

*Many pathogens* ns 0.05-0.1 g AI L-1 Ishii et al., 1999

**ASM** 

0.0015-0.075 g AI L-1

PR-1 and PR-5 0.05 g AI L-1 Ziadi et al., 2001

AI L-1

ns 0.025 or 0.05 g

ns 12-30 g AI ha-1 Ruess et al., 1996

**concentration Reference**

0.1-0.2 g AI L-1 Brisset et al.,

32.4 g AI L-1 Narusaka et al.,

2000

1999

1999

Huang et al.,

2000

Godard et al.,

induce host immune systems have offered a new prospect for disease management.

eliminate the risk of the development of resistant strains by the pathogen.

The efficacy of ASM has been tested in field, glasshouse and pot trials (Table 2).

**ester; CGA 245704; benzothiadiazole or BTH)** 

Apple seedlings cv. Golden Delicious

Cereals, tobacco

Cauliflower

Cucumber (*Cucumis sativus* 

Cucumber (*Cucumis sativus* L.) and Japanese pear (*Pyrus pyrifolia* Nakai var. culta Nakai)

Cauliflower

Grapevine cv

Melons cv. Early Yellow Hami

L.)

**Host Pathogen Induced** 

(*Brassica oleracea*) *Peronospora parasitica* ns

*Erwinia amylovora* 

*Erysiphe graminis, Septoria* spp.*, Puccinia*  spp.*, Peronospora hyoscyami* f. sp. *tabacina*

> *Cladosporium cucumerinum*

(*Brassica oleracea*) *P. parasitica* -1,3-glucanase.

*Fusarium* spp.*, Alternaria* spp.*, Rhizopus* spp. *Trichothecium* sp.

**1.3 Elicitation of defence responses with chemical activators** 

antagonistic regulation of the two distinct pathways (Pena-Cortes et al., 1993; Lawton et al., 1995; Xu et al., 1994; Doares et al., 1995; O'Donnell et al., 1996; Niki et al., 1998; Gupta et al., 2000; Rao et al., 2000).

In the rhizobacteria-mediated induced systemic resistance (ISR) pathway, components from the JA/ethylene response acted in sequence in activating a systemic resistance response that, like pathogen induced SAR, was dependent on the regulatory protein NPR1 (Pieterse & van Loon, 1999). The ISR pathway shares signalling events with pathways initiated upon pathogen infection, but is not associated with the activation of genes encoding plant defensins, thionins or PR-proteins (Pieterse & van Loon, 1999) (Fig. 2, pathway 3). This observation indicates that ISR inducing rhizobacteria, such as P. fluorescens strain WCS417r, trigger a novel signalling pathway leading to the production of so far unidentified defense compounds (Pieterse et al., 1996; Pietrese et al., 1998). Protection of NahG Arabidopsis plants by gaseous MeJA suggested that induction of a SA non-dependent systemic pathway was regulated by JA (Thomma et al., 2000) (Fig. 2, pathway 3). Protection was provided against two necrotrophic fungi*, A. brassicicola* and *B. cinerea*.

Fig. 2. Model showing systemic signalling pathways that can be induced in plants by nonpathogenic rhizobacteria, pathogen infection and wounding, such as caused by foraging insects. 1: ISR is induced in NPR1 Arabidopsis plants as a result of JA and ethylene responses. 2: SAR is induced in NPR1 Arabidopsis plants after necrosis by pathogenic fungi, bacteria or virus. 3: JA/ethylene pathway is up- regulated after fungal infection. JA/ethylene expression leads to genes encoding plant defensins, thionins, proteinase inhibitors and SA-independent PR-proteins. 4 and 6: A number of genes are regulated after mechanical wounding. JA and ethylene levels rise after mechanical wounding. 5: Cross-talk between SA- and JA- dependent pathways exist. Adopted from Pieterse & van Loon (1999).

antagonistic regulation of the two distinct pathways (Pena-Cortes et al., 1993; Lawton et al., 1995; Xu et al., 1994; Doares et al., 1995; O'Donnell et al., 1996; Niki et al., 1998; Gupta et al.,

In the rhizobacteria-mediated induced systemic resistance (ISR) pathway, components from the JA/ethylene response acted in sequence in activating a systemic resistance response that, like pathogen induced SAR, was dependent on the regulatory protein NPR1 (Pieterse & van Loon, 1999). The ISR pathway shares signalling events with pathways initiated upon pathogen infection, but is not associated with the activation of genes encoding plant defensins, thionins or PR-proteins (Pieterse & van Loon, 1999) (Fig. 2, pathway 3). This observation indicates that ISR inducing rhizobacteria, such as P. fluorescens strain WCS417r, trigger a novel signalling pathway leading to the production of so far unidentified defense compounds (Pieterse et al., 1996; Pietrese et al., 1998). Protection of NahG Arabidopsis plants by gaseous MeJA suggested that induction of a SA non-dependent systemic pathway was regulated by JA (Thomma et al., 2000) (Fig. 2, pathway 3). Protection was provided

Non-pathogenic rhizobacteria Pathogen infection Wounding

Necrosis

**- + 5**

SAR

SA inducible PRs

Fig. 2. Model showing systemic signalling pathways that can be induced in plants by nonpathogenic rhizobacteria, pathogen infection and wounding, such as caused by foraging insects. 1: ISR is induced in NPR1 Arabidopsis plants as a result of JA and ethylene

responses. 2: SAR is induced in NPR1 Arabidopsis plants after necrosis by pathogenic fungi,

bacteria or virus. 3: JA/ethylene pathway is up- regulated after fungal infection. JA/ethylene expression leads to genes encoding plant defensins, thionins, proteinase inhibitors and SA-independent PR-proteins. 4 and 6: A number of genes are regulated after mechanical wounding. JA and ethylene levels rise after mechanical wounding. 5: Cross-talk between SA- and JA- dependent pathways exist. Adopted from Pieterse & van Loon (1999).

SA production JA + Ethylene production

SA non-inducible PRs Plant defensins,

thionins

Proteinase inhibitors

Choline kinase WR3 genes

**6**

**4**

against two necrotrophic fungi*, A. brassicicola* and *B. cinerea*.

**1 2 3**

2000; Rao et al., 2000).

JA response

Unknown defensive

NPR1

compounds

ISR

Ethylene response

#### **1.3 Elicitation of defence responses with chemical activators**

Disease management in the past has been achieved by various methods including resistant cultivars, biological control, crop rotation, tillage, and chemical pesticides (Kessmann et al., 1994). Recently, the use of abiotic and/or biotic agents, as well as, synthetic compounds that induce host immune systems have offered a new prospect for disease management.

A chemical is generally characterized as a plant activator when it induces natural and/or systemic defence responses, activate gene expression and provide protection on the same spectrum of diseases exerted by a wild type host (Kessmann et al., 1996; Ruess et al., 1996). Plant activators, normally, do not exert direct antimicrobial activity against pathogens when used for disease control, but rather work through their mutagenic elicitation effect and help eliminate the risk of the development of resistant strains by the pathogen.

#### **1.3.1 Acibenzolar-S-methyl (ASM; benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl ester; CGA 245704; benzothiadiazole or BTH)**


The efficacy of ASM has been tested in field, glasshouse and pot trials (Table 2).

Novel Elicitors Induce Defense Responses in Cut Flowers 97

**response** 

ns

chitinase and β-1,3 glucanase

βglucuronidase (GUS), osmotin protein

βglucuronidase (GUS)

PPO, POD,

Τable 3. Effects of MeJA on different host-pathogen interactions. (ns: not shown, na: not

*A. brassicicola* PDF1.2 45 μM Penninckx et

*digitatum* ns 1-50 μM Droby et al.,

*infestans* phytoalexins 1-10 μM Il'inskaya et

glucanase 0.2 mM Yao & Tian,

LOX and PIs 0.1-10 mM Thaler et al.,

PPO 1 mM Thaler et al.,

**MeJA** 

0.5-50 μM and 0.001-1 μL L-1 air

**concentration Reference** 

<sup>10</sup>μmol L-1 Cao et al.,

0.045-4550 μM Xu et al.,

<sup>45</sup>μM Mitter et al.,

Thomma et al., 2000

al., 1996

1999

2008

2005

1994

1998

1996

1999

al., 1996

Constabel and Ryan, 1998

**1.3.2 Jasmonates (plant hormones produced through the octadecanoid pathway)**  The efficacy of jasmonates has been tested in field, glasshouse and pot trials (Table 3).

**Host Pathogen Induced** 

*B. cinerea, A.brassicicola, Plectosphaerella cucumerina* 

*Penicillium* 

*acutatum* 

*Phytophthora* 

*Phytophthora parasitica var. nicotianae, Cercospora nicotianae, TMV* 

*Helicoverpa zea, Spodoptera exigua* 

*Spodoptera exigua, Pseudomonas syringae* pv. *tomato* 

Sweet cherry *Monilinia fructicola* PAL, β-1,3-

of species na PPO na

Arabidopsis (*Arabidopsis thaliana*)

Arabidopsis (*Arabidopsis thaliana*)

Grapefruit (*Citrus paradisi*) var. 'Marsh Seedless

Large number

Potato plants (*Solanum tuberosum*)

Tobacco cell

Tobacco cv. Xanthi-nc

Tomato plants (*Lycopersicon esculentum*)

Tomato plants (*Lycopersicon esculentum*)

applicable).

Loquat fruit *Colletotrichum* 

cultures na


Table 2. Effects of ASM on different host-pathogen interactions (ns: not shown, na: not applicable).

Although, most of ASM application were carried out preharvest, there is number of published research on ASM postharvest applications (i.e. Cao et al., 2010). Additionally, a considerable work on postharvest application of ASM on ornamentals has been published the recent years (i.e. Darras et al., 2007). ASM was introduced as a potent inducer of SAR and treated plants were resistant to the same spectrum of diseases as plants activated naturally (Kessmann et al., 1996; Friedrich et al., 1996). Although ASM and its metabolites exhibited no direct antimicrobial activity towards plant pathogens tested, they induced the same biochemical processes in the plant as those observed after natural activation of SAR (Friedrich et al., 1996; Lawton et al., 1996). The compound, which was inactive in plants that do not express the SAR-signaling pathway, required a lag time of approximately 30 days between application and protection (Lawton et al., 1996).

**response** 

ascorbate peroxidase, guaiacol peroxidase, PAL, β-1,3 glucanase

(Pmg) PAL, coumarins 0.32-6.48 g

pv. *vesicatoria* ns 1.25-5 g

*B. cinerea* ns 0.25-2 g

Callose enriched wall appositions phenolic compounds

Table 2. Effects of ASM on different host-pathogen interactions (ns: not shown, na: not

Although, most of ASM application were carried out preharvest, there is number of published research on ASM postharvest applications (i.e. Cao et al., 2010). Additionally, a considerable work on postharvest application of ASM on ornamentals has been published the recent years (i.e. Darras et al., 2007). ASM was introduced as a potent inducer of SAR and treated plants were resistant to the same spectrum of diseases as plants activated naturally (Kessmann et al., 1996; Friedrich et al., 1996). Although ASM and its metabolites exhibited no direct antimicrobial activity towards plant pathogens tested, they induced the same biochemical processes in the plant as those observed after natural activation of SAR (Friedrich et al., 1996; Lawton et al., 1996). The compound, which was inactive in plants that do not express the SAR-signaling pathway, required a lag time of approximately 30 days

**ASM** 

AI L-1

0.035-0.375 g

AI L-1

1,3-glucanase 0.05-0.5 g.L-1 Cao et al., 2010

97.2 g AI L-1

ns 0.05-30 g

*(CMV)* ns 0.1 mM Anfoka, 2000

*tritici* WCI genes (1-5) 0.3 mM Gorlach et al.,

**concentration Reference**

AI L-1 Katz et al., 1998

AI L-1 Dann et al., 1998

AI L-1 Cole, 1999

Gondim et al.,

Buonaurio et al.,

Terry and Joyce,

Benhamou & Belanger, 1998

1996

2008

2002

2000

**Host Pathogen Induced** 

With or without elicitor

*Xanthomonas campestris* 

Strawberry Microbial populations chitinase and β-

*Pseudomonas syringae* pv *tabaci, Thanatephorus cucumeris, Cercospora nicotianae*

*Fusarium oxysporum*  f.sp. *radicis-lycopersici* 

*Cucumber mosaic virus*

between application and protection (Lawton et al., 1996).

Wheat *Erysiphe graminis* f.sp.

*Sclerotinia sclerotiorum* ns

Melon fruit *Fusarium pallidoroseum*

Parsley cells (*Petroselinum crispum* L.)

Pepper (*Capsicum annuum* L.)

Soybean seedlings

Strawberry plants cvs. Elsanta and Andana

Tobacco plants cv. Kutsaga Mammoth 10

Tomato plants (*Lycopersicon esculentum*)

Tomato plants cv. Vollendung

applicable).

#### **1.3.2 Jasmonates (plant hormones produced through the octadecanoid pathway)**


The efficacy of jasmonates has been tested in field, glasshouse and pot trials (Table 3).

Τable 3. Effects of MeJA on different host-pathogen interactions. (ns: not shown, na: not applicable).

Novel Elicitors Induce Defense Responses in Cut Flowers 99

*jamesonii*) UV-C *B. cinerea* Postharvest Darras et al.,

*Epicoccum* sp.

ASM *B. cinerea* Spray – pre- and

MeJA *B. cinerea* Gas - postharvest Eyre et al., 2006

Peonies (*Paeonia lactiflora)* MeJA *B. cinerea* Gas - postharvest Gast, 2001

*Fusarium oxysporum* f. sp. *cyclaminis* 

*infestans* 

*Fusarium oxysporum* f. sp. *albedinis* 

*Fusarium oxysporum* f. sp. *gladioli*

Table 4. Chemical and biological elicitors tested on cut flowers and ornamental pot plants

ASM *B. cinerea* Spray -

*B. cinerea* Spray -

**Application method and timing** 

preharvest

postharvest

preharvest


preharvest

*helianthi Spray - preharvest Tosi et al., 1999*

preharvest Beasley, 2001

preharvest Elmer, 2006a

trunk Jaiti et al., 2009

Dip Elmer, 2006b

Spray -

postharvest

Spray -

Spray preharvest

Injection in the

**Reference**

Suo & Leung,

Darras et al., 2006b

Darras et al., 2005; 2007

Dimitriev et al.,

Dinh et al., 2007; 2008

Becktell et al.,

2005

Meir et al., 1998;

2002

2005

2012

2003

**Host Elicitor Target pathogen** 

Rose (*Rosa hybrida*) ASM *Diplocarpon rosae* Spray -

MeJA *B. cinerea* Pulse, spray -

MeJA *B. cinerea* Pulse, spray, gas

ASM, MeJA, SA, INA

MeJA &

against various pathogens infecting either pre- or postharvest.

*(Helianthus annuus) ASM Plasmopara* 

(*Chamelaucium uncinatum*) SA *Alternaria* sp.,

Petunia (*Petunia hybrida*) *Phytophthora* 

a. Cut flowers

Gerbera (*Gerbera* 

Sunflower plants (*Helianthus annuus*)

*Sunflower plants* 

b. Pot plants

plants

Cyclamen (*Cyclamen* 

c. Landscape architecture

d. Propagation material

Gladiolus corms

*persicum*) ASM

Date palm ASM

(*Gladiolus x hortulanus*) ASM

Geraldton waxflower

Freesia (*Freesia hybrida*) MeJA &

Although, firstly tested preharvest, JA or MeJA has been extensively used postharvest at different hosts (i.e. fruits, vegetables, cut flowers), application modes (i.e. spray, pulse, gas) and incubation environments (i.e. storage or ambient temperatures). For example, JA and MeJA were tested on grapefruit for suppressing postharvest green mold decay [*Penicillium digitatum* (Pers.:Fr.) Sacc.] (Droby et al., 1999). Studies showed that 50 μM and 1 μM MeJA concentrations were effective against the disease and that the reduction in the decay was the same at incubation temperatures of 2 or 20°C. Moreover, as the in-vitro tests showed no direct antifungal activity of JA and MeJA, it was suggested that the disease suppression was achieved via natural resistance induction (Droby et al., 1999). Treatment of Arabidopsis plants with MeJA reduced *A. brassicicola, B. cinerea* and *Plectosphaerella cucumerina* disease development (Thomma et al., 2000). Application of gaseous MeJA to plants resulted in a greater disease reduction compared to that on plants sprayed with MeJA or treated with INA. Gaseous MeJA protected SA-degrading transformant NahG plants, suggesting that gaseous MeJA induced a non-SA dependent systemic response (Thomma et al., 2000). Combination of ASM and JA was tested against bacterial and insect attack on field grown tomato plants (Thaler et al., 1999). Two signaling pathways, one involving SA and another involving JA were proposed to provide resistance against pathogens and insect herbivores, respectively (Thaler et al., 1999).

#### **1.4 Elicitation of defence responses in floriculture**

The efficacy of ASM and MeJA on ornamental pot plants and on cut flowers has been tested pre- and postharvest, respectively (Table 4). Most of such tests were carried out in the very recent years and still increasing. For example, pre- and postharvest treatments with MeJA or ASM on cut flowers conferred a variable measure of protection against postharvest infections by *B. cinerea* (Dinh et al., 2007).

JA and MeJA provided systemic protection to various rose cultivars (e.g. Mercedes, Europa, Lambada, Frisco, Sacha and Eskimo) against *B. cinerea* (Meir et al., 1998). MeJA applied as postharvest pulse, significantly reduced *B. cinerea* lesion size on detached rose petals. In the same study, MeJA at concentrations of 100-400 μM showed in-vitro antifungal activity on *B. cinerea* spore germination and germ-tube elongation. Similarly, a postharvest pulse, spray, or vapour treatment with MeJA 200 μΜ, 600 μΜ or 1 μL L-1, respectively, significantly reduced petal specking by *B. cinerea* on cut inflorescences of *Freesia hybrida* 'Cote d'Azur' (Darras et al., 2005; 2007). Moreover, 1-100 μL L-1 MeJA postharvest vapour treatment reduced *B. cinerea* development on cut Geraldton waxflower 'Purple Pride' and 'Mullering Brook' sprigs (Eyre et al., 2006). Application of gaseous MeJA to fresh cut peonies resulted in the lowest disease severity and in an improvement of vase life compared to the untreated controls (Gast, 2001).

MeJA and ASM, applied preharvest had variable responses against postharvest infection by *B. cinerea*. ASM was not as effective as MeJA in suppressing the development of postharvest *B. cinerea* disease for glasshouse grown freesias (Darras et al., 2006b). Dinh et al. (2007) reported that multiple sprays of ≤1000 μM MeJA to field grown plants significantly reduced *B. cinerea* on Geraldton waxflower 'My Sweet Sixteen' cut sprigs, that were un-inoculated or artificially inoculated with *B. cinerea* (Dinh et al., 2007).

Although, firstly tested preharvest, JA or MeJA has been extensively used postharvest at different hosts (i.e. fruits, vegetables, cut flowers), application modes (i.e. spray, pulse, gas) and incubation environments (i.e. storage or ambient temperatures). For example, JA and MeJA were tested on grapefruit for suppressing postharvest green mold decay [*Penicillium digitatum* (Pers.:Fr.) Sacc.] (Droby et al., 1999). Studies showed that 50 μM and 1 μM MeJA concentrations were effective against the disease and that the reduction in the decay was the same at incubation temperatures of 2 or 20°C. Moreover, as the in-vitro tests showed no direct antifungal activity of JA and MeJA, it was suggested that the disease suppression was achieved via natural resistance induction (Droby et al., 1999). Treatment of Arabidopsis plants with MeJA reduced *A. brassicicola, B. cinerea* and *Plectosphaerella cucumerina* disease development (Thomma et al., 2000). Application of gaseous MeJA to plants resulted in a greater disease reduction compared to that on plants sprayed with MeJA or treated with INA. Gaseous MeJA protected SA-degrading transformant NahG plants, suggesting that gaseous MeJA induced a non-SA dependent systemic response (Thomma et al., 2000). Combination of ASM and JA was tested against bacterial and insect attack on field grown tomato plants (Thaler et al., 1999). Two signaling pathways, one involving SA and another involving JA were proposed to provide resistance against pathogens and insect herbivores,

The efficacy of ASM and MeJA on ornamental pot plants and on cut flowers has been tested pre- and postharvest, respectively (Table 4). Most of such tests were carried out in the very recent years and still increasing. For example, pre- and postharvest treatments with MeJA or ASM on cut flowers conferred a variable measure of protection against postharvest

JA and MeJA provided systemic protection to various rose cultivars (e.g. Mercedes, Europa, Lambada, Frisco, Sacha and Eskimo) against *B. cinerea* (Meir et al., 1998). MeJA applied as postharvest pulse, significantly reduced *B. cinerea* lesion size on detached rose petals. In the same study, MeJA at concentrations of 100-400 μM showed in-vitro antifungal activity on *B. cinerea* spore germination and germ-tube elongation. Similarly, a postharvest pulse, spray, or vapour treatment with MeJA 200 μΜ, 600 μΜ or 1 μL L-1, respectively, significantly reduced petal specking by *B. cinerea* on cut inflorescences of *Freesia hybrida* 'Cote d'Azur' (Darras et al., 2005; 2007). Moreover, 1-100 μL L-1 MeJA postharvest vapour treatment reduced *B. cinerea* development on cut Geraldton waxflower 'Purple Pride' and 'Mullering Brook' sprigs (Eyre et al., 2006). Application of gaseous MeJA to fresh cut peonies resulted in the lowest disease severity and in an improvement of vase life compared to the untreated

MeJA and ASM, applied preharvest had variable responses against postharvest infection by *B. cinerea*. ASM was not as effective as MeJA in suppressing the development of postharvest *B. cinerea* disease for glasshouse grown freesias (Darras et al., 2006b). Dinh et al. (2007) reported that multiple sprays of ≤1000 μM MeJA to field grown plants significantly reduced *B. cinerea* on Geraldton waxflower 'My Sweet Sixteen' cut sprigs, that were un-inoculated or

respectively (Thaler et al., 1999).

controls (Gast, 2001).

infections by *B. cinerea* (Dinh et al., 2007).

**1.4 Elicitation of defence responses in floriculture** 

artificially inoculated with *B. cinerea* (Dinh et al., 2007).


Table 4. Chemical and biological elicitors tested on cut flowers and ornamental pot plants against various pathogens infecting either pre- or postharvest.

Novel Elicitors Induce Defense Responses in Cut Flowers 101

induced a range of defence mechanisms to halt infection development. MeJA applied postharvest as vapour at 1-100 μL L-1 significantly reduced the development of *B. cinerea* on cut Geraldton waxflower 'Purple Pride' and 'Mullering Brook' sprigs (Eyre et al., 2006). In a very recent study, Darras et al. (2011) demonstrated that gaseous MeJA at 0.1 μL L-1 significantly increased polythenol oxidase (PPO) activities 24 and 36 h post-treatment. This observation suggests that MeJA-induced defence mechanisms might be associated with the production of quinones (Constabel and Ryan, 1998), which probably helped in *B. cinerea* disease reduction. The effects of PPO in *B. cinerea* disease control have been confirmed for gerbera flowers (Darras et al., 2012). A low dose of UV-C irradiation increased PPO activity and was positively correlated with the reduction of *B. cinerea* disease symptoms on the florets (Darras et al., 2012). This indicates that PPO might play an important role in *B. cinerea*

Fig. 3. *B. cinerea* necrotic lesions on artificially inoculated freesia cv. 'Cote d'Azur' flowers treated with 0.1 μL L-1 gaseous MeJA (left) or left un-treated (control) (right) and incubated

disease control on cut flowers.

for 48 h at 20°C (Darras, 2003).

Chemical elicitors such as ASM have been applied in pot ornamentals such as petunia (Becktell et al., 2005), cyclamen (Elmer, 2006a) and in gladiolus corms (Elmer, 2006b), but effectiveness varied within the different experimental designs and conditions. In cyclamen, infection by *Fusarium oxysporum* f.sp. *cyclaminis* was reduced with increasing ASM doses (Elmer, 2006a). Additionally, the dry mass of ASM treated cyclamen plants increased with increasing ASM rates. However, as no further assays were carried out to assess possible induction of defence responses, it was not clear whether ASM reduced *F. oxysporum* f.sp. *cyclaminis* via induction of defence mechanisms or via a profound fungitoxic effect. It has been demonstrated in other research that ASM may exert direct toxic activity against *B. cinerea* (Darras et al., 2006b). In addition, ASM did not confer a significant level of protection on gladiolus corms against *F. oxysporum* f. sp. *gladioli*, and compared to conventional fungicides, although, the number of emerging flower spikes increased significantly compared to the ASM-untreated corms (Elmer, 2006b).

#### **2. Elicitation of defence responses in cut** *Freesia hybrida* **flowers – A typical example**

#### **2.1 Background**

Infection problems by *Botrytis cinerea* are typical to most geographical areas around the world and concern cut flower industry. Infection of cut flowers by the fungus results in visible lesions on flower petals (petal spotting or petal specking). According to Darras et al. (2004) freesia flower rejections at certain periods of the year (viz. April, May, October) lead in severe economic losses to growers, importers and sellers. Infection by *B. cinerea* of most cut flowers occurs in the glasshouse when a single conidium germinates and penetrates petal epidermal cells. A necrotic lesion appears postharvest after a brief incubation period under favourable environmental conditions (Darras et al., 2006a). Infection is difficult to control as it appears later in handling chain under various conditions during transport or storage.

In most cases, *B. cinerea* disease is controlled by conventional fungicides. However, extensive use of fungicides such as dicarboximides in the glasshouse has led to appearance of fungicide resistance (Pappas 1997). Alternative management methods within the concept of IDM can help overcome such problems.

For this reason, plant defence inducers (i.e. elicitors) such as ASM and MeJA have been tested with applications at various intervals, pre- or postharvest to activate systemic defence responses of the host (Kessmann et al., 1994; Meir et al., 1998; Thomma et al., 2000). For cut freesia flowers postharvest pulse, spray, or gaseous MeJA treatment at 200 μM, 600 μM, or 1 μL L–1, respectively, significantly reduced petal specking by *B. cinerea* on cv. 'Cote d'Azur' inflorescences (Darras et al., 2005; 2007). An apparent induced defence response was recorded by both ASM and MeJA treatment. However, only MeJA conferred constant and significant disease reductions. MeJA vapour at 1 μL L–1 significantly reduced lesion numbers and diameters on freesia petals by up to 56% and 50%, respectively (Darras et al., 2005).

#### **2.2 Overview of published research and further discussion**

Freesia inflorescences cv 'Cote d'Azur' gassed with 0.1 μL L-1 MeJA showed significantly smaller lesions after artificial inoculation with *B. cinerea* (Fig. 3). Gaseous MeJA might have

Chemical elicitors such as ASM have been applied in pot ornamentals such as petunia (Becktell et al., 2005), cyclamen (Elmer, 2006a) and in gladiolus corms (Elmer, 2006b), but effectiveness varied within the different experimental designs and conditions. In cyclamen, infection by *Fusarium oxysporum* f.sp. *cyclaminis* was reduced with increasing ASM doses (Elmer, 2006a). Additionally, the dry mass of ASM treated cyclamen plants increased with increasing ASM rates. However, as no further assays were carried out to assess possible induction of defence responses, it was not clear whether ASM reduced *F. oxysporum* f.sp. *cyclaminis* via induction of defence mechanisms or via a profound fungitoxic effect. It has been demonstrated in other research that ASM may exert direct toxic activity against *B. cinerea* (Darras et al., 2006b). In addition, ASM did not confer a significant level of protection on gladiolus corms against *F. oxysporum* f. sp. *gladioli*, and compared to conventional fungicides, although, the number of emerging flower spikes increased significantly

**2. Elicitation of defence responses in cut** *Freesia hybrida* **flowers – A typical** 

Infection problems by *Botrytis cinerea* are typical to most geographical areas around the world and concern cut flower industry. Infection of cut flowers by the fungus results in visible lesions on flower petals (petal spotting or petal specking). According to Darras et al. (2004) freesia flower rejections at certain periods of the year (viz. April, May, October) lead in severe economic losses to growers, importers and sellers. Infection by *B. cinerea* of most cut flowers occurs in the glasshouse when a single conidium germinates and penetrates petal epidermal cells. A necrotic lesion appears postharvest after a brief incubation period under favourable environmental conditions (Darras et al., 2006a). Infection is difficult to control as it appears

In most cases, *B. cinerea* disease is controlled by conventional fungicides. However, extensive use of fungicides such as dicarboximides in the glasshouse has led to appearance of fungicide resistance (Pappas 1997). Alternative management methods within the concept

For this reason, plant defence inducers (i.e. elicitors) such as ASM and MeJA have been tested with applications at various intervals, pre- or postharvest to activate systemic defence responses of the host (Kessmann et al., 1994; Meir et al., 1998; Thomma et al., 2000). For cut freesia flowers postharvest pulse, spray, or gaseous MeJA treatment at 200 μM, 600 μM, or 1 μL L–1, respectively, significantly reduced petal specking by *B. cinerea* on cv. 'Cote d'Azur' inflorescences (Darras et al., 2005; 2007). An apparent induced defence response was recorded by both ASM and MeJA treatment. However, only MeJA conferred constant and significant disease reductions. MeJA vapour at 1 μL L–1 significantly reduced lesion numbers and diameters on freesia petals by up to 56% and 50%, respectively (Darras et al., 2005).

Freesia inflorescences cv 'Cote d'Azur' gassed with 0.1 μL L-1 MeJA showed significantly smaller lesions after artificial inoculation with *B. cinerea* (Fig. 3). Gaseous MeJA might have

later in handling chain under various conditions during transport or storage.

**2.2 Overview of published research and further discussion** 

compared to the ASM-untreated corms (Elmer, 2006b).

of IDM can help overcome such problems.

**example** 

**2.1 Background** 

induced a range of defence mechanisms to halt infection development. MeJA applied postharvest as vapour at 1-100 μL L-1 significantly reduced the development of *B. cinerea* on cut Geraldton waxflower 'Purple Pride' and 'Mullering Brook' sprigs (Eyre et al., 2006). In a very recent study, Darras et al. (2011) demonstrated that gaseous MeJA at 0.1 μL L-1 significantly increased polythenol oxidase (PPO) activities 24 and 36 h post-treatment. This observation suggests that MeJA-induced defence mechanisms might be associated with the production of quinones (Constabel and Ryan, 1998), which probably helped in *B. cinerea* disease reduction. The effects of PPO in *B. cinerea* disease control have been confirmed for gerbera flowers (Darras et al., 2012). A low dose of UV-C irradiation increased PPO activity and was positively correlated with the reduction of *B. cinerea* disease symptoms on the florets (Darras et al., 2012). This indicates that PPO might play an important role in *B. cinerea* disease control on cut flowers.

Fig. 3. *B. cinerea* necrotic lesions on artificially inoculated freesia cv. 'Cote d'Azur' flowers treated with 0.1 μL L-1 gaseous MeJA (left) or left un-treated (control) (right) and incubated for 48 h at 20°C (Darras, 2003).

Novel Elicitors Induce Defense Responses in Cut Flowers 103

Induction of PPO, Suppression of PAL

**Treatment Response Disease parameters**

At 20°C, 0.1μL L-1

respectively.

respectively.

respectively.

respectively.

At 5°C, 0.15 g A.I. L-1

30 and 43%, respectively.

MeJA gas reduced disease severity,

lesion numbers and lesion diameters by 68, 56 and 50%,

UV-C irradiation with 1 kj m-2, reduced disease severity, lesion numbers and lesion diameters by 74 68 and 14%,

At 20°C, 200μM MeJA pulse reduced disease severity, lesion numbers and lesion diameters by.43, 29 and 18%,

At 20°C, 600μM MeJA spray reduced disease severity, lesion numbers and lesion diameters by 42, 35 and 0%,

severity, lesion numbers and lesion diameters by. 18,

acibenzolar reduced disease

Inactivation of *B. cinerea* conidia

No effect on PAL activity

Fig. 4. Ranking, in terms of relative efficacy, of postharvest biological (i.e. UV-C) and chemical (i.e. ASM, MeJA) elicitors tested on cut freesia inflorescences to control *B. cinerea*

**Elicitor treatment ranking** (ie. ranked according to their relative efficacy)

**MeJA gas** Postharvest treatment before artificial inoculation

**UV-C** Postharvest treatment before and after artificial inoculation

**MeJA pulse** Postharvest treatment before artificial inoculation

**MeJA spray** Postharvest treatment before artificial inoculation

**Acibenzolar** Postharvest treatment before artificial inoculation

infection starting with the most effective (Darras, 2003).

Lesion diameters on the detached freesia petals were significantly reduced with increasing MeJA spray, pulse or gaseous concentrations (Darras et al., 2007). The first published evidence of postharvest MeJA spray treatments enhancing protection of cut flowers against *B. cinerea* was the work by Meir et al. (2005) on cut roses. According to Meir et al. (2005), simultaneous MeJA pulsing and spraying under handling conditions resulted in suppression of gray mold in seven rose cultivars ('Eskimo', 'Profita', 'Tamara', 'Sun Beam', 'Pink Tango', 'Carmen', 'Golden Gate'). In an earlier study MeJA applied as a pulse variably reduced *B. cinerea* lesion numbers and diameters (Meir et al., 1998). Our findings are in agreement with those by Meir et al. (1998) that disease severity in both artificially inoculated and naturally infected rose flowers was reduced by a MeJA pulse at 0.2 mM at 20°C. On cut Geraldton waxflower 'Purple Pride' and 'Mullering Brook' sprigs, 1-100 μL L-1 MeJA postharvest vapour treatment significantly reduced the development of *B. cinerea* (Eyre et al., 2006). However, it also induced flower fall incidence, which was correlated with a systemic resistance-associated up-regulation of ethylene biosynthesis.

Irrespective to the concentration tested, ASM provided no protection to artificially inoculated freesia flowers (Darras et al., 2007). However, natural infection was significantly (*P* < 0.05) reduced after ASM treatment during storage at 5 and at 12°C. On the contrary, postharvest treatments of strawberry cv. Camarosa fruit with ASM failed to reduce natural infection by *B. cinerea* at 5°C (Terry & Joyce, 2004b). Generally, ASM tended to provide protection on freesia flowers at lower incubation temperatures (Darras et al., 2007). However, it was not clear whether such disease reductions were the result of the induction of host's defence responses or a direct fungitoxic activity measured in the same study. Likewise, Terry & Joyce (2000) showed that ASM reduced in-vitro *B. cinerea* mycelial growth on ASM-amended agar. It is possible that the limited disease control on freesia flowers at 5°C was due to direct toxic effect of ASM rather than via SAR induction.

Elicitation of defence responses in cut flowers is an interesting prospect for *B. cinerea* disease control especially as it may offer alternatives to fungicide application. In series of postharvest experiments with freesia inflorescences the potential to induce natural defence mechanisms or directly controlling *B. cinerea* disease by application of biological and chemical elicitors was investigated. Postharvest treatments with ASM, MeJA or UV-C irradiation markedly suppressed *B. cinerea* specking on freesia petals by reducing disease severity, lesion numbers and lesion diameters. However, attempts to further minimise disease damage caused by *B. cinerea* using combined treatments with different plant activators (i.e. both ASM and MeJA), were not successful (Darras et al., 2011).

In summary, ASM was the least effective in reducing *B. cinerea* specking on cut freesia flowers (Fig. 4). In addition, it remained unclear as to whether or not SAR was induced. In contrast, gaseous MeJA reduced disease severity most probably by inducing JAdependant biochemical responses. These contrasting results tend to concur with observations by Pieterse & van Loon (1999) and Thomma et al. (2001) that SA- versus JAdependent pathways are effective against different pathogens. The results of a most recent paper (i.e. Darras et al., 2011)*)* suggested that, SA-dependant pathway and consequently the SAR response was not effective in freesia flowers against *B. cinerea* infection. In contrast, the JA-dependant pathway was apparently induced and suppressive of *B. cinerea* infection (Darras et al., 2011).

Lesion diameters on the detached freesia petals were significantly reduced with increasing MeJA spray, pulse or gaseous concentrations (Darras et al., 2007). The first published evidence of postharvest MeJA spray treatments enhancing protection of cut flowers against *B. cinerea* was the work by Meir et al. (2005) on cut roses. According to Meir et al. (2005), simultaneous MeJA pulsing and spraying under handling conditions resulted in suppression of gray mold in seven rose cultivars ('Eskimo', 'Profita', 'Tamara', 'Sun Beam', 'Pink Tango', 'Carmen', 'Golden Gate'). In an earlier study MeJA applied as a pulse variably reduced *B. cinerea* lesion numbers and diameters (Meir et al., 1998). Our findings are in agreement with those by Meir et al. (1998) that disease severity in both artificially inoculated and naturally infected rose flowers was reduced by a MeJA pulse at 0.2 mM at 20°C. On cut Geraldton waxflower 'Purple Pride' and 'Mullering Brook' sprigs, 1-100 μL L-1 MeJA postharvest vapour treatment significantly reduced the development of *B. cinerea* (Eyre et al., 2006). However, it also induced flower fall incidence, which was correlated with a

Irrespective to the concentration tested, ASM provided no protection to artificially inoculated freesia flowers (Darras et al., 2007). However, natural infection was significantly (*P* < 0.05) reduced after ASM treatment during storage at 5 and at 12°C. On the contrary, postharvest treatments of strawberry cv. Camarosa fruit with ASM failed to reduce natural infection by *B. cinerea* at 5°C (Terry & Joyce, 2004b). Generally, ASM tended to provide protection on freesia flowers at lower incubation temperatures (Darras et al., 2007). However, it was not clear whether such disease reductions were the result of the induction of host's defence responses or a direct fungitoxic activity measured in the same study. Likewise, Terry & Joyce (2000) showed that ASM reduced in-vitro *B. cinerea* mycelial growth on ASM-amended agar. It is possible that the limited disease control on freesia flowers at

Elicitation of defence responses in cut flowers is an interesting prospect for *B. cinerea* disease control especially as it may offer alternatives to fungicide application. In series of postharvest experiments with freesia inflorescences the potential to induce natural defence mechanisms or directly controlling *B. cinerea* disease by application of biological and chemical elicitors was investigated. Postharvest treatments with ASM, MeJA or UV-C irradiation markedly suppressed *B. cinerea* specking on freesia petals by reducing disease severity, lesion numbers and lesion diameters. However, attempts to further minimise disease damage caused by *B. cinerea* using combined treatments with different plant

In summary, ASM was the least effective in reducing *B. cinerea* specking on cut freesia flowers (Fig. 4). In addition, it remained unclear as to whether or not SAR was induced. In contrast, gaseous MeJA reduced disease severity most probably by inducing JAdependant biochemical responses. These contrasting results tend to concur with observations by Pieterse & van Loon (1999) and Thomma et al. (2001) that SA- versus JAdependent pathways are effective against different pathogens. The results of a most recent paper (i.e. Darras et al., 2011)*)* suggested that, SA-dependant pathway and consequently the SAR response was not effective in freesia flowers against *B. cinerea* infection. In contrast, the JA-dependant pathway was apparently induced and suppressive of *B. cinerea*

systemic resistance-associated up-regulation of ethylene biosynthesis.

5°C was due to direct toxic effect of ASM rather than via SAR induction.

activators (i.e. both ASM and MeJA), were not successful (Darras et al., 2011).

infection (Darras et al., 2011).

Fig. 4. Ranking, in terms of relative efficacy, of postharvest biological (i.e. UV-C) and chemical (i.e. ASM, MeJA) elicitors tested on cut freesia inflorescences to control *B. cinerea* infection starting with the most effective (Darras, 2003).

Novel Elicitors Induce Defense Responses in Cut Flowers 105

warranted to help interpret the MeJA mode of action in cut flowers. Also, additional inplanta trials on extra freesia varieties and a wider range of MeJA concentrations may help in

In view to the promising results using MeJA, it is likely that elicitor based strategies within IDM could be used for the control of Botrytis or other pathogens on freesias and ornamental pot plants, as well as on various cut flowers. In turn, IDM would minimise the risk of pathogens developing resistance to fungicides and also reduce public concerns over

More research could be undertaken into potential synergistic effects of combined pre- and postharvest treatments with plant activators and/or abiotic biological agents (i.e. UV-C irradiation). In due course, pre and/or postharvest use of plant activators could have commercial potential for postharvest disease suppression (Kessmann et al., 1994; Kessmann et al., 1996; Thaler et al., 1996; Meir et al., 1998; Huang et al., 2000; (Darras et

Anfoka, G.H. (2000). Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester induces

Anonymous, (2011). FloraHolland reposts 7% increase in turnover. In *Floraculture* 

<http://www.floracultureinternational.com/index.php?option=com\_content&vie

Atkinson, M.M. (1993). Molecular mechanisms of pathogen recognition by plants. *Advances* 

Beasley, D.R. (2001). Strategies for control of *Botrytis cinerea* on Geraldton waxflower

Becktell, M.C.; Daughtrey, M.L. & Fry, W.E. (2005). Epidemiology and management of

Beier, R.C. & Oertli, E.H. (1983). Psoralen and other linear furocoumarins as phytoalexins in

Benhamou, N. & Bélanger, R.R. (1998). Benzothiadiazole-mediated induced resistance to

Bostock, R.; Kuć, J., & Laine, R. (1981). Eicosapentaenoic and arachidonic acids from

Bolwell, G.P. (1999). Role of active oxygen species and NO in plant defence responses.

petunia and tomato late blight in the greenhouse. *Plant Disease*, Vol.89(9), pp. 1000-

Fusarium oxysporum f.sp. radicis-lycopersici in tomato. *Plant Physiology*, Vol.118,

*Phytophthora infestans* elicit fungitoxic sesquiterpenes in potato. *Science*, Vol.212, pp.

systemic resistance in tomato (*Lycopersicon esculentum*. Mill cv. Vollendung) to

Agrios, G. N. 1997. *Plant Pathology* (4th edition), Academic Press, London UK

Cucumber mosaic virus. *Crop Protection*, Vol.19, pp. 401-405

flowers. PhD thesis. The University of Queensland, Australia

*International*, 30/08/2011, Available from:

celery. *Phytochemistry,* Vol.22, pp. 2595-2597

*Current Opinion in Plant Biology*, Vol.2, pp. 287-294

*in Plant Pathology*, Vol.10, pp. 35-64

w=article&id=2196:floraholland-reports-7-increaseinturnover&catid=52:bussiness&Itemid=376>

better understanding MeJA efficacy.

al., 2011).

**4. References** 

1008.

67-69

pp. 1203-1212

extensive fungicide use (Jacobsen & Backman, 1993).

#### **3. Conclusions and recommendations for future research**

Management of postharvest infection of cut freesia flowers by *B. cinerea*, was, in most cases, successful. ASM was somewhat effective compared to untreated controls mostly when applied preharvest at 1.43 μΜ. In-vitro studies showed direct antifungal activity of ASM against *B. cinerea* colony growth and conidial germination. Inconsistency of ASM applied pre- or postharvest may be explained by: 1) variability of environmental conditions in the glasshouse, which may affected defence enhancement (Herms & Mattson, 1992; Terry, 2002); and, 2) infection by *B. cinerea* might not necessarily be sensitive to induced SAR responses, and thus ASM treatments may not correspond to *B. cinerea* disease suppression (Thomma et al., 1998; Govrin & Levine, 2002). Friedrich et al. (1996) reported that ASM failed to control *B. cinerea* in tobacco, but was effective against other pathogens. The apparent inability of ASM to control *B. cinerea* was seemingly supported by the observation that PAL activity in ASM treated freesia inflorescences was not higher compared to the untreated controls. Therefore, ASM did not induce biochemical defence processes, such as the production of antifungal secondary metabolites like phytoalexins through the phenylpropanoid pathway (Kombrink & Somssich, 1995; Kuć, 1995).

In contrast to inconsistent effects of ASM, MeJA was markedly effective in suppressing *B. cinerea* specking on cut freesia flowers when applied either pre- or postharvest. MeJA effectiveness was application method and concentration dependent. MeJA applied as gas was more effective compared to pulsing or spraying. It is possible that MeJA may function as an airborne signal which activated disease resistance and the expression of defence related genes in plant tissue (Shulaev et al., 1997). This finding agrees with earlier findings in Arabidopsis presented by Thomma et al. (2000). In Arabidopsis, this effect was mediated via the JA-dependent defence responses (Thomma et al., 2000). MeJA did not exert any direct antifungal activity in-vitro except at the concentration of 600 μM and therefore it is possible that MeJA reduced *B. cinerea* disease on freesia flowers by inducing responses correlated with the JA-dapendent pathway (Darras et al., 2005). PPO levels in freesia flowers after MeJA gaseous treatment increased by 47 and 57% compared to the untreated controls (Darras et al., 2011). However, PAL activity decreased markedly compared 12 h post MeJA application and maintained at minimum level (i.e. ≈ 0). These findings suggest that MeJA might suppress the action of PAL in the phenylpropanoid pathway and consequently reduce or block SA production. Antagonistic regulation of JA- and SAdependent pathways has been documented in the past by Pena-Cortes et al. (1993), Conconi et al. (1996), Niki et al. (1998), Gupta et al. (2000), and Rao et al. (2000). The apparent suppression of PAL in freesia flowers by MeJA constitute additional evidence of a JA- and SA- antagonistic response.

MeJA applied to freesia plants 28 days before harvest suppressed postharvest flower specking caused by *B. cinerea* in both a temperature and variety dependent fashion. MeJA was highly effective when flowers were incubated at 20°C compared to incubation at 5 or 12°C. It is likely that low incubation temperatures slow down plant's metabolism and also the production of defence related compounds (Jarvis, 1980). Overall, MeJA provided a considerable level of protection against *B. cinerea* when applied preharvest and, thus, could be considered a promising tool in an IDM context. Further study at the molecular level is warranted to help interpret the MeJA mode of action in cut flowers. Also, additional inplanta trials on extra freesia varieties and a wider range of MeJA concentrations may help in better understanding MeJA efficacy.

In view to the promising results using MeJA, it is likely that elicitor based strategies within IDM could be used for the control of Botrytis or other pathogens on freesias and ornamental pot plants, as well as on various cut flowers. In turn, IDM would minimise the risk of pathogens developing resistance to fungicides and also reduce public concerns over extensive fungicide use (Jacobsen & Backman, 1993).

More research could be undertaken into potential synergistic effects of combined pre- and postharvest treatments with plant activators and/or abiotic biological agents (i.e. UV-C irradiation). In due course, pre and/or postharvest use of plant activators could have commercial potential for postharvest disease suppression (Kessmann et al., 1994; Kessmann et al., 1996; Thaler et al., 1996; Meir et al., 1998; Huang et al., 2000; (Darras et al., 2011).

#### **4. References**

104 Plant Pathology

Management of postharvest infection of cut freesia flowers by *B. cinerea*, was, in most cases, successful. ASM was somewhat effective compared to untreated controls mostly when applied preharvest at 1.43 μΜ. In-vitro studies showed direct antifungal activity of ASM against *B. cinerea* colony growth and conidial germination. Inconsistency of ASM applied pre- or postharvest may be explained by: 1) variability of environmental conditions in the glasshouse, which may affected defence enhancement (Herms & Mattson, 1992; Terry, 2002); and, 2) infection by *B. cinerea* might not necessarily be sensitive to induced SAR responses, and thus ASM treatments may not correspond to *B. cinerea* disease suppression (Thomma et al., 1998; Govrin & Levine, 2002). Friedrich et al. (1996) reported that ASM failed to control *B. cinerea* in tobacco, but was effective against other pathogens. The apparent inability of ASM to control *B. cinerea* was seemingly supported by the observation that PAL activity in ASM treated freesia inflorescences was not higher compared to the untreated controls. Therefore, ASM did not induce biochemical defence processes, such as the production of antifungal secondary metabolites like phytoalexins through the phenylpropanoid pathway

In contrast to inconsistent effects of ASM, MeJA was markedly effective in suppressing *B. cinerea* specking on cut freesia flowers when applied either pre- or postharvest. MeJA effectiveness was application method and concentration dependent. MeJA applied as gas was more effective compared to pulsing or spraying. It is possible that MeJA may function as an airborne signal which activated disease resistance and the expression of defence related genes in plant tissue (Shulaev et al., 1997). This finding agrees with earlier findings in Arabidopsis presented by Thomma et al. (2000). In Arabidopsis, this effect was mediated via the JA-dependent defence responses (Thomma et al., 2000). MeJA did not exert any direct antifungal activity in-vitro except at the concentration of 600 μM and therefore it is possible that MeJA reduced *B. cinerea* disease on freesia flowers by inducing responses correlated with the JA-dapendent pathway (Darras et al., 2005). PPO levels in freesia flowers after MeJA gaseous treatment increased by 47 and 57% compared to the untreated controls (Darras et al., 2011). However, PAL activity decreased markedly compared 12 h post MeJA application and maintained at minimum level (i.e. ≈ 0). These findings suggest that MeJA might suppress the action of PAL in the phenylpropanoid pathway and consequently reduce or block SA production. Antagonistic regulation of JA- and SAdependent pathways has been documented in the past by Pena-Cortes et al. (1993), Conconi et al. (1996), Niki et al. (1998), Gupta et al. (2000), and Rao et al. (2000). The apparent suppression of PAL in freesia flowers by MeJA constitute additional evidence of

MeJA applied to freesia plants 28 days before harvest suppressed postharvest flower specking caused by *B. cinerea* in both a temperature and variety dependent fashion. MeJA was highly effective when flowers were incubated at 20°C compared to incubation at 5 or 12°C. It is likely that low incubation temperatures slow down plant's metabolism and also the production of defence related compounds (Jarvis, 1980). Overall, MeJA provided a considerable level of protection against *B. cinerea* when applied preharvest and, thus, could be considered a promising tool in an IDM context. Further study at the molecular level is

**3. Conclusions and recommendations for future research** 

(Kombrink & Somssich, 1995; Kuć, 1995).

a JA- and SA- antagonistic response.

Agrios, G. N. 1997. *Plant Pathology* (4th edition), Academic Press, London UK


Novel Elicitors Induce Defense Responses in Cut Flowers 107

Darras, A.I.; Joyce, D.C.; Terry, L.A. & Vloutoglou I. (2006a). Postharvest infections of

Darras, A.I.; Joyce, D.C. & Terry, L.A. (2006b). Acibenzolar-S-methyl and methyl jasmonate

Darras, A.I.; Joyce, D.C.; Terry, L.A.; Pompodakis, N.E. & Dimitriadis, C.I. (2007). Efficacy of

Darras, A.I.; Joyce, D.C. & Terry, L.A. (2011). MeJA and ASM protect cut *Freesia hybrida*

Darras, A.I.; Demopoulos, V. & Tiniakou, C.A. (2012). UV-C irradiation induces defence

Dinh, S.-Q.; Joyce, D.C.; Irving, D.E. & Wearing, A.H. (2007). Field applications of three

Dinh, S.-Q.; Joyce, D.C.; Irving, D.E. & Wearing, A.H. (2008). Effects of multiple applications

Delaney, T.P.; Uknes, S.; Vernooij, B.; Friedrich, L.; Weymann, K.; Negretto, D.; Gaffney, T.;

Dimitriev, A.; Tena, M. & Jorrin, J. (2003). Systemic acquired resistance in sunflower

Dirkse, FB. (1982). Preharvest treatment of chrysanthemum against *Botrytis cinerea*. *Acta* 

Dixon, R.A.; Lamb, C.J. & Harrison, M.J. (1994). Early events in the activation of plant defense responses. *Annual Review of Phytopathology*, Vol.32, pp. 479-501 Doares, S.H.; Syrovers, T.; Weiler, E. & Ryan C.A. (1995). Oligogalacturonides and chitosan

Dong, X. (1998). SA, JA, ethylene, and disease resistance in plants. *Current Opinions in Plant* 

Droby, S.; Porat, R.; Cohen, L.; Weiss, B.; Shapiro, S.; Philisoph-Hadas, S. & Meir, S. (1999).

Ebel, J. (1986). Phytoalexin synthesis: the biochemical analysis of the induction process.

*Horticultural Science & Biotechnology*, Vol.86(1), pp. 74-78

plant disease resistance. *Science*, Vol.266, pp. 1247-1249

(*Helianthus annus* L.). *TSitologiia i Genetika*, Vol.37, pp. 9-15

*National Academy of Science U.S.A.*, Vol.92, pp. 4095-4098

*Annual Review of Phytopathology*, Vol.24, pp. 235-264

55-63

1051

142-148

Vol.36, pp. 332-340

*Technology* Vol.64, pp. 168-174

*Plant Pathology,* Vol.37, pp. 87-94

*Horticulturae*, Vol.125, pp. 221–226.

*Biology*, Vol.1, pp. 316-323

184-188

*Freesia hybrida* L. flowers by *Botrytis cinerea. Australasian Plant Pathology*, Vol.35, pp.

of glasshouse-grown freesias suppress postharvest petal specking caused by *Botrytis cinerea. Journal of Horticultural Science & Biotechnology*, Vol.81(6), pp. 1043-

postharvest treatments with acibenzolar-S-methyl and methyl jasmonate against *Botrytis cinerea* infecting cut *Freesia hybrida* L. flowers. *Australasian Plant Pathology*,

inflorescences against *Botrytis cinerea*, but do not act synergistically. *Journal of* 

responses and improves vase life of cut gerbera flowers. *Postharvest Biology and* 

different classes of known host plant defence elicitors did not suppress infection of Geraldton waxflower by *Botrytis cinerea. Australasian Plant Pathology*, Vol.36, pp.

of chemical elicitors on *Botrytis cinerea* infecting Geraldton waxflower. *Australasian* 

Gut-Rella, M.; Kessmann, H. & Ward, E. (1994). A central role of salicylic acid in

activate plant defensive genes through the octadecanoid pathway. *Proceedings of the* 

Suppressing green mold decay in grapefruit with postharvest jasmonate application. *Journal of the American Society for Horticultural Science*, Vol.124(2), pp.


Brisset, M-N.; Cesbron, S.; Thomson S.V. & Paulin, J-P. (2000). Acibenzolar-S-methyl induces

Buonaurio, R.; Scarponi, L.; Ferrara, M.; Sidoti, P. & Bertona, A. (2002). Induction of systemic

Cao, S.; Zheng, Y.; Yang Z.; Tang, S.; Jin, P.; Wang, K. & Wang, X. (2008). Effect of methyl

Cao, S.; Hu, Z.C.; Zheng, Y.H.; Li, X.W.; Wang, H.O. & Pang, B. (2010). Effect of post-harvest

Chalutz, E. & Stahmann, M. (1969). Induction of pisatin by ethylene. *Phytopathology*, Vol.59,

Chappell, J. & Hahlbrock, K. (1984). Transcription of plant defence genes in response to UV

Cole, D.L. (1999). The efficacy of acibenzolar-S-methyl, an inducer of systemic acquired

Conconi, A.; Smerdon, M.J.; Howe, G.A. & Ryan, C.A. (1996). The octadecanoid signaling

Constabel, C.P. & Ryan C.A. (1998). A survey of wound and methyl jasmonate induced leaf polyphenol oxidase in crop plants. *Phytochemistry*, Vol.47, pp. 507-511 Creelman, R.A.; Tierney, M.L. & Mullet, J.E. (1992). Jasmonic acid/methyl jasmonate

Creelman R.A. & Mullet J.E. (1997). Biosynthesis and action of jasmonates in plants. *Annual Review of Plant Physiology and Plant Molecular Biology*, Vol.48, pp. 355-381 Dann, E.; Diers, B.; Byrum, J. & Hammerschmidt, R. (1998). Effect of treating soybean with

Darras, A.I. (2003). Biology and management of freesia flower specking caused by *Botrytis* 

Darras, A.I.; Joyce, D.C. & Terry, L.A. (2004). A survey of possible associations between pre-

Darras, A.I.; Terry, L.A. & Joyce, D.C. (2005). Methyl jasmonate vapour treatment

studies. *European Journal of Plant Pathology*, Vol.104, pp. 271-278

*Australian Journal of Experimental Agriculture*, Vol.44, pp. 103-108

*Postharvest Biology and Technology*, Vol.38, pp. 175-182

*cinerea*. PhD Thesis, Cranfield University, UK

*European Journal of Plant Pathology*, Vol.106, pp. 529-536

light or fungal elicitor. *Nature*, Vol.311, pp. 76-78

307.

185-190

pp. 1972-1973

pp. 267-273

226-229

4941

disease. *European Journal of Plant Pathology*, Vol.108, pp. 41-49

the accumulation of defense-related enzymes in apple and protects from fire blight.

acquired resistance in pepper plants by acibenzolar-S-methyl against bacterial spot

jasmonate on the inhibition of *Colletotrichum acutatum* infection in loquat fruit and the possible mechanisms. *Postharvest Biology and Technology,* Vol.49, pp. 301-

treatment with BTH on fruit decay, microbial populations, and the maintenance of quality in strawberry*. Journal of Horticultural Science & Biotechnology*, Vol.85(3), pp.

resistance, against bacterial and fungal diseases of tobacco. *Crop Protection*, Vol.18,

pathway in plants mediates a response to ultraviolet radiation. *Nature*, Vol.383, pp.

accumulate in wounded soybean hypocotyls and modulated wound gene expression. *Proceedings of the National Academy for Science U.S.A.*, Vol.89, pp. 4938-

2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH) on seed yields and the level of disease caused by *Sclerotinia sclerotiorum* in field and greenhouse

harvest environment conditions and postharvest rejections of cut freesia flowers.

suppresses specking caused by *Botrytis cinerea* on cut *Freesia hybrida* L. flowers.


Novel Elicitors Induce Defense Responses in Cut Flowers 109

Gondim, D.M.F.; Terao, D.; Martins-Miranda, A.S.; Vansconcelos, I.M. & Oliveira, J.T.A.

Gorlach, J.; Volrath, S.; Knauf-Beiter, G.; Hengy, G.; Beckhove, U.; Kogel, K.-H.; Oostendorp,

Govrin, E.M. & Levine, A. (2002). Infection of Arabidopsis with a necrotrophic pathogen,

Gundlach, H.; Muller, M.J.; Kutchan, T.M. & Zenk, M.H. (1992). Jasmonic acid is a signal

Gupta, V.; Willits, M.G. & Glazebrook, J. (2000). *Arabidopsis thaliana* EDS4 contributes to

Hahlbrock, K. & Scheel, D. (1989). Physiology and molecular biology of phenylpropanoid

Herms, D.A. & Mattson, W.J. (1992). The dilemma of plants: to grow or defend. *The* 

Huang, Y.; Deverall, B.J.; Tang, W.H.; Wang, W. & Wu, F.W. (2000). Foliar application of

Iriti, M.; Rossoni, M.; Borgo, M. & Faoro, F. (2004). Benzothiadiazole enhanves

Ishii, H.; Tomita, Y.; Horio, T.; Narusaka, Y.; Nakazawa, Y.; Nishimura, K. & Iwamoto, S.

Japanese pear diseases. *European Journal of Plant Pathology*, Vol.105, pp. 77-85 Jacobsen, B.J. & Backman, P.A. (1993). Biological and cultural plant disease controls:

from disease. *European Journal of Plant Pathology*, Vol.106, pp. 651-656 Il'inskaya, L.I.; Goenburg, E.V.; Chalenko, G.I. & Ozeretskovskaya, O.L. (1996). Involvement

*Russian Journal of Plant Physiology*, Vol.43(5), pp. 622-628

Isaac, S. (1992). *Fungal-Plant Interactions*. Chapman & Hall, London, UK

acquired resistance (SAR). *Plant Molecular Biology*, Vol. 48, pp. 267-276 Grisebach, H. (1981). *Lignins*. In 'The Biochemistry of Plants' (Ed. E.E. Conn). Chapter 7, pp.

melon seedlings. *Journal of Phytopathology*, Vol.156(10), pp. 607-614

disease resistance in wheat. *Plant Cell*, Vol.8, pp. 629-643

457-478, Academic Press, New York, USA

*Quarterly Review of Biology*, Vol.67, pp. 283-335

*of Science USA*, Vol.89, pp. 2389-2393

Vol.13, pp. 503-5011

347- 369

pp.4406-4413

311-315

397-405

mildew of crucifers caused by *Peronospora parasitica*. *Crop Protection*, Vol.18, pp.

(2008). Benzo-thiadiazole-7-carbothioic acid S-methyl ester does not protect melon fruits against *Fusarium pallidoroseum* infection but induces defence responses in

M.; Staub, T.; Ward, E.; Kessmann, H. & Ryals, J. (1996). Benzothiadiazole, a novel class of inducers of systemic acquired resistance, activates gene expression and

*Botrytis cinerea*, elicits various defense responses but does not induce systemic

transducer in elicitor-induced plant cell cultures. *Proceedings of the National Academy* 

salicylic acid (SA)-dependent expression of defense responses: evidence for inhibition of jasmonic acid signaling by SA. *Molecular Plant Microbe Interactions*,

pathway. *Annual Review of Plant Physiology and Plant Molecular Biology*, Vol.40, pp.

acibenzolar-S-methyl and protection of postharvest rock melons and Hami melons

of jasmonic acid in the induction of potato resistance to Phytophthora infection.

resveratrol and anthocyanin biosynthesis in grapevine, meanwhile improving resistanve to *Botrytis cinerea. Journal of Agricultural and Food Chemistry,* Vol.52,

(1999). Induced resistance of acibenzolar-S-methyl (CGA 245704) to cucumber and

alternative and supplements to chemicals in IPM systems. *Plant Disease*, Vol.77, pp.


Ebel, J. & Cosio, E.G. (1994). Elicitors of plant defense responses. *International Review of* 

Elad, Y. (1988). Latent infection of *Botrytis cinerea* in rose flowers and combined chemical and physiological control of the disease. *Crop Protection*, Vol.7, pp. 361-366 Elad, Y.; Kirshner, B. & Gotlib, Y. (1993). Attempts to control *Botrytis cinerea* on roses by pre-

Elad, Y. (1997). Responses of plants to infection by *Botrytis cinerea* and novel means

Elmer, W.H. (2006a). Efficacy of preplant treatments of gladiolus corms with combinations

Elmer, W.H. (2006b). Effects of acibenzolar-S-methyl on the suppression of Fusarium wilt on

Epple P.; Apel, K. & Bohlmann, H. (1997). Overexpression of an endogenous thionin

Evans, A. & van der Ploeg, R. (2008). Auctions around the world. *FloraCulture International*,

Eyre, J.X.; Faragher, J.; Joyce, D.C. & Franz, P.R. (2006). Effects of postharvest jasmonate

Farmer, E.E. & Ryan, C.A. (1992). Octadecanoid precursors of jasmonic acid activate the synthesis of wound-inducible proteinase inhibitors. *Plant Cell*, Vol.4, pp. 129-134 Flor, H.H. (1971). Current status of the gene-for-gene concept. *Annual Review of* 

Friedrich, L.; Lawton, K.; Ruess, W.; Masner, P.; Specker, N.; Gut Rella, M.; Meiers, B.;

Fritzemeier, K-H.; Cretin, C.; Kombrink, E.; Rohwer, F.; Taylor, J.; Scheel, D. & Hahlbrock, K.

Gast, K. (2001). Methyl jasmonate and long term storage of fresh cut peony flowers. *Acta* 

Garrod, B.; Lewis, B.G.; Brittain, M.J. & Davies, W.P. (1982). Studies on the contribution of

Godard, J.-F.; Ziadi, S.; Monot, C.; Le Corre, D. & Silue, D. (1999). Benzothiadiazole

*uncinatum*). *Australasian Plant Pathology*, Vol.46, pp. 717-723

*Phytophthora infestans. Plant Physiology*, Vol.85, pp. 34-41

and postharvest treatments with biological and chemical agents. *Crop Protection*

involved in reducing their susceptibility to infection. *Biological Reviews*, Vol.72,

of acibenzolar-S-methyl and biological or chemical fungicides for suppression of fusarium corm rot [*Fusarium oxysporum* f. sp. *gladioli*]. *Canadian Journal of Plant* 

enhances resistance of Arabidopsis against *Fusarium oxysporum*. *The Plant Cell*,

treatments against *Botrytis cinerea* on Geraldton waxflower (*Chamelaucium* 

Dincher, S.; Staub, T.; Uknes, S.; Metraux, J-P.; Kessmann, H. & Ryals, J. (1996). A Benzothiadiazole derivative induces systemic acquired resistance in tobacco. *Plant* 

(1987). Transient induction of phenylalanine ammonia-lyase and 4-coumarate:CoA legase mRNAs in potato leaves infected with virulent or avirulent races of

lignin and suberin to the impedance of wounded carrot toot tissue to fungal

(BTH) induces resistance in cauliflower (*Brassica oleracea* var *botrytis*) to downy

*Cytology*, Vol.148, pp. 1-36

*Pathology*, Vol.28(4), pp. 609-624

*Phytopathology*, Vol.9, pp. 275-296

*Horticulturae*, Vol.543, pp. 327-330.

invasion. *New Phytologist*, Vol.90, pp. 99-108

*Journal*, Vol.10, pp. 61-72

cyclamen. *Crop Protection*, Vol.25, pp. 671-676

Vol.12, pp. 69-73

Vol.9, pp. 509-520

Vol.5, pp. 8-9

pp. 381-342

mildew of crucifers caused by *Peronospora parasitica*. *Crop Protection*, Vol.18, pp. 397-405


Novel Elicitors Induce Defense Responses in Cut Flowers 111

Kuć, J. (1995). Phytoalexins, stress metabolism and disease resistance in plants. *Annual* 

Lawton, K.A.; Weymann, K.; Friedrich, L.; Vernooij, B.; Uknes, S. & Ryals, J. (1995). Systemic

Lawton, K.; Friedrich, L.; Hunt, M.; Weymann, K.; Delaney, T.; Kessmann, H.; Staub, T. &

Matern, U. & Kneusel, R. (1988). Phenolic compounds in plant disease resistance.

Mauch, F.; Hadwiger, L.A. & Boller, T. (1988). Antifungal hydrolases in pea tissue. 2.

Meir, S.; Droby, S.; Davidson, H.; Alsvia, S.; Cohen, L.; Horev, B. & Philosoph-Hadas, S.

Métraux, J-P. (2001). Systemic acquired resistance and salicylic acid: current state of

Mitter, N.; Kazan, K.; Way, H.M.; Broekaert, W.F. & Manners, J.M. (1998). Systemic

and jasmonic acid in transgenic tobacco. *Plant Science*, Vol.136, pp. 169-180 Narusaka, Y.; Narusaka, M.; Horio, T. & Ishii, H. (1999). Induction of disease resistance in

Nasser, W.; De Tapia, M.; Kauffmann, S.; Montasser-Kouhsari, S. & Burkard, G. (1988).

maize PR proteins are chitinases. *Plant Molecular Biology*, Vol.11, pp. 529-538 Niki, T.; Mitsuhara, I.; Seo, S.; Ohtsubo, N. & Ohashi, Y. (1998). Antagonistic effect of

Nojiri, H.; Sugimori, M.; Yamane, H.; Nishimura, Y.; Yamada, A.; Shibuya, N.; Kodama, O.;

knowledge. *European Journal of Plant Pathology*, Vol.107, pp. 13-18

*Annals of the Phytopathological Society of Japan*, Vol.65, pp. 116-122

methyl jasmonate. *Postharvest Biology and Technology*, Vol.13, pp. 235-243 Meir, S.; Droby, S.; Kochanek, S.; Salim, S. & Philosoph-Hadas, S. (2005) Use of methyl

pods by pathogens and elicitors*. Plant Physiology*, Vol.76, pp. 607-611 Mauch-Mani, B. & Slusarenko, A.J. (1996). Production of salicylic acid precursors is a major

*Peronospora parasitica. The Plant Cell*, Vol.8, pp. 203-212

Lucas, J.A. (1997). *Plant Pathology and Plant Pathogens*. 3rd edition, Blackwell Science, UK Martinez, J.A.; Valdes, R.; Vicente, M.J.; & Banon, S. (2008). Phenotypical differences among

acquired resistance in Arabidopsis requires salicylic acid but not ethylene.

Ryals, J. (1996). Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway. *Plant* 

*Botrytis cinerea* isolates from ornamental plants. *Communications in Agricultural and* 

Inhibition of fungal growth by combinations of chitinase and -1,3-glucanase in pea

function of phenylalanine ammonia lyase in the resistance of Arabidopsis to

(1998). Suppression of Botrytis rot in cut rose flowers by postharvest application of

jasmonate for suppression of Botriytis rot in various cultivars of cut rose flowers.

induction of an Arabidopsis plant defensin gene promoter by tobacco mosaic virus

cucumber by acibenzolar-S-methyl and expression of resistance-related genes.

Identification and characterization of maize pathogenesis related proteins. Four

salicylic acid and jasmonic acid on the expression o pathogenesis-related (PR) protein genes in wounded mature tobacco leaves. *Plant Cell Physiology*, Vol.39(5),

Murofushi, N. & Omori, T. (1996). Involvement of jasmonic acid in elicitor-induced

*Review of Phytopathology*, Vol.33, pp. 275-297

*Applied Biological Sciences*, Vol.73, pp. 121-129

*Phytoparasitica*, Vol.16, pp. 153-170

*Acta Horticulturae*, Vol.669, pp. 91-98

pp. 500-507

*Journal*, Vol.10, pp. 71-82

*Molecular Plant-Microbe Interactions*, Vol.8, pp. 863-870


Jaiti, F.; Verdeil, J.L. & El Hadrami, I. (2009). Effect of jasmonic acid on the induction of

Jarvis, W.R. (1977). *Botryotinia and Botrytis species: taxonomy, physiology, and pathogenicity. A* 

Jarvis, W.R. (1980). *Epidemiology*. In 'The Biology of Botrytis' Eds. Coley-Smith, J.R., Verhoeff, K., and Jarvis, W.R. pp. 219-245, Academic Press, York, London Joyce, D.C. (1993). Postharvest floral organ fall in Geraldton waxflowers (*Chamelaucium* 

Joyce, D.C. & Johnson, G.I. (1999). Prospects for exploitation of natural disease resistance

Katz, V.A.; Thulke, O.U. & Conrath, U. (1998). A Benzothiadiazole primes parsley cells for

Keen, N.T. & Yoshikawa, M. (1983). -1,3-endoglucanase from soybean releases elicitoractive carbohydrates from fungus cell walls. *Plant Physiology*, Vol.71, pp. 460-465 Keller, H.; Pamboukdjian, N.; Ponchet, M.; Poupet, A.; Delon, R.; Verrier, J-L.; Roby, D. &

Kendra, D.F. & Hadwiger, L.A. (1984). Characterization of the smallest chitosan oligomer

Kessmann, H.; Staub, T.; Hofmann, C.; Maetzke, T. & Herzog, J. (1994). Induction of

Kessmann, H.; Oostendorp, M.; Ruess, W.; Staub, T.; Kunz, W. & Ryals, J. (1996). Systemic

Kombrink, E. & Hahlbrock, K. (1986). Responses of cultured parsley cells to elicitors fro

Kombrink, E. & Somssich, I. (1995). Defense responses of plants to pathogens. *Advances in* 

Kombrink, E. & Schmelzer, E. (2001). The hypersensitive response and its role in local

Kuć, J. (1991). *Phytoalexins: perspectives and prospects*. In 'Mycotoxins and phytoalexins' (Eds. Sharma, R., and Salunkhe, D.), . pp. 595-603, Boca Raton: CRC, USA

*Pisum sativum*. *Experimental Mycology*, Vol.8, pp. 276-282

*Pathology*, Vol.74(1), pp. 84-90

Monograph No 15

*Cell*, Vol.11, pp. 223-235

*Phytopathology*, Vol.32, pp. 439-459

*Plant Physiology*, Vol.81, pp. 216-221

*Botanical Research*, Vol.21, pp. 2-34

487

48

1339

pp. 10-13

69-78

polyphenoloxidase and peroxidase activities in relation to date palm resistance against *Fusarium oxysporum* f. sp. *albedinis*. *Physiological and Molecular Plant* 

*guide to the literature*. Research Branch Canada Department of Agriculture,

*uncinatum* Schauer). *Australian Journal of Experimental Agriculture*, Vol.33, pp. 481-

in harvested horticultural crops. *Postharvest News and Information*, Vol.10, pp. 45-

augmented elicitation of defense responses. *Plant Physiology*, Vol.117, pp. 1333-

Ricci, P. (1999). Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance. *The Plant* 

that is maximally antifungal to *Fusarium solani* and elicits pisatin formation in

systemic acquired disease resistance in plants by chemicals. *Annual Review of* 

activated resistance – a new technology for plant disease control. *Pesticide Outlook*

phytopathogenic fungi. Timing and dose dependency of elicitor-induced reactions.

and systemic disease resistance. *European Journal of Plant Pathology*, Vol.107, pp.


Novel Elicitors Induce Defense Responses in Cut Flowers 113

Pieterse, C.M.J.; van Wees, S.; van Pelt, J.A.; Knoester, M.; Laan, R.; Gerrits, H.; Weisbeek,

Rao, M.V.; Lee, H-I.; Creelman, R.A.; Mullet, J.E. & Davis, K.R. (2000). Jasmonic acid

Redolfi, P. (1983). Occurrence of pathogenesis related and similar proteins in different plant

Rojo, E.; Titarenko, E.; Leon, J.; Berger, S.; Vancanneyt, G. & Sanchez-Serrano, J.J. (1998).

Rojo, E.; Leon, J. & Sanchez-Serrano, J.J. (1999). Cross-talk between wound signaling

Ross, A.F. (1961). Systemic acquired resistance induced by localized virus infections in

Ruess, W.; Mueller, K.; Knauf-Beiter, G.; Kenz, W. & Staub, T. (1996). Plant activator CGA

Ryals, J.A.; Ward, E. & Metraux J.P. (1992). *Systemic acquired resistance: An inducible defense* 

Ryals, J.A.; Neuenschwander, U.H.; Willits, M.G.; Molina, A.; Steiner, H-Y. & Hunt, M.D. 1996. Systemic Acquired Resistance. *The Plant Cell*, Vol.8, pp. 1809-1819 Salinas, J.; Glandorf, D.C.M.; Picavet, F.D. & Verhoeff, K. (1989). Effects of temperature,

Salinas, J. & Verhoeff, K. (1995). Microscopic studies of the infection of gerbera flowers by *Botrytis cinerea. European Journal of Plant Pathology*, Vol.101, pp. 377-386 Seglie, L.; Spadano, D.; Devecchi, M.; Larcher, F.; Gullino, M.L. (2009). Use of 1-

Schaller, A. & Ryan, C.A. (1995). Systemin – a polypeptide defense signal in plants.

Schroder, M.; Hahlbrock, K. & Kombrink, E. (1992). Temporal and spatial patterns of 1,3-β-

Sembdner, G. & Parthier, B. (1993). The biochemistry and the physiological and molecular

species. *Netherlands Journal of Plant Pathology*, Vol.89, pp. 245-254

systemic resistance in Arabidopsis. *The Plant Cell*, Vol.10, pp. 1571-1580 Pieterse, C.M.J. & van Loon, L.C. (1999). Salicylic acid-independent plant defence pathways.

*Trends in Plant Science*, Vol.4(2), pp. 52-58

pp. 1633-1646

1996

51-64

Cambridge UK

*Journal*, Vol.13, pp. 153-165

*The Plant Journal*, Vol.20, pp. 136-142

plants. *Virology*, Vol.14, pp. 341-358

*Mediterranea*, Vol.48, pp. 253-261

*infestans. Plant Journal*, Vol.2, pp. 161-172

*BioEssays*, Vol.18(1), pp. 27-33

Vol.44, pp. 569-589

P.J. & van Loon L.C. (1998). A novel signalling pathway controlling induced

signaling modulates ozone-induced hypersensitive cell death. *The Plant Cell*, Vol.12,

Reversible protein phosphorylation regulates jasmonic acid-dependent and independent wound signal transduction pathways in *Arabidopsis thaliana. The Plant* 

pathways determines local versus systemic gene expression in *Arabidopsis thaliana.* 

245704: an innovative approach for disease control in cereals and tobacco. *Brighton Crop Protection Conference-Pests and Diseases*, Vol.2A-6, pp. 53-60, 18-21 November

*mechanism in plants*. In 'Inducible Plant Proteins: Their Biochemistry and Molecular Biology', (Ed. J.L. Wray), pp. 205-229, Cambridge University Press,

relative humidity and age of conidia on the incidence of specking on gerbera flowers caused by *Botrytis cinerea. Netherlands Journal of Plant Pathology*, Vol.95, pp.

methylcyclopropene for the control of *Botrytis cinerea* on cut flowers. *Phytopathologia* 

glucanase and chitinase induction in potato leaves infected by *Phytophthora* 

actions of jasmonates. *Annual Review of Plant Physiology and Plant Molecular Biology*,

phytoalexin production in suspension-cultured rice cells. *Plant Physiology*, Vol.110, pp. 387-392


Nurnberger, T.; Nennstiel, D.; Jabs, T.; Sacks, W.R.; Hahlbrock, K. & Scheel, D. (1994). High

O'Donnell, P.J.; Calvert, C.; Atzorn, R.; Wasternack, C.; Leyser, H.M.O. & Bowles, D.J.

O'Neil, T.M.; Hanks, G.R. & Wilson, T.W. (2004). Control of smoulder (*Botrytis narcissicola*) in narcissus with fungicides. *Annals of Applied Biology*, Vol.145, pp. 129-137 Pallas, J.A.; Paiva, N.L.; Lamb, C. & Dixon, R.A. (1996). Tobacco plants epigenetically

Parthier, B. (1991). Jasmonates, new regulators of plant growth and development: many

Pappas, A.C. (1997). Evolution of fungicide resistance in *Botrytis cinerea* in protected crops in

Pearce, G.; Strydom, D.; Johnson, S. & Ryan, C.A. (1991). A polypeptide from tomato leaves

Pena-Cortes, H.; Albrecht, T.; Prat, S.; Weiler, E.W. & Willmitzer, L. (1993). Aspirin prevents

Penninckx, I.A.M.A.; Eggermont, K.; Terras, F.R.G.; Thomma, B.P.H.J.; De Samblanx, G.W.;

Pichay, D.S.; Frantz, J.M.; Locke, J.C.; Krause, C.R. & Fernandez, G.C.J. (2007). Impact of

Pie, K. & de Leeuw, G.T.N. (1991). Histopathology of the initial stages of interaction between

Pieterse, C.M.J.; van Wees, S.; Hoffland, E.; van Pelt, J.A. & van Loon, L.C. (1996). Systemic

salicylic acid-independed pathway. *The Plant Cell*, Vol.8, pp. 2309-2323 Perrin, D.R. & Cruickshank, I.A.M. (1965). Studies on phytoalexins VII. The chemical

induces wound-inducible proteinase inhibitor proteins. *Science*, Vol.253, pp. 895-

wound-induced gene expression in tomato leaves by blocking jasmonic acid

Buchala, A.; Metraux, J-P.; Manners J.M. & Broekaert, W.F. (1996). Pathogeninduced systemic activation of a plant defensin gene in Arabidopsis follows a

stimulation of pisatin formation in *Pisum sativum* L. *Austrian Journal of Biological* 

applied nitrogen concentration on growth of elatior Begonia and new guinea impatiens and susceptibility of Begonia to *Botrytis cinerea*. *Journal of the American* 

rose flowers and *Botrytis cinerea. Netherlands Journal of Plant Pathology*, Vol.97, pp.

resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis related gene expression. *The Plant Cell*, Vol.8,

facts and few hypotheses on their actions. *Botanica Acta* pp. 446-454

triggers multiple defenses responses. *The Plant Cell*, Vol.78, pp. 449-460 Nurnberger, T. & Scheel, D. (2001). Signal transmission in the plant immune response.

*Trends in Plant Science*, Vol.6(8), pp. 372-379

Greece. *Crop Protection*, Vol.16, pp. 257-263

biosynthesis. *Planta,* Vol.191, pp. 123-128

*Society for Horticultural Science*, Vol.132, pp. 193-201

*Science*, Vol.274, pp. 1914-1917

*Journal*, Vol.10, pp. 281-293

*Science*, Vol.18, pp. 803-816

898

335-344

pp. 1225-1237

pp. 387-392

phytoalexin production in suspension-cultured rice cells. *Plant Physiology*, Vol.110,

affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes

(1996). Ethylene as a signal mediating the wound response of tomato plants.

suppressed in phenylalanine ammonia lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus. *The Plant* 


Novel Elicitors Induce Defense Responses in Cut Flowers 115

Thomma, B. P.H.J.; Penninckx, I.A.M.A.; Broekaert, W.F. & Cammue, B.P.A. (2001). The

Titarenko, E.; Rojo, E.; Leon, J. & Sanchez-Serrano, J.J. (1997). Jasmonic acid-dependent and

Tomas, A.; Wearing, A.H. & Joyce, D.C. (1995). *Botrytis cinerea*: a causal agent of premature

Tosi, L. & Zazzerini, A. (1999). Benzothiadiazole induces resistance to *Plasmopara helianthi* in

Van Loon, L.C. & Gerritsen, Y.A.M. (1989). Localization of pathogenesis-related proteins in

Van Loon, L.C.; Pierpoint, W.S.; Boller, T. & Conejero, V. (1994). Recommendations for

Van Loon, L.C. (1997). Induced resistance in plants and the role of pathogenesis-related

Van Meeteren, U. (2009). Causes of quality loss of cut flowers - A critical analysis of

Vernooij, B.; Friedrich, L.; Morse, A.; Reist, R., Kolditz-Jawhar, R.; Ward, E.; Uknes, S.;

Ward, E.R.; Uknes, S.J.; Williams, S.C.; Dincher, S.S.; Wiederhold, D.L.; Alexander, D.C.;

Wegulo, S.N. & Vilchez, M. (2007). Evaluation of lisianthus cultivars for resistance to *Botrytis* 

Wasternack, C. & Parthier, B. (1997). Jasmonate-signalled plant gene expression. *Trends in* 

Williamson, B.; Duncan, G.H.; Harrison, J.G.; Harding, L.A.; Elad, Y. & Zimand, G. (1995).

Xu, Y.; Linda Chang, P-F.; Liu, D.; Narasimhan, M.L.; Raghothama, K.G.; Hasegawa, P.M. &

Yao, H. & Tian, S. (2005). Effects of pre- and post-harvest application of salicylic acid or

Yoshikawa, M.; Yamaoka, N. & Takeuchi, Y. (1993). Elicitors: their significance and primary

*Arabidopsis thaliana. Plant Physiology*, Vol.115, pp. 817-826

sunflower plants*. Journal of Phytopathology*, Vol.147, pp. 365-370

reaction to tobacco mosaic virus. *Plant Science*, Vol.63, pp. 131-140

proteins. *European Journal of Plant Pathology*, Vol.103, pp. 753-765

postharvest treatments. *Acta Horticulturae*, Vol.847, pp. 27-36

transduction. *The Plant Cell*, Vol.6, pp. 959-965

*cinerea*. *Plant Disease*, Vol.91, pp. 997-1001

*cinerea. Mycological Research*, Vol.99, pp. 1303-1310

and methyl jasmonate. *The Plant Cell*, Vol.6, pp. 1077-1085

*Postharvest Biology and Technology*, Vol.35, pp. 253-262

*Plant Science*, Vol.2, pp. 302-307

Vol.34, pp. 1163-1173

Vol.13, pp. 63-68

pp. 26-28

pp. 245-264

1094

complexity of disease signaling in Arabidopsis*. Current Opinion in Immunology*,

independent signaling pathways control wound-induced gene activation in

flower drop in packaged Geraldton waxflower. *Australasian Plant Pathology*, Vol.24,

infected and non-infected leaves of Samsun NN tobacco during the hypersensitive

naming plant pathogenesis-related proteins. *Plant Molecular Biology Reporter*, Vol.12,

Kessmann, H. & Ryals, J. (1994). Salicylic acid is not the translocated signal responsible for inducing systemic acquired resistance but is required in signal

Ahi-Goy, P.; Metraux, J.P. & Ryals, J.A. (1991). Coordinate gene activity in response to agents that induce systemic acquired resistance. *The Plant Cell*, Vol.3, pp. 1085-

Effect of humidity on infection of rose petals by dry-inoculated conidia of *Botrytis* 

Bressan, R.A. (1994). Plant defense genes are synergistically induced by ethylene

methyl jasmonate on inducing disease resistance of sweet cherry fruit in storage.

modes of action in the induction of plant defense reactions. *Plant Cell Physiology*,


Sharkey, T.D. (1996). Emission of low molecular mass hydrocarbons from plants. *Trends in* 

Sharp, J.; Albersheim, P.; Ossowski, O.; Pilotti, A.; Garegg, P. & Lindberg, B. (1984).

Shulaev, V.; Silverman, P. & Raskin, I. (1997). Airborne signaling by methyl salicylate in

Staswick, P.E. (1990). Novel regulation of vegetative storage protein genes. *The Plant Cell*,

Stermer, B.A. (1995). *Molecular regulation of systemic induced resistance*. In "Induced Resistance

Sticher, L.; Mauch-Mani, B. & Metraux, J.P. (1997). Systemic Acquired Resistance. *Annual* 

Taiz, L. & Zeiger, E. (1998). *Plant Physiology.* 2nd ed. pp. 61-80, Sinauer Associates, Inc.,

Terry, L.A. & Joyce, D.C. (2000). *Suppression of grey mould on strawberry fruit with the chemical plant activator acibenzolar.* Pest Management Science*, Vol.56, pp. 989-992*  Terry, L.A. (2002). Natural disease resistance in strawberry fruit and Geraldton waxflower

Terry, L.A. & Joyce, D.C. (2004a) Elicitors of induced disease resistance in postharvest

Terry, L.A. & Joyce, D.C. (2004b) Influence of growing conditions on efficacy of acibenzolar

Thaler, J.S.; Stout, M.J.; Karban, R. & Duffey, S.S. (1996). Exogenous jasmonates simulate

Thaler, J.S.; Fidantsef, A.L.; Duffey, S.S. & Bostock R.M. (1999). Trade-offs in plant defense

Thomma, B. P.H.J.; Eggermont, K.; Broekaert, W. & Cammue, B.P.A. (2000). Disease

methyl jasmonate. *Plant Physiology and Biochemistry*, Vol.38, pp. 421-427.

induced resistance. *Journal of Chemical Ecology*, Vol.25, pp. 1597-1609 Thomma, B. P.H.J.; Eggermont, K.; Penninckx, I.A.M.A.; Mauch-Mani, B.; Vogelsang, R.;

horticultural crops: a brief review. *Postharvest Biology and Technology*, Vol.32, pp. 1-

and botryticides in suppression of *Botrytis cinerea* on strawberry fruit. *Advances in* 

insect wounding in tomato plants (*Lycopersicon esculentum*) in the laboratory and

against pathogens and herbivores: a field demonstration of chemical elicitors of

Cammue, B.P.A. & Broekaert, W. (1998). Separate jasmonate-dependent and salicylate-dependent defense response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. *Proceedings of the National Academy of* 

development of several fungi on Arabidopsis can be reduced by treatment with

Strider, D.L. (1985). *Diseases of Floral Crops*. Vol. 2, Praeger Publishers, New York, USA Suo, Y.; & Leung, D.W.M. (2002). BTH-induced accumulation of extracellular proteins and

blackspot disease in rose. *Biologia Plantarum*, Vol.45, pp. 273-279

plant pathogen resistance. *Nature*, Vol.385, pp. 718-721

*Review of Phytopathology*, Vol.35, pp. 235-270

flowers. PhD Thesis, Cranfield University, UK

field. *Journal of Chemical Ecology*, Vol.22, pp. 1767-1781

*Strawberry Research*, Vol.23, pp. 11-19

*Science USA*, Vol.95, pp. 15107-15111

Comparison of the structure of elicitor activities of a synthetic and mycelial-wallderived hexa-(-D-glucopyranosyl)-D-glusitol. *Journal of Biological Chemistry*,

to Disease in Plants". Eds. R. Hammerscmidt and Kuć, J. pp. 111-140, Kluwer

*Plant Science*, Vol.3, pp. 78-82

Vol.259, pp. 11341-11345

Academic Publishers, USA

Vol.2, pp. 1-6

Publishers, USA

13


**5** 

*China* 

Jing Yang and Chengyun Li

**Functional Identification of Genes Encoding** 

In the course of coevolution of plants and pathogens for many millions of years, plants possessed many kinds of recognition and resistance mechanisms to prevent or limit pathogen infection. At the same time, pathogen also initiated many pathogenicity mechanisms, such as development of specialized infection structures, secretion of hydrolytic enzymes, production of host selective toxins, and detoxification of plant antimicrobial compounds (Idnurm and Howlett, 2001; Talbot, 2003; Randall et al., 2005), to avoid or overcome plant resistance mechanism. However, filamentous pathogen including fungi and oomycete could secrete a diverse array of effector proteins into the plant cell to manipulate the plant innate immunity, which facilitates the pathogen to successfully colonize and reproduce (Birch et al., 2006; Chisholm et al., 2006; Kamoun, 2006; O'Connell and Panstruga, 2006; Catanzariti et al., 2007; Kamoun, 2007). Several studies have shown that effector proteins could play dual role as both toxins and inducers of host resistance. Effector proteins were regarded as functioning primarily in virulence, but they also could elicit innate

Rice blast caused by *Magnaporthe oryzae* (Couch and Kohn, 2002) is the most devastating fungal disease of rice (*Oryza sativa*; Zeigler et al., 1994; Talbot, 2003). Functional identification of *M. oryzae* effectors can elucidate some pathogenicity mechanisms of the blast fungus, providing a clue to better manage blast disease. Several *Avr* genes have been cloned and characterized from *M.oryzae*, such as *Avr-Pita* (Orbach et al., 2000; Valent et al., 1991), *Avr1-CO39* (Farman and Leong, 1998), *Ace1* (Bohnert et al., 2004; Collemare et al., 2008) and the *Pwl* effectors (Kang et al.,1995; Sweigard et al., 1995). The *Avr-Pita* effector appears homologous to fungal zinc-dependent metalloproteases and is dispensable for virulence on rice (Jia et al., 2001; Orbach et al., 2000). *Avr-Pita* interacts with the cognate resistance protein *Pi-ta* (Jia et al., 2000). *Avr-Pita1* (*Avr-Pita*) including *Avr-Pita2* and *Avr-Pita3* are *Avr-Pita* family (Khang et al., 2008). *Avr-Pita2* acts as an elicitor of defense responses mediated by *Pi-t*a, while *Avr-Pita3* does not. Members of *Avr-P*ita family are detected among blast isolates isolated from different kinds of hosts by PCR-based method. *Ace1* effector is a putative cytoplasmic fusion polypeptide containing a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS), two distinct classes of enzymes that are involved in the production of microbial secondary metabolites (Bohnert et al., 2004;

immunity in plant varieties carrying corresponding resistance protein.

**1. Introduction** 

**Effector Proteins in** *Magnaporthe oryzae*

*Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming, Yunnan* 

Ziadi, S.; Barbedette, S.; Godard, J.F.; Monot, C.; Le Corre, D. & Silue, D. (2001). Production of pathogenesis-related proteins in the cauliflower (*Brassica oleracea* var. *botrytis*) downy mildew (*Peronospora parasitica*) pathosystem treated with acibenzolar-Smethyl. *Plant Pathology*, Vol.50, pp. 579-586

### **Functional Identification of Genes Encoding Effector Proteins in** *Magnaporthe oryzae*

Jing Yang and Chengyun Li

*Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming, Yunnan China* 

#### **1. Introduction**

116 Plant Pathology

Ziadi, S.; Barbedette, S.; Godard, J.F.; Monot, C.; Le Corre, D. & Silue, D. (2001). Production

methyl. *Plant Pathology*, Vol.50, pp. 579-586

of pathogenesis-related proteins in the cauliflower (*Brassica oleracea* var. *botrytis*) downy mildew (*Peronospora parasitica*) pathosystem treated with acibenzolar-S-

> In the course of coevolution of plants and pathogens for many millions of years, plants possessed many kinds of recognition and resistance mechanisms to prevent or limit pathogen infection. At the same time, pathogen also initiated many pathogenicity mechanisms, such as development of specialized infection structures, secretion of hydrolytic enzymes, production of host selective toxins, and detoxification of plant antimicrobial compounds (Idnurm and Howlett, 2001; Talbot, 2003; Randall et al., 2005), to avoid or overcome plant resistance mechanism. However, filamentous pathogen including fungi and oomycete could secrete a diverse array of effector proteins into the plant cell to manipulate the plant innate immunity, which facilitates the pathogen to successfully colonize and reproduce (Birch et al., 2006; Chisholm et al., 2006; Kamoun, 2006; O'Connell and Panstruga, 2006; Catanzariti et al., 2007; Kamoun, 2007). Several studies have shown that effector proteins could play dual role as both toxins and inducers of host resistance. Effector proteins were regarded as functioning primarily in virulence, but they also could elicit innate immunity in plant varieties carrying corresponding resistance protein.

> Rice blast caused by *Magnaporthe oryzae* (Couch and Kohn, 2002) is the most devastating fungal disease of rice (*Oryza sativa*; Zeigler et al., 1994; Talbot, 2003). Functional identification of *M. oryzae* effectors can elucidate some pathogenicity mechanisms of the blast fungus, providing a clue to better manage blast disease. Several *Avr* genes have been cloned and characterized from *M.oryzae*, such as *Avr-Pita* (Orbach et al., 2000; Valent et al., 1991), *Avr1-CO39* (Farman and Leong, 1998), *Ace1* (Bohnert et al., 2004; Collemare et al., 2008) and the *Pwl* effectors (Kang et al.,1995; Sweigard et al., 1995). The *Avr-Pita* effector appears homologous to fungal zinc-dependent metalloproteases and is dispensable for virulence on rice (Jia et al., 2001; Orbach et al., 2000). *Avr-Pita* interacts with the cognate resistance protein *Pi-ta* (Jia et al., 2000). *Avr-Pita1* (*Avr-Pita*) including *Avr-Pita2* and *Avr-Pita3* are *Avr-Pita* family (Khang et al., 2008). *Avr-Pita2* acts as an elicitor of defense responses mediated by *Pi-t*a, while *Avr-Pita3* does not. Members of *Avr-P*ita family are detected among blast isolates isolated from different kinds of hosts by PCR-based method.

> *Ace1* effector is a putative cytoplasmic fusion polypeptide containing a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS), two distinct classes of enzymes that are involved in the production of microbial secondary metabolites (Bohnert et al., 2004;

Functional Identification of Genes Encoding Effector Proteins in *Magnaporthe oryzae* 119

Fig. 1. Length distribution of predicted secretory (Sec-type) signal peptide of short protein in

Because signal peptide has common application value in exogenous gene expression, it was necessary to analyze their composition and structure. We analyzed amino acid composition of 116 Sec-type signal peptides sequence. Frequency of 20 kinds of amino acids in 116 signal peptides sequences of secreted proteins were analyzed (Figure 2). Result showed that nonpolar amino acids such as alanine, leucine, proline and valine have the highest frequency(45.79%), followed by negatively charged acidic amino acids including aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, threonine, methionine, tryptophan and tyrosine (33.49%). The frequency of polar amino acids (glycine, asparagines, glutarnine, serine) positively charged basic amino acids such as arginine and lysine had the lowest

Fig. 2. Frequency of amino acid in predicted signal-peptide-containing short proteins.

*M. oryzae*.

frequency (15.83%).

Collemare et al., 2008). *Ace1* is thought to function as avirulence indirectly by producing a secondary metabolite that activates Pi33. *Ace1* is only expressed in appressoria, suggesting that the secondary metabolite produced might have a role in virulence (Bohnert et al., 2004; Fudal et al., 2005).The *Pwl* effectors, encoded by the *Pwl* (pathogenicity on weeping lovegrass, *Eragrostis curvula*) gene family, are rapidly evolving, and they are small glycine-rich secreted proteins that commonly distributed in *M.oryzae*. Presently, four genes such as *Pwl1*-*Pwl4* have been determined in *M. oryzae*, and the four genes confer species-specific avirulence on weeping lovegrass and finger millet, but not on rice (Kang et al., 1995; Sweigard et al., 1995). Yoshida *et al*. (2009) examined DNA polymorphisms of 1032 putative secreted proteins from the genome sequence of isolate 70-15 among 46 isolates, and found no association with Avr function on a set of differential rice cultivars carrying different *R* genes, indicating that the isolate 70-15 might have lost several functional *Avr* genes through sexual recombination.

After fungal effector proteins are secreted into plant cells, the question arises: do they mediate virulence or avirulence on host? To discover pathogenicity mechanism of the pathogen, it is indispensable to identify the function of effector proteins. Here we will introduce our studies on functional identification of effector proteins from *M. oryzae*.

#### **2. Screening candidate effector-encoding genes from** *M. oryzae*

Whole-genome sequence of fungal pathogens has provided an enormous amount of data that can be analyzed for mining putative secreted effector proteins. *M. oryzae* genome sequence has been available online, which contribute many novel effector-encoding genes. Some online softwares could be aided to predict some features such as secretion, domain and homology of effector proteins. Presently, secreted proteins are categorized into two classes based on their secreted pathway, one is classically secreted proteins, with N-terminal signal peptide, and the other is non-classically secreted proteins, whose secreted pathway is known as leaderless secretion (Nickel, 2003). Combination of SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 (http://www.cbs.dtu.dk/ services/) are used to predict classically secreted effector proteins. Non-classical secreted proteins were further predicted using SecretomeP 2.0 Server (http://www.cbs.dtu.dk/services/).

Total of 12,595 putative proteins including 1,486 small proteins from *M. oryzae* genome database were predicted. Of which, 1,134 putative proteins were predicted for classically secreted proteins with N-terminal signal peptide. Here, we will focus on small secreted proteins (amino acid length <100), there were 119 classically secreted proteins among 1,486 small proteins. Among 119 effector proteins, 116 effectors had a Sec-type signal peptides, and had common A-X-A motif, X stand for any amino acid residue, C-domain of the signal peptide could be cleaved by one of the various type I SPase of *B.Subtilis* (Tjalsma et al., 1997; 1998; 1999). In C-domain, uncharged residues were present at the -1 and -3 positions, high frequency of leucine (29%) was at -2 position. Frequency of alanine at -1 position was 71%, and the other 19 amino acid residues occurred at +1 position except cysteine (C).Most of secretory proteins with this signal peptide are secreted into the extracellular environment. Length of signal peptides of 116 secretory proteins centralized in 16~22 amino acid residues, signal peptides with 18 amino acid residues reached the highest amounts, the second was signal peptide with 19 amino acid residues. Signal peptide with the most length was composed of 36 amino acid residues, and the shortest signal peptide was composed of 15 amino acid residues (Figure 1).

Collemare et al., 2008). *Ace1* is thought to function as avirulence indirectly by producing a secondary metabolite that activates Pi33. *Ace1* is only expressed in appressoria, suggesting that the secondary metabolite produced might have a role in virulence (Bohnert et al., 2004; Fudal et al., 2005).The *Pwl* effectors, encoded by the *Pwl* (pathogenicity on weeping lovegrass, *Eragrostis curvula*) gene family, are rapidly evolving, and they are small glycine-rich secreted proteins that commonly distributed in *M.oryzae*. Presently, four genes such as *Pwl1*-*Pwl4* have been determined in *M. oryzae*, and the four genes confer species-specific avirulence on weeping lovegrass and finger millet, but not on rice (Kang et al., 1995; Sweigard et al., 1995). Yoshida *et al*. (2009) examined DNA polymorphisms of 1032 putative secreted proteins from the genome sequence of isolate 70-15 among 46 isolates, and found no association with Avr function on a set of differential rice cultivars carrying different *R* genes, indicating that the isolate 70-15 might have lost several functional *Avr* genes through sexual recombination.

After fungal effector proteins are secreted into plant cells, the question arises: do they mediate virulence or avirulence on host? To discover pathogenicity mechanism of the pathogen, it is indispensable to identify the function of effector proteins. Here we will

Whole-genome sequence of fungal pathogens has provided an enormous amount of data that can be analyzed for mining putative secreted effector proteins. *M. oryzae* genome sequence has been available online, which contribute many novel effector-encoding genes. Some online softwares could be aided to predict some features such as secretion, domain and homology of effector proteins. Presently, secreted proteins are categorized into two classes based on their secreted pathway, one is classically secreted proteins, with N-terminal signal peptide, and the other is non-classically secreted proteins, whose secreted pathway is known as leaderless secretion (Nickel, 2003). Combination of SignalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 (http://www.cbs.dtu.dk/ services/) are used to predict classically secreted effector proteins. Non-classical secreted proteins were further predicted

Total of 12,595 putative proteins including 1,486 small proteins from *M. oryzae* genome database were predicted. Of which, 1,134 putative proteins were predicted for classically secreted proteins with N-terminal signal peptide. Here, we will focus on small secreted proteins (amino acid length <100), there were 119 classically secreted proteins among 1,486 small proteins. Among 119 effector proteins, 116 effectors had a Sec-type signal peptides, and had common A-X-A motif, X stand for any amino acid residue, C-domain of the signal peptide could be cleaved by one of the various type I SPase of *B.Subtilis* (Tjalsma et al., 1997; 1998; 1999). In C-domain, uncharged residues were present at the -1 and -3 positions, high frequency of leucine (29%) was at -2 position. Frequency of alanine at -1 position was 71%, and the other 19 amino acid residues occurred at +1 position except cysteine (C).Most of secretory proteins with this signal peptide are secreted into the extracellular environment. Length of signal peptides of 116 secretory proteins centralized in 16~22 amino acid residues, signal peptides with 18 amino acid residues reached the highest amounts, the second was signal peptide with 19 amino acid residues. Signal peptide with the most length was composed of 36 amino acid residues, and the shortest signal peptide was composed of 15

introduce our studies on functional identification of effector proteins from *M. oryzae*.

**2. Screening candidate effector-encoding genes from** *M. oryzae* 

using SecretomeP 2.0 Server (http://www.cbs.dtu.dk/services/).

amino acid residues (Figure 1).

Fig. 1. Length distribution of predicted secretory (Sec-type) signal peptide of short protein in *M. oryzae*.

Because signal peptide has common application value in exogenous gene expression, it was necessary to analyze their composition and structure. We analyzed amino acid composition of 116 Sec-type signal peptides sequence. Frequency of 20 kinds of amino acids in 116 signal peptides sequences of secreted proteins were analyzed (Figure 2). Result showed that nonpolar amino acids such as alanine, leucine, proline and valine have the highest frequency(45.79%), followed by negatively charged acidic amino acids including aspartic acid, glutamic acid, phenylalanine, histidine, isoleucine, threonine, methionine, tryptophan and tyrosine (33.49%). The frequency of polar amino acids (glycine, asparagines, glutarnine, serine) positively charged basic amino acids such as arginine and lysine had the lowest frequency (15.83%).

Fig. 2. Frequency of amino acid in predicted signal-peptide-containing short proteins.

Functional Identification of Genes Encoding Effector Proteins in *Magnaporthe oryzae* 121

microorganisms (Clare et al., 2004). The plasmids of pMALMgNIP04 and pMAL were

MGS0253.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0255.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0274.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0338.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0662.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0703.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0718.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0992.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0997.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1033.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1035.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1195.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1242.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1298.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1344.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1382.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS1473.1 + + + + + + + + + + + + + + + + + + + + + 100 MGS0351.1 II + + + + + + + + + + + + + + + + + + + + + 100

**1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 (%)**

+ + + + + + + + + + + + + + + + + + + + + 100

+ + - + + + + + - - + + + + + + + - + + - 76.2

MGS0004.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS0074.1 + + + - + + + + + + + + + + + + + + + + - 90.5 MGS0123.1 + - + - + + + - + - - - - + + - - + + - + 52.4 MGS0140.1 + + + + - + + - + + + + + + + + + + + + + 90.5 MGS0149.1 + + + + - + + - + + + + + + + + + + + + + 90.5 MGS0398.1 + + + - + + + + + + + + + + + + + + + + + 95.2 MGS0415.1 + + - + + + + + + + + + + + + + + + + + + 95.2 MGS0431.1 + + - + + - + + + + + - + + + + + + + + + 85.7 MGS0621.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS0698.1 - + + + + - + + - + + + + + + + + + + + + 85.7 MGS0879.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS1011.1 + + + + + + + + + + + + + + + + + + - - - 85.7 MGS1041.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS1070.1 + + + + + + + + + + + + + + + + + + + - - 90.5 MGS1078.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS1117.1 + + + + + + + + + + + + + + + + - - - - - 76.2 MGS1172.1 + + + + + + + + + + + + + + + + + + + - - 90.5 MGS1276.1 + + + + + + + + + + + + + + + + + + + - - 90.5 MGS1322.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS1361.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS1392.1 + + + + + + + + + + + + + + + + + + + - - 90.5 MGS1439.1 + + + + + + + + + - + + + + + + + + + - - 90.5 MGS1460.1 + + + + + + + + + + + + + + + + + + + + - 95.2 MGS1470.1 + + + + + + + + + + + + - + + + + + + + - 90.5 MGS1477.1 + + + + + + + + + + + + + + + + + + + + - 95.2 Frequency (%) **97.7 97.7 93.3 93.3 95.6 95.6 100 93.3 95.6 93.3 97.7 95.6 95.6 100 100 97.7 95.6 95.6 97.7 82.2 57.8** -

"+" mean gene was examined in isolates, "-"mean gene was not examined in isolates. Table 1. Frequency of 45 genes in 21 isolates of M. oryzae from Yunnan, China.

**Twenty-one isolates Fre-**

**quency** 

induced to express of fusion proteins of MBP-MgNIP04 andMBP.

Gene code Type

I

III

MGS0011.1

MGS0001.1

Subcellular location of secreted effector proteins can provide important information to explain plant pathogen interactions. SubLoc v1.0 was used to predict subcellular location of 116 secreted effector proteins. Result showed that 50 proteins were secreted extracellularly, 30 proteins were transported into nucleus, 25 effectors were transferred into mitochondria and 11 were translocated into cytoplasm.

#### **3. Polymorphism of effector-encoding genes in blast isolates from Yunnan, China**

To analyze polymorphism distribution of effector-encoding genes in blast isolates from Yunnan, 45 ones from 116 genes were selected as candidates for analyzing polymorphism in 21 isolates from Yunnan. The result showed that each gene appeared in different distribution among 21 isolates, For example, MGS0001.1 was presented in 16 isolates, but not in five isolates. MGS0011.1 was distributed in 21 isolates. Although MGS0351.1 was distributed in 21 isolates with the PCR product size ranging from 350 to 400 base pairs, the reference sequence size of the gene was 412 bp. To explain sequence difference between PCR product and reference sequence of gene, PCR products of MGS0001.1 and MGS0351.1 from three different isolates were cloned and sequenced, respectively, the sequence analysis showed that PCR products sequences of MGS0001.1 from three different isolates appeared high identical with the reference sequence. While PCR product sequences of MGS0351.1 from three different isolates showed fragment- deletion of GTTGTTTTGTTGTT and GTTGTT, comparing with reference sequence, but the deletion occurred in intron region of the gene.

There was three-type polymorphism distribution of 45 genes in 21 blast isolates. The type I included 18 ones among 45 genes, which distributed in 21 isolates, the type II consisted only of MGS0351.1 which was present in 21 isolates, but PCR products showed fragment deletion comparing to reference sequence. The type III consisted of 26 genes that were randomly present in 21 isolates, while not all genes were distributed in 21 isolates. Among 45 effectorencoding genes, MGS0123.1 had the lowest frequency of 52.4%. Many genes could be examined in each isolate, except in isolate 21. Nineteen genes were not determined in the isolate 21. More than 40 genes could be determined in other 44 isolates, and all the 45 genes distributed in isolate 7, 14 and 15 (Table 1).

The results indicated that 45 effector-encoding genes not only had the polymorphism distribution but also appeared conserved in 21 blast isolates. Some genes were not determined in isolates, the reason might be the result of gene evolution during plant-pathogen interaction. Thus, it is essential to analyze their function for conserved or varied genes.

#### **4. Effector-encoding gene cloning and** *in vitro* **functional identification**

We selected 10 effector genes as candidates to clone and functionally identify. The cloned nucleotide sequence of MgNIP04 from Y99-63c was aligned with the short protein, MGS0004previously sequenced.MgNIP04 was identical to MGS0004.MgNIP04 encoding a 96 amino acid protein with unknown domains or motifs. MgNIP04 contained a signal peptide of 20 amino acids at the N-terminus. Subcellular localization prediction suggested that it was a cytoplasmic protein. However, it is not homologous to Nep1-like proteins, a novel class of necrosis-inducing proteins found in a variety of taxonomically unrelated

Subcellular location of secreted effector proteins can provide important information to explain plant pathogen interactions. SubLoc v1.0 was used to predict subcellular location of 116 secreted effector proteins. Result showed that 50 proteins were secreted extracellularly, 30 proteins were transported into nucleus, 25 effectors were transferred into mitochondria

**3. Polymorphism of effector-encoding genes in blast isolates from Yunnan,** 

To analyze polymorphism distribution of effector-encoding genes in blast isolates from Yunnan, 45 ones from 116 genes were selected as candidates for analyzing polymorphism in 21 isolates from Yunnan. The result showed that each gene appeared in different distribution among 21 isolates, For example, MGS0001.1 was presented in 16 isolates, but not in five isolates. MGS0011.1 was distributed in 21 isolates. Although MGS0351.1 was distributed in 21 isolates with the PCR product size ranging from 350 to 400 base pairs, the reference sequence size of the gene was 412 bp. To explain sequence difference between PCR product and reference sequence of gene, PCR products of MGS0001.1 and MGS0351.1 from three different isolates were cloned and sequenced, respectively, the sequence analysis showed that PCR products sequences of MGS0001.1 from three different isolates appeared high identical with the reference sequence. While PCR product sequences of MGS0351.1 from three different isolates showed fragment- deletion of GTTGTTTTGTTGTT and GTTGTT, comparing with reference sequence, but the deletion occurred in intron region of

There was three-type polymorphism distribution of 45 genes in 21 blast isolates. The type I included 18 ones among 45 genes, which distributed in 21 isolates, the type II consisted only of MGS0351.1 which was present in 21 isolates, but PCR products showed fragment deletion comparing to reference sequence. The type III consisted of 26 genes that were randomly present in 21 isolates, while not all genes were distributed in 21 isolates. Among 45 effectorencoding genes, MGS0123.1 had the lowest frequency of 52.4%. Many genes could be examined in each isolate, except in isolate 21. Nineteen genes were not determined in the isolate 21. More than 40 genes could be determined in other 44 isolates, and all the 45 genes

The results indicated that 45 effector-encoding genes not only had the polymorphism distribution but also appeared conserved in 21 blast isolates. Some genes were not determined in isolates, the reason might be the result of gene evolution during plant-pathogen interaction.

We selected 10 effector genes as candidates to clone and functionally identify. The cloned nucleotide sequence of MgNIP04 from Y99-63c was aligned with the short protein, MGS0004previously sequenced.MgNIP04 was identical to MGS0004.MgNIP04 encoding a 96 amino acid protein with unknown domains or motifs. MgNIP04 contained a signal peptide of 20 amino acids at the N-terminus. Subcellular localization prediction suggested that it was a cytoplasmic protein. However, it is not homologous to Nep1-like proteins, a novel class of necrosis-inducing proteins found in a variety of taxonomically unrelated

Thus, it is essential to analyze their function for conserved or varied genes.

**4. Effector-encoding gene cloning and** *in vitro* **functional identification** 

and 11 were translocated into cytoplasm.

distributed in isolate 7, 14 and 15 (Table 1).

**China** 

the gene.


microorganisms (Clare et al., 2004). The plasmids of pMALMgNIP04 and pMAL were induced to express of fusion proteins of MBP-MgNIP04 andMBP.

"+" mean gene was examined in isolates, "-"mean gene was not examined in isolates.

Table 1. Frequency of 45 genes in 21 isolates of M. oryzae from Yunnan, China.

Functional Identification of Genes Encoding Effector Proteins in *Magnaporthe oryzae* 123

All expression levels of candidate genes were normalized by *actin* housekeeping gene and quantified by both the comparative threshold method and standard curve method. The results showed that expression levels of all candidate genes were significantly different in isolates of 94-64-1b Y99-63, 95-23-4a, Y98-16 and 94-64-1b. When two isolates of Y98-16 and Y99-63 grown under complete medium and nitrogen-starvation medium, relative expression quantity of genes was different. Expression of more genes was detected when two isolates grew under nitrogen starvation for 24 h, than when the two isolates grew under complete

Fig. 3. Expression pattern of some predicted effector protein-encoding genes from *M.oryzae.*  a: Relative expression quantity of target gene of Y99-63 and Y98-16 cultured in different

We detected expression level of all candidate genes at 24 hpi, 48 hpi, 72 hpi, 96 hpi and 168 hpi. Results revealed that all gene expression levels were apparently up-regulated, achieved

b: Relative expression quantity of target gene of Y99-63 and Y98-16 cultured in different

the maximum at 48hpi, but decreased after 72 hpi (Table 2 and Figure 4).

medium (Figure 3).

mediums using 2-ᇞᇞC t method

mediums using the standard curve method.

To determine if MgNIP04 could interact with rice proteins, *E. coli*-expressing MBP-MgNIP04 was inoculated on wounded rice leaves. MBP expressed from pMAL-c2X was used as a control. The concentration of expression products of MBP-MgNIP04 and MBP were determined following the procedure of Bradford (1976) using bovine serum albumin as a standard. The expressed products of pMAL-MgNIP04 and pMAL-c2X at a concentration of 2.0 µg/µl were inoculated on wounded rice seedling leaf tissues. Necrotic specks formed around the inoculation site of leaf tissues that were inoculated with fused MBP-Mg04, while no necrotic specks appeared when inoculated with MBP. The results demonstrated that the protein encoded by MgNIP04 could directly interact with rice proteins. We also performed other experiments such as MgNIP04 *in vitro* induced H2O2 production, induced callose deposition in rice leaves and roots which could be automatically transported in rice root cells, when rice suspension-cultured cells were treated by MBP-MgNIP04.

The vector of pCAMbia-MgNIP04 was transformed into blast isolate of Y98-16. The differences of conidiation, germination, appressorium and pathogenicity between wild type strain of Y98- 16 and transformant harboring MgNIP04::GFP were identified. The result revealed =no obvious difference in conidiation, germination and appressorium between Y98-16 and the transformant.The pathogenicity was further assayed for Y98-16 and transformant, rice cultivar Lijiangxintuanheigu that was almost susceptible to all races of blast fungus was challenged with blast strains. The result showed the transformant caused less symptom on Lijiangxintuanheigu than Y98-16. These data indicated *MgNIP04* did not influence blast fungus pathogenicity qualitatively but quantitatively, and virulence level of blast fungus decreased along with increasing of copy number of *MgNIP04* (there was one more copy at least in transformant than in Y98-16 although the gene copy number was not analyzed).

In order to test infecting ability difference of Y98-16 and transformant to rice roots, we used Y98-16 and transformant to inoculate the roots of Lijiangxintuanheigu. Brown symptom appeared on rice roots when blast fungus infected rice roots for 7 days. Brown symptom on the roots caused by Y98-16 regardless of areas and amounts of brown lateral roots regardless of areas and amounts of brown lateral roots was more apparent than by transformant. To determine whether *MgNIP04* was expressed in mycelia colonizing the roots, the roots infected by transformant and Y98-16 were observed using laser scanning confocal microscopy, respectively. The result showed that MgNIP04::GFP was observed in mycelia colonized roots. Along with infecting time elongation, the colonizing mycelia gradually increased. The frozen slices from brown- and no brown-root tissue were observed using laser scanning confocal microscopy. The results showed that mycelia not only had infected the epidermal cell but also colonized along root cell intervals.

#### **5. Expression pattern of effector protein-encoding genes from** *M. oryzae*

Many studies have used quantitative polymerase chain reaction (PCR) to evaluate fungal growth during the infection process (Hu et al., 1993; Mahuku et al., 1995; Groppe and Boller, 1997; Judelson and Tooley, 2000).Therefore, we detected the expression pattern of candidate novel genes *MGNIP10*, *MGNIP18*, *MGNIP24*, *MGNIP34*, *MGNIP38*, *MGNIP53*, *MGNIP74*, *MGNIP97* and *MgNIP04* in different isolates from Yunnan, China*,* the same isolate grown under nitrogen-starvation medium and complete medium and different time points when Lijiangxintuanheigu was challenged with blast fungus using real-time fluorescence quantitative PCR.

To determine if MgNIP04 could interact with rice proteins, *E. coli*-expressing MBP-MgNIP04 was inoculated on wounded rice leaves. MBP expressed from pMAL-c2X was used as a control. The concentration of expression products of MBP-MgNIP04 and MBP were determined following the procedure of Bradford (1976) using bovine serum albumin as a standard. The expressed products of pMAL-MgNIP04 and pMAL-c2X at a concentration of 2.0 µg/µl were inoculated on wounded rice seedling leaf tissues. Necrotic specks formed around the inoculation site of leaf tissues that were inoculated with fused MBP-Mg04, while no necrotic specks appeared when inoculated with MBP. The results demonstrated that the protein encoded by MgNIP04 could directly interact with rice proteins. We also performed other experiments such as MgNIP04 *in vitro* induced H2O2 production, induced callose deposition in rice leaves and roots which could be automatically transported in rice root

The vector of pCAMbia-MgNIP04 was transformed into blast isolate of Y98-16. The differences of conidiation, germination, appressorium and pathogenicity between wild type strain of Y98- 16 and transformant harboring MgNIP04::GFP were identified. The result revealed =no obvious difference in conidiation, germination and appressorium between Y98-16 and the transformant.The pathogenicity was further assayed for Y98-16 and transformant, rice cultivar Lijiangxintuanheigu that was almost susceptible to all races of blast fungus was challenged with blast strains. The result showed the transformant caused less symptom on Lijiangxintuanheigu than Y98-16. These data indicated *MgNIP04* did not influence blast fungus pathogenicity qualitatively but quantitatively, and virulence level of blast fungus decreased along with increasing of copy number of *MgNIP04* (there was one more copy at least in transformant than in Y98-16 although the gene copy number was not analyzed).

In order to test infecting ability difference of Y98-16 and transformant to rice roots, we used Y98-16 and transformant to inoculate the roots of Lijiangxintuanheigu. Brown symptom appeared on rice roots when blast fungus infected rice roots for 7 days. Brown symptom on the roots caused by Y98-16 regardless of areas and amounts of brown lateral roots regardless of areas and amounts of brown lateral roots was more apparent than by transformant. To determine whether *MgNIP04* was expressed in mycelia colonizing the roots, the roots infected by transformant and Y98-16 were observed using laser scanning confocal microscopy, respectively. The result showed that MgNIP04::GFP was observed in mycelia colonized roots. Along with infecting time elongation, the colonizing mycelia gradually increased. The frozen slices from brown- and no brown-root tissue were observed using laser scanning confocal microscopy. The results showed that mycelia not only had infected

**5. Expression pattern of effector protein-encoding genes from** *M. oryzae*

Many studies have used quantitative polymerase chain reaction (PCR) to evaluate fungal growth during the infection process (Hu et al., 1993; Mahuku et al., 1995; Groppe and Boller, 1997; Judelson and Tooley, 2000).Therefore, we detected the expression pattern of candidate novel genes *MGNIP10*, *MGNIP18*, *MGNIP24*, *MGNIP34*, *MGNIP38*, *MGNIP53*, *MGNIP74*, *MGNIP97* and *MgNIP04* in different isolates from Yunnan, China*,* the same isolate grown under nitrogen-starvation medium and complete medium and different time points when Lijiangxintuanheigu was challenged with blast fungus using real-time fluorescence

cells, when rice suspension-cultured cells were treated by MBP-MgNIP04.

the epidermal cell but also colonized along root cell intervals.

quantitative PCR.

All expression levels of candidate genes were normalized by *actin* housekeeping gene and quantified by both the comparative threshold method and standard curve method. The results showed that expression levels of all candidate genes were significantly different in isolates of 94-64-1b Y99-63, 95-23-4a, Y98-16 and 94-64-1b. When two isolates of Y98-16 and Y99-63 grown under complete medium and nitrogen-starvation medium, relative expression quantity of genes was different. Expression of more genes was detected when two isolates grew under nitrogen starvation for 24 h, than when the two isolates grew under complete medium (Figure 3).

Fig. 3. Expression pattern of some predicted effector protein-encoding genes from *M.oryzae.*  a: Relative expression quantity of target gene of Y99-63 and Y98-16 cultured in different mediums using 2-ᇞᇞC t method

b: Relative expression quantity of target gene of Y99-63 and Y98-16 cultured in different mediums using the standard curve method.

We detected expression level of all candidate genes at 24 hpi, 48 hpi, 72 hpi, 96 hpi and 168 hpi. Results revealed that all gene expression levels were apparently up-regulated, achieved the maximum at 48hpi, but decreased after 72 hpi (Table 2 and Figure 4).

Functional Identification of Genes Encoding Effector Proteins in *Magnaporthe oryzae* 125

Standard curve Fold change

> 7660± 1802

3.63± 0.37

414± 95.7

77± 3.26

879± 44.2

354± 79.7

2198± 209

> 8± 1.87

325± 59.7

Table 2. Relative expression quantity of target gene in infected rice leaves at different stages

Based on the results, we knew that *MgNIP04 in vitro* expression products could induce callose deposition of rice suspension cells, up-taken by rice root without presence of pathogen and quantitatively influence virulence level of blast fungus Y98-16. Are there differences of defense-related gene expression pattern between Y98-16 and transformant infecting Lijiangxintuanheigu, respectively? We used RT-PCR to analysis gene expression of selected defense-related genes when rice cultivar of Lijiangxintuanheigu was inoculated by Y98-16 and transformant, respectively. The genes of *APXa* (AY254495.1), *Chia4a* (AB096140.1), *CHS* (AB058397.1), *OsPAL* (AX16099.1), *OsPHGPX* (AJ270955.1), *OsPR1a* (AJ278436.1) and *PR-10a* (AF274850.1) were expressed from 24hpi to 168hpi when Lijiangxintuanheigu was challenged with Y98-16 and transformant, respectively, and rice βactin gene (CT831215.1) was control. The expression of *OsGST2* (AJ486976.1), *PR-10c* (AF274852.1) and *Npr1* (DQ450949.1) was not detected at any time points regardless of Y98- 16 or transformant inoculating Lijiangxintuanheigu. The expression of *PR-10b* was detected at all selected time points during transformant infecting rice, but it was not detected during Y98-16 infecting rice. The expression of the ethylene synthesase gene was only detected at 0, 24, 48 and 96hpi when Lijiangxintuanheigu was challenged with Y98-16, while the gene

Y99-63 24 h-I 48 h-I 72 h-I 96 h-I 168 h-I

54388± 14248

> 0.92± 0.04

359± 36.3

68± 9.84

773± 8.74

282± 20.7

1931± 87.3

> 5± 0.39

337± 34.3 Standard curve Fold change

> 7101± 1626

> > 0.6± 0.02

95± 7.37

25± 2.95

192± 1.99

78± 4.98

501± 22.5

3± 0.19

89± 7.86

2 -△△Ct Fold change

44878± 10524

> 0.29± 0.01

> > 76± 4.36

> > 19± 0.89

> > 88± 1.51

100± 17.0

277± 14.2

3± 0.17

130± 9.00 Standard curve Fold change

> 6812± 1360

0.25± 0.00

> 29± 1.26

> 10± 0.36

> 32± 0.51

> 36± 5.35

101± 5.10

2± 0.09

45± 2.65

2 -△△Ct Fold change

> 243± 76.8

0.1± 0.01

5± 0.48

1± 0.06

3± 0.11

8± 0.57

3± 0.27

0.1± 0.01

3± 0.19 Standard curve Fold change

> 291± 237

0.13± 0.02

> 3± 0.24

0.6± 0.04

2± 0.08

5± 0.35

3± 0.22

0.1± 0.01

2± 0.13

2 -△△Ct Fold change

Isolates

Control

*MGNIP04* 1 37602±

*MGNIP10* 1 4.88±

*MGNIP18* 1 1799±

*MGNIP24* 1 236±

*MGNIP34* 1 2012±

*MGNIP38* 1 1323±

*MGNIP53* 1 4117±

*MGNIP74* 1 9±

*MGNIP97* 1 634±

post inoculation.

2 -△△Ct Fold change

6231

0.22

201

37.5

68.3

121

306

2.00

98.7

Standard curve Fold change

> 4800± 854

2.24± 0.08

334± 28.1

77± 15.49

376± 11.7

263± 21.4

776± 56

5± 0.82

135± 18.5

**6. Rice defense-related gene expression pattern** 

2 -△△Ct Fold change

71139± 19038

> 8.92± 1.04

2386± 743

287± 14.8

5401± 297

1959± 512

12888± 1242

> 17± 5.22

1825± 385

Targeted gene

Fig. 4. Expression pattern of some predicted effector protein-encoding genes in infected rice leaves.

a: Relative expression amount of target gene in infected rice leaves at different stages post inoculation using 2-ᇞᇞCt method

b: Relative expression amount of target gene in infected rice leaves at different stages post inoculation using stand curve method.


a

b

Fig. 4. Expression pattern of some predicted effector protein-encoding genes in infected rice

a: Relative expression amount of target gene in infected rice leaves at different stages post

b: Relative expression amount of target gene in infected rice leaves at different stages post

leaves.

inoculation using 2-ᇞᇞCt method

inoculation using stand curve method.


Table 2. Relative expression quantity of target gene in infected rice leaves at different stages post inoculation.

#### **6. Rice defense-related gene expression pattern**

Based on the results, we knew that *MgNIP04 in vitro* expression products could induce callose deposition of rice suspension cells, up-taken by rice root without presence of pathogen and quantitatively influence virulence level of blast fungus Y98-16. Are there differences of defense-related gene expression pattern between Y98-16 and transformant infecting Lijiangxintuanheigu, respectively? We used RT-PCR to analysis gene expression of selected defense-related genes when rice cultivar of Lijiangxintuanheigu was inoculated by Y98-16 and transformant, respectively. The genes of *APXa* (AY254495.1), *Chia4a* (AB096140.1), *CHS* (AB058397.1), *OsPAL* (AX16099.1), *OsPHGPX* (AJ270955.1), *OsPR1a* (AJ278436.1) and *PR-10a* (AF274850.1) were expressed from 24hpi to 168hpi when Lijiangxintuanheigu was challenged with Y98-16 and transformant, respectively, and rice βactin gene (CT831215.1) was control. The expression of *OsGST2* (AJ486976.1), *PR-10c* (AF274852.1) and *Npr1* (DQ450949.1) was not detected at any time points regardless of Y98- 16 or transformant inoculating Lijiangxintuanheigu. The expression of *PR-10b* was detected at all selected time points during transformant infecting rice, but it was not detected during Y98-16 infecting rice. The expression of the ethylene synthesase gene was only detected at 0, 24, 48 and 96hpi when Lijiangxintuanheigu was challenged with Y98-16, while the gene

Functional Identification of Genes Encoding Effector Proteins in *Magnaporthe oryzae* 127

gel electrophoresis.

Fig. 5. 2-DE maps of Y99-63 and Y98-16.Protein spots labeled with arrow (1 to 5)are coexpressed in two strains of Y99-63 and Y98-16 with the pI ranging from 5.5 to 6.0, but the expression level of Y99-63 is over five times more than Y98-16. Protein spots indicated by arrow (6 to 9) are proteins which are specifically expressed in the strain Y99-63, with their MW ranging from 10 to 20 kDa and pI from 5.0 to 6.0. MW is indicated on the right side in KD. IEF is abbreviation for isoelectric focusing and SDS-PAGE is for SDS-polyacrylamide

expression was detected at all time points when transformant inoculating rice. The expression of *OsPR4* was only detected at 168hpi during transformant inoculating rice. Based on these data, the most tested defense-related genes expression pattern was not any different between Y98-16 and transformant of MgNIP04::GFP, which indicated *MgNIP04*  quantitatively influenced pathogenicity of blast fungus.

#### **7. Expression difference of effector-encoding genes from blast isolates with different virulence determined using two-dimensional gel electrophoresis**

Fungi maintained their cell living and even growth through material reutilization when they were in nutrition-stress environment. Some research showed that expression quantity of pathogenicity-related genes increased when rice blast strains grew under nitrogen-starvation medium, which enhanced the pathogenicity of blast strains (Talbot et al., 1997).

The two isolates of Y99-63 and Y98-16 were from Yunnnan, China. Virulence test of two isolates of Y98-16 and Y99-63 on rice isogenic lines of IRBL1-24 had been previously performed in our lab, and virulence of Y99-63 was more intensive than Y98-16. To analyze the virulence of extracellularly secreted proteins on rice varieties such as susceptible variety of Lijiangxintuanheigu, resistant variety of Tetep and rice isogenic lines of IRBL1- 24, we separated the extracellularly secreted proteins when Y98-16 and Y99-63 grew under nitrogen starvation for 48h, and the wounded rice leaves were inoculated with extracellularly secreted proteins. The result showed that necrosis speck occurred around the wounded leaves and wounded stems of rice when secreted proteins were inoculated on leaves or stems for 48h, and speck diameter of leaves or stems treated with secreted proteins was 2 to 4 fold larger than leaves or stems treated with sterilized water.

We compared difference of extracellularly secreted proteins from Y99-63 and Y98-16 growing under nitrogen-starvation medium for 48h using two-dimensional electrophoresis technology (Figure 5). The result showed that more proteins spots were detected from Y99- 63 growing under nitrogen-starvation medium than Y98-16 (Table 3). And pI and molecular weight of secreted proteins had an apparent difference between Y99-63 and Y98-16 (Figure 6 and Figure 7).


Note: \* mean master reference gel; Match rate 1 for the match-point block of gel protein spots representing the ratio; Match rate 2 is the ratio of match point to total master.

Table 3. Comparison of 2-DE maps of two strains in *M.oryzae.* 

expression was detected at all time points when transformant inoculating rice. The expression of *OsPR4* was only detected at 168hpi during transformant inoculating rice. Based on these data, the most tested defense-related genes expression pattern was not any different between Y98-16 and transformant of MgNIP04::GFP, which indicated *MgNIP04* 

**7. Expression difference of effector-encoding genes from blast isolates with different virulence determined using two-dimensional gel electrophoresis** 

Fungi maintained their cell living and even growth through material reutilization when they were in nutrition-stress environment. Some research showed that expression quantity of pathogenicity-related genes increased when rice blast strains grew under nitrogen-starvation medium, which enhanced the pathogenicity of blast strains (Talbot et

The two isolates of Y99-63 and Y98-16 were from Yunnnan, China. Virulence test of two isolates of Y98-16 and Y99-63 on rice isogenic lines of IRBL1-24 had been previously performed in our lab, and virulence of Y99-63 was more intensive than Y98-16. To analyze the virulence of extracellularly secreted proteins on rice varieties such as susceptible variety of Lijiangxintuanheigu, resistant variety of Tetep and rice isogenic lines of IRBL1- 24, we separated the extracellularly secreted proteins when Y98-16 and Y99-63 grew under nitrogen starvation for 48h, and the wounded rice leaves were inoculated with extracellularly secreted proteins. The result showed that necrosis speck occurred around the wounded leaves and wounded stems of rice when secreted proteins were inoculated on leaves or stems for 48h, and speck diameter of leaves or stems treated with secreted

proteins was 2 to 4 fold larger than leaves or stems treated with sterilized water.

We compared difference of extracellularly secreted proteins from Y99-63 and Y98-16 growing under nitrogen-starvation medium for 48h using two-dimensional electrophoresis technology (Figure 5). The result showed that more proteins spots were detected from Y99- 63 growing under nitrogen-starvation medium than Y98-16 (Table 3). And pI and molecular weight of secreted proteins had an apparent difference between Y99-63 and Y98-16 (Figure 6

Strain Replicate group Protein spots Protein matched spots Match Rate 1 Match Rate 2

Y98-16 3 253±10 253±10 100% 100% Y99-63 3 262±10 132±8 50.4% 52.2%

Note: \* mean master reference gel; Match rate 1 for the match-point block of gel protein spots

representing the ratio; Match rate 2 is the ratio of match point to total master.

Table 3. Comparison of 2-DE maps of two strains in *M.oryzae.* 

quantitatively influenced pathogenicity of blast fungus.

al., 1997).

and Figure 7).

\*

Functional Identification of Genes Encoding Effector Proteins in *Magnaporthe oryzae* 129

Bradford, M. M. (1976). A Rapid and Sensitive Method for the Quantitation of Microgram

Catanzariti, A.M., Dodds, P.N. & Ellis, J.G. (2007). Avirulence Proteins from Haustoria-

Chisholm, S.T., Coaker, G., Day, B. & Staskawicz, B. J. (2006). Host-Microbe Interactions: Shaping the Evolution of the Plant Immune Response. Cell. 124:803-814. Clare, L., Salmond & Gorge P.C. (2004). The Nep1-like Proteins – a Growing Family of

Collemare, J., Pianfetti, M., Houlle, A. E., Morin, D., Camborde, L., Gagey, M.J., Barbisan, C.,

Couch, B. C. & Kohn L.M. (2002). A multilocus gene genealogy concordant with host

Farman, M. L. & Leong, S. A.(1998). Chromosome Walking to the AVR1-CO39 Avirulence

Fudal, I., Bohnert, H. U., Tharreau, D & Lebrun, M. H. (2005). Transposition of MINE, a

Idnurm, A. & Howlett, B.J. (2001).Pathogen city Genes of Phytopathogenic Fungi. Mol. Plant

Jia Y, Correll, J.C., Lee, F.N., Eizenga, G.C., Yang, Y. , Gealy, D.R., Valent, B.,& Zhu, Q. 2001.

Kamoun, S. (2006). A Catalogue of the Effector Secretome of Plant Pathogenic Oomycetes.

Kamoun, S. (2007). Groovy Times: Filamentous Pathogen Effectors Revealed. Curr. Opin.

Kang, S., Sweigard, J. A., & Valent, B. (1995). The PWL Host Specificity Gene Family in the Blast Fungus Magnaporthe grisea. Mol. Plant-Microbe Interact, 8:939-948 Khang, C.H., Park, S. Y., Lee, Y. H., Valent, B., & Kang, S.(2008). Genome Organization and

Mahuku, G.S., Goodwin, P.H. & Hall, R. (1995). A Competitive Polymerase Chain Reaction

Nickel, W. (2003). The mystery of nonclassical protein secretion. *Eur. J. Biochem*. 270, 2109-

and the rice blast pathogen. Phytopathology 91(Suppl. 6):S44(abstr.). Judelson, H.S., Tooley, P.W. (2000). Enhanced Polymerase Chain Reaction Methods for

Fudal, I., Lebrun, M.H. & Böhnert, H.U.(2008). *Magnaporthe grisea* Avirulence Gene *ACE1* Belongs to an Infection-Specific Gene Cluster Involved in Secondary

preference indicates segregation of a new species, *Magnaporthe oryzae*, from *M.* 

Gene of Magnaporthe grisea: Discrepancy between the Physical and Genetic Maps.

Composite Retrotransposon, in the Avirulence Gene *ACE1* of the Rice Blast Fungus

Understanding molecular interaction mechanisms of the *Pi-ta* rice resistance genes

Detecting and Quantifying Phytophthora infestans in Plants. Phytopathology

Evolution of the *AVR-Pita* Avirulence Gene Family in the *Magnaporthe grisea*

to Quantify DNA of Leptosphaeria maculans During Blackleg Development in

Microbial Elicitors of Plant Necrosis. Mol Plant Pathol, 5:353–359.

Forming Pathogens. FEMS Microbiol. Lett. 269:181-188.

Magnaporthe grisea. Fungal. Genet. Biol., 42:761-772

Annual Review of Phytopathology. 44: 41–60.

Species Complex. Mol. Plant-Microbe Interact, 21:658-670.

Oilseed Rape. Mol Plant-Microbe Interact 8:761–767.

Metabolism. New Phytol. 179:196-208.

*grisea*. Mycologia 94(4): 683-693.

Genetics, 150:1049-1058.

Pathol.2:241-255.

90:1112–1119.

2119.

Plant Biol.10:358-365.

72:249–254.

Quantities of Protein Utilizing the Principle of Protein-dye Binding. Anal Biochem,

Fig. 6. Comparison of the distribution of the proteins spots based on molecular weight.

Fig. 7. Comparison of the distribution of the proteins spots based on pI.

#### **8. Acknowledgment**

This work was supported by the National Basic Research Program (No. 2011CB100400) from The Ministry of Science and Technology of China and the National Natural Science Funds, China (30860161), respectively.

#### **9. References**

Birch, P. R., Rehmany, A. P., Pritchard, L., Kamoun, S. & Beynon, J. L. (2006). Trafficking Arms: Oomycete Effectors Enter Host Plant Cells. Trends Microbiol. 14:8-11

Bohnert, H. U., Fudal, I., Dior, W., Tharreau, D., Notteghem J. L. & Lebrun, M. H.(2004). A Putative Polyketide Synthase/Peptide Synthetase from *Magnaporthe grisea* Signals Pathogen Attack to Resistance Rice. Plant Cell, 16:2499-2513.

Fig. 6. Comparison of the distribution of the proteins spots based on molecular weight.

Fig. 7. Comparison of the distribution of the proteins spots based on pI.

This work was supported by the National Basic Research Program (No. 2011CB100400) from The Ministry of Science and Technology of China and the National Natural Science Funds,

Birch, P. R., Rehmany, A. P., Pritchard, L., Kamoun, S. & Beynon, J. L. (2006). Trafficking Arms: Oomycete Effectors Enter Host Plant Cells. Trends Microbiol. 14:8-11 Bohnert, H. U., Fudal, I., Dior, W., Tharreau, D., Notteghem J. L. & Lebrun, M. H.(2004). A

Pathogen Attack to Resistance Rice. Plant Cell, 16:2499-2513.

Putative Polyketide Synthase/Peptide Synthetase from *Magnaporthe grisea* Signals

**8. Acknowledgment** 

**9. References** 

China (30860161), respectively.


**6** 

*Kobe University* 

*Japan* 

**The Role of the Extracellular** 

Kenichi Ikeda, Kanako Inoue, Hiroko Kitagawa, Hiroko Meguro, Saki Shimoi and Pyoyun Park

**Matrix (ECM) in Phytopathogenic Fungi:** 

Crop yield loss as a result of disease has an economic impact on many people. To protect against disease, plant pathologists have developed various fungicides and disease resistant cultivars. In parallel, pathogens have evolved to escape from disease protection measures through, for example, the emergence of fungicide resistant isolates and the breakdown of disease resistant cultivars. To resolve these issues, we should develop various disease protection strategies for different types of target sites. Therefore, we must improve our understanding of the infection mechanism of pathogens. Pathogens possess several classes of genes that are essential for causing disease, i.e., pathogenicity genes or virulence genes, the products of which are called pathogenicity factors. Phytopathogenic fungi have developed various pathogenic factors, e.g., adhesion molecules to the host cells, sensor machineries against host plants, invasion machineries into the host cells, adaptation ability on the host cells, and so on. These factors comprise potential targets for disease control. Fungal adhesion to host cells is an initial important step to establish infection, which is considered to be a universal mechanism across plant pathogenic fungi. In this chapter, we provide a review of the components required for fungal adhesion to the host cell, and

Pathogenic fungi have various strategies for propagation. Some fungi produce asexual spores, sexual spores, sclerotia, budding progenies, or just extending mycelia. These propagules comprise minimal compartmental units to transmit genetic information, and are highly replicable. These small propagules are able to travel great distances and colonize novel niches. Dispersing propagules may land on various types of environment. Each pathogenic fungus is capable of adapting to establish individuals on a specific environment. The mechanism of pathogenic fungal adhesion is considered to occur in a number of ways. For example, the hydrophobic interaction is a universal system of adhesion, i.e., most fungal surfaces are covered with hydrophobic components, such as hydrophobin. In addition,

propose fungal adhesion as a potential target for disease control.

either protein-protein or protein-carbohydrate interaction also occurs.

**2. Importance of fungal adhesion to the host cell** 

**1. Introduction** 

**A Potential Target for Disease Control** 


### **The Role of the Extracellular Matrix (ECM) in Phytopathogenic Fungi: A Potential Target for Disease Control**

Kenichi Ikeda, Kanako Inoue, Hiroko Kitagawa, Hiroko Meguro, Saki Shimoi and Pyoyun Park *Kobe University Japan* 

#### **1. Introduction**

130 Plant Pathology

O'Connell, R.J. & Panstruga, R. (2006). Tete a tete inside a Plant Cell: Establishing

Orbach, M. J., Farrall, L., Sweigard, J.A., Chumley, F. G. & Valent, B. (2000). A Telomeric

Randall, T.A. et al. (2005). Large-Scale Gene Discovery in the Oomycete *Phytophthora* 

Sweigard, J. A., Carroll, A. M., Kang, S., Farrall, L., Chumley, F. G. & Valent, B. (1995).

Talbot NJ,McCafeny HRK, Ma M et a1. (1997). Nitrogen Starvation of the Rice Blast Fungus

Tjalsma, H., Noback, M. A., Bron, S., Venema, G., Yamane, K., & van Dijl J. M. (1997).*Bacillus* 

Tjalsma, H., Bolhuis, A., van Roosmalen, M. L., Wiegert, T., Schumann, W., Broekhuizen, C.

Tjalsma, H., van den Dolder, J., Meijer, W. J. J., Venema, G., Bron, S., & van Dijl, J. M. (1999).

Valent, B., Farrall, L., & Chumley, F. G.(1991). Magnaporthe grisea Genes for Pathogenicity and Virulence Identified Through a Series of Backcrosses. Genetics, 127:87-101. Yoshida, K., Saitoh, H., Fujisawa, S., Kanzaki, H., Matsumura, H., Yoshida, K., Tosa, Y.,

Pathogen *Magnaporthe oryzae*. Plant Cell, 21:1573-1591

Expression. Physiological and Molecular Plant Pathology.50:179-195 Talbot, N. J. (2003). On the Trail of a Cereal Killer: Exploring the Biology of *Magnaporthe* 

Different *sip* Genes. Journal Biological Chemistry, 272:25983–25992.

Fungi. Mol. Plant Microbe Interact. 18:229-243.

*grisea.* Annu. Rev. Microbiol. 57:177-202.

*Developement*, 12:2318–2331.

181:2448–2454.

Specificity in the Rice Blast Fungus. Plant Cell, 7:1221-1233.

Phytol.171:699-718.

Cell, 12:2019-2032

Compatibility between Plants and Biotrophic Fungi and Oomycetes. New

Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta. Plant

*infestans* Reveals Likely Components of Phytopathogenicity Shared with True

Identification, Cloning, and Characterization of *PWL2,* a Gene for Host Species

*Magnaporthe grisea* may Act as an Environmental Cue for Disease Symptom

*subtilis* Contains Four Closely Related Type I Signal Peptidases with Overlapping Substrate Specificities: Constitutive and Temporally Controlled Expression of

P., Quax, W., Venema, G., Bron, S., & van Dijl, J. M. (1998).Functional analysis of the secretory precursor processing machinery of *Bacillus subtilis*: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases. *Genes* 

The plasmid-encoded type I signal peptidase SipP can functionally replace the major signal peptidases SipS and SipT of *Bacillus subtilis*. *Journal Bacteriology,* 

Chuma, I., Takano,Y., Win, J., Kamoun, S., & Terauchi, R. (2009). Association Genetics Reveals Three Novel Avirulence Genes from the Rice Blast Fungal Crop yield loss as a result of disease has an economic impact on many people. To protect against disease, plant pathologists have developed various fungicides and disease resistant cultivars. In parallel, pathogens have evolved to escape from disease protection measures through, for example, the emergence of fungicide resistant isolates and the breakdown of disease resistant cultivars. To resolve these issues, we should develop various disease protection strategies for different types of target sites. Therefore, we must improve our understanding of the infection mechanism of pathogens. Pathogens possess several classes of genes that are essential for causing disease, i.e., pathogenicity genes or virulence genes, the products of which are called pathogenicity factors. Phytopathogenic fungi have developed various pathogenic factors, e.g., adhesion molecules to the host cells, sensor machineries against host plants, invasion machineries into the host cells, adaptation ability on the host cells, and so on. These factors comprise potential targets for disease control. Fungal adhesion to host cells is an initial important step to establish infection, which is considered to be a universal mechanism across plant pathogenic fungi. In this chapter, we provide a review of the components required for fungal adhesion to the host cell, and propose fungal adhesion as a potential target for disease control.

#### **2. Importance of fungal adhesion to the host cell**

Pathogenic fungi have various strategies for propagation. Some fungi produce asexual spores, sexual spores, sclerotia, budding progenies, or just extending mycelia. These propagules comprise minimal compartmental units to transmit genetic information, and are highly replicable. These small propagules are able to travel great distances and colonize novel niches. Dispersing propagules may land on various types of environment. Each pathogenic fungus is capable of adapting to establish individuals on a specific environment. The mechanism of pathogenic fungal adhesion is considered to occur in a number of ways. For example, the hydrophobic interaction is a universal system of adhesion, i.e., most fungal surfaces are covered with hydrophobic components, such as hydrophobin. In addition, either protein-protein or protein-carbohydrate interaction also occurs.

The Role of the Extracellular Matrix (ECM)

1998; Rauceo *et al.*, 2006).

(Sundstrom, 1999).

adhesion in animal and plant pathogenic fungi.

**3. Components of adhesion strategies 3.1 Animal pathogenic fungal adhesion** 

two different steps, depending on morphological switching.

in Phytopathogenic Fungi: A Potential Target for Disease Control 133

adhesins (Sundstrom, 1999). In the next section, we describe various components of

*Candida albicans* causes severe oropharyngeal and esophageal mucositis in patients that have human immunodeficiency virus (HIV), and is the most intensively studied animal pathogenic fungi. *C. albicans* normally propagates budding yeast cells (blastospores). When the blastospores attach to the animal cells, the blastospores transform into a filamentous form to invade underlying tissues. The cell wall components between the blastospore and filamentous hypha are different. Therefore, the adhesion of *C. albicans* may be divided into

Blastospore adhesion is the first step of adhesion to the host epithelial and endothelial cells, especially to the extracellular matrix, and is important for the morphological switching of yeast cells from blastospore to filamentous forms (Gale *et al*., 1998; Klotz *et al*., 1993). This adhesion step is dependent on the presence of calcium ions (Klotz *et al*., 1993). Ultrastructure analysis has revealed that the cell wall of blastospores is surrounded by a fibrillar reticulated layer (fimbriae) that contains mannose sugar, suggesting the importance of glycoprotein in this process (Bobichon *et al.*, 1994). The molecular approach elucidated the presence of several adhesive proteins, called adhesins. Since the blastospore adheres to the extracellular matrix of the animal cell, the involvement of the integrin-like component was suspected to be a component of adhesion. The homologous gene of vertebrate leukocyte integrins was cloned as *INT1* and characterized (Gale *et al*., 1996; Gale *et al.*, 1998). When the *INT1* gene was introduced into *Saccharomyces cerevisiae*, the transformants producing INT1 exhibited enhanced aggregation (Gale *et al*., 1996). Furthermore, the disruption of *INT1* suppressed blastospore adhesion to epithelial cells, hyphal growth, and virulence in mice (Gale *et al*., 1998). In addition to INT1, agglutinin family proteins, ALA1 and ALS1, and the fibronectin binding protein, FLS5, were also involved in blastospore adhesion (Fu *et al.*,

Once the blastospores settle on the host cells, morphological switching occurs to form filamentous growth (germ tubes, pseudohyphae, and hyphae). Proline and glutamine rich protein-encoding genes have been abundantly expressed in hyphae but not yeast forms, and were designated as *HWP1* (Staab *et al*., 1996). The disruption of the *HWP1* gene reduced the stable attachment of blastospores to human buccal epithelial cells, and reduced their capacity to cause systemic candidiasis in mice (Staab *et al*., 1999). The HWP1 protein also served as a substrate for mammalian transglutaminases, suggesting that the blastospore adherence mechanism may be involved in the cross-linking of HWP1 to unidentified proteins through transglutaminase activity (Staab *et al.*, 1999). Moreover, adhesin family proteins, ALS3 and HYR1, were also found to be involved in filamentous growth adhesion

Other adhesion components of animal pathogenic fungi have also been also studied. In *Aspergillus fumigatus*, a hydrophobin RODA was involved in adhesion (Thau *et al.*, 1994). In *Rhizopups oryzae*, the spores adhered to laminin and type IV collagen, but not to fibronectin,

with adhesion decreasing during the elongation of germ tubes (Bouchara *et al.*, 1996).

The plant surface is coated with a cuticle (wax) to prevent desiccation, particularly when the environment is highly hydrophobic. When the spores of pathogens land on the plant surface, the spores elongate into germ tubes and have elaborate infection machineries, such as appressorium to infect plant cells. However, the spores must first settle on the plant surface to accomplish infection. If the spores are not able to adhere to the plant surface, they are easily dislodged by the wind or rain fall. Moreover, the spore germlings encounter the counterforce of infection pressure from the plant surface. Therefore, firm adhesion enables spore germlings to anchor tightly onto the host surface, and is important for the differentiation of specialized infection structures. Adhesion is required for infection in most plant pathogens, and this mechanism is also developing in phytopathogenic fungi. We showed that adhesion ability was variable depending on the living strategies of different fungal species (Fig. 1). For example, the saprotrophic fungus *Neurospora crassa* has weak adhesion ability on plastic surface. In contrast, phytopathogenic fungi, such as *Alternaria alternata* Japanese pear pathotype and *Magnaporthe oryzae*, adhered strongly to plastic surfaces. Since plastic surfaces are highly hydrophobic, they mimic the plant surface. Hence, phytopathogenic fungi might have evolved to settle on the surfaces of plants. However, the precise molecular mechanism of fungal adhesion on plant surfaces remains undetermined.

Fig. 1. Variation in the ability of fungal adhesion to plastic surfaces. The saprotrophic fungus *Neurospora crassa* has a weak adhesion ability. The phytopathogenic fungi, *Alternaria alternata* Japanese pear pathotype and *Magnaporthe oryzae*, have high adhesion ability. Adhesion ability was determined by total number of germlings was first counted under the microscope and then washed by dipping in distilled water 100 times vertically to remove the detached germlings.

In animal pathogenic fungi, adhesion to host cells is also important to manifest pathogenicity. However, because the animal cell has no cell wall and no wax layer, adhesion mechanisms to the host cell are supposed to be different to that of phytopathogenic fungi. Moreover, the environment within the animal body is different to that of the atmosphere, i.e., low oxygen concentrations, high carbon dioxide concentrations, as well as being filled with serum. Animal pathogenic fungi have evolved various adhesive proteins called adhesins (Sundstrom, 1999). In the next section, we describe various components of adhesion in animal and plant pathogenic fungi.

#### **3. Components of adhesion strategies**

#### **3.1 Animal pathogenic fungal adhesion**

132 Plant Pathology

The plant surface is coated with a cuticle (wax) to prevent desiccation, particularly when the environment is highly hydrophobic. When the spores of pathogens land on the plant surface, the spores elongate into germ tubes and have elaborate infection machineries, such as appressorium to infect plant cells. However, the spores must first settle on the plant surface to accomplish infection. If the spores are not able to adhere to the plant surface, they are easily dislodged by the wind or rain fall. Moreover, the spore germlings encounter the counterforce of infection pressure from the plant surface. Therefore, firm adhesion enables spore germlings to anchor tightly onto the host surface, and is important for the differentiation of specialized infection structures. Adhesion is required for infection in most plant pathogens, and this mechanism is also developing in phytopathogenic fungi. We showed that adhesion ability was variable depending on the living strategies of different fungal species (Fig. 1). For example, the saprotrophic fungus *Neurospora crassa* has weak adhesion ability on plastic surface. In contrast, phytopathogenic fungi, such as *Alternaria alternata* Japanese pear pathotype and *Magnaporthe oryzae*, adhered strongly to plastic surfaces. Since plastic surfaces are highly hydrophobic, they mimic the plant surface. Hence, phytopathogenic fungi might have evolved to settle on the surfaces of plants. However, the precise molecular mechanism of

Fig. 1. Variation in the ability of fungal adhesion to plastic surfaces. The saprotrophic fungus

In animal pathogenic fungi, adhesion to host cells is also important to manifest pathogenicity. However, because the animal cell has no cell wall and no wax layer, adhesion mechanisms to the host cell are supposed to be different to that of phytopathogenic fungi. Moreover, the environment within the animal body is different to that of the atmosphere, i.e., low oxygen concentrations, high carbon dioxide concentrations, as well as being filled with serum. Animal pathogenic fungi have evolved various adhesive proteins called

*Neurospora crassa* has a weak adhesion ability. The phytopathogenic fungi, *Alternaria alternata* Japanese pear pathotype and *Magnaporthe oryzae*, have high adhesion ability. Adhesion ability was determined by total number of germlings was first counted under the microscope and then washed by dipping in distilled water 100 times vertically to remove

fungal adhesion on plant surfaces remains undetermined.

the detached germlings.

*Candida albicans* causes severe oropharyngeal and esophageal mucositis in patients that have human immunodeficiency virus (HIV), and is the most intensively studied animal pathogenic fungi. *C. albicans* normally propagates budding yeast cells (blastospores). When the blastospores attach to the animal cells, the blastospores transform into a filamentous form to invade underlying tissues. The cell wall components between the blastospore and filamentous hypha are different. Therefore, the adhesion of *C. albicans* may be divided into two different steps, depending on morphological switching.

Blastospore adhesion is the first step of adhesion to the host epithelial and endothelial cells, especially to the extracellular matrix, and is important for the morphological switching of yeast cells from blastospore to filamentous forms (Gale *et al*., 1998; Klotz *et al*., 1993). This adhesion step is dependent on the presence of calcium ions (Klotz *et al*., 1993). Ultrastructure analysis has revealed that the cell wall of blastospores is surrounded by a fibrillar reticulated layer (fimbriae) that contains mannose sugar, suggesting the importance of glycoprotein in this process (Bobichon *et al.*, 1994). The molecular approach elucidated the presence of several adhesive proteins, called adhesins. Since the blastospore adheres to the extracellular matrix of the animal cell, the involvement of the integrin-like component was suspected to be a component of adhesion. The homologous gene of vertebrate leukocyte integrins was cloned as *INT1* and characterized (Gale *et al*., 1996; Gale *et al.*, 1998). When the *INT1* gene was introduced into *Saccharomyces cerevisiae*, the transformants producing INT1 exhibited enhanced aggregation (Gale *et al*., 1996). Furthermore, the disruption of *INT1* suppressed blastospore adhesion to epithelial cells, hyphal growth, and virulence in mice (Gale *et al*., 1998). In addition to INT1, agglutinin family proteins, ALA1 and ALS1, and the fibronectin binding protein, FLS5, were also involved in blastospore adhesion (Fu *et al.*, 1998; Rauceo *et al.*, 2006).

Once the blastospores settle on the host cells, morphological switching occurs to form filamentous growth (germ tubes, pseudohyphae, and hyphae). Proline and glutamine rich protein-encoding genes have been abundantly expressed in hyphae but not yeast forms, and were designated as *HWP1* (Staab *et al*., 1996). The disruption of the *HWP1* gene reduced the stable attachment of blastospores to human buccal epithelial cells, and reduced their capacity to cause systemic candidiasis in mice (Staab *et al*., 1999). The HWP1 protein also served as a substrate for mammalian transglutaminases, suggesting that the blastospore adherence mechanism may be involved in the cross-linking of HWP1 to unidentified proteins through transglutaminase activity (Staab *et al.*, 1999). Moreover, adhesin family proteins, ALS3 and HYR1, were also found to be involved in filamentous growth adhesion (Sundstrom, 1999).

Other adhesion components of animal pathogenic fungi have also been also studied. In *Aspergillus fumigatus*, a hydrophobin RODA was involved in adhesion (Thau *et al.*, 1994). In *Rhizopups oryzae*, the spores adhered to laminin and type IV collagen, but not to fibronectin, with adhesion decreasing during the elongation of germ tubes (Bouchara *et al.*, 1996).

The Role of the Extracellular Matrix (ECM)

mechanisms of fungal adhesion in *M. oryzae*.

in Phytopathogenic Fungi: A Potential Target for Disease Control 135

spores, and disperses to distant areas. Successful infection by this fungus requires the following steps: (1) the spore to land on the host surface and elongate the germ tube from the apical spore cell and (2) the tip of the germ tube to differentiate into the appressorium, which is the infection machinery. Subsequently, the fungi penetrate into the host cuticle and generate a penetration peg from the bottom of the appressorium (Howard *et al*., 1991). Although the conidia itself has low adhesion ability, the hydrated conidia attains strong adhesion ability within 30 minutes of incubation (Hamer *et al.*, 1988). Ultrastructural analysis has revealed that the spore tip mucilage (STM) is preserved at the apical spore and released after hydration (Braun and Howard, 1994). The attached spores on the surface start to germinate and secrete ECM. The secreted ECM supports the enhanced adhesion ability of the fungi on the plant surface. It is not known whether the STM and the ECM are the same component. During the infection process, some essential environmental signals have been identified as appressorium (infection)-inducing factors, such as the hardness (Xiao *et al*., 1994) and the hydrophobicity of the attachment surface (Jelitto *et al*., 1994; Lee & Dean, 1994), and the chemical components from the plant surface (Gilbert *et al*., 1996). In addition, a number of up-regulating genes have also been characterized (Talbot, 2003). However, the available information was not sufficient to identify the principal components and regulation

While the components of STM remain unclear, it was found that STM was reactive with lectin concanabalin A (ConA; α-D-glucose and α-(1,3)-/α-(1,6)-D-mannose binding), suggesting that STM consists of mannose containing glycoprotein (Hamer *et al.*, 1988). The ECM was also ConA-positive (Xiao *et al.*, 1994). The effect of three different specific sugarbinding lectins, namely ConA, PSA (α-D-glucose and α-(1,6)-D-mannose binding), and WGA (chitin binding), on appressorium formation and the adhesion ability of the *M. oryzae* germlings was evaluated (Fig. 3). High concentrations (50 μg ml-1) of lectins, ConA, and WGA were found to inhibit appressorium formation and adhesion. These results indicate that these lectin treatments covered the cell wall surface, and inhibited the development of the cell wall architecture (data not shown). In contrast, low concentrations (10 μg ml-1) of these lectins only affected adhesion, suggesting that low concentrations of lectins are preferentially bound to the adhesion components (Fig. 3). Moreover, although PSA was observed to bind to the cell wall surface, it did not affect the adhesion. ConA and PSA commonly bind to the α-D-glucose and the α-(1,6)-D-mannose moieties; however, only ConA binds to α-(1,3)-D-mannose, suggesting that α-(1,3)-D-mannose is a potential adhesion component (Fig. 3). Our unpublished study revealed that the incubation of *M. oryzae* spores in modified nutrient conditions produced less adherent spore germlings. Furthermore, the less adherent spore germlings were ConA-negative (K. Inoue, unpublished

Protein secretion via the Golgi pathway might be responsible for ECM production. In the treatment with cycloheximide at the early step of germination, the germ tube and the appressorium were not formed, and the germlings were easily removed (Fig. 4). In the treatments with monensin and tunicamycin, the adhesion rate was significantly lower than for the water control (Fig. 4). Hence, N-glycosylation of the mannose sugar moiety might be important for the maturation of the adhesive protein(s). This result also agreed with the

data), suggesting that mannose sugar may be important for adhesion.

conclusion of the lectin treatment experiments.

#### **3.2 Plant pathogenic fungal adhesion**

Plant pathogenic fungi have several adhesion strategies. This review mainly focuses on the spore dispersal of plant pathogenic fungi. When the spores land on the plant surface, they germinate and develop appressoria to enter into the plant cell (Fig. 2). Appressoria adhere tightly to the plant surface. This process is divided into two steps, i.e., spore adhesion and germ tube adhesion. During germ tube elongation, the fungal surface is covered with *de novo* synthesized compounds collectively called the extracellular matrix (ECM), which might be involved in adhesion (Beckett *et al.*, 1990). The term ECM is confusing because the outer surface components of animal cells are also called the extracellular matrix (ECM). In this chapter, ECM corresponds to the secreted products from plant pathogenic fungi only. The adhesion strength and the adhesion components in each step are different for each plant pathogenic fungus. Here, we provide an overview of the typical adhesion mechanisms in plant pathogenic fungi.

Fig. 2. Scanning electron microscope image of appressoria of phytopathogenic fungi on the host plant surface. (A) Appressorium of *Magnaporthe oryzae* on a wheat leaf. (B) Appressorium of *Venturia nashicola* on a Japanese pear leaf. Infection structure (C) and appressorium (D) of *Alternaria alternata* Japanese pear pathotype on a Japanese pear leaf.

#### **3.2.1** *Magnaporthe oryzae*

The blast fungus *M. oryzae* is one of the most destructive fungal diseases in gramineous crop plants, especially rice, barley, and wheat. *M. oryzae* produces millions of conidia, asexual

Plant pathogenic fungi have several adhesion strategies. This review mainly focuses on the spore dispersal of plant pathogenic fungi. When the spores land on the plant surface, they germinate and develop appressoria to enter into the plant cell (Fig. 2). Appressoria adhere tightly to the plant surface. This process is divided into two steps, i.e., spore adhesion and germ tube adhesion. During germ tube elongation, the fungal surface is covered with *de novo* synthesized compounds collectively called the extracellular matrix (ECM), which might be involved in adhesion (Beckett *et al.*, 1990). The term ECM is confusing because the outer surface components of animal cells are also called the extracellular matrix (ECM). In this chapter, ECM corresponds to the secreted products from plant pathogenic fungi only. The adhesion strength and the adhesion components in each step are different for each plant pathogenic fungus. Here, we provide an overview of the typical adhesion mechanisms in

Fig. 2. Scanning electron microscope image of appressoria of phytopathogenic fungi on the

The blast fungus *M. oryzae* is one of the most destructive fungal diseases in gramineous crop plants, especially rice, barley, and wheat. *M. oryzae* produces millions of conidia, asexual

host plant surface. (A) Appressorium of *Magnaporthe oryzae* on a wheat leaf. (B) Appressorium of *Venturia nashicola* on a Japanese pear leaf. Infection structure (C) and appressorium (D) of *Alternaria alternata* Japanese pear pathotype on a Japanese pear leaf.

**3.2 Plant pathogenic fungal adhesion** 

plant pathogenic fungi.

**3.2.1** *Magnaporthe oryzae* 

spores, and disperses to distant areas. Successful infection by this fungus requires the following steps: (1) the spore to land on the host surface and elongate the germ tube from the apical spore cell and (2) the tip of the germ tube to differentiate into the appressorium, which is the infection machinery. Subsequently, the fungi penetrate into the host cuticle and generate a penetration peg from the bottom of the appressorium (Howard *et al*., 1991). Although the conidia itself has low adhesion ability, the hydrated conidia attains strong adhesion ability within 30 minutes of incubation (Hamer *et al.*, 1988). Ultrastructural analysis has revealed that the spore tip mucilage (STM) is preserved at the apical spore and released after hydration (Braun and Howard, 1994). The attached spores on the surface start to germinate and secrete ECM. The secreted ECM supports the enhanced adhesion ability of the fungi on the plant surface. It is not known whether the STM and the ECM are the same component. During the infection process, some essential environmental signals have been identified as appressorium (infection)-inducing factors, such as the hardness (Xiao *et al*., 1994) and the hydrophobicity of the attachment surface (Jelitto *et al*., 1994; Lee & Dean, 1994), and the chemical components from the plant surface (Gilbert *et al*., 1996). In addition, a number of up-regulating genes have also been characterized (Talbot, 2003). However, the available information was not sufficient to identify the principal components and regulation mechanisms of fungal adhesion in *M. oryzae*.

While the components of STM remain unclear, it was found that STM was reactive with lectin concanabalin A (ConA; α-D-glucose and α-(1,3)-/α-(1,6)-D-mannose binding), suggesting that STM consists of mannose containing glycoprotein (Hamer *et al.*, 1988). The ECM was also ConA-positive (Xiao *et al.*, 1994). The effect of three different specific sugarbinding lectins, namely ConA, PSA (α-D-glucose and α-(1,6)-D-mannose binding), and WGA (chitin binding), on appressorium formation and the adhesion ability of the *M. oryzae* germlings was evaluated (Fig. 3). High concentrations (50 μg ml-1) of lectins, ConA, and WGA were found to inhibit appressorium formation and adhesion. These results indicate that these lectin treatments covered the cell wall surface, and inhibited the development of the cell wall architecture (data not shown). In contrast, low concentrations (10 μg ml-1) of these lectins only affected adhesion, suggesting that low concentrations of lectins are preferentially bound to the adhesion components (Fig. 3). Moreover, although PSA was observed to bind to the cell wall surface, it did not affect the adhesion. ConA and PSA commonly bind to the α-D-glucose and the α-(1,6)-D-mannose moieties; however, only ConA binds to α-(1,3)-D-mannose, suggesting that α-(1,3)-D-mannose is a potential adhesion component (Fig. 3). Our unpublished study revealed that the incubation of *M. oryzae* spores in modified nutrient conditions produced less adherent spore germlings. Furthermore, the less adherent spore germlings were ConA-negative (K. Inoue, unpublished data), suggesting that mannose sugar may be important for adhesion.

Protein secretion via the Golgi pathway might be responsible for ECM production. In the treatment with cycloheximide at the early step of germination, the germ tube and the appressorium were not formed, and the germlings were easily removed (Fig. 4). In the treatments with monensin and tunicamycin, the adhesion rate was significantly lower than for the water control (Fig. 4). Hence, N-glycosylation of the mannose sugar moiety might be important for the maturation of the adhesive protein(s). This result also agreed with the conclusion of the lectin treatment experiments.

The Role of the Extracellular Matrix (ECM)

germling adhesion (S. Inoue *et al.*, 1987).

in Phytopathogenic Fungi: A Potential Target for Disease Control 137

Ultrastrucutural analysis was used to elucidate the ECM components, and revealed that the ECM was an electron dense and fibrous structure (Fig. 5; K. Inoue *et al.*, 2007). This fibrous structure was supposed to be similar to the components of the cell adhesion factors (extracellular matrix in animal cells), such as collagen, fibronectin, and laminin. We then performed immunohistochemical analysis, using specific antibodies against mammal cell adhesion factors. Positive signals were detected from the treatments with antibodies against collagen, vitronectin, fibronectin, laminin, and integrin (K. Inoue *et al.*, 2007). Similar results have been reported by other research groups (Bae *et al.*, 2007; Dean *et al.*, 1994), and for other plant pathogenic fungi (Celerin *et al.*, 1996; Corrêa *et al.*, 1996; Gale *et al.*, 1998; Hyon *et al.*, 2009; Jian *et al.*, 2007; Kaminskyj and Heath, 1995; Manning *et al.*, 2004; Sarma *et al.*, 2005). Cell to cell adhesion mediated by integrin was involved in Arg-Gly-Asp (RGD) motifs (Giancotti & Ruoslahti, 1999). The germlings treated with RGD peptide reduced adhesion ability and appressorium formation (Bae *et al*., 2007). The suppression effect by the RGD treatment was reversed by supplementing with cAMP, suggesting that RGD recognition may be involved in the signaling cascade for appressorium formation (Bae *et al.*, 2007). Based on this evidence, the functions and the components of fungal ECM resemble animal ECM. However, a similar nucleotide sequence to that of the animal ECM component was not detected from the fungal genome sequences. This suggests that only the restricted tertiary protein structure had a

Melanin is also one of the cell surface components. The fungicide treatments, chlobenthiazone and tricyclazole, that inhibit melanin synthesis, have been shown to reduce

Fig. 5. Transmission electron microscopy images of *M. oryzae* appressoria on the leaf surface of wheat. Note: fibrous components were observed at the interface between the plant and

Hydrophobicity is one of the important factors for appressorium formation in *M. oryzae* (Jelitto *et al*., 1994; Lee & Dean, 1994). To determine whether substrate hydrophobicity is essential for fungal adhesion and appressorium formation, fungal differentiation and

fungal appressorium. Adapted from Inoue *et al*. (2007), with permission.

resemblance, with the amino acids sequences being highly diverged.

Fig. 3. Effects of lectins on appressorium formation and adhesion of *M. oryzae* germlings on the hydrophobic surface. The rate of appressorium formation (upper) and adhesion (lower) in *M. oryzae* germlings treated with the water control (a), 10 μg ml-1 ConA (b), 10 μg ml-1 PSA (c), and 10 μg ml-1 WGA (d) at 0 hour post-inoculation (hpi; white bars), 1 hpi (grey bars), and 6 hpi (black bars). Bars indicate standard deviation. \* indicates a significant difference from the control (*p* < 0.05).

Fig. 4. The effects of inhibitors on appressorium formation and the adhesion of *M. oryzae*  germlings on the hydrophobic surface. The rate of appressorium formation (upper) and adhesion (lower) of *M. oryzae* on the hydrophobic surface by inhibitor treatments with the water control (a), 50 μg ml-1 colchicine (b), 0.1 μg ml-1 cycloheximide (c), 1 μg ml-1 monensin (d), and 10 μg ml-1 tunicamycin (e) at 0 hpi (white bars), 1 hpi (grey bars), and 6 hpi (black bars). Bars indicate standard deviation. \*, indicates a significant difference from the control (*p* < 0.05).

One of the candidate ECM components in *M. oryzae, Emp1*, was isolated from the cDNA library, and was homologous to the extracellular matrix protein *Fem1* in *Fusarium oxysporum* (Ahn *et al.*, 2004). The *emp1* disruption mutants showed a slight reduction in adhesion ability, but were largely involved in appressorium formation, suggesting that EMP1 was involved in surface recognition (Ahn *et al*., 2004).

Fig. 3. Effects of lectins on appressorium formation and adhesion of *M. oryzae* germlings on the hydrophobic surface. The rate of appressorium formation (upper) and adhesion (lower) in *M. oryzae* germlings treated with the water control (a), 10 μg ml-1 ConA (b), 10 μg ml-1 PSA (c), and 10 μg ml-1 WGA (d) at 0 hour post-inoculation (hpi; white bars), 1 hpi (grey bars), and 6 hpi (black bars). Bars indicate standard deviation. \* indicates a significant

Fig. 4. The effects of inhibitors on appressorium formation and the adhesion of *M. oryzae*  germlings on the hydrophobic surface. The rate of appressorium formation (upper) and adhesion (lower) of *M. oryzae* on the hydrophobic surface by inhibitor treatments with the water control (a), 50 μg ml-1 colchicine (b), 0.1 μg ml-1 cycloheximide (c), 1 μg ml-1 monensin (d), and 10 μg ml-1 tunicamycin (e) at 0 hpi (white bars), 1 hpi (grey bars), and 6 hpi (black bars). Bars indicate standard deviation. \*, indicates a significant difference from the control

One of the candidate ECM components in *M. oryzae, Emp1*, was isolated from the cDNA library, and was homologous to the extracellular matrix protein *Fem1* in *Fusarium oxysporum* (Ahn *et al.*, 2004). The *emp1* disruption mutants showed a slight reduction in adhesion ability, but were largely involved in appressorium formation, suggesting that EMP1 was

difference from the control (*p* < 0.05).

involved in surface recognition (Ahn *et al*., 2004).

(*p* < 0.05).

Ultrastrucutural analysis was used to elucidate the ECM components, and revealed that the ECM was an electron dense and fibrous structure (Fig. 5; K. Inoue *et al.*, 2007). This fibrous structure was supposed to be similar to the components of the cell adhesion factors (extracellular matrix in animal cells), such as collagen, fibronectin, and laminin. We then performed immunohistochemical analysis, using specific antibodies against mammal cell adhesion factors. Positive signals were detected from the treatments with antibodies against collagen, vitronectin, fibronectin, laminin, and integrin (K. Inoue *et al.*, 2007). Similar results have been reported by other research groups (Bae *et al.*, 2007; Dean *et al.*, 1994), and for other plant pathogenic fungi (Celerin *et al.*, 1996; Corrêa *et al.*, 1996; Gale *et al.*, 1998; Hyon *et al.*, 2009; Jian *et al.*, 2007; Kaminskyj and Heath, 1995; Manning *et al.*, 2004; Sarma *et al.*, 2005). Cell to cell adhesion mediated by integrin was involved in Arg-Gly-Asp (RGD) motifs (Giancotti & Ruoslahti, 1999). The germlings treated with RGD peptide reduced adhesion ability and appressorium formation (Bae *et al*., 2007). The suppression effect by the RGD treatment was reversed by supplementing with cAMP, suggesting that RGD recognition may be involved in the signaling cascade for appressorium formation (Bae *et al.*, 2007). Based on this evidence, the functions and the components of fungal ECM resemble animal ECM. However, a similar nucleotide sequence to that of the animal ECM component was not detected from the fungal genome sequences. This suggests that only the restricted tertiary protein structure had a resemblance, with the amino acids sequences being highly diverged.

Melanin is also one of the cell surface components. The fungicide treatments, chlobenthiazone and tricyclazole, that inhibit melanin synthesis, have been shown to reduce germling adhesion (S. Inoue *et al.*, 1987).

Fig. 5. Transmission electron microscopy images of *M. oryzae* appressoria on the leaf surface of wheat. Note: fibrous components were observed at the interface between the plant and fungal appressorium. Adapted from Inoue *et al*. (2007), with permission.

Hydrophobicity is one of the important factors for appressorium formation in *M. oryzae* (Jelitto *et al*., 1994; Lee & Dean, 1994). To determine whether substrate hydrophobicity is essential for fungal adhesion and appressorium formation, fungal differentiation and

The Role of the Extracellular Matrix (ECM)

adhesion is required.

**3.2.2** *Colletotrichum* **species** 

and are involved in germling adhesion.

**3.2.3** *Botrytis cinerea* 

in Phytopathogenic Fungi: A Potential Target for Disease Control 139

The class II hydrophobin *Mhp1* disruption mutants affected pleiotropically, hydrophobicity, pathogenicity, and spore viability (Kim *et al.,* 2005). In our unpublished data (K. Ikeda), *mhp1* null mutants showed no alteration, except for reducing hydrophobicity. The class II hydrophobin cerato-ulmin in *Ophiostoma novo-ulmi* not only functioned in adhesion, but was also toxic to the host plant, as well as providing tolerance against desiccation (Temple *et al.,* 1997). Paradoxically, the *Cladosporium fulvum* hydrophobin HCf-6 suppressed adhesion ability (Lacroix *et al.,* 2008). Further analysis of the relationship between hydrophobin and

The genus *Colletotrichum* is one of the most important genera of plant pathogens. The genus *Colletotrichum* encompasses numerous species, and the key criterion for their identification is mainly based on plant host determination. The ungerminated conidium of *Colletotrichum*  species possesses adhesion ability. Adhesion is more effective on hydrophobic than hydrophilic surfaces, and is required for *de novo* protein synthesis in *C. graminicola* and *C. musae* (Mercure *et al.*, 1994a; Sela-Buurlage *et al.*, 1991). However, the adhesion strength of *C. graminicola* conidia was not strong, with up to 30% of conidia adhering only, and was influenced by corn leaf age (Mercure *et al.*, 1994a). Attached conidia were ConA positive and were effectively detached by pronase E treatment (Mercure *et al.*, 1994b; Sela-Buurlge *et al.*, 1991). Ultrastructural analysis revealed that the ungerminated conidia of *C. lindemuthianum* and *C. truncatum* were coated with fibrillar ECM, suggesting that the conidia of *Colletrotrichum* species may be covered with adhesive ECM that is dissimilar to *M. oryzae* (Hamer *et al.*, 1988; O'Connell *et al*., 1996; Van Dyke and Mims, 1991). To study the ECM molecular components in *C. lindemuthianum*, monoclonal antibodies (MAbs) were raised against conidia (Pain *et al.*, 1992). A MAb UB20 specifically recognized the conidium surface and UB20 treatment inhibited conidium adhesion (Hughes *et al.*, 1999). However, UB20 treatment did not affect appressorium formation and pathogenicity. The conidium adhesion deficiency was compensated after germination, suggesting that germ tube adhesion was

involved in appressorium formation and pathogenicity (Rawlings *et al.*, 2007).

MAbs were also raised against conidium germlings of *C. lindemuthianum* (Pain *et al.*, 1994a,b). MAbs UB26 and UB31 were observed to bind to the ECM surrounding germ tubes and appressoria, but not to the conidia, suggesting that ECM compounds were different for conidia and germ tubes (Hutchison *et al*., 2002). Moreover, the distribution of UB26 and UB31 positive signals was similar, but spatial distribution was different, i.e., UB31 antigens were located close to the cell wall, while UB26 antigens extended further from the cell wall (Hutchison *et al.*, 2002). These results suggest that multiple components exist in the ECM,

*B. cinerea* is an important fungal pathogen of a number of food and ornamental crops. Conidium adhesion to host surfaces is an important early event in the infection process. Adhesion ability is correlated with the water contact angle of the substrate, suggesting that hydrophobicity is important for conidium adhesion (Doss *et al.*, 1993). However, the percent adhesion of germinated conidia is larger than that of ungerminated conidia (Doss *et al.*,

adhesion were compared on hydrophilic and hydrophobic surfaces. On the hydrophobic surface, most of germlings produced appressoria, with ca. 100% adherence over a six hour period (Fig. 6). On the hydrophilic surface, appressoria were not formed; however, over a six hour period ca. 100% of germlings adhered to the surface (Fig. 6). This result suggests that adhesion ability is dispensable for appressorium formation. The spore germlings may sense hydrophobicity, hardness, or chemical signals to form appressoria.

Fig. 6. The effect of surface characteristics on appressorium formation and the adhesion of *M. oryzae* germlings. (a) The rates of appressorium formation of *M. oryzae* germlings on hydrophobic (white bar) and hydrophilic (black bar) surfaces at 6 hpi. (b) The rates of adhesion to hydrophobic (white bars) and hydrophilic (black bars) surfaces at 0, 0.5, 3, and 6 hpi. Bars indicate standard deviation.

Fungal hydrophobins are cell-surface hydrophobic proteins with four disulphide bonds that are ubiquitous in filamentous fungi (Kershaw *et al.,* 2005; Wösten, 2001). Hydrophobins exist in multiple copies, and these proteins are divided into two groups based on their physical properties (Wessels, 1994). Hydrophobins seem to form part of the ECM. Class I hydrophobins form highly insoluble polymers, whereas class II hydrophobins form polymers that are soluble in some organic solvents (Sunde *et al.,* 2008). These hydrophobins are differentially expressed during the growth stage, and seem to have different functions (Nielsen *et al.,* 2001; Segers *et al.,* 1999; Wessels *et al.,* 1991; Whiteford *et al.,* 2004). In *M. oryzae*, two hydrophobins are well characterized: class I *Mpg1* (Talbot *et al.,* 1993) and class II *Mhp1* (Kim *et al.,* 2005).

In the *mpg1* null mutant, appressorium formation was reduced, suggesting the involvement of host-sensing (Beckerman and Ebbole, 1996; Talbot *et al.,* 1993; Talbot *et al.,* 1996). In a previous study, adhesion in the *mpg1* null mutant was not significantly affected in a conventional wash experiment, but was affected in a boiling-SDS wash experiment (Talbot *et al.,* 1996). In contrast, an RNA silencing experiment of *Mpg1* reduced adhesion (K. Ikeda, unpublished data). The *Mpg1* silencing mutants reduced ConA-positives (K. Ikeda, unpublished data), suggesting that Mpg1 is a ConA-positive glycoprotein, or that Mpg1 recruits ConA-positive glycoproteins to the cell surface. The complex formations involved in Mpg1 may strengthen adhesion and contribute to pathogenicity. The hydrophobin RolA in *Aspergillus oryzae* was associated with cutinase and allowed the substrate to degrade on the hydrophobic surface (Takahashi *et al.*, 2005). One of the cutinase genes *Cut2* in *M. oryzae* was involved in appressorium formation, but not involved in adhesion (Skamnioti & Gurr, 2007). Other cutinase genes may also be involved in adhesion.

The class II hydrophobin *Mhp1* disruption mutants affected pleiotropically, hydrophobicity, pathogenicity, and spore viability (Kim *et al.,* 2005). In our unpublished data (K. Ikeda), *mhp1* null mutants showed no alteration, except for reducing hydrophobicity. The class II hydrophobin cerato-ulmin in *Ophiostoma novo-ulmi* not only functioned in adhesion, but was also toxic to the host plant, as well as providing tolerance against desiccation (Temple *et al.,* 1997). Paradoxically, the *Cladosporium fulvum* hydrophobin HCf-6 suppressed adhesion ability (Lacroix *et al.,* 2008). Further analysis of the relationship between hydrophobin and adhesion is required.

#### **3.2.2** *Colletotrichum* **species**

138 Plant Pathology

adhesion were compared on hydrophilic and hydrophobic surfaces. On the hydrophobic surface, most of germlings produced appressoria, with ca. 100% adherence over a six hour period (Fig. 6). On the hydrophilic surface, appressoria were not formed; however, over a six hour period ca. 100% of germlings adhered to the surface (Fig. 6). This result suggests that adhesion ability is dispensable for appressorium formation. The spore germlings may

Fig. 6. The effect of surface characteristics on appressorium formation and the adhesion of *M. oryzae* germlings. (a) The rates of appressorium formation of *M. oryzae* germlings on hydrophobic (white bar) and hydrophilic (black bar) surfaces at 6 hpi. (b) The rates of adhesion to hydrophobic (white bars) and hydrophilic (black bars) surfaces at 0, 0.5, 3, and 6

Fungal hydrophobins are cell-surface hydrophobic proteins with four disulphide bonds that are ubiquitous in filamentous fungi (Kershaw *et al.,* 2005; Wösten, 2001). Hydrophobins exist in multiple copies, and these proteins are divided into two groups based on their physical properties (Wessels, 1994). Hydrophobins seem to form part of the ECM. Class I hydrophobins form highly insoluble polymers, whereas class II hydrophobins form polymers that are soluble in some organic solvents (Sunde *et al.,* 2008). These hydrophobins are differentially expressed during the growth stage, and seem to have different functions (Nielsen *et al.,* 2001; Segers *et al.,* 1999; Wessels *et al.,* 1991; Whiteford *et al.,* 2004). In *M. oryzae*, two hydrophobins are well characterized: class I *Mpg1* (Talbot *et al.,* 1993) and class II

In the *mpg1* null mutant, appressorium formation was reduced, suggesting the involvement of host-sensing (Beckerman and Ebbole, 1996; Talbot *et al.,* 1993; Talbot *et al.,* 1996). In a previous study, adhesion in the *mpg1* null mutant was not significantly affected in a conventional wash experiment, but was affected in a boiling-SDS wash experiment (Talbot *et al.,* 1996). In contrast, an RNA silencing experiment of *Mpg1* reduced adhesion (K. Ikeda, unpublished data). The *Mpg1* silencing mutants reduced ConA-positives (K. Ikeda, unpublished data), suggesting that Mpg1 is a ConA-positive glycoprotein, or that Mpg1 recruits ConA-positive glycoproteins to the cell surface. The complex formations involved in Mpg1 may strengthen adhesion and contribute to pathogenicity. The hydrophobin RolA in *Aspergillus oryzae* was associated with cutinase and allowed the substrate to degrade on the hydrophobic surface (Takahashi *et al.*, 2005). One of the cutinase genes *Cut2* in *M. oryzae* was involved in appressorium formation, but not involved in adhesion (Skamnioti & Gurr,

2007). Other cutinase genes may also be involved in adhesion.

sense hydrophobicity, hardness, or chemical signals to form appressoria.

hpi. Bars indicate standard deviation.

*Mhp1* (Kim *et al.,* 2005).

The genus *Colletotrichum* is one of the most important genera of plant pathogens. The genus *Colletotrichum* encompasses numerous species, and the key criterion for their identification is mainly based on plant host determination. The ungerminated conidium of *Colletotrichum*  species possesses adhesion ability. Adhesion is more effective on hydrophobic than hydrophilic surfaces, and is required for *de novo* protein synthesis in *C. graminicola* and *C. musae* (Mercure *et al.*, 1994a; Sela-Buurlage *et al.*, 1991). However, the adhesion strength of *C. graminicola* conidia was not strong, with up to 30% of conidia adhering only, and was influenced by corn leaf age (Mercure *et al.*, 1994a). Attached conidia were ConA positive and were effectively detached by pronase E treatment (Mercure *et al.*, 1994b; Sela-Buurlge *et al.*, 1991). Ultrastructural analysis revealed that the ungerminated conidia of *C. lindemuthianum* and *C. truncatum* were coated with fibrillar ECM, suggesting that the conidia of *Colletrotrichum* species may be covered with adhesive ECM that is dissimilar to *M. oryzae* (Hamer *et al.*, 1988; O'Connell *et al*., 1996; Van Dyke and Mims, 1991). To study the ECM molecular components in *C. lindemuthianum*, monoclonal antibodies (MAbs) were raised against conidia (Pain *et al.*, 1992). A MAb UB20 specifically recognized the conidium surface and UB20 treatment inhibited conidium adhesion (Hughes *et al.*, 1999). However, UB20 treatment did not affect appressorium formation and pathogenicity. The conidium adhesion deficiency was compensated after germination, suggesting that germ tube adhesion was involved in appressorium formation and pathogenicity (Rawlings *et al.*, 2007).

MAbs were also raised against conidium germlings of *C. lindemuthianum* (Pain *et al.*, 1994a,b). MAbs UB26 and UB31 were observed to bind to the ECM surrounding germ tubes and appressoria, but not to the conidia, suggesting that ECM compounds were different for conidia and germ tubes (Hutchison *et al*., 2002). Moreover, the distribution of UB26 and UB31 positive signals was similar, but spatial distribution was different, i.e., UB31 antigens were located close to the cell wall, while UB26 antigens extended further from the cell wall (Hutchison *et al.*, 2002). These results suggest that multiple components exist in the ECM, and are involved in germling adhesion.

#### **3.2.3** *Botrytis cinerea*

*B. cinerea* is an important fungal pathogen of a number of food and ornamental crops. Conidium adhesion to host surfaces is an important early event in the infection process. Adhesion ability is correlated with the water contact angle of the substrate, suggesting that hydrophobicity is important for conidium adhesion (Doss *et al.*, 1993). However, the percent adhesion of germinated conidia is larger than that of ungerminated conidia (Doss *et al.*,

The Role of the Extracellular Matrix (ECM)

by using enzymatic activity.

universality in filamentous fungi.

**host leaf surface** 

*M. oryzae* **germlings** 

and entomopathogenicity (Wang & Legar, 2007).

**4. Detachment of germlings from the host surface** 

in Phytopathogenic Fungi: A Potential Target for Disease Control 141

insect surface, blastospore production, and virulence to caterpillars. In comparison, MAD2 was involved in adhesion to the plant surface, but showed no effect on fungal differentiation

Fungal adhesion to the host cells is expected to be one of the pathogenicity factors (K. Inoue *et al*., 2007). The regulation of fungal adhesion is expected to lead to disease control. The adhesion mechanisms of pathogenic fungi are common, with specific features existing in each fungus. We attempted to detach pathogenic fungi from the host or artificial substrate

Various types of hydrolytic enzymes were tested for detachment activity. In enzyme treatments of 1 hpi, germlings were detached without affecting appressorium formation, when using α-mannosidase, β-mannosidase, pronase E, collagenase N-2, collagenase S-1, and gelatinase B (K. Inoue *et al.*, 2011). In the enzyme treatments of 6 hpi, most germlings produced appressoria, with the inhibition of ECM production being difficult. In this situation, pronase E and all types of collagenase and gelatinase caused a significant detachment of germlings (K. Inoue *et al.*, 2011). Pronase E is known to be a mucoproteindegrading enzyme, and had the ability of moderate removal effect in *B. sorokiniana* (Apoga *et al*., 2001). In particular, collagenase type S-1 and gelatinase B seemed to be the most effective ECM target-specific enzymes, and had a minimal effect on appressorium formation, even at the early stages of application. Mannose moiety was also a target for ECM degradation. However, there are some discrepancies with a previous study; Xiao *et al*. (1994) reported that α-glucosidase and α-mannosidase are effective in removing germlings; however, we found that β-mannosidase is more effective (K. Inoue *et al.*, 2011). These timelapse experiments clearly show that the timing of adding enzymes influenced the results, which might explain the discrepancies between the two studies. The mannose-degrading enzymes were effective at the early stage, but became ineffective with time. Most of the other substrate-specific enzymes, such as glycan-, protein-, and lipid-degrading enzymes, were difficult to degrade (K. Inoue *et al.*, 2011). These results suggest that the adhesive compounds of ECM consist of glycoproteins with mannose sugars, which gradually accumulate with time. In the pathogenicity test on the host plant, lesion formation was remarkably suppressed on the treatment with crude collagenase, collagenase S-1, and gelatinase B (K. Inoue *et al.*, 2011). A similar detachment effect by crude collagenase was also reported in the *Alternaria alternata* Japanese pear pathotype (Hyon *et al.*, 2009), suggesting

**4.2 SEM analysis of enzyme effects on the adhesion of** *M. oryzae* **germlings to the** 

To examine the effect of the enzymes on germling detachment to the leaf surface, each enzyme was applied to *M. oryzae* germlings at the appropriate time, and incubated for up to 25 hpi. The specimens were then observed by using SEM. It was difficult to ascertain

**4.1 Effects of hydrolytic enzymes on appressorium formation and adhesion of** 

1993). The adhesion ability of *B. cinerea* seems to be strengthened with the differentiation of germlings. Germling attachment was resistant to removal by boiling or by treatment with hydrolytic enzymes, periodic acid, or sulfuric acid, but was readily removed by a strong base, sodium hydrate (Doss *et al.*, 1995). The ECM components of germ tubes detected the presence of enzymatic activities such as polygalacturonase, laccase, and cutinase (Doss, 1999). Moreover, the ECM of germlings contained melanin (Doss *et al.*, 2003). These components were similar to *M. oryzae* and are assumed to be involved in adhesion. The class I hydrophobin *BcHpb1* was also partially involved in adhesion, although the disruption mutant of *BcHpb1* retained pathogenicity (Izumitsu *et al.,* 2010).

#### **3.2.4** *Ustilago violacea* **(***Microbotryum violacea***)**

The anther smut fungus *Ustilago* (*Microbotryum*) *violacea* propagates yeast-like sporidial cells. The cell walls of smut contained a hair-like appendage (fungal fimbriae) that was similar to the Gram-negative bacteria cell wall appendage, fimbriae (Poon & Day, 1974). The fungal fimbriae might be essential for the later stages of conjugation (Poon & Day, 1975). The component of fungal fimbriae contained protein with motifs similar to collagen (Celerin *et al*., 1996). Collagen is characteristic of the glycine-rich repeat motif, and functions in the cell adhesion of animal connective tissues. Collagen family proteins seem to have a conserved conformation between animal and fungi, although amino acid sequences are divergent. The relationship between the fungal fimbriae of smut and the fibrillar ECM of spore coat in other fungal species remains unclear.

#### **3.2.5 Other fungi**

Conidium adhesion was analyzed, and differences were found in various plant pathogenic fungi. In *Bipolaris sorokiniana* and *Venturia inaequalis*, conidium adhesion to the hydrophobic surface is weak, due to the hydrophobic interaction (Apoga *et al.*, 2001; Schumacher *et al.*, 2008). In *Nectria haematococca*, 90 kDa glycoprotein containing mannose sugar was associated with the development of the adhesiveness of macroconidia (Kwon & Epstein, 1997). In *V. inaequalis*, conidia released adhesive material that was termed spore tip glue (STG) (Schumacher *et al.*, 2008). The STG was ConA-negative, suggesting that it was different to STM found in *M. oryzae* (Hamer *et al.*, 1988; Schumacher *et al.*, 2008). These adhesion molecules of ungerminated conidia were pre-synthesized and preserved.

After the germination of conidia in most plant pathogenic fungi, the adhesion ability is increased by the production of ECM from germ tubes. In *Uromyces vicia-fabae* and *Blumeria graminis*, cutinase and esterase in the extracellular matrix (ECM) appeared to play a function in adhesion, either on the hydrophobic or hydrophilic surface, even during pre-germination (Deising *et al.*, 1992; Tucker & Talbot, 2001). Treatment with serine-esterase inhibitor diisopropyl fluorophosphate prevented adhesion (Deising *et al.*, 1992). This evidence suggests that adhesion may be partially involved in the combination of hydrophobin with cutinase as a molecular mechanism in *A. oryzae* (Takahashi *et al.*, 2005). However, the components of other ECM remain undetermined.

In the insect pathogenic fungus *Metarhizium anisopliae*, two proteins, MAD1 and MAD2, were involved in adhesion (Wang & Legar, 2007). MAD1 was involved in adhesion to the

1993). The adhesion ability of *B. cinerea* seems to be strengthened with the differentiation of germlings. Germling attachment was resistant to removal by boiling or by treatment with hydrolytic enzymes, periodic acid, or sulfuric acid, but was readily removed by a strong base, sodium hydrate (Doss *et al.*, 1995). The ECM components of germ tubes detected the presence of enzymatic activities such as polygalacturonase, laccase, and cutinase (Doss, 1999). Moreover, the ECM of germlings contained melanin (Doss *et al.*, 2003). These components were similar to *M. oryzae* and are assumed to be involved in adhesion. The class I hydrophobin *BcHpb1* was also partially involved in adhesion, although the disruption

The anther smut fungus *Ustilago* (*Microbotryum*) *violacea* propagates yeast-like sporidial cells. The cell walls of smut contained a hair-like appendage (fungal fimbriae) that was similar to the Gram-negative bacteria cell wall appendage, fimbriae (Poon & Day, 1974). The fungal fimbriae might be essential for the later stages of conjugation (Poon & Day, 1975). The component of fungal fimbriae contained protein with motifs similar to collagen (Celerin *et al*., 1996). Collagen is characteristic of the glycine-rich repeat motif, and functions in the cell adhesion of animal connective tissues. Collagen family proteins seem to have a conserved conformation between animal and fungi, although amino acid sequences are divergent. The relationship between the fungal fimbriae of smut and the fibrillar ECM of spore coat in other

Conidium adhesion was analyzed, and differences were found in various plant pathogenic fungi. In *Bipolaris sorokiniana* and *Venturia inaequalis*, conidium adhesion to the hydrophobic surface is weak, due to the hydrophobic interaction (Apoga *et al.*, 2001; Schumacher *et al.*, 2008). In *Nectria haematococca*, 90 kDa glycoprotein containing mannose sugar was associated with the development of the adhesiveness of macroconidia (Kwon & Epstein, 1997). In *V. inaequalis*, conidia released adhesive material that was termed spore tip glue (STG) (Schumacher *et al.*, 2008). The STG was ConA-negative, suggesting that it was different to STM found in *M. oryzae* (Hamer *et al.*, 1988; Schumacher *et al.*, 2008). These adhesion

After the germination of conidia in most plant pathogenic fungi, the adhesion ability is increased by the production of ECM from germ tubes. In *Uromyces vicia-fabae* and *Blumeria graminis*, cutinase and esterase in the extracellular matrix (ECM) appeared to play a function in adhesion, either on the hydrophobic or hydrophilic surface, even during pre-germination (Deising *et al.*, 1992; Tucker & Talbot, 2001). Treatment with serine-esterase inhibitor diisopropyl fluorophosphate prevented adhesion (Deising *et al.*, 1992). This evidence suggests that adhesion may be partially involved in the combination of hydrophobin with cutinase as a molecular mechanism in *A. oryzae* (Takahashi *et al.*, 2005). However, the

In the insect pathogenic fungus *Metarhizium anisopliae*, two proteins, MAD1 and MAD2, were involved in adhesion (Wang & Legar, 2007). MAD1 was involved in adhesion to the

molecules of ungerminated conidia were pre-synthesized and preserved.

components of other ECM remain undetermined.

mutant of *BcHpb1* retained pathogenicity (Izumitsu *et al.,* 2010).

**3.2.4** *Ustilago violacea* **(***Microbotryum violacea***)**

fungal species remains unclear.

**3.2.5 Other fungi** 

insect surface, blastospore production, and virulence to caterpillars. In comparison, MAD2 was involved in adhesion to the plant surface, but showed no effect on fungal differentiation and entomopathogenicity (Wang & Legar, 2007).

#### **4. Detachment of germlings from the host surface**

Fungal adhesion to the host cells is expected to be one of the pathogenicity factors (K. Inoue *et al*., 2007). The regulation of fungal adhesion is expected to lead to disease control. The adhesion mechanisms of pathogenic fungi are common, with specific features existing in each fungus. We attempted to detach pathogenic fungi from the host or artificial substrate by using enzymatic activity.

#### **4.1 Effects of hydrolytic enzymes on appressorium formation and adhesion of**  *M. oryzae* **germlings**

Various types of hydrolytic enzymes were tested for detachment activity. In enzyme treatments of 1 hpi, germlings were detached without affecting appressorium formation, when using α-mannosidase, β-mannosidase, pronase E, collagenase N-2, collagenase S-1, and gelatinase B (K. Inoue *et al.*, 2011). In the enzyme treatments of 6 hpi, most germlings produced appressoria, with the inhibition of ECM production being difficult. In this situation, pronase E and all types of collagenase and gelatinase caused a significant detachment of germlings (K. Inoue *et al.*, 2011). Pronase E is known to be a mucoproteindegrading enzyme, and had the ability of moderate removal effect in *B. sorokiniana* (Apoga *et al*., 2001). In particular, collagenase type S-1 and gelatinase B seemed to be the most effective ECM target-specific enzymes, and had a minimal effect on appressorium formation, even at the early stages of application. Mannose moiety was also a target for ECM degradation. However, there are some discrepancies with a previous study; Xiao *et al*. (1994) reported that α-glucosidase and α-mannosidase are effective in removing germlings; however, we found that β-mannosidase is more effective (K. Inoue *et al.*, 2011). These timelapse experiments clearly show that the timing of adding enzymes influenced the results, which might explain the discrepancies between the two studies. The mannose-degrading enzymes were effective at the early stage, but became ineffective with time. Most of the other substrate-specific enzymes, such as glycan-, protein-, and lipid-degrading enzymes, were difficult to degrade (K. Inoue *et al.*, 2011). These results suggest that the adhesive compounds of ECM consist of glycoproteins with mannose sugars, which gradually accumulate with time. In the pathogenicity test on the host plant, lesion formation was remarkably suppressed on the treatment with crude collagenase, collagenase S-1, and gelatinase B (K. Inoue *et al.*, 2011). A similar detachment effect by crude collagenase was also reported in the *Alternaria alternata* Japanese pear pathotype (Hyon *et al.*, 2009), suggesting universality in filamentous fungi.

#### **4.2 SEM analysis of enzyme effects on the adhesion of** *M. oryzae* **germlings to the host leaf surface**

To examine the effect of the enzymes on germling detachment to the leaf surface, each enzyme was applied to *M. oryzae* germlings at the appropriate time, and incubated for up to 25 hpi. The specimens were then observed by using SEM. It was difficult to ascertain

The Role of the Extracellular Matrix (ECM)

*et al*. (2010), with permission.

**4.3 Screening of gelatinolytic bacteria for biological control** 

would allow the enrichment of high gelatinolytic genera (Shimoi *et al.*, 2010).

Ca2+ or 1 mM Zn2+, and to a lesser extent with 2 mM Mn2+ (Shimoi *et al.*, 2010).

Fig. 8. Screening method to isolate gelatinolytic bacteria from nature. Adapted from Shimoi

The effects of bacterial gelatinolytic activity on the germling adhesion of *M. oryzae* were evaluated. Treatment with each of the five bacterial isolates significantly decreased the percentage of germling adhesion (Fig. 9). In particular, the effects of Ac1 and Ch1 on germling detachment were comparable to the effects of commercial collagenase placed simultaneously on spore suspensions (Fig. 9). The detachment of germlings decreased when bacterial cultures were added 6 h after the inoculation of the spore suspension. Germling

in Phytopathogenic Fungi: A Potential Target for Disease Control 143

To screen collagenolytic/gelatinolytic bacteria, we examined two different screening methods, i.e., (1) utilization of a leaf-associating bacteria library and (2) direct screening from the field, and incubation of samples with collagen (Fig. 8). Collagen is fibrous and difficult to catabolize for nutrient acquisition; therefore, we expected that direct screening with collagen incubation

The leaf-associated library was screened for gelatinolytic activity on a 96-microtiter plate (first screening) and Petri dish (second screening). In the case of direct screening samples from the field, five bacterial isolates, which showed especially high gelatinolytic activity, were further characterized as *Acidovorax* sp. (Ac1), *Chryseobacterium* sp. (Ch1 and Ch2), *Sphingomonas* sp. (Sp1), and *Pseudomonas* sp. (Ps1) (Shimoi *et al.*, 2010). The effects of the proteinase inhibitors, EDTA, antipain, aprotinin, and PMSF on bacterial gelatinolytic activity were evaluated. The gelatinolytic activities of Ac1, Ch1, Ch2, and Sp1 were inhibited by EDTA in a dose-dependent manner (Shimoi *et al.*, 2010). In contrast, gelatinolytic activity in Ps1 was not inhibited by EDTA (Shimoi *et al.*, 2010). Bacterial gelatinolytic activities were not inhibited by antipain, aprotinin, or PMSF. These enzymatic characters suggested that all isolates, except Ps1, produced metalloproteinases. Therefore, we examined the effects of each metal cation on gelatinolytic activity in the presence of 5 mM EDTA. The gelatinolytic activities of Sp1 and Ac1 were restored by supplementation with 10 mM Ca2+ (Shimoi *et al.*, 2010). In contrast, the gelatinolytic activity of Ch1 and Ch2 were restored by supplementation with either 10 mM

whether the absence of spores was the result of the enzyme treatments or the lack of spores at the onset of the experiment. *M. grisea* reportedly produces cutinases (Skamnioti & Gurr, 2007; Sweigard *et al*., 1992). Therefore, this pathogen can degrade the wax of plant surfaces. The detached infection structure would be recognizable as vestiges of degraded wax on the wheat surface. In the treatment with cellulase or protease, the infection structures tightly adhered onto the surface (K. Inoue *et al.*, 2011). In contrast, the treatment with crude collagenase or gelatinase B (matrix metalloproteinases; MMPs) resulted in the detachment of germlings, and vestiges of the presence of germlings were observed (K. Inoue *et al.*, 2011). The detachment effect caused by the treatment with crude collagenase was validated at 18 hpi. In contrast, the detachment effect was cancelled out at 24 hpi, at which time fungal penetration into the host was established.

The SEM observation clearly shows the effects of the enzymes on the degradation of the interface between host plant and germlings. We demonstrated that the reduction in pathogenicity is attributable to the detachment of germlings based on treatment with effective enzymes. In this study, MMPs were confirmed to be useful for protecting wheat from *M. oryzae*.

Fig. 7. Detachment effects of *M. oryzae* germlings following treatment with hydrolytic enzymes. Left photos: light microscopy; right photos: scanning electron microscopy. The treatments with collagenase were detached, and only the vestiges of germlings were observed (lower photos). Other hydrolytic enzymes, such as glycosidase were, not affected by germling detachment (upper photos).

whether the absence of spores was the result of the enzyme treatments or the lack of spores at the onset of the experiment. *M. grisea* reportedly produces cutinases (Skamnioti & Gurr, 2007; Sweigard *et al*., 1992). Therefore, this pathogen can degrade the wax of plant surfaces. The detached infection structure would be recognizable as vestiges of degraded wax on the wheat surface. In the treatment with cellulase or protease, the infection structures tightly adhered onto the surface (K. Inoue *et al.*, 2011). In contrast, the treatment with crude collagenase or gelatinase B (matrix metalloproteinases; MMPs) resulted in the detachment of germlings, and vestiges of the presence of germlings were observed (K. Inoue *et al.*, 2011). The detachment effect caused by the treatment with crude collagenase was validated at 18 hpi. In contrast, the detachment effect was cancelled out at 24 hpi, at which time fungal

The SEM observation clearly shows the effects of the enzymes on the degradation of the interface between host plant and germlings. We demonstrated that the reduction in pathogenicity is attributable to the detachment of germlings based on treatment with effective enzymes. In this study, MMPs were confirmed to be useful for protecting wheat

Fig. 7. Detachment effects of *M. oryzae* germlings following treatment with hydrolytic enzymes. Left photos: light microscopy; right photos: scanning electron microscopy. The treatments with collagenase were detached, and only the vestiges of germlings were observed (lower photos). Other hydrolytic enzymes, such as glycosidase were, not affected

penetration into the host was established.

by germling detachment (upper photos).

from *M. oryzae*.

#### **4.3 Screening of gelatinolytic bacteria for biological control**

To screen collagenolytic/gelatinolytic bacteria, we examined two different screening methods, i.e., (1) utilization of a leaf-associating bacteria library and (2) direct screening from the field, and incubation of samples with collagen (Fig. 8). Collagen is fibrous and difficult to catabolize for nutrient acquisition; therefore, we expected that direct screening with collagen incubation would allow the enrichment of high gelatinolytic genera (Shimoi *et al.*, 2010).

The leaf-associated library was screened for gelatinolytic activity on a 96-microtiter plate (first screening) and Petri dish (second screening). In the case of direct screening samples from the field, five bacterial isolates, which showed especially high gelatinolytic activity, were further characterized as *Acidovorax* sp. (Ac1), *Chryseobacterium* sp. (Ch1 and Ch2), *Sphingomonas* sp. (Sp1), and *Pseudomonas* sp. (Ps1) (Shimoi *et al.*, 2010). The effects of the proteinase inhibitors, EDTA, antipain, aprotinin, and PMSF on bacterial gelatinolytic activity were evaluated. The gelatinolytic activities of Ac1, Ch1, Ch2, and Sp1 were inhibited by EDTA in a dose-dependent manner (Shimoi *et al.*, 2010). In contrast, gelatinolytic activity in Ps1 was not inhibited by EDTA (Shimoi *et al.*, 2010). Bacterial gelatinolytic activities were not inhibited by antipain, aprotinin, or PMSF. These enzymatic characters suggested that all isolates, except Ps1, produced metalloproteinases. Therefore, we examined the effects of each metal cation on gelatinolytic activity in the presence of 5 mM EDTA. The gelatinolytic activities of Sp1 and Ac1 were restored by supplementation with 10 mM Ca2+ (Shimoi *et al.*, 2010). In contrast, the gelatinolytic activity of Ch1 and Ch2 were restored by supplementation with either 10 mM Ca2+ or 1 mM Zn2+, and to a lesser extent with 2 mM Mn2+ (Shimoi *et al.*, 2010).

Fig. 8. Screening method to isolate gelatinolytic bacteria from nature. Adapted from Shimoi *et al*. (2010), with permission.

The effects of bacterial gelatinolytic activity on the germling adhesion of *M. oryzae* were evaluated. Treatment with each of the five bacterial isolates significantly decreased the percentage of germling adhesion (Fig. 9). In particular, the effects of Ac1 and Ch1 on germling detachment were comparable to the effects of commercial collagenase placed simultaneously on spore suspensions (Fig. 9). The detachment of germlings decreased when bacterial cultures were added 6 h after the inoculation of the spore suspension. Germling

The Role of the Extracellular Matrix (ECM)

**5. Conclusion** 

foliar environment.

**7. References** 

**6. Acknowledgments** 

105: 1251-1260.

*Interaction,* 9:450-456.

surfaces. *Mycological Research,* 94:865-875.

cell wall. *Cryo-Letters*, 15:161-172.

surfaces. *Protoplasma,* 181:202-212.

in Phytopathogenic Fungi: A Potential Target for Disease Control 145

Germling adhesion is a general feature of plant pathogenic fungi, but its components are complicated. This adhesion ability is considered to be a promising a target for protection against disease. We found that glycoprotein degrading enzymes effectively detach germlings, while gelatinolytic bacteria showed a protective effect against disease. If these gelatinolytic bacteria can produce cell lytic enzymes, such as chitinase or glucanase, efficiency against disease protection would increase synergistically. Our future goals are to screen specific bacteria that produce multiple enzymes and provide a stable habitat in the

This research was supported by Grants-in-Aid for Scientific Research B (No. 18380033), Grants-in-Aid for Young Scientists B (No. 19780036), and Grants-in Aid for Young Scientists

Ahn, N., Kim, S., Choi, W., Im, H. & Lee, Y. H. (2004). Extracellular matrix protein gene,

Apoga, D., Jansson, H. B. & Tunlid, A. (2001). Adhesion of conidia and germlings of the

Bae, C.-Y., Kim, S., Choi, W. B. & Lee, Y.-H. (2007). Involvement of extracellular matrix and

*Magnaporthe oryzae*. *Journal of Microbiology and Biotechnology*, 17:1198-1203. Beckerman, J. L. & Ebbole, D. J. (1996). MPG1, a gene encoding a fungal hydrophobin of

Beckett, A., Tatnell, J. A. & Taylor, N. (1990). Adhesion and pre-invasion behaviour of

Bobichon, H., Gache, D. & Bouchet, P. (1994). Ultrarapid cryofixation of *Candida albicans*:

Bouchara, J.-P., Oumeziane, N. A., Lissitzky, J.-C., Larcher, G., Tronchin, G. & Ghabasse, D.

extracellular matrix components. *European Journal of Cell Biology*, 70:76-83. Braun, E. J. & Howard, R. J. (1994). Adhesion of fungal spores and germlings to host plant

Celerin, M., Ray, J. M., Schisler, N. J., Day, A. W., Steter-Stevenson, W. G. & Laudenbach, D. E. (1996). Fungal fimbriae are composed of collagen. *EMBO Journal*, 15:4445-4453. Corrêa, A., Staples, R. C. & Hoch, H. C. (1996). Inhibition of thigmostimulated cell differentiation with RGD-peptides in *Uromyces* germlings. *Protoplasma,* 194:91-102.

*EMP1*, is required for appressorium formation and pathogenicity of the rice blast

plant pathogenic fungus *Bipolaris sorokiniana* to solid surfaces. *Mycological Research*,

integrin-like proteins on conidial adhesion and appressorium differentiation in

*Magnaporthe grisea*, is involved in surface recognition. *Molecular Plant-Microbe* 

urediniospores of *Uromyces viciae-fabae* during germination on host and synthetic

evidence for a fibrillar reticulated external layer and mannan channels within the

(1996). Attachment of spores of the human pathogenic fungus *Rhizopus oryzae* to

A (No. 23688006) from the Japan Society for the Promotion of Science.

fungus, *Magnaporthe grisea*. *Molecules and cells*, 17:166-173.

adhesion was restored when EDTA was added to the mixtures of bacteria and spore suspension, except in the case of Ps1.

Fig. 9. Detachment effect of *M. oryzae* germlings treated with gelatinolytic bacteria or commercial collagenase. Gelatinolytic bacteria and collagenase were treated concurrently black bars) or 6 hours after inoculation (white bars) with *M. oryzae* spores. Adapted from Shimoi *et al*. (2010), with permission.

We evaluated the protective effects of the selected bacterial isolates on rice blast disease. Spore suspensions of *M. oryzae* and each bacterial culture were inoculated onto rice leaves (cv. Lijiangxintuanheigu; LTH). The treatment with all the bacterial isolates significantly decreased disease indices compared to the control (Fig. 10, Shimoi *et al.*, 2010).

Fig. 10. Disease symptoms of rice blast inoculated with *M. oryzae* and gelatinolytic bacteria. Control: *M. oryzae* spore suspension alone. Adapted from Shimoi *et al*. (2010), with permission.

#### **5. Conclusion**

144 Plant Pathology

adhesion was restored when EDTA was added to the mixtures of bacteria and spore

Fig. 9. Detachment effect of *M. oryzae* germlings treated with gelatinolytic bacteria or commercial collagenase. Gelatinolytic bacteria and collagenase were treated concurrently black bars) or 6 hours after inoculation (white bars) with *M. oryzae* spores. Adapted from

decreased disease indices compared to the control (Fig. 10, Shimoi *et al.*, 2010).

We evaluated the protective effects of the selected bacterial isolates on rice blast disease. Spore suspensions of *M. oryzae* and each bacterial culture were inoculated onto rice leaves (cv. Lijiangxintuanheigu; LTH). The treatment with all the bacterial isolates significantly

Fig. 10. Disease symptoms of rice blast inoculated with *M. oryzae* and gelatinolytic bacteria.

Control: *M. oryzae* spore suspension alone. Adapted from Shimoi *et al*. (2010), with

suspension, except in the case of Ps1.

Shimoi *et al*. (2010), with permission.

permission.

Germling adhesion is a general feature of plant pathogenic fungi, but its components are complicated. This adhesion ability is considered to be a promising a target for protection against disease. We found that glycoprotein degrading enzymes effectively detach germlings, while gelatinolytic bacteria showed a protective effect against disease. If these gelatinolytic bacteria can produce cell lytic enzymes, such as chitinase or glucanase, efficiency against disease protection would increase synergistically. Our future goals are to screen specific bacteria that produce multiple enzymes and provide a stable habitat in the foliar environment.

#### **6. Acknowledgments**

This research was supported by Grants-in-Aid for Scientific Research B (No. 18380033), Grants-in-Aid for Young Scientists B (No. 19780036), and Grants-in Aid for Young Scientists A (No. 23688006) from the Japan Society for the Promotion of Science.

#### **7. References**


The Role of the Extracellular Matrix (ECM)

6968.2011.02353.x)

194:471-477.

*Microbiology,* 56:117-125.

*Microbiology,* 57:1224-1237.

*Molecular Plant Pathology*, 51:63-74.

14:133-147.

286:136-144.

*Medicine and Biology,* 23:1-8.

*Journal of General Plant Pathology,* 73:388-398.

in Phytopathogenic Fungi: A Potential Target for Disease Control 147

Inoue, K., Suzuki, T., Ikeda, K., Jiang, S., Hosogi, N., Hyon, G.-S., Hida, S., Yamada, T. &

Inoue, K., Onoe, T., Park, P. & Ikeda, K. (2011). Enzymatic detachment of spore germlings in

Inoue, S. Kato, T., Jordan, V. W. L. & Brent, K. J. (1987). Inhibition of appressorial adhesion of *Pyricularia oryzae* to barley leaves by fungicides. *Pesticide Science*, 19:145-152. Izumitsu, K., Kimura, S., Kobayashi, H., Morita, A., Saitoh, Y. & Tanaka, C. (2010). Class I

Jelito, T. C., Page, H. A. & Read, N. D. (1994). Role of external signals in regulating the pre-

Jian, S., Hyon, G.-S., Inoue, K., Park, P. & Ishii, H. (2007). Immunohistochemical and

Kaminskyj, S. G. W. & Heath, I. B. (1995). Integrin and spectrin homologues, and cytoplasm-

Kershaw, M. J., Thornton, C. R., Wakley, G. E. & Talbot, N. J. (2005). Four conserved

Kim, S., Ahn, I. P., Rho, H. S. & Lee, Y. H. (2005). *MHP1*, a *Magnaporthe grisea* hydrophobin

Klotz, S. A., Rutten, M. J., Smith, R. L., Babcock, S. R. & Cunningham, M. D. (1993).

Kwon, Y. H. & Epstein, L. (1997). Isolation and composition of the 90 kDa glycoprotein

Lacroix, H., Whiteford, J. R. & Spanu, P. D. (2008). Localization of *Cladosporium fulvum*

Lee, Y. H. & Dean, R. A. (1994). Hydrophobicity of contact surface induces appressorium formation in *Magnaporthe grisea*. *FEMS Microbiology Letters*, 115:71-75. Manning, V. A., Andrie, R. M., Trippe, A. F. & Ciuffetti, L. M. (2004). Ptr ToxA requires

*Botrytis cinerea*. *Journal of General Plant Pathology,* 76:254-260.

*Microscopy Technology for Medicine and Biology*, 21:7-11.

wall adhesion in tip growth. *Journal of Cell Science,* 108:849-856.

of host leaves during plant infection. *Journal of Electron Microscopy Technology for* 

Park, P. (2007). Extracellular matrix of *Magnaporthe oryzae* may have a role in host adhesion during fungal penetration and is digested by matrix metalloproteinases.

*Magnaporthe oryzae*. *FEMS Microbiology Letters*, 323:13-19 (doi: 10.1111/j.1574-

hydrophobin BcHpb1 is important for adhesion but not for later infection of

penetration phase of infection by the rice blast fungus, *Magnaporthe grisea*. *Planta*,

cytochemical analysis of extracellular matrix produced from *Venturia nashicola*, scab fungus, on the surfaces of susceptible Japanese pear leaves. *Journal of Electron* 

intramolecular disulphide linkages are required for secretion and cell wall localization of a hydrophobin during fungal morphogenesis. *Molecular* 

gene, is required for fungal development and plant colonization. *Molecular* 

Adherence of *Candida albicans* to immobilized extracellular matrix proteins is mediated by calcium-dependent surface glycoproteins. *Microbial Pathogenesis*,

associated with adhesion of *Nectria haematococca* macroconidia. *Physiological and* 

hydrophobins reveals a role for HCf-6 in adhesion. *FEMS Microbiology Letters,*

multiple motifs for complete activity. *Molecular Plant-Microbe Interaction,* 17:491-501.


Dean, R. A., Lee, Y. H., Mitchell, T. K. & Whitehead, D. S. (1994). Signalling systems and

Doss, R. P. (1999). Composition and enzymatic activity of the extracellular matrix secreted by germlings of *Botrytis cinerea*. *Applied and Environmental Microbiology*, 65:404-408. Doss, R. P., Deisenhofer, J., von Nidda, H.-A. K., Soeldner, A. H. & McGuire, R. P. (2003).

Doss, R. P., Potter, S. W., Chastagner, G. A. & Christian, J. K. (1993). Adhesion of

Doss, R. P., Potter, S. W., Soeldner, A. H., Christian, J. K. & Fukunaga, L. E. (1995). Adhesion of germlings of *Botrytis cinerea*. *Applied and Environmental Microbiology*, 61:260-265. Fu, Y., Rieg, G., Fonzi, W. A., Belanger, P. H., Edwards Jr, J. E. & Filler, S. G. (1998).

Gale, C. A., Finkel, D., Tao, N., Meinke, M., McClellan, M., Olson, J., Kendrick, K. &

Gilbert, R. D., Johnson, A. M. & Dean, R. A. (1996). Chemical signals responsible for

Hamer, J. E., Howard, R. J., Chumley, F. G. & Valent, B. (1988). A mechanism for surface attachment in spores of a plant pathogenic fungus. *Science,* 239: 288–290. Howard, R. J., Ferrari, M. A., Roach, D. H. & Money, N. P. (1991). Penetration of hard

*National Academy of Sciences of the United States of America*, 88:11281-11284. Hughes, H. B., Carzaniga, R., Rawlings, S. L., Green, J. R. & O'Connell, R. J. (1999). Spore

Hutchison, K. A., Green, J. R., Wharton, P. S. & O'Connell, R. J. (2002). Identification and

appressoria of *Colletotrichum* species. *Mycological Research*, 106:729-736. Hyon, G.-S., Muranaka, Y., Ikeda, K., Inoue, K., Hosogi, N., Meguro, H., Yamada, T., Hida,

uredospores to the host cuticle. *The Plant Cell*, 4:1101-1111.

*Environmental Microbiology*, 59:1786-1791.

*United States of America*, 93:357-361.

*and Molecular Plant Pathology*, 48:335-346.

*albicans* to a single gene, *INT1*. *Science,* 279:1355-1358.

Giancotti, F. G. & Ruoslahti, E. (1999). Integrin signaling. *Science*, 285:1028-1032.

63:687-691.

145:1927-1936.

gene expression regulating appressorium formation in *Magnaporthe grisea*. In *Rice blast disease*, R. S. Zeigler et al. (eds), CAB International, Wallingford, pp. 23-34. Deising, H., Nicholson, R. L., Haug, M., Howard, R. J. & Mendgen, K. (1992). Adhesion pad

formation and the involvement of cutinase and esterases in the attachment of

Melanin in the extracellular matrix of germlings of *Botrytis cinerea*. *Phytochemistry*,

nongerminated *Botrytis cinerea* conidia to several substrata. *Applied and* 

Expression of the *Candida albicans* gene *ALS1* in *Saccharomyces cerevisiae* induces adherence to endothelial and epithelial cells. *Infection and Immunity*, 66:1783-1786. Gale, C. A., Bendel, C. M., McClellan, M., Hauser, M., Becker, J. M., Berman, J. & Hostetter,

M. K. (1998). Linkage of adhesion, filamentous growth, and virulence in *Candida* 

Hostetter, M. (1996). Cloning and expression of a gene encoding an integrin-like protein in *Candida albicans*. *Proceedings of the National Academy of Sciences of the* 

appressorium formation in the rice blast fungus *Magnaporthe grisea*. *Physiological* 

substrates by a fungus employing enormous turgor pressures. *Proceedings of the* 

surface glycoproteins of *Colletotrichum lindemuthianum* are recognized by a monoclonal antibody which inhibits adhesion to polystyrene. *Microbiology*,

localisation of glycoproteins in the extracellular matrices around germ-tubes and

S., Suzuki, T. & Park, P. (2009). The extracellular matrix produced from *Alternaria alternata* Japanese pear pathotype plays a possible role of adhesion on the surfaces of host leaves during plant infection. *Journal of Electron Microscopy Technology for Medicine and Biology,* 23:1-8.


The Role of the Extracellular Matrix (ECM)

19:2674-2689.

1538.

in Phytopathogenic Fungi: A Potential Target for Disease Control 149

Shimoi, S., Inoue, K., Kitagawa, H., Yamasaki, M., Tsushima, S., Park, P. & Ikeda, K. (2010).

Skamnioti, P. & Gurr, S. J. (2007). *Magnaporthe grisea* cutinase2 mediates appressorium

Staab, J. F., Bradway, S. D., Fidel, P. L. & Sundstrom, P. (1999). Adhesive and mammalian

Staab, J. F., Ferrer, C. A. & Sundstrom, P. (1996). Developmental expression of a tandemly

Sunde, M., Kwan, A. H., Templeton, M. D., Beever, R. E. & Mackay, J.P. (2008). Structural

Sundstrom P. (1999). Adhesins in *Candida albicans*. *Current Opinion in Microbiology*, 2:353-357. Sweigard, J. A., Chumley, F. G. & Valent, B. (1992). Cloning and analysis of *CUT1*, a cutinase gene from *Magnaporthe grisea*. *Molecular and General Genetics*, 232:174-182. Takahashi, T., Maeda, H., Yoneda, S., Ohtaki, S., Yamagata, Y., Hasegawa, F., Gomi, K.,

Talbot, N. J. (2003). On the trail of a cereal killer: exploring the biology of *Magnaporthe grisea*.

Talbot, N. J., Ebbole, D. J. & Hamer, J. E. (1993). Identification and characterization of *MPG1*,

Talbot, N. J., Kershaw, M. J., Weakley, G. E., de Vries, O. M. H., Wessels, J. G. H. & Hamer, J.

Thau, N., Monod, M., Crestani, B., Rolland, C., Tronchin, G., Latgé, J.-P. & Paris, S. (1994). *rodletless* mutants of *Aspergillus fumigatus*. *Infection and Immunity,* 62: 4380-4388. Tucker, S. L. & Talbot, N. J. (2001). Surface attachment and pre-penetration state

Van Dyke, C. G. & Mims, C. W. (1991). Ultrastructure of conidia, conidium germination, and

Wang, C. & Leger, R. J. S. (2007). The MAD1 adhesin of *Metarhizium anisopliae* links adhesion

Wessels, J.G.H. (1994). Developmental regulation of fungal cell formation. *Annual Reviews of* 

*Candida albicans*. *The Journal of Biological Chemistry*, 271:6298-6305.

gelatinolytic bacteria. *Biological Control,* 55:85-91.

analysis of hydrophobins. *Micron,* 39:773-784.

*Annual Review of Microbiology*, 57:177-202.

*Fungal Genetics and Biology*, 22:39-53.

*Phytopathology,* 32:413-437.

*truncatum*. *Canadian Journal of Botany*, 69:2355-2467.

enables attachment to plants. *Eukaryotic Cell*, 6:808-816.

*Plant Cell,* 5:1575-1590.

417.

Biological control for rice blast disease by employing detachment action with

differentiation and host penetration and is required for full virulence. *Plant Cell*,

transglutaminase substrate properties of *Candida albicans* Hwp1. *Science*, 283:1535-

repeated, proline- and glutamine-rich amino acid motif on hyphal surfaces of

Nakajima, T. & Abe, K. (2005). The fungal hydrophobin RolA recruits polyesterase and laterally moves on hydrophobic surfaces. *Molecular Microbiology*, 57:1780-1796.

a gene involved in pathogenicity from the rice blast fungus *Magnaporthe grisea.*

E. (1996). MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of *Magnaporthe grisea*. *Plant Cell,* 8:985-999. Temple, B., Horgen, P. A., Bernier, L. & Hintz, W. E. (1997). Cerato-ulmin, a hydrophobin

secreted by the causal agents of Dutch elm disease, is a parasitic fitness factor.

development by plant pathogenic fungi. *Annual Reviews in Phytopathology*, 39:385-

appressorium development in the plant pathogenic fungus *Colletotrichum* 

with blastospore production and virulence to insects, and the MAD2 adhesin


Mercure, E. W., Leite, B. & Nicholson, R. L. (1994a). Adhesion of ungerminated conidia of

Mercure, E. W., Kunoh, H. & Nicholson, R. L. (1994b). Adhesion of *Colletotrichum graminicola*

Nielsen, P. S., Clark, A. J., Oliver, R. P., Huber, M. & Spanu, P. D. (2001). HCf-6, a novel class II hydrophobin from *Cladosporium fulvum*. *Microbiological Research,* 156:59-63. O'Connell, R. J., Pain, N. A., Hutchison, K. A., Jones, G. L. & Green, J. R. (1996).

Pain, N. A., Green, J. R., Gammie, F. & O'Connell, R. J. (1994a) Immunomagnetic isolation of

Pain, N. A., O'Connell, R. J., Mendgen, K. & Green, J. R. (1994b) Identification of

*Colletotrichum lindemuthianum*-bean interaction. *New Phytologist*, 127:233-242. Pain, N. A., O'Connell, R. J., Bailey, J. A. & Green, J. R. (1992). Monoclonal antibodies which

Poon, N. H. & Day, A. W. (1974). Fimbriae in the fungus, *Ustilago violacea*. *Nature*, 250:648-

Poon, N. H. & Day, A. W. (1975). Fungal fimbriae. I. Structure, origin, and synthesis.

Rauceo, J. M., De Armond, R., Otoo, H., Kahn, P. C., Klotz, S. A., Gaur, N. K. & Lipke, P. N.

Rawlings, S. L., O'Connell, R. J. & Green, J. R. (2007). The spore coat of the bean anthracnose

Sarma, G. N., Manning, V. A., Ciuffetti, L. M. & Karplus, P. A. (2005). Structure of Ptr ToxA:

Schumacher, C. F. A., Steiner, U., Dehne, H. W. & Oerke, E. C. (2008). Localized adhesion of

Segers, G. C., Hamada, W., Oliver, R. P. & Spanu, P. D. (1999). Isolation and characterisation

Sela-Buurlage, M. B., Epstein, L. & Rodriguez, R. J. (1991). Adhesion of ungerminated

*Cladosporium fulvum*. *Molecular and General Genetics,* 261:644-652.

*Molecular Plant Pathology*, 45:407-420.

*Molecular Plant Pathology*, 45:421-440.

*Canadian Journal of Microbiology*, 21:537-546.

adhesin Als5p. *Eukaryotic Cell*, 5:1664-1673.

181:204-212.

127:223-232.

40:111-126.

649.

119.

17:3190-3202.

*Phytopathology*, 98:760-768.

*Colletotrichum graminicola* to artificial hydrophobic surfaces. *Physiological and* 

conidia to corn leaves: a requirement for disease development. *Physiological and* 

Ultrastructure and composition of the cell surfaces of infection structures formed by the fungal plant pathogen *Colletotrichum lindemuthianum*. *Journal of Microscopy*,

viable intracellular hyphae of *Colletotrichum lindemuthianum* (Sacc. & Magn.) Briosi & Cav. from infected bean leaves using a monoclonal antibody. *New Phytologist*,

glycoproteins specific to biotrophic intracellular hyphae formed in the

show restricted binding to four *Colletotrichum* species: *C. lindemuthianum, C. malvarum, C. orbiculare* and *C. trifolii*. *Physiological and Molecular Plant Pathology*,

(2006). Threonine-rich repeats increase fibronectin binding in the *Candida albicans* 

fungus Colletotrichum lindemuthianum is required for adhesion, appressorium development and pathogenicity. *Physiological and Molecular Plant Pathology*, 70:110-

an RGD-containing host-selective toxin from *Pyrenophora tritici-repentis*. *Plant Cell,*

nongerminated *Venturia inaequalis* conidia to leaves and artificial surfaces.

of five different hydrophobin-encoding cDNAs from the fungal tomato pathogen

*Colletotrichum musae* conidia. *Physiological and Molecular Plant Pathology*, 39:345-352.


**7** 

*Spain* 

**Molecular Tools for Detection of Plant** 

*1IFAPA Las Torres-Tomejil, Junta de Andalucía, Alcalá del Río, Sevilla* 

Nieves Capote1, Ana María Pastrana1,

Ana Aguado1 and Paloma Sánchez-Torres2

*2IVIA, Generalitat Valenciana, Moncada, Valencia* 

**Pathogenic Fungi and Fungicide Resistance** 

Plant pathogenic fungi are the causal agents of the most detrimental diseases in plants, including economically important crops, provoking considerable yield losses worldwide. Fungal pathogens can infect a wide range of plant species or be restricted to one or few host species. Some of them are obligate parasites requiring the presence of the living host to grow and reproduce, but most of them are saprophytic and can survive without the presence of the living plant, in the soil, water or air. Isolates of a fungal species can be differentiated by morphological characteristics, host range (*formae speciales*), pathogenic aggressiveness (pathotypes or races) or their ability to form stable vegetative heterokaryons by fusion between genetically different strains (belonging to the same vegetative compatibility group,

Detection and accurate identification of plant pathogens is one of the most important strategies for controlling plant diseases to initiate preventive or curative measures. Special interest should be taken in the early detection of pathogens in seeds, mother plants and propagative plant material to avoid the introduction and further spreading of new pathogens in a growing area where it is not present yet. For that reason, the availability of fast, sensitive and accurate methods for detection and identification of fungal pathogens is increasingly necessary to improve disease control decision making. Traditionally, the most prevalent techniques used to identify plant pathogens relied upon culture-based morphological approaches. These methods, however, are often time-consuming, laborious, and require extensive knowledge of classical taxonomy. Other limitations include the difficulty of some species to be cultured *in vitro*, and the inability to accurately quantify the pathogen (Goud & Termorshuizen, 2003). These limitations have led to the development of molecular approaches with improved accuracy and reliability. A high variety of molecular methods have been used to detect, identify and quantify a long list of plant pathogenic fungi. Molecular methods have also been applied to the study of the genetic variability of pathogen populations, and even for the description of new fungal species. In general, these methods are much faster, more specific, more sensitive, and more accurate, and can be performed and interpreted by personnel with no specialized taxonomical expertise. Additionally, these techniques allow the detection and identification of non-culturable

**1. Introduction** 

VCG).


### **Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance**

Nieves Capote1, Ana María Pastrana1, Ana Aguado1 and Paloma Sánchez-Torres2 *1IFAPA Las Torres-Tomejil, Junta de Andalucía, Alcalá del Río, Sevilla 2IVIA, Generalitat Valenciana, Moncada, Valencia Spain* 

#### **1. Introduction**

150 Plant Pathology

Wessels, J. G. H., de Vries, O. M. H., Ásgeirsdóttir, S. A. & Schuren, F. H. J. (1991).

Whiteford, J. R., Lacroix, H., Talbot, N. J. & Spanu, P. D. (2004). Stage-specific cellular

Wösten, H. A. B. (2001). Hydrophobins: multipurpose proteins. *Annual Reviews in* 

Xiao, J.-Z., Ohshima, A., Kamakura, T., Ishiyama, T. & Yamaguchi, I. (1994). Extracellular

*Cladosporium fulvum*. *Fungal Genetics and Biology,* 41:624-634.

*Molecular and Plant-Microbe Interaction*, 7:639-644.

*Schizophyllum*. *Plant Cell,* 3:793-799.

*Microbiology*, 55:625-646.

Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in

localisation of two hydrophobins during plant infection by the pathogenic fungus,

glycoprotein(s) associated with cellular differentiation in *Magnaporthe grisea*.

Plant pathogenic fungi are the causal agents of the most detrimental diseases in plants, including economically important crops, provoking considerable yield losses worldwide. Fungal pathogens can infect a wide range of plant species or be restricted to one or few host species. Some of them are obligate parasites requiring the presence of the living host to grow and reproduce, but most of them are saprophytic and can survive without the presence of the living plant, in the soil, water or air. Isolates of a fungal species can be differentiated by morphological characteristics, host range (*formae speciales*), pathogenic aggressiveness (pathotypes or races) or their ability to form stable vegetative heterokaryons by fusion between genetically different strains (belonging to the same vegetative compatibility group, VCG).

Detection and accurate identification of plant pathogens is one of the most important strategies for controlling plant diseases to initiate preventive or curative measures. Special interest should be taken in the early detection of pathogens in seeds, mother plants and propagative plant material to avoid the introduction and further spreading of new pathogens in a growing area where it is not present yet. For that reason, the availability of fast, sensitive and accurate methods for detection and identification of fungal pathogens is increasingly necessary to improve disease control decision making. Traditionally, the most prevalent techniques used to identify plant pathogens relied upon culture-based morphological approaches. These methods, however, are often time-consuming, laborious, and require extensive knowledge of classical taxonomy. Other limitations include the difficulty of some species to be cultured *in vitro*, and the inability to accurately quantify the pathogen (Goud & Termorshuizen, 2003). These limitations have led to the development of molecular approaches with improved accuracy and reliability. A high variety of molecular methods have been used to detect, identify and quantify a long list of plant pathogenic fungi. Molecular methods have also been applied to the study of the genetic variability of pathogen populations, and even for the description of new fungal species. In general, these methods are much faster, more specific, more sensitive, and more accurate, and can be performed and interpreted by personnel with no specialized taxonomical expertise. Additionally, these techniques allow the detection and identification of non-culturable

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 153

Schaad, 2003) or amplification of fungal RNA (RT-PCR) (Lee et al., 1989) may solve this problem to same extent. Like this, many PCR variants have been developed to improve sensitivity, specificity, rapidity and throughput, and to allow the quantification of the

Collection and preparation of samples is a critical step for the detection of plant pathogenic fungi. The starting material may be symptomatic plant tissue (roots, leaves, stems, flowers, fruits or seeds), soil, water or air. Also, latent infections can be detected on symptomless

In the case of infected plants, the first step consists in the cultivation of the fungi. After surface sterilisation of the plant tissue (e.g. with 1% sodium hypochlorite or 50% hydrogen peroxide) small pieces are transferred to Petri dishes containing an appropriate nutrient medium (e.g. Potato Dextrosa Agar, PDA) supplemented with antibiotics to prevent bacterial contaminants (usually streptomycin), and incubated at required temperature (25- 30ºC) for pathogen development. If the fungal pathogen has infected deep internal tissues, cutting the plant material to expose core tissues may be necessary. Pure fungal colonies must be obtained either by isolating single spore or single hyphal tips. In the first case, a spore suspension of the fungus is prepared and serial dilutions in sterile water are obtained. An optimal dilution is transferred to Petri dishes containing 2% agar-water and incubated at appropriate temperature to induce spore germination. A single germinating spore is then isolated and a pure colony of the fungus is obtained. To get a single hyphal tip, a small segment of fungal growth is transferred to a new Petri dish containing nutrient medium and incubated at optimum temperature to allow growth of the mycellium. A separate hyphal edge is them transferred to new agar plate to obtain a pure colony of the fungal species. Isolation of the fungus from an infected plant tissue can however obviate the presence in the infected plant of other non-culturable pathogens or fungi having special culture conditions. Competition between distinct fungi can also mask the detection by culturing of an infecting pathogen. For DNA extraction, fungal mycelium must be firstly homogenized in a mortar in the presence of liquid nitrogen. Other homogenisation procedures obviate the use of liquid nitrogen, e.g, grinding the mycellium inside centrifuge tubes with the help of sealed tips or plastic pistils that fit perfectly into the

Alternatively, total DNA from the plant and the fungi can be isolated together from the infected plant tissue. That allows skipping the fungi culture step, although DNA from different fungal species or strains may be obtained. Homogenisation of the infected plant tissues is usually performed in the presence of liquid nitrogen. Then, an extraction buffer is added to obtain a crude extract. Alternatively, plant material may be introduced into individual plastic bags containing a soft net in the case of tender material (Homex, Bioreba; Stomacher, AES Laboratoire) or a heavy net in the case of dry or harder material such as bark tissue or seeds (PlantPrint Diagnostics). Homogenisation can be made by the help of a manual roller or by the use of special apparatus designed to facilitate the homogenisation (e.g. Homex, Bioreba). As in the case of fungal mycellium, small amounts of plant tissue can be homogenised inside centrifuge tubes using pistils coupled to electric drills in the

fungal pathogen in the plant and the environment.

tubes coupled to household drills (González et al., 2008).

presence of extraction buffers.

**2.1.1 Starting material** 

plants.

microorganisms, and due to its high degree of specificity, molecular techniques can distinguish closely related organisms at different taxonomic levels. Here, we review the most important tools for molecular detection of plant pathogenic fungi, their applicability, and their implementation in horticultural and agricultural practices.

On the other hand, once the pathogenic fungus is already established in a given crop growing area, the use of synthetic fungicides constitutes the main strategy to control plant diseases, since these compounds act quickly and effectively. The disadvantages of continued use of fungicides are their limited spectrum and the emergence of resistant fungal isolates. This fact leads to many yield losses as control systems are not longer effective. The development of resistance to fungicides in fungal pathogens and the growing public concern over the health and environmental hazards associated with the high level of pesticide have resulted in a significant interest in knowing more about fungal resistance. The emergence of more stringent regulations regarding pesticide residues means that one of the main priorities is to ensure food security by reducing the use of fungi toxics. For this reason, it is important to identify and characterize the mechanisms involved in the emergence of strains resistant to fungicides used for control diseases and to know the molecular methods currently available to detect them.

#### **2. Molecular methods for detection of plant pathogenic fungi**

#### **2.1 Polymerase Chain Reaction (PCR)**

The polymerase chain reaction **(**PCR) is the most important and sensitive technique presently available for the detection of plant pathogens. PCR allows the amplification of millions of copies of specific DNA sequences by repeated cycles of denaturation, polymerisation and elongation at different temperatures using specific oligonucleotides (primers), deoxyribonucleotide triphosphates (dNTPs) and a thermostable *Taq* DNA polymerase in the adequate buffer (Mullis & Faloona, 1987). The amplified DNA fragments are visualized by electrophoresis in agarose gel stained with EtBr, SYBR Green or other safer molecule able to intercalate in the double stranded DNA, or alternatively by colorimetric (Mutasa et al., 1996) or fluorometric assays (Fraaije et al., 1999). The presence of a specific DNA band of the expected size indicates the presence of the target pathogen in the sample. Advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens in an automated reaction. Main advantages of PCR techniques include high sensitivity, specificity and reliability. Moreover, it is not necessary to isolate the pathogen from the infected material reducing the diagnosis time from weeks to hours, and allowing the detection and identification of non-culturable pathogens. This characteristic has been especially useful in the analysis of symptomless plants. However, the frequent presence of PCR inhibitors in the plant tissues or soil can reduce considerably the sensitivity of the reaction and even result in false negative detection. Many attempts have been carried out to overcome this issue (see below). Another disadvantage of the PCR methodology is the occurrence of false positive results due to the presence of DNA or PCR products (amplicons) contaminants. For this reason, it is advisable to separate physically and temporally pre- and post-PCR analysis. Another failure of PCR-based methods is the inability to discriminate viable from nonviable fungi or fungal structures, which might inform of the real threat for the plant. Development of a prior PCR step involving enrichment culturing (BIO-PCR) (Ozakman & Schaad, 2003) or amplification of fungal RNA (RT-PCR) (Lee et al., 1989) may solve this problem to same extent. Like this, many PCR variants have been developed to improve sensitivity, specificity, rapidity and throughput, and to allow the quantification of the fungal pathogen in the plant and the environment.

#### **2.1.1 Starting material**

152 Plant Pathology

microorganisms, and due to its high degree of specificity, molecular techniques can distinguish closely related organisms at different taxonomic levels. Here, we review the most important tools for molecular detection of plant pathogenic fungi, their applicability,

On the other hand, once the pathogenic fungus is already established in a given crop growing area, the use of synthetic fungicides constitutes the main strategy to control plant diseases, since these compounds act quickly and effectively. The disadvantages of continued use of fungicides are their limited spectrum and the emergence of resistant fungal isolates. This fact leads to many yield losses as control systems are not longer effective. The development of resistance to fungicides in fungal pathogens and the growing public concern over the health and environmental hazards associated with the high level of pesticide have resulted in a significant interest in knowing more about fungal resistance. The emergence of more stringent regulations regarding pesticide residues means that one of the main priorities is to ensure food security by reducing the use of fungi toxics. For this reason, it is important to identify and characterize the mechanisms involved in the emergence of strains resistant to fungicides used for control diseases and to know the

The polymerase chain reaction **(**PCR) is the most important and sensitive technique presently available for the detection of plant pathogens. PCR allows the amplification of millions of copies of specific DNA sequences by repeated cycles of denaturation, polymerisation and elongation at different temperatures using specific oligonucleotides (primers), deoxyribonucleotide triphosphates (dNTPs) and a thermostable *Taq* DNA polymerase in the adequate buffer (Mullis & Faloona, 1987). The amplified DNA fragments are visualized by electrophoresis in agarose gel stained with EtBr, SYBR Green or other safer molecule able to intercalate in the double stranded DNA, or alternatively by colorimetric (Mutasa et al., 1996) or fluorometric assays (Fraaije et al., 1999). The presence of a specific DNA band of the expected size indicates the presence of the target pathogen in the sample. Advances in PCR-based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens in an automated reaction. Main advantages of PCR techniques include high sensitivity, specificity and reliability. Moreover, it is not necessary to isolate the pathogen from the infected material reducing the diagnosis time from weeks to hours, and allowing the detection and identification of non-culturable pathogens. This characteristic has been especially useful in the analysis of symptomless plants. However, the frequent presence of PCR inhibitors in the plant tissues or soil can reduce considerably the sensitivity of the reaction and even result in false negative detection. Many attempts have been carried out to overcome this issue (see below). Another disadvantage of the PCR methodology is the occurrence of false positive results due to the presence of DNA or PCR products (amplicons) contaminants. For this reason, it is advisable to separate physically and temporally pre- and post-PCR analysis. Another failure of PCR-based methods is the inability to discriminate viable from nonviable fungi or fungal structures, which might inform of the real threat for the plant. Development of a prior PCR step involving enrichment culturing (BIO-PCR) (Ozakman &

and their implementation in horticultural and agricultural practices.

molecular methods currently available to detect them.

**2.1 Polymerase Chain Reaction (PCR)** 

**2. Molecular methods for detection of plant pathogenic fungi** 

Collection and preparation of samples is a critical step for the detection of plant pathogenic fungi. The starting material may be symptomatic plant tissue (roots, leaves, stems, flowers, fruits or seeds), soil, water or air. Also, latent infections can be detected on symptomless plants.

In the case of infected plants, the first step consists in the cultivation of the fungi. After surface sterilisation of the plant tissue (e.g. with 1% sodium hypochlorite or 50% hydrogen peroxide) small pieces are transferred to Petri dishes containing an appropriate nutrient medium (e.g. Potato Dextrosa Agar, PDA) supplemented with antibiotics to prevent bacterial contaminants (usually streptomycin), and incubated at required temperature (25- 30ºC) for pathogen development. If the fungal pathogen has infected deep internal tissues, cutting the plant material to expose core tissues may be necessary. Pure fungal colonies must be obtained either by isolating single spore or single hyphal tips. In the first case, a spore suspension of the fungus is prepared and serial dilutions in sterile water are obtained. An optimal dilution is transferred to Petri dishes containing 2% agar-water and incubated at appropriate temperature to induce spore germination. A single germinating spore is then isolated and a pure colony of the fungus is obtained. To get a single hyphal tip, a small segment of fungal growth is transferred to a new Petri dish containing nutrient medium and incubated at optimum temperature to allow growth of the mycellium. A separate hyphal edge is them transferred to new agar plate to obtain a pure colony of the fungal species. Isolation of the fungus from an infected plant tissue can however obviate the presence in the infected plant of other non-culturable pathogens or fungi having special culture conditions. Competition between distinct fungi can also mask the detection by culturing of an infecting pathogen. For DNA extraction, fungal mycelium must be firstly homogenized in a mortar in the presence of liquid nitrogen. Other homogenisation procedures obviate the use of liquid nitrogen, e.g, grinding the mycellium inside centrifuge tubes with the help of sealed tips or plastic pistils that fit perfectly into the tubes coupled to household drills (González et al., 2008).

Alternatively, total DNA from the plant and the fungi can be isolated together from the infected plant tissue. That allows skipping the fungi culture step, although DNA from different fungal species or strains may be obtained. Homogenisation of the infected plant tissues is usually performed in the presence of liquid nitrogen. Then, an extraction buffer is added to obtain a crude extract. Alternatively, plant material may be introduced into individual plastic bags containing a soft net in the case of tender material (Homex, Bioreba; Stomacher, AES Laboratoire) or a heavy net in the case of dry or harder material such as bark tissue or seeds (PlantPrint Diagnostics). Homogenisation can be made by the help of a manual roller or by the use of special apparatus designed to facilitate the homogenisation (e.g. Homex, Bioreba). As in the case of fungal mycellium, small amounts of plant tissue can be homogenised inside centrifuge tubes using pistils coupled to electric drills in the presence of extraction buffers.

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 155

to avoid the nucleic acids isolation step. One of them uses few microliters of crude extract loaded and immobilized on small pieces of paper, e.g FTA cards. A subsequent lysis of the cells in appropriate buffer allows the release of nucleic acids that are fixed in the membrane and protected from degradation. DNA can be stored on dry cards for several years in a dry place at room temperature without decreasing the sensitivity of detection (Smith & Burgoyne, 2004). Moreover, membrane immobilised-DNA is suitable of transportation or mail to other laboratories. Suzuki et al. (2006) reported that nucleic acids recovered from FTA cards could be used for the detection of *Aspergillus oryzae*, releasing the DNA from the fungal tissue by treatment in a microwave oven before application to the membranes. Grund et al. (2010) used FTA cards coupled to PCR for the detection of plant pathogens including oomycetes such as

Generally, conserved known genes with enough sequence variation are selected for designing PCR diagnostic assays and performing phylogenetic analysis. The most common region used for these purposes has been the internal transcribed spacer (ITS) region of ribosomal RNA genes. rDNA region consists of multiple copies (up to 200 copies per haploid genome) arranged in tandem repeats comprising the 18S small subunit, the 5.8S, and the 28S large subunit genes separated by internal transcribed spacer regions (ITS1 and ITS2) (Bruns et al., 1991; Liew et al., 1998). This region contains highly conserved areas adequate for genera- o species-consensus primer designing (RNA ribosomal genes), alternate with highly variable areas that allow discrimination over a wide range of taxonomic levels (ITS region) (White et al., 1990). The ITS region is ubiquitous in nature and found in all eukaryotes. In addition, the high copy numbers of rRNA genes in the fungal genome enable a highly sensitive PCR amplification. Furthermore, a large numbers of ribosomal sequences are publicly available in databases, facilitating the validation and the

Traditionally, molecular identification of plant pathogenic fungi is accomplished by PCR amplification of ITS region followed by either restriction analysis (Durán et al., 2010) or direct sequencing and BLAST searching against GenBank or other databases (White et al., 1990). Identification could be a challenge when using BLAST analysis with ITS sequences because there can be minimal or no differences between some species or, in some cases, intraspecific variation can confuse the boundaries between species (e.g., *P. fragariae* var. *fragariae* and *P. fragariae* var. *rubi* have identical ITS sequences). The sequence analysis of the ITS region has additionally served to proposed new species. Abad et al. (2008) aligned ITS regions of *Phytophthora* spp. associated with root rot from different geographic origins and hosts with GenBank sequences from other *Phytophthora* species and proposed a new isolate *Phytophthora bisheria* sp. nov. Just as, Burgess et al. (2009) also distinguished new and undescribed taxa of *Phytophthora* from natural ecosystems. This alignment analysis has also been used for the identication of new species of *Pythium* (de Cock & Lévesque, 2004;

Nechwatal & Mendgen, 2006; Paul, 2006, 2009; Paul et al., 2005; Paul & Bala, 2008).

The ITS region has also been widely used in fungal taxonomy and it is known to show variation between species e.g., between *Phytium ultimum* and *P. helicoides* (Kageyama et al., 2007); *Peronospora arborescens* and *P. cristata* (Landa et al., 2007); *Colletotrichum gloeosporioides* and *C. acutatum* (Kim J. T. et al., 2008), and within species e.g allowing differentiation of

*Phytophthora* and filamentous fungi such as *Fusarium*.

**2.1.3 Selection of target DNA to amplify** 

reliability of the detection assays.

When analysing soil samples, the main focus for phytopathologists is the isolation of DNA from different microorganisms and then, the specific detection and monitoring of the fungus(i) of interest. Classical approach consists of cultivating the soil fungi in different media and screen for the desired pathogen. However, many microorganisms from the soil community can not be isolated by this procedure. An alternative method is to isolate DNA directly from the soil sample without prior culturing. Protocols using enzymatic (e.g. protease, chitinase, glucanase) or mechanical lysis (glass-beads beating, freeze-thawing, vigorous shaking, microwave or grinding in liquid nitrogen) have been reported, but a combination of both procedures seems to be the more effective (Anderson I. C. & Parkin, 2007, Jiang et al., 2011). In the same way, improved protocols for an efficient isolation of DNA from water for detection and monitoring of plant pathogens have been reported (Pereira et al., 2010).

During the homogenisation process, polysaccharides and phenolic compounds from plants or humic and fulvic acids from soils can be released that can inhibit the *Taq* DNA polymerase leading to the occurrence of false negatives (Munford et al., 2006; Tebbe & Vahjen, 1993; Wilson, 1997). This problem may be partially overcome by the use in the extraction buffer of some compounds such as polyvinylpyrrolidone (PVP) or cetyltrimethyl ammonium bromide (CTAB) for plant extracts, and bovine sero albumine (BSA) for soil samples (Anderson I.C. & Cairney, 2004), or by removing inhibitors by the use of spin/vacuum columns. Some PCR variants such as Magnetic Capture-Hybridisation PCR have been developed to remove the presence of PCR inhibitors in plant extracts (see below). In addition, it is increasingly common to use an internal positive control of the PCR reaction either by the amplification of a conserved plant gene (e.g. cytochrome oxidase I, *cox* I) in multiplex (Bilodeau, et al., 2009) or in a parallel assay (Garrido et al., 2009), or by the addition of an exogenous DNA and their corresponding primers to each reaction (Cruz-Pérez et al., 2001).

#### **2.1.2 DNA extraction methods**

There are no universally validated nucleic-acids extraction protocols for fungi, infected plant material or soil. Many published protocols are available to ensure an efficient and reproducible method for DNA extraction from plants (revised by Demeke and Jenkins, 2010; Biswas and Biswas, 2011); from fungi (Chi et al., 2009; Feng et al., 2010; González-Mendoza et al., 2010; Niu et al., 2008; Zelaya-Molina et al., 2011; Zhang, Y. J. et al., 2010); and from soil (revised by Hirsch et al., 2010).

The use of commercial kits for nucleic acids extraction, either general or specifically designed for plant material, fungi or soil is gaining acceptance because they are easy to use and are enable to efficiently remove inhibitory compounds during the purification process. They are generally based on magnetic beads or spin columns, although quicker protocols are also available (e.g. QuickExtract™ Plant DNA Extraction Solution, Epicentre). Automated or semi-automated systems have also been developed to allow the isolation of nucleic acids from different samples, among others, QIAxtractor, QIAgen; 6700 Automated Nucleic Acid Workstation, Life Technologies; Magna Pure LC extraction system, Roche; Solucion m2000, Abbott.

Purification of nucleic acids is labour intensive, costly, time-consuming and not applicable when a large number of samples need to be analysed. Several attempts have been undertaken to avoid the nucleic acids isolation step. One of them uses few microliters of crude extract loaded and immobilized on small pieces of paper, e.g FTA cards. A subsequent lysis of the cells in appropriate buffer allows the release of nucleic acids that are fixed in the membrane and protected from degradation. DNA can be stored on dry cards for several years in a dry place at room temperature without decreasing the sensitivity of detection (Smith & Burgoyne, 2004). Moreover, membrane immobilised-DNA is suitable of transportation or mail to other laboratories. Suzuki et al. (2006) reported that nucleic acids recovered from FTA cards could be used for the detection of *Aspergillus oryzae*, releasing the DNA from the fungal tissue by treatment in a microwave oven before application to the membranes. Grund et al. (2010) used FTA cards coupled to PCR for the detection of plant pathogens including oomycetes such as *Phytophthora* and filamentous fungi such as *Fusarium*.

#### **2.1.3 Selection of target DNA to amplify**

154 Plant Pathology

When analysing soil samples, the main focus for phytopathologists is the isolation of DNA from different microorganisms and then, the specific detection and monitoring of the fungus(i) of interest. Classical approach consists of cultivating the soil fungi in different media and screen for the desired pathogen. However, many microorganisms from the soil community can not be isolated by this procedure. An alternative method is to isolate DNA directly from the soil sample without prior culturing. Protocols using enzymatic (e.g. protease, chitinase, glucanase) or mechanical lysis (glass-beads beating, freeze-thawing, vigorous shaking, microwave or grinding in liquid nitrogen) have been reported, but a combination of both procedures seems to be the more effective (Anderson I. C. & Parkin, 2007, Jiang et al., 2011). In the same way, improved protocols for an efficient isolation of DNA from water for detection and monitoring of plant pathogens have been reported

During the homogenisation process, polysaccharides and phenolic compounds from plants or humic and fulvic acids from soils can be released that can inhibit the *Taq* DNA polymerase leading to the occurrence of false negatives (Munford et al., 2006; Tebbe & Vahjen, 1993; Wilson, 1997). This problem may be partially overcome by the use in the extraction buffer of some compounds such as polyvinylpyrrolidone (PVP) or cetyltrimethyl ammonium bromide (CTAB) for plant extracts, and bovine sero albumine (BSA) for soil samples (Anderson I.C. & Cairney, 2004), or by removing inhibitors by the use of spin/vacuum columns. Some PCR variants such as Magnetic Capture-Hybridisation PCR have been developed to remove the presence of PCR inhibitors in plant extracts (see below). In addition, it is increasingly common to use an internal positive control of the PCR reaction either by the amplification of a conserved plant gene (e.g. cytochrome oxidase I, *cox* I) in multiplex (Bilodeau, et al., 2009) or in a parallel assay (Garrido et al., 2009), or by the addition of an exogenous DNA and their

There are no universally validated nucleic-acids extraction protocols for fungi, infected plant material or soil. Many published protocols are available to ensure an efficient and reproducible method for DNA extraction from plants (revised by Demeke and Jenkins, 2010; Biswas and Biswas, 2011); from fungi (Chi et al., 2009; Feng et al., 2010; González-Mendoza et al., 2010; Niu et al., 2008; Zelaya-Molina et al., 2011; Zhang, Y. J. et al., 2010); and from soil

The use of commercial kits for nucleic acids extraction, either general or specifically designed for plant material, fungi or soil is gaining acceptance because they are easy to use and are enable to efficiently remove inhibitory compounds during the purification process. They are generally based on magnetic beads or spin columns, although quicker protocols are also available (e.g. QuickExtract™ Plant DNA Extraction Solution, Epicentre). Automated or semi-automated systems have also been developed to allow the isolation of nucleic acids from different samples, among others, QIAxtractor, QIAgen; 6700 Automated Nucleic Acid Workstation, Life Technologies; Magna Pure LC extraction system, Roche;

Purification of nucleic acids is labour intensive, costly, time-consuming and not applicable when a large number of samples need to be analysed. Several attempts have been undertaken

corresponding primers to each reaction (Cruz-Pérez et al., 2001).

(Pereira et al., 2010).

**2.1.2 DNA extraction methods** 

(revised by Hirsch et al., 2010).

Solucion m2000, Abbott.

Generally, conserved known genes with enough sequence variation are selected for designing PCR diagnostic assays and performing phylogenetic analysis. The most common region used for these purposes has been the internal transcribed spacer (ITS) region of ribosomal RNA genes. rDNA region consists of multiple copies (up to 200 copies per haploid genome) arranged in tandem repeats comprising the 18S small subunit, the 5.8S, and the 28S large subunit genes separated by internal transcribed spacer regions (ITS1 and ITS2) (Bruns et al., 1991; Liew et al., 1998). This region contains highly conserved areas adequate for genera- o species-consensus primer designing (RNA ribosomal genes), alternate with highly variable areas that allow discrimination over a wide range of taxonomic levels (ITS region) (White et al., 1990). The ITS region is ubiquitous in nature and found in all eukaryotes. In addition, the high copy numbers of rRNA genes in the fungal genome enable a highly sensitive PCR amplification. Furthermore, a large numbers of ribosomal sequences are publicly available in databases, facilitating the validation and the reliability of the detection assays.

Traditionally, molecular identification of plant pathogenic fungi is accomplished by PCR amplification of ITS region followed by either restriction analysis (Durán et al., 2010) or direct sequencing and BLAST searching against GenBank or other databases (White et al., 1990). Identification could be a challenge when using BLAST analysis with ITS sequences because there can be minimal or no differences between some species or, in some cases, intraspecific variation can confuse the boundaries between species (e.g., *P. fragariae* var. *fragariae* and *P. fragariae* var. *rubi* have identical ITS sequences). The sequence analysis of the ITS region has additionally served to proposed new species. Abad et al. (2008) aligned ITS regions of *Phytophthora* spp. associated with root rot from different geographic origins and hosts with GenBank sequences from other *Phytophthora* species and proposed a new isolate *Phytophthora bisheria* sp. nov. Just as, Burgess et al. (2009) also distinguished new and undescribed taxa of *Phytophthora* from natural ecosystems. This alignment analysis has also been used for the identication of new species of *Pythium* (de Cock & Lévesque, 2004; Nechwatal & Mendgen, 2006; Paul, 2006, 2009; Paul et al., 2005; Paul & Bala, 2008).

The ITS region has also been widely used in fungal taxonomy and it is known to show variation between species e.g., between *Phytium ultimum* and *P. helicoides* (Kageyama et al., 2007); *Peronospora arborescens* and *P. cristata* (Landa et al., 2007); *Colletotrichum gloeosporioides* and *C. acutatum* (Kim J. T. et al., 2008), and within species e.g allowing differentiation of

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 157

mitochondrial *cox* I gene, a SNP method has been developed to detect and differentiate isolates of *Phytophthora ramorum* from Europe and those originating in the United States (Kroon et al., 2004). In other experiment, polymorphisms detected in the microsatellite flanking regions of *Phytophthora infestans* allowed the development of a SNP genetic marker system for typing this pathogen (Abbott et al., 2010). Many of the examples described below

PCR methods are based on the use of specific oligonucleotides or primers that specifically hybridise with the DNA target and that are required to initiate the synthesis of the new DNA chain. In some real-time PCR methods additional specific oligonucleotides are used, named probes, that hybridise with the target DNA between the two primers. The design of primers and probes is crucial for PCR to be specific and efficient. Primer specificity relies on its sequence, length and GC content, which determines its melting temperature (*Tm,* the temperature at which 50% of primer-target duplex are hybridized). DNA amplified fragments (amplicons) size must be short enough to ensure efficiency of the reaction and high sensitivity (Singh and Singh, 1997). Real-time PCR usually requires shorter amplicons than conventional PCR. Balancing of primers concentration is necessary especially in multiplex PCR reactions and in real-time PCR using non-specific fluorescent dyes. Also, formation of hairpin structures or complementarities between primers must be avoided.

The first step for designing primers and probes consists in the aligment of the sequences of interest by the blastn program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al*.*, 1997) using sequences from the GenBank, EMBL and DDBJ databases. Partial or complete nucleotide sequences of many fungal genes are available at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/Genbank/) (Bethesda, MD, USA). Consensus sequences are used to design primers for detection of members of a same genus or species. If the consensus is not possible, degenerate primers can be used, although this may severely affect the overall sensitivity of the PCR reaction. On the other hand, variable sequences are useful for the differentiation of pathogens at lower taxonomic levels and for the

Software packages for primer and probe design are available, among others Primer Express, Applied Biosystems; LightCycler Probe Design, Roche; Primer Explorer, Eiken Chemical Co.; Beacon Designer, Premier Biosoft International; Primer Premier, Premier Biosoft; Primer Analysis Software, OLIGO; Oligo perfect designer, Life Technologies/Invitrogen; or

Identification of fungal pathogens by conventional PCR may be achieved at different taxonomic levels (genus, species or strain) depending on the specificity of the primers. As recent examples, PCR methods for identification of *Sclerotium rolfsii* (Jeeva et al., 2010) and *Colletotrichum capsici* (Torres-Calzada et al., 2011) have been developed based in specific sequences of the ITS region. Improved variants of PCR have emerged with higher

analysis of the molecular variability of fungal population in phylogenetic studies.

Primer3 (http://frodo.wi.mit.edu/primer3/).

**2.1.6 PCR-based methods 2.1.6.1 Conventional PCR** 

take advantage of SNPs for designing highly specific detection assays.

**2.1.5 Design of primers and probes** 

*Puccinia striiformis* f. sp. *tritici* (Zhao et al., 2007), and distinguishing between weakly and high virulent isolates of *Leptosphaeria maculans* (Xue et al., 1992), or difining anastomosis subgroups of *Rhizoctonia solani* (Budge, 2009; Godoy-Lutz et al., 2008).

The ITS region remains an important locus for molecular identification of fungi. However, as more sequence data is collected from a wider range of fungal isolates, the utility of alternative loci for accurate species identification is increasing. The intergenic spacer sequence (IGS) placed between the 28S and 18S rRNA genes is the region with the greatest amount of sequence variation in rDNA. It is frequently used in PCR-based methods when there are not enough differences available across the ITS. Primers in this region have been designed to detect and identify *Verticillium dahliae* and *V. alboatrum* (Schena et al., 2004) and to distinguish pathogenic and non-pathogenic *Fusarium oxysporum* in tomato (Validov et al., 2011). As another example, Inami et al. (2010) differentiated *Fusarium oxysporum f. sp lycopersici*, and its races using primers and TaqMan-MGB probes based on IGS and avirulent SIX genes.

Other housekeeping genes with higher variability are being more extensively used to develop diagnostics for fungi, including nuclear genes such as -tubulin (Aroca et al., 2008; Fraaije et al., 2001; Mostert et al., 2006), translation elongation factor 1 alpha (*TEF* 1 (Geiser et al., 2004; Knutsen et al., 2004, Kristensen et al., 2005), calmodulin (Mulè et al., 2004), avirulence genes (Lievens et al., 2009), and mitochondrial genes such as the multicopy *cox* I and *cox* II and their intergenic region (Martin & Tooley, 2003; Nguyen & Seifert, 2008; Seifert et al., 2007). Mating type genes also show high diversity and fast evolutionary rate and could be used for inter- and intra-species differentiation, e.g. Foster et al. (2002) distinguished between the two mating types of *Pyrenopeziza brassicae*. Moreover, Martínez-Espinoza et al. (2003) used mating type genes to specifically detect *Ustilago maydis* in maize cultivars. To enhance the specificity of a diagnostic assay, a combination of multiple diagnostic regions is recommended. Many authors have followed this multi-locus diagnostic strategy, e.g. Collado-Romero et al. (2008) studied the evolutionary relationships among *Verticillium dahliae* vegetative compatibility groups by AFLP fingerprints and sequence analysis of actin, -tubulin, calmodulin, and histone 3 genes, the ITS region, and a *V. dahliae*specific sequence; Dixon et al. (2009) demonstrated the host specialisation and phylogenetic diversity of *Corynespora cassiicola* using the ITS region, actine gene and two random hypervariable loci; Glienke et al. (2011) performed sequence analysis of the ITS region and partial *TEF* 1, actin and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes to study the genetic diversity of *Phyllosticta* spp. allowing differentiation of pathogenic and non pathogenic species and describing two new *Phyllosticta* species; Inderbitzin et al. (2010) verified a high species diversity in *Botryosphaeriaceae* species performing phylogenetic analyses based on six loci, including the ITS region and *TEF* 1, GPDH, a heat shock protein, histone-3 and tubulin genes; Shimomoto et al. (2011) used RAPDs and sequences from tubulin, *TEF* 1 calmodulin and actin genes to detect pathogenic and genetic variation among isolates of *Corynespora cassiicola*.

#### **2.1.4 Single nucleotide polymorphisms (SNPs)**

Closely related pathogens showing different host ranges or pathogenicity often differ in only a single to a few base pairs in target genes commonly used for identification. Therefore, the ability to discriminate single nucleotide polymorphisms (SNPs) should be pursued in any diagnostic assay. Based on the DNA nucleotide sequence difference in the mitochondrial *cox* I gene, a SNP method has been developed to detect and differentiate isolates of *Phytophthora ramorum* from Europe and those originating in the United States (Kroon et al., 2004). In other experiment, polymorphisms detected in the microsatellite flanking regions of *Phytophthora infestans* allowed the development of a SNP genetic marker system for typing this pathogen (Abbott et al., 2010). Many of the examples described below take advantage of SNPs for designing highly specific detection assays.

#### **2.1.5 Design of primers and probes**

156 Plant Pathology

*Puccinia striiformis* f. sp. *tritici* (Zhao et al., 2007), and distinguishing between weakly and high virulent isolates of *Leptosphaeria maculans* (Xue et al., 1992), or difining anastomosis

The ITS region remains an important locus for molecular identification of fungi. However, as more sequence data is collected from a wider range of fungal isolates, the utility of alternative loci for accurate species identification is increasing. The intergenic spacer sequence (IGS) placed between the 28S and 18S rRNA genes is the region with the greatest amount of sequence variation in rDNA. It is frequently used in PCR-based methods when there are not enough differences available across the ITS. Primers in this region have been designed to detect and identify *Verticillium dahliae* and *V. alboatrum* (Schena et al., 2004) and to distinguish pathogenic and non-pathogenic *Fusarium oxysporum* in tomato (Validov et al., 2011). As another example, Inami et al. (2010) differentiated *Fusarium oxysporum f. sp lycopersici*, and its

races using primers and TaqMan-MGB probes based on IGS and avirulent SIX genes.

Other housekeeping genes with higher variability are being more extensively used to develop diagnostics for fungi, including nuclear genes such as -tubulin (Aroca et al., 2008; Fraaije et al., 2001; Mostert et al., 2006), translation elongation factor 1 alpha (*TEF* 1 (Geiser et al., 2004; Knutsen et al., 2004, Kristensen et al., 2005), calmodulin (Mulè et al., 2004), avirulence genes (Lievens et al., 2009), and mitochondrial genes such as the multicopy *cox* I and *cox* II and their intergenic region (Martin & Tooley, 2003; Nguyen & Seifert, 2008; Seifert et al., 2007). Mating type genes also show high diversity and fast evolutionary rate and could be used for inter- and intra-species differentiation, e.g. Foster et al. (2002) distinguished between the two mating types of *Pyrenopeziza brassicae*. Moreover, Martínez-Espinoza et al. (2003) used mating type genes to specifically detect *Ustilago maydis* in maize cultivars. To enhance the specificity of a diagnostic assay, a combination of multiple diagnostic regions is recommended. Many authors have followed this multi-locus diagnostic strategy, e.g. Collado-Romero et al. (2008) studied the evolutionary relationships among *Verticillium dahliae* vegetative compatibility groups by AFLP fingerprints and sequence analysis of actin, -tubulin, calmodulin, and histone 3 genes, the ITS region, and a *V. dahliae*specific sequence; Dixon et al. (2009) demonstrated the host specialisation and phylogenetic diversity of *Corynespora cassiicola* using the ITS region, actine gene and two random hypervariable loci; Glienke et al. (2011) performed sequence analysis of the ITS region and partial *TEF* 1, actin and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes to study the genetic diversity of *Phyllosticta* spp. allowing differentiation of pathogenic and non pathogenic species and describing two new *Phyllosticta* species; Inderbitzin et al. (2010) verified a high species diversity in *Botryosphaeriaceae* species performing phylogenetic analyses based on six loci, including the ITS region and *TEF* 1, GPDH, a heat shock protein, histone-3 and tubulin genes; Shimomoto et al. (2011) used RAPDs and sequences from tubulin, *TEF* 1 calmodulin and actin genes to detect pathogenic and genetic variation

Closely related pathogens showing different host ranges or pathogenicity often differ in only a single to a few base pairs in target genes commonly used for identification. Therefore, the ability to discriminate single nucleotide polymorphisms (SNPs) should be pursued in any diagnostic assay. Based on the DNA nucleotide sequence difference in the

subgroups of *Rhizoctonia solani* (Budge, 2009; Godoy-Lutz et al., 2008).

among isolates of *Corynespora cassiicola*.

**2.1.4 Single nucleotide polymorphisms (SNPs)** 

PCR methods are based on the use of specific oligonucleotides or primers that specifically hybridise with the DNA target and that are required to initiate the synthesis of the new DNA chain. In some real-time PCR methods additional specific oligonucleotides are used, named probes, that hybridise with the target DNA between the two primers. The design of primers and probes is crucial for PCR to be specific and efficient. Primer specificity relies on its sequence, length and GC content, which determines its melting temperature (*Tm,* the temperature at which 50% of primer-target duplex are hybridized). DNA amplified fragments (amplicons) size must be short enough to ensure efficiency of the reaction and high sensitivity (Singh and Singh, 1997). Real-time PCR usually requires shorter amplicons than conventional PCR. Balancing of primers concentration is necessary especially in multiplex PCR reactions and in real-time PCR using non-specific fluorescent dyes. Also, formation of hairpin structures or complementarities between primers must be avoided.

The first step for designing primers and probes consists in the aligment of the sequences of interest by the blastn program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al*.*, 1997) using sequences from the GenBank, EMBL and DDBJ databases. Partial or complete nucleotide sequences of many fungal genes are available at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/Genbank/) (Bethesda, MD, USA). Consensus sequences are used to design primers for detection of members of a same genus or species. If the consensus is not possible, degenerate primers can be used, although this may severely affect the overall sensitivity of the PCR reaction. On the other hand, variable sequences are useful for the differentiation of pathogens at lower taxonomic levels and for the analysis of the molecular variability of fungal population in phylogenetic studies.

Software packages for primer and probe design are available, among others Primer Express, Applied Biosystems; LightCycler Probe Design, Roche; Primer Explorer, Eiken Chemical Co.; Beacon Designer, Premier Biosoft International; Primer Premier, Premier Biosoft; Primer Analysis Software, OLIGO; Oligo perfect designer, Life Technologies/Invitrogen; or Primer3 (http://frodo.wi.mit.edu/primer3/).

#### **2.1.6 PCR-based methods**

#### **2.1.6.1 Conventional PCR**

Identification of fungal pathogens by conventional PCR may be achieved at different taxonomic levels (genus, species or strain) depending on the specificity of the primers. As recent examples, PCR methods for identification of *Sclerotium rolfsii* (Jeeva et al., 2010) and *Colletotrichum capsici* (Torres-Calzada et al., 2011) have been developed based in specific sequences of the ITS region. Improved variants of PCR have emerged with higher

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 159

Another multiplexing method that allows simultaneous detection and identification of multiple oomycetes and fungi in complex plant or environmental samples is the use of ligation detection (LD) system using padlock probes (PLPs). PLPs are long oligonucleotide probes containing asymmetric target complementary regions at their 5' and 3' ends. Padlock probes also incorporate a desthiobiotin moiety for specific capture and release, an internal endonuclease IV cleavage site for linearization, and a unique sequence identifier, the socalled ZipCode, for standardised microarray hybridisation. DNA samples are PCR amplified and subjected to PLP ligation. Under perfectly hybridisation with the target, the PLPs are circularized by enzymatic ligation. Then, the probes are captured with streptavidin-coupled magnetic beads, cut at the internal cleavage site, allowing only the originally ligated PLPs to be visualized by hybridisation on a universal complementary ZipCode microarray. Padlock probes have been used for the simultaneous detection of *Phytophthora cactorum*, *P. nicotianae*, *Pythium ultimum*, *P. aphanidermatum*, *P. undulatum*, *Rhizoctonia solani*, *Fusarium oxysporum* f. sp. *radicis-lycopersici*, *F. solani*, *Myrothecium roridum*, *M. verrucaria*, *Verticillium dahliae* and *V. alboatrum* in samples collected from horticultural

This technique was developed to circumvent the presence of PCR inhibitors in plant extracts. Magnetic beads are coated with a biotinilated oligonucleotide that is specific of a DNA region of the fungus of interest. Hybridisation takes place between the fungal DNA and magnetic beads-oligomer and the conjugate is recovery separated from inhibitory compounds. After the magnetic capture-hybridisation, PCR amplification was carried out using species-specific primers. Langrell & Barbara, (2001) used this method to detect *Nectria* 

This serological-based PCR method uses forward and reverse primers carrying at their 5' end biotin and an antigenic group (e.g. fluorescein), respectively (Landgraf et al., 1991). PCR amplified DNA can be immobilized on avidin or streptavidin-coated microtiter plates via the biotin moiety of the forward primer and then can be quantified by an ELISA specific for the antigenic group of the reverse primer (e.g. anti-fluorescein antibody detected by colorimetric reactions). PCR-ELISA method is as sensitive as nested PCR. In addition, it does not require electrophoretic separation and/or hybridisation, and can be easily automated. All reactions can be performed in 96-well microtiter plates for mass screening of PCR products making them very suitable for routine diagnostic purposes. This procedure has been used for detection and differentiation of *Didymella bryoniae* from related *Phoma* species in cucurbits (Somai et al., 2002) and for detection of several species of *Phytophtora* and *Pythium* (Bailey et al., 2002).

An important limitation of molecular methods is the inability to distinguish living or dead fungi or fungal structures. So, results from detection and identification of fungal plant pathogens should be validated by pathogenicity tests. Since mRNA is degraded rapidly in dead cells, the detection of mRNA by RT–PCR is considered an accurate indicator of cell viability (Sheridan et al., 1998). In RT-PCR the RNA is reverse transcribed using the enzyme reverse transcriptase. The resulting cDNA is then amplified using conventional or any other

water circulation systems in a single assay (van Doorn et al., 2009).

**2.1.6.4 Magnetic Capture-Hybridisation (MCH)-PCR** 

*galligena* in apple and pear trees.

**2.1.6.6 Reverse Transcription (RT)-PCR** 

**2.1.6.5 PCR-ELISA** 

sensitivity, specificity and throughput and allowing the quantification of fungi in infected plants or environment.

#### **2.1.6.2 Nested-PCR and Cooperational-PCR (Co-PCR)**

Nested PCR approach is used when an improvement of the sensitivity and/or specificity of detection is necessary. This method consists in two consecutive rounds of amplification in which two external primers amplify a large amplicon that is then used as a target for a second round of amplification using two internal primers (Porter-Jordan et al., 1990). The two reactions are usually performed in separated tubes involving time and effort and increasing the risks of false positives due to cross contamination. However, some improvements in the relative concentrations of the external and internal primers have permitted to perform the two reactions in a single closed tube supporting high throughput. This method has been widely used for detection and/or further characterisation of numerous fungi (Aroca & Raposo, 2007; Grote et al., 2002; Hong et al., 2010; Ippolito et al., 2002; Langrell et al., 2008; Meng and Wang 2010; Mercado-Blanco et al., 2001; Qin et al., 2011; Wu et al., 2011).

An alternative PCR method that enhances sensitivity and minimise contamination risks is the Co-operational PCR. In Co-PCR a single reaction containing the four primers, one pair internal to other, enhances the production of the longest fragment by the co-operational action of all amplicons (Olmos et al., 2002). Co-PCR is usually coupled with dot blot hybridisation by using a specic probe to enhance the specificity of the detection and provide a sensitivity level similar to nested PCR method. Martos et al. (2011) used this method for sensitive and specific detection of grapevine fungi. In both nested- and Co-PCR methods, the use of external primers can be used for generic amplification and the internal primers for further and more specific characterisation of the amplified product at species or strain level.

#### **2.1.6.3 Multiplex PCR**

Multiplex PCR is based on the use of several PCR primers in the same reaction allowing the simultaneous and sensitive detection of different DNA targets, reducing time and cost. This method is useful in plant pathology since plants are usually infected by more than one pathogen. Different fragments specific to the target fungi were simultaneously amplified and identified on the basis of their molecular sizes on agarose gels. Although the efficiency of amplification is strongly influenced by amplicon size (shorter amplicons may be amplified preferentially over longer ones), an accurate and careful design of primers and the optimisation of their relative concentrations are required to overcome this drawback and get an equilibrate detection of all target fungi. Multiplex PCR technique has been used for the simultaneous detection and differentiation of *Podosphaera xanthii* and *Golovinomyces cichoracearum* in sunflower (Chen et al., 2008); for detecting *Phytophthora lateralis* in cedar trees and water samples, including detection of an internal control in the same reaction (Winton & Hansen, 2001); for determining the mating type of the pathogens *Tapesia yallundae* and *T. acuformis* (Dyer et al., 2001); for differentiating two pathotypes of *Verticilliun albo-atrum* infecting hop (Radiek et al., 2004) and for distinguishing among eleven taxons of wood decay fungi infecting hardwood trees (Guglielmo et al., 2007). Due to the complexity of the design this technique has recently been replaced by other multiplexing techniques including multiplex real-time PCR (see below).

Another multiplexing method that allows simultaneous detection and identification of multiple oomycetes and fungi in complex plant or environmental samples is the use of ligation detection (LD) system using padlock probes (PLPs). PLPs are long oligonucleotide probes containing asymmetric target complementary regions at their 5' and 3' ends. Padlock probes also incorporate a desthiobiotin moiety for specific capture and release, an internal endonuclease IV cleavage site for linearization, and a unique sequence identifier, the socalled ZipCode, for standardised microarray hybridisation. DNA samples are PCR amplified and subjected to PLP ligation. Under perfectly hybridisation with the target, the PLPs are circularized by enzymatic ligation. Then, the probes are captured with streptavidin-coupled magnetic beads, cut at the internal cleavage site, allowing only the originally ligated PLPs to be visualized by hybridisation on a universal complementary ZipCode microarray. Padlock probes have been used for the simultaneous detection of *Phytophthora cactorum*, *P. nicotianae*, *Pythium ultimum*, *P. aphanidermatum*, *P. undulatum*, *Rhizoctonia solani*, *Fusarium oxysporum* f. sp. *radicis-lycopersici*, *F. solani*, *Myrothecium roridum*, *M. verrucaria*, *Verticillium dahliae* and *V. alboatrum* in samples collected from horticultural water circulation systems in a single assay (van Doorn et al., 2009).

#### **2.1.6.4 Magnetic Capture-Hybridisation (MCH)-PCR**

This technique was developed to circumvent the presence of PCR inhibitors in plant extracts. Magnetic beads are coated with a biotinilated oligonucleotide that is specific of a DNA region of the fungus of interest. Hybridisation takes place between the fungal DNA and magnetic beads-oligomer and the conjugate is recovery separated from inhibitory compounds. After the magnetic capture-hybridisation, PCR amplification was carried out using species-specific primers. Langrell & Barbara, (2001) used this method to detect *Nectria galligena* in apple and pear trees.

#### **2.1.6.5 PCR-ELISA**

158 Plant Pathology

sensitivity, specificity and throughput and allowing the quantification of fungi in infected

Nested PCR approach is used when an improvement of the sensitivity and/or specificity of detection is necessary. This method consists in two consecutive rounds of amplification in which two external primers amplify a large amplicon that is then used as a target for a second round of amplification using two internal primers (Porter-Jordan et al., 1990). The two reactions are usually performed in separated tubes involving time and effort and increasing the risks of false positives due to cross contamination. However, some improvements in the relative concentrations of the external and internal primers have permitted to perform the two reactions in a single closed tube supporting high throughput. This method has been widely used for detection and/or further characterisation of numerous fungi (Aroca & Raposo, 2007; Grote et al., 2002; Hong et al., 2010; Ippolito et al., 2002; Langrell et al., 2008; Meng and Wang 2010; Mercado-Blanco et al., 2001; Qin et al.,

An alternative PCR method that enhances sensitivity and minimise contamination risks is the Co-operational PCR. In Co-PCR a single reaction containing the four primers, one pair internal to other, enhances the production of the longest fragment by the co-operational action of all amplicons (Olmos et al., 2002). Co-PCR is usually coupled with dot blot hybridisation by using a specic probe to enhance the specificity of the detection and provide a sensitivity level similar to nested PCR method. Martos et al. (2011) used this method for sensitive and specific detection of grapevine fungi. In both nested- and Co-PCR methods, the use of external primers can be used for generic amplification and the internal primers for further and more specific characterisation of the amplified product at species or

Multiplex PCR is based on the use of several PCR primers in the same reaction allowing the simultaneous and sensitive detection of different DNA targets, reducing time and cost. This method is useful in plant pathology since plants are usually infected by more than one pathogen. Different fragments specific to the target fungi were simultaneously amplified and identified on the basis of their molecular sizes on agarose gels. Although the efficiency of amplification is strongly influenced by amplicon size (shorter amplicons may be amplified preferentially over longer ones), an accurate and careful design of primers and the optimisation of their relative concentrations are required to overcome this drawback and get an equilibrate detection of all target fungi. Multiplex PCR technique has been used for the simultaneous detection and differentiation of *Podosphaera xanthii* and *Golovinomyces cichoracearum* in sunflower (Chen et al., 2008); for detecting *Phytophthora lateralis* in cedar trees and water samples, including detection of an internal control in the same reaction (Winton & Hansen, 2001); for determining the mating type of the pathogens *Tapesia yallundae* and *T. acuformis* (Dyer et al., 2001); for differentiating two pathotypes of *Verticilliun albo-atrum* infecting hop (Radiek et al., 2004) and for distinguishing among eleven taxons of wood decay fungi infecting hardwood trees (Guglielmo et al., 2007). Due to the complexity of the design this technique has recently been replaced by other multiplexing techniques

plants or environment.

2011; Wu et al., 2011).

strain level.

**2.1.6.3 Multiplex PCR** 

including multiplex real-time PCR (see below).

**2.1.6.2 Nested-PCR and Cooperational-PCR (Co-PCR)** 

This serological-based PCR method uses forward and reverse primers carrying at their 5' end biotin and an antigenic group (e.g. fluorescein), respectively (Landgraf et al., 1991). PCR amplified DNA can be immobilized on avidin or streptavidin-coated microtiter plates via the biotin moiety of the forward primer and then can be quantified by an ELISA specific for the antigenic group of the reverse primer (e.g. anti-fluorescein antibody detected by colorimetric reactions). PCR-ELISA method is as sensitive as nested PCR. In addition, it does not require electrophoretic separation and/or hybridisation, and can be easily automated. All reactions can be performed in 96-well microtiter plates for mass screening of PCR products making them very suitable for routine diagnostic purposes. This procedure has been used for detection and differentiation of *Didymella bryoniae* from related *Phoma* species in cucurbits (Somai et al., 2002) and for detection of several species of *Phytophtora* and *Pythium* (Bailey et al., 2002).

#### **2.1.6.6 Reverse Transcription (RT)-PCR**

An important limitation of molecular methods is the inability to distinguish living or dead fungi or fungal structures. So, results from detection and identification of fungal plant pathogens should be validated by pathogenicity tests. Since mRNA is degraded rapidly in dead cells, the detection of mRNA by RT–PCR is considered an accurate indicator of cell viability (Sheridan et al., 1998). In RT-PCR the RNA is reverse transcribed using the enzyme reverse transcriptase. The resulting cDNA is then amplified using conventional or any other

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 161

Real-time PCR is currently considered the gold standard method for detection of plant pathogens. This technique allows the monitoring of the reaction during the amplification process by the use of a fluorescent signal that increases proportionally to the number of amplicons generated and to the number of targets present in the sample (Wittwer et al., 1997). Many are the advantages of real time PCR over conventional PCR, including that this system does not require the use of post PCR processing (electrophoresis, colorimetric reaction or hybridisation), avoiding the risk of carryover contamination and reducing assay labour and material costs. In addition to its improved sensitivity and specificity, this technique allows the accurate quantification of the target pathogen, by interpolating the quantity measured to a standard curve with known amounts of target copies. This quantification characteristic is very useful in phytopathology in order to correlate the amount of fungus in a biological sample with the disease state, or to monitor the progress of the disease in an infected plant (Garrido et al., 2009). In addition, real-time PCR is a high throughput method for the analysis of a large number of samples due to the use of a platebased system which permits the analysis of 96 or 384 samples at the same time. This characteristic facilitates the robotisation of the nucleic acids extraction and master mix preparation steps, reducing personal and gaining time. Portable real-time PCR machines are emerging as diagnostic tools for *on site* detection under field conditions providing a realistic option to perform molecular tests at the same place of the collection of samples (e.g. portable Cepheid SmartCycler). This is especially interesting in situations where a rapid diagnostic test is needed. Another advantage of real-time PCR is the capability to perform multiplex

detection of two or more pathogens in the same reaction (see below).

phytopathogenic fungi even in multiplex assays (Tabla 1).

Real-time RT-PCR chemistry can be based on the use of doubled-stranded DNA binding dyes, such as SYBR Green, specific fluorescent labelled probes such as TaqMan, Molecular Beacons,

SYBR Green I is a fluorescence intercalating dye with a high affinity for double-stranded DNA. The overall fluorescent signal from a reaction is proportional to the amount of double-stranded DNA (dsDNA) present in the sample, and increase as the target is amplified. The main advantage of the use of intercalator dyes is that no probe is required, which reduces assay setup and running costs. Binding dyes are also attractive because protocols using established primers and PCR conditions can readily be converted to the realtime method. However, intercalating dyes detect accumulation of both specific and nonspecific PCR products. So, to assess the specificity of the reaction, it is necessary a further step of melting curve analysis that allows the identification of the PCR product by its *T*m. Additionally, a fine optimisation of primers concentration is crucial to avoid formation of also detected primer-dimers. Quantification of targets using SYBR Green is not very accurate, since the amount of fluorescent signal is proportional to the mass of dsDNA produced in the reaction (amplification of a longer product will generate more signal than a shorter one). Generally, small amplicons must be selected (between 50 and 200 bp) for optimal efficiency. SYBR Green real-time PCR with melting curve analysis has been described as a simple, rapid, and reliable technique for the detection and identification of

or Scorpions, or dye-primer based systems, such as hairpin primers or Plexor system.

**2.1.6.9 Real-time PCR** 

*a. Non-specific fluorescent dyes* 

PCR-based method. RT-PCR has been used to detect viable populations of *Mycosphaerella graminicola* in wheat (Guo et al., 2005). A RT-nested-PCR method was applied for detection of *Oidium neolycopersici* in tomato (Matsuda et al., 2005). Even so, the most frequent application of this technique in phytopathology is the analysis of plant and fungal gene expression during disease development (Yang et al., 2010).

#### **2.1.6.7** *in situ* **PCR**

This technique allows the amplification of specific gene sequences within intact cells or tissues combining two technologies: PCR and *in situ* hybridisation (ISH) (Long, 1993; Nuovo, 1992). The improved sensitivity of this technique allows the localization of one target copy per cell (Haase et al., 1990; Nuovo et al., 1991). However, background detection is usually high because nonspecific DNA synthesis during *in situ* PCR on tissue sections may occur (Nuovo et al., 1994). In addition, it is a time-consuming technique due to the need for a hybridisation step and technically demanding procedures such as light microscopy. Bindsley et al. (2002) used *in situ* PCR technique to identify *Blumeria graminis* spores and mycelia on barley leaves.

#### **2.1.6.8 PCR-DGGE**

This method is mainly applied for the analysis of the genetic diversity of microbial communities without the need of any prior knowledge of the species (Muyzer, 1999; Gothwal et al., 2007; Portillo et al., 2011). DGGE (Denaturing Gradient Gel Electrophoresis) and its variant TGGE (Temperature Gradient Gel Electrophoresis) use chemical gradient such as urea (DGGE) or temperature (TGGE) to denature and separate DNA samples when they are moving across an acrylamide gel. In PCR-DGGE target DNA from plant or environmental samples are firstly amplified by PCR and then subjected to denaturing electrophoresis. Sequence variants of particular fragments migrate at different positions in the denaturing gradient gel, allowing a very sensitive detection of polymorphisms in DNA sequences. In addition, PCR-DGGE primers contain a GC rich tail in their 5' end to improve the detection of small variations (Myers et al., 1985). The bands obtained in the gel can be extracted, cloned or reamplified and sequenced for identification, being even possible to identify constituents that represent only 1% of the total microbial community. These techniques are very suitable for the identification of novel or unknown organisms and the most abundant species can be readily detected.

This method is however time-consuming, poorly reproducible and provides relative information about the abundance of detected species. Interpretation of the results may be difficult since the microheterogeneity present in some target genes may appear as multiple bands in the gel for a single species, leading to an overestimation of the community diversity. Furthermore, fragments with different sequences but similar melting behaviour are not always correctly separated. In other cases, the analysis of complex communities of microorganisms may result in blurred gels due to the large number of bands obtained.

A PCR-DGGE detection tool based in the amplification of the ITS region has been recently applied to detect multiple species of *Phytophthora* from plant material and environmental samples (Rytkönen et al., 2011). Other authors have used this technique to compare the structure of fungal communities growing in different conditions or environments, e.g. to study the impact of culture management such as biofumigation, chemifumigation or fertilisation on the relative abundance of soil fungal species (Omirou et al., 2011; Wakelin et al., 2008).

#### **2.1.6.9 Real-time PCR**

160 Plant Pathology

PCR-based method. RT-PCR has been used to detect viable populations of *Mycosphaerella graminicola* in wheat (Guo et al., 2005). A RT-nested-PCR method was applied for detection of *Oidium neolycopersici* in tomato (Matsuda et al., 2005). Even so, the most frequent application of this technique in phytopathology is the analysis of plant and fungal gene

This technique allows the amplification of specific gene sequences within intact cells or tissues combining two technologies: PCR and *in situ* hybridisation (ISH) (Long, 1993; Nuovo, 1992). The improved sensitivity of this technique allows the localization of one target copy per cell (Haase et al., 1990; Nuovo et al., 1991). However, background detection is usually high because nonspecific DNA synthesis during *in situ* PCR on tissue sections may occur (Nuovo et al., 1994). In addition, it is a time-consuming technique due to the need for a hybridisation step and technically demanding procedures such as light microscopy. Bindsley et al. (2002) used *in* 

This method is mainly applied for the analysis of the genetic diversity of microbial communities without the need of any prior knowledge of the species (Muyzer, 1999; Gothwal et al., 2007; Portillo et al., 2011). DGGE (Denaturing Gradient Gel Electrophoresis) and its variant TGGE (Temperature Gradient Gel Electrophoresis) use chemical gradient such as urea (DGGE) or temperature (TGGE) to denature and separate DNA samples when they are moving across an acrylamide gel. In PCR-DGGE target DNA from plant or environmental samples are firstly amplified by PCR and then subjected to denaturing electrophoresis. Sequence variants of particular fragments migrate at different positions in the denaturing gradient gel, allowing a very sensitive detection of polymorphisms in DNA sequences. In addition, PCR-DGGE primers contain a GC rich tail in their 5' end to improve the detection of small variations (Myers et al., 1985). The bands obtained in the gel can be extracted, cloned or reamplified and sequenced for identification, being even possible to identify constituents that represent only 1% of the total microbial community. These techniques are very suitable for the identification of novel or unknown organisms and the

This method is however time-consuming, poorly reproducible and provides relative information about the abundance of detected species. Interpretation of the results may be difficult since the microheterogeneity present in some target genes may appear as multiple bands in the gel for a single species, leading to an overestimation of the community diversity. Furthermore, fragments with different sequences but similar melting behaviour are not always correctly separated. In other cases, the analysis of complex communities of microorganisms may result in blurred gels due to the large number of bands obtained.

A PCR-DGGE detection tool based in the amplification of the ITS region has been recently applied to detect multiple species of *Phytophthora* from plant material and environmental samples (Rytkönen et al., 2011). Other authors have used this technique to compare the structure of fungal communities growing in different conditions or environments, e.g. to study the impact of culture management such as biofumigation, chemifumigation or fertilisation on

the relative abundance of soil fungal species (Omirou et al., 2011; Wakelin et al., 2008).

*situ* PCR technique to identify *Blumeria graminis* spores and mycelia on barley leaves.

expression during disease development (Yang et al., 2010).

most abundant species can be readily detected.

**2.1.6.7** *in situ* **PCR** 

**2.1.6.8 PCR-DGGE** 

Real-time PCR is currently considered the gold standard method for detection of plant pathogens. This technique allows the monitoring of the reaction during the amplification process by the use of a fluorescent signal that increases proportionally to the number of amplicons generated and to the number of targets present in the sample (Wittwer et al., 1997).

Many are the advantages of real time PCR over conventional PCR, including that this system does not require the use of post PCR processing (electrophoresis, colorimetric reaction or hybridisation), avoiding the risk of carryover contamination and reducing assay labour and material costs. In addition to its improved sensitivity and specificity, this technique allows the accurate quantification of the target pathogen, by interpolating the quantity measured to a standard curve with known amounts of target copies. This quantification characteristic is very useful in phytopathology in order to correlate the amount of fungus in a biological sample with the disease state, or to monitor the progress of the disease in an infected plant (Garrido et al., 2009). In addition, real-time PCR is a high throughput method for the analysis of a large number of samples due to the use of a platebased system which permits the analysis of 96 or 384 samples at the same time. This characteristic facilitates the robotisation of the nucleic acids extraction and master mix preparation steps, reducing personal and gaining time. Portable real-time PCR machines are emerging as diagnostic tools for *on site* detection under field conditions providing a realistic option to perform molecular tests at the same place of the collection of samples (e.g. portable Cepheid SmartCycler). This is especially interesting in situations where a rapid diagnostic test is needed. Another advantage of real-time PCR is the capability to perform multiplex detection of two or more pathogens in the same reaction (see below).

Real-time RT-PCR chemistry can be based on the use of doubled-stranded DNA binding dyes, such as SYBR Green, specific fluorescent labelled probes such as TaqMan, Molecular Beacons, or Scorpions, or dye-primer based systems, such as hairpin primers or Plexor system.

#### *a. Non-specific fluorescent dyes*

SYBR Green I is a fluorescence intercalating dye with a high affinity for double-stranded DNA. The overall fluorescent signal from a reaction is proportional to the amount of double-stranded DNA (dsDNA) present in the sample, and increase as the target is amplified. The main advantage of the use of intercalator dyes is that no probe is required, which reduces assay setup and running costs. Binding dyes are also attractive because protocols using established primers and PCR conditions can readily be converted to the realtime method. However, intercalating dyes detect accumulation of both specific and nonspecific PCR products. So, to assess the specificity of the reaction, it is necessary a further step of melting curve analysis that allows the identification of the PCR product by its *T*m. Additionally, a fine optimisation of primers concentration is crucial to avoid formation of also detected primer-dimers. Quantification of targets using SYBR Green is not very accurate, since the amount of fluorescent signal is proportional to the mass of dsDNA produced in the reaction (amplification of a longer product will generate more signal than a shorter one). Generally, small amplicons must be selected (between 50 and 200 bp) for optimal efficiency. SYBR Green real-time PCR with melting curve analysis has been described as a simple, rapid, and reliable technique for the detection and identification of phytopathogenic fungi even in multiplex assays (Tabla 1).

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 163

TaqMan chemistry (Heid et al., 1996) is based on the use of an oligonucleotide probe located between the two PCR primers and labelled with a fluorophore covalently attached to the 5' end (reporter) and a quencher on the 3'end. When the reporter and the quencher are close, the emission of fluorescence is inhibited. After the PCR denaturation step, primers and probes specifically hybridise to the complementary target. The probe is then cleaved by the 5'-3' exonuclease activity of the *Taq* DNA polymerase causing the separation of the fluorophore and the quencher and allowing the reporter dye to emit fluorescence. The fluorescence detected in the real-time PCR thermal cycler is directly proportional to the

TaqMan probes were designed to increase the specificity of the reaction because detection and accurate quantification require high complementarity with the target sequence. The number of phytopathogenic fungi detected by this method has exponentially increased in

Modified probes have been designed to improve the TaqMan reaction specificity such as MGB probes that include a Minor Groove Binding group at the 3' end raising the *Tm* of the hybrid. That allows the use of shorter and more specific probes. The high specificity of MGB probes make them very suitable for specific detection of fungal species based on SNPs (Massart et al., 2005). For the same purpose, primers or probes can be synthesized with a lock nucleic acid (LNA), which are modified nucleotides that form methylene bridges after binding to the target DNA (Braasch & Corey, 2001). That provokes the lock of the duplex structure, improving their binding affinity and stability and allowing the use of higher annealing temperatures. LNA primers have been used for the specific detection of *P. ramorum* (Tomlinson et al., 2007) and the multiplex detection of species of *Phytophthora* (Bilodeau et al., 2009). The high specificity of TaqMan probes may compromise however the accuracy of the detection and quantification due to the existence of interstrain variability in the target sequence that may result in failure to

In addition to its high specificity, one of the advantages of the TaqMan chemistry is that probes can be labeled with different reporter dyes (FAM, VIC, TET, TAMRA, HEX, JOE, ROX, Cy5, Texas Red, etc) which allows multiplexing detection of two or more distinct pathogens in the same reaction increasing throughput (Aroca et al., 2008; Bilodeau et al., 2009). However, for this porpose the synthesis of different probes is required making the multiplex detection analysis more expensive. The types and number of fluorescent labels that can be used depend upon the detection capabilities of the real-time instrument used. Additionally, designing of real-time multiplex assays may be difficult. Special attention must be focus on avoiding primer competition that could strongly drop the levels of specificity and sensitivity. In fact, TaqMan

Molecular Beacons (MB) (Tyagi & Kramer, 1996) are specific oligonucleotide probes (15-40 mer) flanked by two complementary 5-7-mer arms sequences, with a fluorescent dye covalently attached to the 5' end and a quencher dye at the 3' end. When the molecular beacon is in an unbound state the arms form a stem/loop structure in which the fluorophore

fluorophore released and the amount of DNA template present in the PCR.

detect or underestimation of the amount of DNA targets in the sample.

multiplex assays ussually show lower detection sensitivity than single reactions.

recent years. Some examples are detailed in Table 2.

*b. Specific fluorescent labelled probes* 

*b.1 Hydrolysis probes* 

*b.2 Hairpin probes* 


Table 1. Examples of SYBR Green real-time PCR assays for detection of plant pathogenic fungi (last 6 years).

#### *b. Specific fluorescent labelled probes*

#### *b.1 Hydrolysis probes*

162 Plant Pathology

SYBR Green Moradi et al., 2010 Wheat

SYBR Green Moradi et al., 2010 Wheat

al., 2010

al., 2011

Brandfass & Karlovsky,

Brandfass & Karlovsky,

<sup>2006</sup> Cereals

<sup>2006</sup> Cereals

<sup>2006</sup> Cotton

Chickpea Melon Pea Soil

Chickpea Soil

Chickpea Soybean Pigeon pea

Wheat

Wheat Barley Soil

Wheat Barley Soil

**Pathogen Real-time chemistry Reference Host plant** *Botrytis cinerea* SYBR Green Diguta el al., 2010 Grape *Cladosporium fulvum* SYBR Green Yan et al., 2008 Tomato *Colletotrichum acutatum* SYBR Green Samuelian et al., 2011 Grape *Fusarium avenaceum* SYBR Green Moradi et al., 2010 Wheat

> SYBR Green multiplex

> SYBR Green multiplex

*Fusarium oxysporum* SYBR Green Jiménez-Fernández et

sp. *vasinfectum* SYBR Green Abd-Elsalam et al.,

*phaseolina* SYBR Green Babu et al., 2011

*Pythium irregular* SYBR Green Schroeder et al., 2006

*Pythium ultimum* SYBR Green Schroeder et al., 2006

sp. *ciceris* SYBR Green Jiménez-Fernández et

*Fusarium poae* SYBR Green Moradi et al., 2010 Wheat *Fusarium verticillioides* SYBR Green Kurtz et al., 2010 Corn *Greeneria uvicola* SYBR Green Samuelian et al., 2011 Grape

*Phoma sclerotioides* SYBR Green Larsen et al., 2007 Alfalfa

*Phoma tracheiphila* SYBR Green Demontis et al., 2008 Citrus *Phytophthora capsici* SYBR Green Silvar et al., 2005 Pepper *Phytophthora cryptogea* SYBR Green Minerdi et al., 2008 Gerbera *Plasmodiophora brassicae* SYBR Green Sundelin et al., 2010 Oilseed rape *Puccinia horiana* SYBR Green Alaei et al., 2009 Chrysanthemum

*Rhizoctonia oryzae* SYBR Green Okubara et al., 2008 Cereals *Rhizoctonia solani* SYBR Green Okubara et al., 2008 Cereals *Rhynchosporium secalis* SYBR Green Fountaine et al. 2007 Barley *Sclerotinia sclerotiorum* SYBR Green Yin et al., 2009 Oilseed rape

Table 1. Examples of SYBR Green real-time PCR assays for detection of plant pathogenic

SYBR Green, Plexor Attallah et al., 2007 Potato SYBR Green Gayoso et al., 2007 Hot pepper SYBR Green Markakis et al., 2009 Olive

*Fusarium culmorum* 

*Fusarium graminearum* 

*Fusarium oxysporum* f.

*Fusarium oxysporum* f.

*Macrophomina* 

*Verticillium dahliae* 

fungi (last 6 years).

TaqMan chemistry (Heid et al., 1996) is based on the use of an oligonucleotide probe located between the two PCR primers and labelled with a fluorophore covalently attached to the 5' end (reporter) and a quencher on the 3'end. When the reporter and the quencher are close, the emission of fluorescence is inhibited. After the PCR denaturation step, primers and probes specifically hybridise to the complementary target. The probe is then cleaved by the 5'-3' exonuclease activity of the *Taq* DNA polymerase causing the separation of the fluorophore and the quencher and allowing the reporter dye to emit fluorescence. The fluorescence detected in the real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.

TaqMan probes were designed to increase the specificity of the reaction because detection and accurate quantification require high complementarity with the target sequence. The number of phytopathogenic fungi detected by this method has exponentially increased in recent years. Some examples are detailed in Table 2.

Modified probes have been designed to improve the TaqMan reaction specificity such as MGB probes that include a Minor Groove Binding group at the 3' end raising the *Tm* of the hybrid. That allows the use of shorter and more specific probes. The high specificity of MGB probes make them very suitable for specific detection of fungal species based on SNPs (Massart et al., 2005). For the same purpose, primers or probes can be synthesized with a lock nucleic acid (LNA), which are modified nucleotides that form methylene bridges after binding to the target DNA (Braasch & Corey, 2001). That provokes the lock of the duplex structure, improving their binding affinity and stability and allowing the use of higher annealing temperatures. LNA primers have been used for the specific detection of *P. ramorum* (Tomlinson et al., 2007) and the multiplex detection of species of *Phytophthora* (Bilodeau et al., 2009). The high specificity of TaqMan probes may compromise however the accuracy of the detection and quantification due to the existence of interstrain variability in the target sequence that may result in failure to detect or underestimation of the amount of DNA targets in the sample.

In addition to its high specificity, one of the advantages of the TaqMan chemistry is that probes can be labeled with different reporter dyes (FAM, VIC, TET, TAMRA, HEX, JOE, ROX, Cy5, Texas Red, etc) which allows multiplexing detection of two or more distinct pathogens in the same reaction increasing throughput (Aroca et al., 2008; Bilodeau et al., 2009). However, for this porpose the synthesis of different probes is required making the multiplex detection analysis more expensive. The types and number of fluorescent labels that can be used depend upon the detection capabilities of the real-time instrument used. Additionally, designing of real-time multiplex assays may be difficult. Special attention must be focus on avoiding primer competition that could strongly drop the levels of specificity and sensitivity. In fact, TaqMan multiplex assays ussually show lower detection sensitivity than single reactions.

#### *b.2 Hairpin probes*

Molecular Beacons (MB) (Tyagi & Kramer, 1996) are specific oligonucleotide probes (15-40 mer) flanked by two complementary 5-7-mer arms sequences, with a fluorescent dye covalently attached to the 5' end and a quencher dye at the 3' end. When the molecular beacon is in an unbound state the arms form a stem/loop structure in which the fluorophore

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 165

multiplex Tooley et al., 2006

multiplex Tooley et al., 2006

*Plasmopara viticola* TaqMan Valsesia et al., 2005 Grapevine

*Puccinia coronata* TaqMan Jackson et al., 2006 Oat *Puccinia graminis* TaqMan Barnes & Szabo, 2007 Cereals

*Puccinia recondita* TaqMan Barnes & Szabo, 2007 Cereals

*Puccinia striiformis* TaqMan Barnes & Szabo, 2007 Cereal

*Puccinia triticina* TaqMan Barnes & Szabo, 2007 Cereals

*Pyrenophora teres* TaqMan MGB Leisova et al., 2006 Barley *Pyrenophora teres* f*. maculata* TaqMan MGB Leisova et al., 2006 Barley *Pyrenophora teres* f*. teres* TaqMan MGB Leisova et al., 2006 Barley *Pythium vexans* TaqMan Tewoldemedhin et al., 2011 Apple *Rhynchosporium secalis* TaqMan, LNA Fountaine et al., 2007 Barley *Rosellinia necatrix* Scorpion Ruano-Rosa et al., 2007 Avocado *Thielaviopsis basicola* TaqMan Huang & Kang, 2010 Tobacco

*Ustilaginoidea virens* TaqMan Ashizawa et al., 2010 Rice

for detection of plant pathogenic fungi (last 6 years).

Table 2. Examples of real-time PCR based on specific fluorescent labelled probes or primers

 **Reference Host plant** 

multiplex Schena et al., 2006 Forest trees

TaqMan Hughes et al., 2006 *Parrotia persica* 

multiplex Schena et al., 2006 Forest trees

Tomlinson et al., 2007

TaqMan Tomlinson et al., 2005 Rhododendron

multiplex Bilodeau et al., 2009 Oak

Beacon Macía-Vicente et al., 2009 Barley

multiplex Bilodeau et al., 2009 Oak

Rododendron and other host species

Rododendron and other host species

Grasses

Grasses

Grasses

Grasses

Soil

Soil

**chemistry**

TaqMan

TaqMan

TaqMan

TaqMan

Scorpion and Molecular Beacon

**Pathogen Real-time**

*Phytophthora pseudosyringae* TaqMan

*Phytophthora quercina* TaqMan

*Pochonia chlamydosporia* Molecular

*Phytophthora ramorum* 

*Phytophthora* spp.


 **Reference Host plant** 

<sup>2007</sup> Citrus fruit

Barley

Chickpea Soybean Pigeon pea

wood

wood

wood

wood

Soil

Beacons Macía-Vicente et al., 2009 Barley

multiplex Aroca et al., 2008 Grapevine

multiplex Aroca et al., 2008 Grapevine

multiplex Aroca et al., 2008 Grapevine

multiplex Aroca et al., 2008 Grapevine

multiplex Schena et al., 2006 Forest trees

multiplex Schena et al., 2006 Forest trees

**chemistry**

*Biscogniauxia mediterranea* TaqMan Luchi et al., 2005 Oak *Botrytis squamosa* TaqMan Carisse et al., 2009 Onion *Chalara fraxinea* TaqMan Ioos et al., 2009 Ash trees *Colletotrichum acutatum* TaqMan Garrido et al., 2009 Strawberry *Colletotrichum gloesporioides* TaqMan Garrido et al., 2009 Strawberry *Colletotrichum* spp. TaqMan Garrido et al., 2009 Strawberry *Discula destructiva* TaqMan Zhang, N. et al., 2011 Dogwood *Fusarium avenaceum* TaqMan MGB Kulik et al., 2011 Cereals

*Fusarium foetens* TaqMan De Weerdt et al., 2006 Begonia *Fusarium graminearum* TaqMan Demeke et al., 2010 Wheat

*lycopersici* and its races TaqMan MGB Inami et al., 2010 Tomato *Fusarium poae* TaqMan MGB Kulik et al., 2011 Cereals *Fusarium tricinctum* TaqMan MGB Kulik et al., 2011 Cereals *Fuscoporia torulosa* Scorpions Campanile et al., 2008 Oak *Gremmeniella abietina* TaqMan Børja et al., 2006 Spruce

*Mycosphaerella graminicola* TaqMan MGB Bearchell et al., 2005 Wheat

*Phaeospora nodorum* TaqMan MGB Bearchell et al., 2005 Wheat *Phialophora gregata* TaqMan Malvick & Impullitti, 2007 Soybean

*Phoma tracheiphila* TaqMan Demontis et al., 2008 Citrus *Phoma tracheiphila* TaqMan Licciardello et al., 2006 Citrus *Phomopsis* sp. TaqMan Børja et al., 2006 Spruce

*Phytophthora erythroseptica* TaqMan Nanayakkara et al., 2009 Potato

*Guignardia citricarpa* TaqMan van Gent-Pelzer et al.,

*Macrophomina phaseolina* TaqMan MGB Babu et al., 2011

**Pathogen Real-time**

*Fusarium equiseti* Molecular

*Phaeoacremonium aleophilum* Taqman

*Phaeoacremonium mortoniae* Taqman

*Phaeoacremonium parasiticum* Taqman

*Phaeoacremonium viticola* Taqman

*Phytophthora citricola* TaqMan

*Phytophthora kernoviae* TaqMan

*Fusarium oxysporum* f. sp.


Table 2. Examples of real-time PCR based on specific fluorescent labelled probes or primers for detection of plant pathogenic fungi (last 6 years).

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 167

amounts of magnesium pyrophosphate), as well as by electrophoresis on agarose gel. LAMP method is very suitable for field testing and potentially valuable to laboratories without PCR facilities. This isothermal method has been applied for the rapid detection of *Fusarium graminearum* in contaminated wheat seeds (Abd-Elsalam et al., 2011) and for the detection of

Fingerprinting approaches allow the screening of random regions of the fungal genome for identifying species-specic sequences when conserved genes have not enough variation to successfully identify species (McCartney et al., 2003). Fingerprinting analyses are generally used to study the phylogenetic structure of fungal populations. However, these techniques have been also useful for identifying specific sequences used for the detection of fungi at very low taxonomic level, and even for differentiate strains of the same species with

RFLP involves restriction enzyme digestion of the pathogen DNA, followed by separation of the fragments by electrophoresis in agarose or polyacrilamide gels to detect differences in the size of DNA fragments. Polymorphisms in the restriction enzyme cleavage sites are used to distinguish fungal species. Although DNA restriction profile can be directly observed by staining the gels, Southern blot analysis is usually necessary. DNA must be transferred to adequate membranes and hybridised with an appropriate probe. However, the Southern blot technique is laborious, and requires large amounts of undegraded DNA. RFLPs have been largely used for the study of the diversity of micorrhizal and soil fungal communities (Thies, 2007; Kim Y. T. et al., 2010; Martínez-García et al., 2011). Although used for differentiation of pathogenic fungi (Hyakumachi et al., 2005) this early technique has been

PCR-RFLP combines the amplification of a target region with the further digestion of the PCR products obtained. PCR primers specific to the genus *Phytophthota* were used to amplify and further digest the resulting amplicons yielding a specific restriction pattern of 27 different *Phytophthora* species (Drenth et al., 2006). PCR-RFLP analysis of the ITS region demonstrated the presence of different anastomosis group (AG) within isolates of *Rhizoctonia solani* (Pannecoucque & Höfte, 2009); It also allowed the differentiation of pathogenic and non pathogenic strains of *Pythium myriotolum* (Gómez-Alpizar et al., 2011). In other cases, the analysis of the ITS region by this technique failed in differentiating closely related species (e.g., clade 1c species such as *Phytophthora infestans* and *P. mirabilis*)

RAPD analyses rely on PCR amplification of the pathogen genome with short arbitrary sequences (usually decamers) that are used as primers. These primers are probably able to find distinct complementary sequences in the genome producing specific banding patterns. The resulting PCR fragments are then separated by electrophoresis to obtain fingerprints

*Phytophthora ramorum* and *P. kernoviae* in field samples (Tomlinson et al., 2007, 2010).

different host range, virulence, compatibility group or mating type.

progressively supplanted by other fingerprint techniques based in PCR.

**2.3.2 Random Amplified Polymorphic DNA (RAPD)** 

**2.3.1 Restriction fragment length polymorphism (RFLP)** 

**2.3 Fingerprinting** 

(Grünwald et al., 2011).

and the quencher are in close proximity and fluorescence is quenched. When the probe hibridises to the target sequence the complementary arms separate, thus allowing the emission of fluorescence and hence making possible the detection and quantification of the target sequence. Because the stem/loop structure is very thermostable molecular beacons must have a high specificity to hybridise to a target. This makes the chemistry appropriate for the detection of single nucleotide differences in mutation and SNP analyses. Molecular beacons have allowed real-time specific quantification of *Fusarium equiseti* and *Pochonia chlamydosporia* (a nematode parasitic fungus) in barley roots (Macía-Vicente et al., 2009).

Scorpions® are bifunctional molecules in which an upstream hairpin probe is covalently linked to a downstream primer sequence (Whitcombe et al., 1999). The hairpin probe contains a fluorophore at the 5' end and a quencher at the 3' end. The loop portion of the scorpion probe is complementary to the target sequence. During the amplification reaction the probe becomes attached to the target region synthesized in the first PCR cycle. Following the second cycle of denaturation and annealing, the probe and the target hybridise resulting in separation of the fluorophore from the quencher and an increase in the fluorescence emitted. Improvement of Scorpions sensitivity has been achieved by placing the quencher in a separate oligonucleotide (Scorpions bi-probes) allowing greater separation of fluorophore and quencher and giving stronger signals. As with all dyeprobe based methods, Scorpion probes follow strict design considerations for secondary structure and primer sequence to ensure that a secondary reaction will not compete with the correct probing event. Scorpion technology has been used for the detection of *Rosellinia necatrix* in roots of different plant host species and soils (Ruano-Rosa et al., 2007; Schena & Ippolito, 2003), and for the detection of *Fuscoporia torulosa* in holm oaks (Campanile et al., 2008).

#### *c. Specific fluorescent labelled primers*

Unlike other real-time chemistries, in which the incorporation of the fluorescent dye increases with the increasing number of copies of the DNA target, Plexor system measures the decrease of the fluorescence over time by the quenching of the dye. One of the two primers is labelled with a fluorescent dye and modified with methylisocytosine (isodC) residue at the 5′ end, whereas the other primer is not. The dabcyl-iso dGTP (iso-dG) present in the real-time PCR reaction cocktail is incorporated at the position complementary to the iso-dC label acting as a quencher and reducing the fluorescence over time. A quantitative real-time using Plexor primers has been developed for the detection and quantification of *Verticillium dahliae* in potato (Atallah et al., 2007).

#### **2.2 Isothermal amplification methods**

An efficient and cost-effective alternative to PCR is the possibility of isothermal amplification that does not require thermocycler aparatus. Loop-Mediated Isothermal Amplification (LAMP) (Notomi et al., 2000) uses a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from *Geobacillus stearothermophilus* for DNA amplification. This technique can specifically amplify the DNA target using only a heated block in less than 1 hour. Amplification products can be detected directly by visual inspection in vials using SYBR Green I, or by measuring the increased turbidity (due to the production of large amounts of magnesium pyrophosphate), as well as by electrophoresis on agarose gel. LAMP method is very suitable for field testing and potentially valuable to laboratories without PCR facilities. This isothermal method has been applied for the rapid detection of *Fusarium graminearum* in contaminated wheat seeds (Abd-Elsalam et al., 2011) and for the detection of *Phytophthora ramorum* and *P. kernoviae* in field samples (Tomlinson et al., 2007, 2010).

#### **2.3 Fingerprinting**

166 Plant Pathology

and the quencher are in close proximity and fluorescence is quenched. When the probe hibridises to the target sequence the complementary arms separate, thus allowing the emission of fluorescence and hence making possible the detection and quantification of the target sequence. Because the stem/loop structure is very thermostable molecular beacons must have a high specificity to hybridise to a target. This makes the chemistry appropriate for the detection of single nucleotide differences in mutation and SNP analyses. Molecular beacons have allowed real-time specific quantification of *Fusarium equiseti* and *Pochonia chlamydosporia* (a nematode parasitic fungus) in barley roots (Macía-Vicente et al., 2009).

Scorpions® are bifunctional molecules in which an upstream hairpin probe is covalently linked to a downstream primer sequence (Whitcombe et al., 1999). The hairpin probe contains a fluorophore at the 5' end and a quencher at the 3' end. The loop portion of the scorpion probe is complementary to the target sequence. During the amplification reaction the probe becomes attached to the target region synthesized in the first PCR cycle. Following the second cycle of denaturation and annealing, the probe and the target hybridise resulting in separation of the fluorophore from the quencher and an increase in the fluorescence emitted. Improvement of Scorpions sensitivity has been achieved by placing the quencher in a separate oligonucleotide (Scorpions bi-probes) allowing greater separation of fluorophore and quencher and giving stronger signals. As with all dyeprobe based methods, Scorpion probes follow strict design considerations for secondary structure and primer sequence to ensure that a secondary reaction will not compete with the correct probing event. Scorpion technology has been used for the detection of *Rosellinia necatrix* in roots of different plant host species and soils (Ruano-Rosa et al., 2007; Schena & Ippolito, 2003), and for the detection of *Fuscoporia torulosa* in holm oaks

Unlike other real-time chemistries, in which the incorporation of the fluorescent dye increases with the increasing number of copies of the DNA target, Plexor system measures the decrease of the fluorescence over time by the quenching of the dye. One of the two primers is labelled with a fluorescent dye and modified with methylisocytosine (isodC) residue at the 5′ end, whereas the other primer is not. The dabcyl-iso dGTP (iso-dG) present in the real-time PCR reaction cocktail is incorporated at the position complementary to the iso-dC label acting as a quencher and reducing the fluorescence over time. A quantitative real-time using Plexor primers has been developed for the detection and quantification of

An efficient and cost-effective alternative to PCR is the possibility of isothermal amplification that does not require thermocycler aparatus. Loop-Mediated Isothermal Amplification (LAMP) (Notomi et al., 2000) uses a set of six oligonucleotide primers with eight binding sites hybridizing specifically to different regions of a target gene, and a thermophilic DNA polymerase from *Geobacillus stearothermophilus* for DNA amplification. This technique can specifically amplify the DNA target using only a heated block in less than 1 hour. Amplification products can be detected directly by visual inspection in vials using SYBR Green I, or by measuring the increased turbidity (due to the production of large

(Campanile et al., 2008).

*c. Specific fluorescent labelled primers* 

*Verticillium dahliae* in potato (Atallah et al., 2007).

**2.2 Isothermal amplification methods** 

Fingerprinting approaches allow the screening of random regions of the fungal genome for identifying species-specic sequences when conserved genes have not enough variation to successfully identify species (McCartney et al., 2003). Fingerprinting analyses are generally used to study the phylogenetic structure of fungal populations. However, these techniques have been also useful for identifying specific sequences used for the detection of fungi at very low taxonomic level, and even for differentiate strains of the same species with different host range, virulence, compatibility group or mating type.

#### **2.3.1 Restriction fragment length polymorphism (RFLP)**

RFLP involves restriction enzyme digestion of the pathogen DNA, followed by separation of the fragments by electrophoresis in agarose or polyacrilamide gels to detect differences in the size of DNA fragments. Polymorphisms in the restriction enzyme cleavage sites are used to distinguish fungal species. Although DNA restriction profile can be directly observed by staining the gels, Southern blot analysis is usually necessary. DNA must be transferred to adequate membranes and hybridised with an appropriate probe. However, the Southern blot technique is laborious, and requires large amounts of undegraded DNA. RFLPs have been largely used for the study of the diversity of micorrhizal and soil fungal communities (Thies, 2007; Kim Y. T. et al., 2010; Martínez-García et al., 2011). Although used for differentiation of pathogenic fungi (Hyakumachi et al., 2005) this early technique has been progressively supplanted by other fingerprint techniques based in PCR.

PCR-RFLP combines the amplification of a target region with the further digestion of the PCR products obtained. PCR primers specific to the genus *Phytophthota* were used to amplify and further digest the resulting amplicons yielding a specific restriction pattern of 27 different *Phytophthora* species (Drenth et al., 2006). PCR-RFLP analysis of the ITS region demonstrated the presence of different anastomosis group (AG) within isolates of *Rhizoctonia solani* (Pannecoucque & Höfte, 2009); It also allowed the differentiation of pathogenic and non pathogenic strains of *Pythium myriotolum* (Gómez-Alpizar et al., 2011). In other cases, the analysis of the ITS region by this technique failed in differentiating closely related species (e.g., clade 1c species such as *Phytophthora infestans* and *P. mirabilis*) (Grünwald et al., 2011).

#### **2.3.2 Random Amplified Polymorphic DNA (RAPD)**

RAPD analyses rely on PCR amplification of the pathogen genome with short arbitrary sequences (usually decamers) that are used as primers. These primers are probably able to find distinct complementary sequences in the genome producing specific banding patterns. The resulting PCR fragments are then separated by electrophoresis to obtain fingerprints

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 169

Depending on the primers used and on the reaction conditions, random amplification of fungal genomes produces genetic polymorphisms specific at the genus, species or strain levels (Liu et al., 2009). As a result, AFLP has been used to differentiate fungal isolates at several taxonomic levels e.g. to distinguish *Cladosporium fulvurn* from *Pyrenopeziuz brassicae* species (Majer et al., 1996), *Aspergillus carbonarius* from *A. ochraceus* (Schmidt et al., 2004), and *Colletotrichum gossypii* from *C. gossypii* var. *cephalosporioides* (Silva et al., 2005); also to differentiate *Monilinia laxa* that infect apple trees from isolates infecting other host plants (Gril et al., 2008); and to separate non-pathogenic strains of *Fusarium oxysporum* from those of *F. commune* (Stewart et al., 2006). AFLP markers have also been used to construct genetic linkage maps e.g. of *Phytophthora infestans* (Van der Lee et al., 1997). Specific AFLP bands may also be used for SCAR markers development used in PCR-based diagnostic tests. Using SCAR markers Cipriani et al. (2009) could distinguish isolates of *Fusarium oxysporum* that specifically infect the weed *Orobanche ramose*. AFLP profiles have also been widely used for the phylogenetic analysis of *Fusarium oxysporum* complex (Baayen et al., 2000; Fourie et al.,

Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are motifs of one to six nucleotides repeated several times in all eukaryotic genomes (generally in non-coding regions). These nucleotide units can differ in repeat number among individuals and their distribution in the genome is almost random. Using primers flanking such variable regions PCR products of different lengths can be obtained. So, the microsatellites are highly versatile genetic markers that have been widely exploited for DNA fingerprinting. The advantages of SSRs are that they are multiallelic, codominant, highly polymorphic and several thousand potentially polymorphic markers are available. Moreover, it is possible the analysis of samples with limited DNA amounts or degraded DNA with high reproducibility. The microsatellites have a high mutation rate and are able to gain and lose repeat units by DNA-replication slippage, a mutation mechanism that is specific to tandemly repeated sequences (Schlöetterer, 2000). This characteristic can create difficulties for populations-genetic analyses. Other drawbacks of the SSRs include the requirement of a prior knowledge of the DNA sequences of the flanking regions and their cost and low throughput because of difficulties for automation and data management. Moreover, a high number of microsatellite loci are necessary for a reliable phylogenetic reconstruction. However, the next-generation sequencing technologies and multiplexing

Microsatellites have been used for the study of the genetic diversity of plant pathogenic fungi within species e.g. *Ascochyta rabiei* (Bayraktar et al., 2007), *Ceratocystis fimbriata* (Rizatto et al., 2010), *Macrophomina phaseolina* (Jana et al., 2005), *Puccinia graminis* and *P. triticina* (Szabo, 2007; Szabo & Kolmer, 2007), *Sclerotinia subarctica* and *S. sclerotiorum* (Winton et al., 2007); and for genetic map construction, e.g. Zheng et al. (2008) constructed a genetic map of *Magnaporthe grisea* consisting of 176 SSR markers. In other experiment, microsatellite markers specific for *Phytophthora ramorum* were employed to distinguish between A1 and A2 mating types isolates

To reduce the cost of developing microsatellites a novel technique has emerged based on sequence tagged microsatellites (STMs). Each STM is amplified by PCR using a single

of this pathogen from two different geographic origins (Prospero et al., 2004).

2011; Groenewald et al., 2006).

microsatellites solve, in part, these problems.

**2.3.4 Microsatellites** 

that may distinguish fungal species varieties or strains (Welsh & McClelland, 1990; Williams et al., 1990). Some of the specific DNA fragments detected in a profile may be cut out of the gel and sequenced to obtain a SCAR (Sequence-characterized amplied region), into which specific primers can be designed for a more precise PCR detection. SCAR primers have been used for instance to specically identify *Phytophthora cactorum* (Causin et al., 2005), *Fusarium subglutinans* (Zaccaro et al., 2007) and *Guignardia citricarpa* (Stringari et al., 2009) in infected plant material; to distinguish among several *formae speciales* of *Fusarium oxysporum* (Lievens et al., 2008); to differentiate the bioherbicidal strain of *Sclerotinia minor* from like organisms (Pan et al., 2010); and to establish two different groups in *Gaeumannomyces graminis* var. *tritici* (Daval et al., 2010).

RAPD results are also useful for the analysis of the genetic diversity among populations. Fingerprints are scored for the presence (1) or absence (0) of bands of various molecular weight sizes in the form of binary matrices. Data are analyzed to obtain statistic coefficients among the isolates that are then clustered to generate dendrograms. RAPDs have been used to analyze the genetic diversity among different species and races of *Fusarium* spp. (Lievens et al., 2007; Arici & Koc 2010) and different pathotypes of *Elsinoë* spp. (Hyun et al., 2009). This technique has also been applied to differentiate fungi isolates according to their host plant (Midorikawa et al., 2008), enzyme production profiles (Saldanha et al., 2007) or geographical origin and chemotypes (Zheng et al., 2009).

The RAPD technique is rapid, inexpensive and does not require any prior knowledge of the DNA sequence of the target organism. Results obtained from RAPD profiles are easy to interpret because they are based on amplification or non amplification of specific DNA sequences. In addition, RAPD analyses can be carried out on large numbers of isolates without the need for abundant quantities of high-quality DNA (Nayaka et al., 2011). Disadvantages of this technique include poor reproducibility between laboratories, and the inability to differentiate non-homologous co-migrating bands. In addition, RAPDs are dominant markers so, they cannot measure the genetic diversity affected by the number of alleles at a locus, nor differentiate homozygotes and heterozygotes individuals. This is not an issue with haploid fungi, but it can be a problem with many basidiomycetes and oomycetes that are heterokaryons, diploids or polyploids (Fourie et al., 2011).

#### **2.3.3 Amplied fragment length polymorphism (AFLP)**

AFLP analysis (Vos et al., 1995) consists in the use of restriction enzymes to digest total genomic DNA followed by ligation of restriction half-site specific adaptors to all restriction fragments. Then, a selective amplification of these restriction fragments is performed with PCR primers that have in their 3' end the corresponding adaptor sequence and selective bases. The band pattern of the amplified fragments is visualized on denaturing polyacrilamide gels. The AFLP technology has the capability to amplify between 50 and 100 fragments at one time and to detect various polymorphisms in different genomic regions simultaneously. It is also highly sensitive and reproducible. As with other fingerprinting techniques, no prior sequence information is needed for amplification (Meudt & Clarke 2007). The disadvantages of AFLPs are that they require high molecular weight DNA, more technical expertise than RAPDs (ligations, restriction enzyme digestions, and polyacrylamide gels), and that AFLP analyses suffer the same analytical limitations of RAPDs (McDonald et al., 1997).

Depending on the primers used and on the reaction conditions, random amplification of fungal genomes produces genetic polymorphisms specific at the genus, species or strain levels (Liu et al., 2009). As a result, AFLP has been used to differentiate fungal isolates at several taxonomic levels e.g. to distinguish *Cladosporium fulvurn* from *Pyrenopeziuz brassicae* species (Majer et al., 1996), *Aspergillus carbonarius* from *A. ochraceus* (Schmidt et al., 2004), and *Colletotrichum gossypii* from *C. gossypii* var. *cephalosporioides* (Silva et al., 2005); also to differentiate *Monilinia laxa* that infect apple trees from isolates infecting other host plants (Gril et al., 2008); and to separate non-pathogenic strains of *Fusarium oxysporum* from those of *F. commune* (Stewart et al., 2006). AFLP markers have also been used to construct genetic linkage maps e.g. of *Phytophthora infestans* (Van der Lee et al., 1997). Specific AFLP bands may also be used for SCAR markers development used in PCR-based diagnostic tests. Using SCAR markers Cipriani et al. (2009) could distinguish isolates of *Fusarium oxysporum* that specifically infect the weed *Orobanche ramose*. AFLP profiles have also been widely used for the phylogenetic analysis of *Fusarium oxysporum* complex (Baayen et al., 2000; Fourie et al., 2011; Groenewald et al., 2006).

#### **2.3.4 Microsatellites**

168 Plant Pathology

that may distinguish fungal species varieties or strains (Welsh & McClelland, 1990; Williams et al., 1990). Some of the specific DNA fragments detected in a profile may be cut out of the gel and sequenced to obtain a SCAR (Sequence-characterized amplied region), into which specific primers can be designed for a more precise PCR detection. SCAR primers have been used for instance to specically identify *Phytophthora cactorum* (Causin et al., 2005), *Fusarium subglutinans* (Zaccaro et al., 2007) and *Guignardia citricarpa* (Stringari et al., 2009) in infected plant material; to distinguish among several *formae speciales* of *Fusarium oxysporum* (Lievens et al., 2008); to differentiate the bioherbicidal strain of *Sclerotinia minor* from like organisms (Pan et al., 2010); and to establish two different groups in *Gaeumannomyces graminis* var.

RAPD results are also useful for the analysis of the genetic diversity among populations. Fingerprints are scored for the presence (1) or absence (0) of bands of various molecular weight sizes in the form of binary matrices. Data are analyzed to obtain statistic coefficients among the isolates that are then clustered to generate dendrograms. RAPDs have been used to analyze the genetic diversity among different species and races of *Fusarium* spp. (Lievens et al., 2007; Arici & Koc 2010) and different pathotypes of *Elsinoë* spp. (Hyun et al., 2009). This technique has also been applied to differentiate fungi isolates according to their host plant (Midorikawa et al., 2008), enzyme production profiles (Saldanha et al., 2007) or

The RAPD technique is rapid, inexpensive and does not require any prior knowledge of the DNA sequence of the target organism. Results obtained from RAPD profiles are easy to interpret because they are based on amplification or non amplification of specific DNA sequences. In addition, RAPD analyses can be carried out on large numbers of isolates without the need for abundant quantities of high-quality DNA (Nayaka et al., 2011). Disadvantages of this technique include poor reproducibility between laboratories, and the inability to differentiate non-homologous co-migrating bands. In addition, RAPDs are dominant markers so, they cannot measure the genetic diversity affected by the number of alleles at a locus, nor differentiate homozygotes and heterozygotes individuals. This is not an issue with haploid fungi, but it can be a problem with many basidiomycetes and

AFLP analysis (Vos et al., 1995) consists in the use of restriction enzymes to digest total genomic DNA followed by ligation of restriction half-site specific adaptors to all restriction fragments. Then, a selective amplification of these restriction fragments is performed with PCR primers that have in their 3' end the corresponding adaptor sequence and selective bases. The band pattern of the amplified fragments is visualized on denaturing polyacrilamide gels. The AFLP technology has the capability to amplify between 50 and 100 fragments at one time and to detect various polymorphisms in different genomic regions simultaneously. It is also highly sensitive and reproducible. As with other fingerprinting techniques, no prior sequence information is needed for amplification (Meudt & Clarke 2007). The disadvantages of AFLPs are that they require high molecular weight DNA, more technical expertise than RAPDs (ligations, restriction enzyme digestions, and polyacrylamide gels), and that AFLP analyses suffer the same analytical limitations of

oomycetes that are heterokaryons, diploids or polyploids (Fourie et al., 2011).

*tritici* (Daval et al., 2010).

geographical origin and chemotypes (Zheng et al., 2009).

**2.3.3 Amplied fragment length polymorphism (AFLP)** 

RAPDs (McDonald et al., 1997).

Microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are motifs of one to six nucleotides repeated several times in all eukaryotic genomes (generally in non-coding regions). These nucleotide units can differ in repeat number among individuals and their distribution in the genome is almost random. Using primers flanking such variable regions PCR products of different lengths can be obtained. So, the microsatellites are highly versatile genetic markers that have been widely exploited for DNA fingerprinting. The advantages of SSRs are that they are multiallelic, codominant, highly polymorphic and several thousand potentially polymorphic markers are available. Moreover, it is possible the analysis of samples with limited DNA amounts or degraded DNA with high reproducibility. The microsatellites have a high mutation rate and are able to gain and lose repeat units by DNA-replication slippage, a mutation mechanism that is specific to tandemly repeated sequences (Schlöetterer, 2000). This characteristic can create difficulties for populations-genetic analyses. Other drawbacks of the SSRs include the requirement of a prior knowledge of the DNA sequences of the flanking regions and their cost and low throughput because of difficulties for automation and data management. Moreover, a high number of microsatellite loci are necessary for a reliable phylogenetic reconstruction. However, the next-generation sequencing technologies and multiplexing microsatellites solve, in part, these problems.

Microsatellites have been used for the study of the genetic diversity of plant pathogenic fungi within species e.g. *Ascochyta rabiei* (Bayraktar et al., 2007), *Ceratocystis fimbriata* (Rizatto et al., 2010), *Macrophomina phaseolina* (Jana et al., 2005), *Puccinia graminis* and *P. triticina* (Szabo, 2007; Szabo & Kolmer, 2007), *Sclerotinia subarctica* and *S. sclerotiorum* (Winton et al., 2007); and for genetic map construction, e.g. Zheng et al. (2008) constructed a genetic map of *Magnaporthe grisea* consisting of 176 SSR markers. In other experiment, microsatellite markers specific for *Phytophthora ramorum* were employed to distinguish between A1 and A2 mating types isolates of this pathogen from two different geographic origins (Prospero et al., 2004).

To reduce the cost of developing microsatellites a novel technique has emerged based on sequence tagged microsatellites (STMs). Each STM is amplified by PCR using a single

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 171

confirmed by real-time PCR analysis (Lievens et al., 2005b), the monitoring of *Phytophthora* species diversity in soil and water samples (Chimento et al., 2005), and the identification and differentiation of toxin producing and non-producing *Fusarium* species in cereal grains (Nicolaisen et al., 2005). Using a *cox* I high density oligonucleotide microarray Chen et al. (2009) could identify *Penicillium* species. Moreover, Lievens et al. (2007) could detect and differentiate *F. oxysporum* f. sp. *cucumerinum* and *F. oxysporum* f. sp. *radicis-cucumerinum* pathogens by a DNA array containing genus-, species- and *forma specialis*-specific

Additionally, A DNA array for simultaneous detection of over 40 different plant pathogenic soilborne fungi and 10 bacteria that frequently occur in greenhouse crops has been developed. This array, called DNA Multiscan® (http://www.dnamultiscan.com/), is routinely used worldwide by companies that offer disease diagnostic services and advice to

As discussed above, morphological characteristics are not always enough to identify a pathogen. One of the most direct approaches to do that consists in the PCR amplification of a target gene with universal primers, followed by sequencing and comparison with the available publicly databases. In addition, new fungal species have been described by using sequencing approaches. However, the use of sequence databases to identify organisms based on DNA similarity may have some pitfalls including erroneous and incomplete sequences, sequences associated with misidentified organisms, the inability to easily change or update data, and problems associated with defining species boundaries, all of them leading to erroneous interpretation of search results. An effort for generating and archiving high quality data by the researchers community should be the remedy of this drawback (Kang et al., 2010). Other limitation of sequencing as diagnostic tool is the need to sequence more than one locus for the robustness of the result, and the impractical of this method in cases when rapid results are needed such as for the control or eradication of serious plant disease outbreaks. Nevertheless, the increase of sequencing capacity and the decrease of costs have allowed the accumulation of a high numbers of fungal sequences in publicly accessible sequence databases, and sequences of selected genes have been widely used for the identification of specific pathogens and the

The Sanger sequencing method has been partially supplanted by several "next-generation" sequencing technologies able to produce a high number of short sequences from multiple organisms in short time. Massive sequencing technologies offer dramatic increases in costeffective sequence throughput, having a tremendous impact on genomic research. They have been used for standard sequencing applications, such as genome sequencing and resequencing, and quantification of sequence variation. The next-generation technologies commercially available today include the 454 GS20 pyrosequencing-based instruments (Roche Applied Science), the Solexa 1G analyzer (Illumina, Inc.), and the SOLiD instrument

Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. The technique is built on a four enzyme real-time monitoring of DNA synthesis by

oligonucleotides.

commercial growers.

development of sequence-based diagnostic methods.

**2.5.1 Massive sequencing techniques** 

(Applied Biosystems).

**2.5 Sequencing** 

primer specific to the conserved DNA sequence flanking the microsatellite repeat in combination with a universal primer that anchors to the 5´-ends of the microsatellites (Hayden et al., 2002). STMs have been developed for the plant pathogens *Rhynchosporium secalis* (Keipfer et al., 2006) and *Pyrenophora teres* (Keipfer et al., 2007).

#### **2.4 DNA hybridisation technology**

The use of Southern blot or dot blot hybridisation techniques using selected probes from DNA libraries was a strategy for the identification of plant pathogens prior to the introduction of PCR-based methods with greater sensitivity, simplicity and speed (Takamatsu et al., 1998; Levesque et al., 1998; Xu et al., 1999). Nevertheless, new and revolutionary methods based in hybridisation have been recently developed for detection and differentiation of phytopathogenic fungi:

#### **2.4.1 DNA arrays**

A DNA array is a collection of species-specific oligonucleotides or cDNAs (known as probes) immobilized on a solid support that is subjected to hybridisation with a labelled target DNA. Macroarrays are membrane-based arrays containing spotted samples of 300 μm in diameter or more. Microarrays uses higher density chips such as glass or silicon, or microscopic beads in where thousands of sample spots (less than 200 μm in diameter) are immobilised via robotisation. The target DNA is a labelled PCR fragment, amplified with universal primers, spanning a genomic region that includes species-specific sequences. Probe-target hybridisation is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets and the relative abundance of nucleic acids sequences in the target can be determined. DNA micro- and macro-arrays are generally used for gene expression profiling but are also powerful tools for identification and differentiation of plant pathogens (Anderson N. et al., 2006; Lievens & Thomma, 2005). Currently, it is one of the most suitable techniques to detect and quantify multiple pathogens present in a sample (plant, soil, or water) in a single assay (Lievens et al., 2005a). The specificity of the DNA array technology allows an accurate SNP detection. This characteristic is crucial for diagnostic application since closely related pathogens may differ in only a single base pair polymorphism for a target gene. Specificity of the assay depends on the number and position of the mismatch(es) in the oligonucleotide probes, the oligonucleotide sequence and the lenght of the amplicon target. For instance, mismatches at the 3' end of the oligonucleotide must be avoided and center mismatches are usually the most discriminatory sites (Lievens et al., 2006). Furthermore, using longer amplicons as targets increases the sensitivity but decreases the specificity of the array hybridisation. For improving specificity and robustness, the use of multiple oligonucleotides for a single pathogen and the use of multiple diagnostic regions are desirable. One of the main drawbacks of this technique is however, the lack of sensitivity. To reach sensitive detections, PCR amplification before array hybridisation is required, biasing the results through the species that are more represented in the sample.

This technology has been applied for detecting oomycete plant pathogens by using specific oligonucleotides designed on the ITS region (Anderson N. et al., 2006; Izzo & Mazzola, 2009). Another ITS and rRNA genes-based microarrays allowed the multiple detection and quantification of tomato pathogens (*Verticillium*, *Fusarium*, *Pythium* and *Rhizoctonia*) confirmed by real-time PCR analysis (Lievens et al., 2005b), the monitoring of *Phytophthora* species diversity in soil and water samples (Chimento et al., 2005), and the identification and differentiation of toxin producing and non-producing *Fusarium* species in cereal grains (Nicolaisen et al., 2005). Using a *cox* I high density oligonucleotide microarray Chen et al. (2009) could identify *Penicillium* species. Moreover, Lievens et al. (2007) could detect and differentiate *F. oxysporum* f. sp. *cucumerinum* and *F. oxysporum* f. sp. *radicis-cucumerinum* pathogens by a DNA array containing genus-, species- and *forma specialis*-specific oligonucleotides.

Additionally, A DNA array for simultaneous detection of over 40 different plant pathogenic soilborne fungi and 10 bacteria that frequently occur in greenhouse crops has been developed. This array, called DNA Multiscan® (http://www.dnamultiscan.com/), is routinely used worldwide by companies that offer disease diagnostic services and advice to commercial growers.

#### **2.5 Sequencing**

170 Plant Pathology

primer specific to the conserved DNA sequence flanking the microsatellite repeat in combination with a universal primer that anchors to the 5´-ends of the microsatellites (Hayden et al., 2002). STMs have been developed for the plant pathogens *Rhynchosporium* 

The use of Southern blot or dot blot hybridisation techniques using selected probes from DNA libraries was a strategy for the identification of plant pathogens prior to the introduction of PCR-based methods with greater sensitivity, simplicity and speed (Takamatsu et al., 1998; Levesque et al., 1998; Xu et al., 1999). Nevertheless, new and revolutionary methods based in hybridisation have been recently developed for detection

A DNA array is a collection of species-specific oligonucleotides or cDNAs (known as probes) immobilized on a solid support that is subjected to hybridisation with a labelled target DNA. Macroarrays are membrane-based arrays containing spotted samples of 300 μm in diameter or more. Microarrays uses higher density chips such as glass or silicon, or microscopic beads in where thousands of sample spots (less than 200 μm in diameter) are immobilised via robotisation. The target DNA is a labelled PCR fragment, amplified with universal primers, spanning a genomic region that includes species-specific sequences. Probe-target hybridisation is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets and the relative abundance of nucleic acids sequences in the target can be determined. DNA micro- and macro-arrays are generally used for gene expression profiling but are also powerful tools for identification and differentiation of plant pathogens (Anderson N. et al., 2006; Lievens & Thomma, 2005). Currently, it is one of the most suitable techniques to detect and quantify multiple pathogens present in a sample (plant, soil, or water) in a single assay (Lievens et al., 2005a). The specificity of the DNA array technology allows an accurate SNP detection. This characteristic is crucial for diagnostic application since closely related pathogens may differ in only a single base pair polymorphism for a target gene. Specificity of the assay depends on the number and position of the mismatch(es) in the oligonucleotide probes, the oligonucleotide sequence and the lenght of the amplicon target. For instance, mismatches at the 3' end of the oligonucleotide must be avoided and center mismatches are usually the most discriminatory sites (Lievens et al., 2006). Furthermore, using longer amplicons as targets increases the sensitivity but decreases the specificity of the array hybridisation. For improving specificity and robustness, the use of multiple oligonucleotides for a single pathogen and the use of multiple diagnostic regions are desirable. One of the main drawbacks of this technique is however, the lack of sensitivity. To reach sensitive detections, PCR amplification before array hybridisation is required, biasing the results through the

This technology has been applied for detecting oomycete plant pathogens by using specific oligonucleotides designed on the ITS region (Anderson N. et al., 2006; Izzo & Mazzola, 2009). Another ITS and rRNA genes-based microarrays allowed the multiple detection and quantification of tomato pathogens (*Verticillium*, *Fusarium*, *Pythium* and *Rhizoctonia*)

*secalis* (Keipfer et al., 2006) and *Pyrenophora teres* (Keipfer et al., 2007).

**2.4 DNA hybridisation technology** 

**2.4.1 DNA arrays** 

and differentiation of phytopathogenic fungi:

species that are more represented in the sample.

As discussed above, morphological characteristics are not always enough to identify a pathogen. One of the most direct approaches to do that consists in the PCR amplification of a target gene with universal primers, followed by sequencing and comparison with the available publicly databases. In addition, new fungal species have been described by using sequencing approaches. However, the use of sequence databases to identify organisms based on DNA similarity may have some pitfalls including erroneous and incomplete sequences, sequences associated with misidentified organisms, the inability to easily change or update data, and problems associated with defining species boundaries, all of them leading to erroneous interpretation of search results. An effort for generating and archiving high quality data by the researchers community should be the remedy of this drawback (Kang et al., 2010). Other limitation of sequencing as diagnostic tool is the need to sequence more than one locus for the robustness of the result, and the impractical of this method in cases when rapid results are needed such as for the control or eradication of serious plant disease outbreaks. Nevertheless, the increase of sequencing capacity and the decrease of costs have allowed the accumulation of a high numbers of fungal sequences in publicly accessible sequence databases, and sequences of selected genes have been widely used for the identification of specific pathogens and the development of sequence-based diagnostic methods.

#### **2.5.1 Massive sequencing techniques**

The Sanger sequencing method has been partially supplanted by several "next-generation" sequencing technologies able to produce a high number of short sequences from multiple organisms in short time. Massive sequencing technologies offer dramatic increases in costeffective sequence throughput, having a tremendous impact on genomic research. They have been used for standard sequencing applications, such as genome sequencing and resequencing, and quantification of sequence variation. The next-generation technologies commercially available today include the 454 GS20 pyrosequencing-based instruments (Roche Applied Science), the Solexa 1G analyzer (Illumina, Inc.), and the SOLiD instrument (Applied Biosystems).

Pyrosequencing is a DNA sequencing technology based on the sequencing-by-synthesis principle. The technique is built on a four enzyme real-time monitoring of DNA synthesis by

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 173

DNA barcoding is a taxonomic method that uses a short genetic marker in the DNA to identify an organism as belonging to a particular species. It has facilitated the description of numerous new species and the characterisation of species complexes. Current fungal species identification platforms are available: *Fusarium*-ID was created as a simple, web-accessible BLAST server that consisted of sequences of the *TEF 1* gene from representative species of *Fusarium* (Geiser et al., 2004). Sequences of multiple marker loci from almost all known *Fusarium* species have been progressively included for supporting strain identification and phylogenetic analyses. Two additional platforms have been constructed: the Fusarium Comparative Genomics Platform (FCGP), which keeps five genomes from four species, supports genome browsing and analysis, and shows computed characteristics of multiple gene families and functional groups; and the Fusarium Community Platform (FCP), an online research and education forum. All together, these platforms form the Cyber

infrastructure for *Fusarium* (CiF; http://www.fusariumdb.org/) (Park, B. et al., 2011).

For *Phytophthora* identification two web-based databases have been created: (i) *Phytophthora* Database (http://www.phytophthoradb.org/) based in nine loci sequences including the ITS region and the 5' portion of the large subunit of rRNA genes; nuclear genes encoding 60S ribosomal protein L10, β-tubulin, enolase, heat shock protein 90, TigA fusion protein, and TEF 1; the mitochondrial gene *cox* II and spacer region between *cox* I and *cox* II genes (Park, J. et al., 2008); and (ii) Phytophthora-ID (http://phytophthora-id.org/) based on sequences of the ITS and the *cox* I and *cox* II spacer regions (Grunwald et al., 2011). Additional web-based databases are available including UNITE (http://unite.ut.ee/index.php), an ITS database supporting the identification of ectomycorrhizal fungi (Koljalg et al., 2005); TrichOKey (http://www.isth.info/tools/molkey/index.php), a database supporting the identification of *Hypocrea* and *Trichoderma* species (Druzhinina et al., 2005); and BOLD (http://www.boldsystems.org/) containing ITS and *cox* I databases from oomycetes (Ratnasingham et al., 2007; Robideau et al., 2011). Consortium for the Barcode of Life (CBOL) is an international collaborative effort which aims to use DNA barcoding to generate a unique genetic barcode for every species of life on earth. The *cox* I mitochondrial gene is emerging as

Despite extensive fungicide use in the previous 90 years, resistance emerged as a practical problem as recently as 1970. The incidence of resistance has been restricted largely to systemic fungicides that operate to biochemical targets (single-site inhibitors). These included several of major groups of fungicides: sterol demethylation, bezimidazoles,

The resistance to toxic compounds is a genetic adaptation of the fungus to one or more fungicides that leads to a reduction in sensitivity to these compounds. This phenomenon, described as genotypic or acquired resistance, is found in numerous fungi that have been sensitive to the fungicide prior to exposure. Fungicide resistance can be acquired by single mutations in genes of the pathogen or by increasing the frequency of subpopulations that are naturally less sensitive. We have to differentiate between resistance and natural or intrinsic insensitivity of species that are not sensitive to the action of these compounds (Delp & Bekker,

pyrimidines, phenylamines, dicarboximides, carboxanilides and morpholines.

**2.5.2 DNA barcoding** 

the standard barcode region for eukariotes.

**3. Fungicide resistance** 

chemiluminescence and a fifth protein, SSB, which can be included to enhance the quality of the obtained sequences and thereby prolong the read length. The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA strand. Thereby, the signal can be quantitatively connected to the number of bases added (Ahmadian et al., 2006). The pyrosequencing principle is used by the 454 platform, the rst next-generation sequencing technology released to the market by Roche Applied Science. 454 technology is based in emulsion PCR (Tawfik et al., 1998), which uses fixing adapterligated-DNA fragments to streptavidin beads in water-in-oil emulsion droplets. In each droplet the DNA fixed to these beads is then amplified by PCR producing about 107 copies of a unique DNA template per bead. Each DNA-bound bead is placed into a ~29 μm well on a PicoTiterPlate, a fiber optic chip, and analyzed using a pyrosequencing reaction. The use of the picotiter plate allows hundreds of thousands of pyrosequencing reactions to be carried out in parallel, massively increasing the sequencing throughput. 454 platform is capable of generating 80–120 Mb of sequence in 200 to 300 bp reads in a 4 h run. This technology enables a rapid and accurate quantification of sequence variation, including mutation detection, SNP genotyping, estimation of allele frequency and gene copy number, allelic imbalance and methylation status. Pyrosequencing can be applied to any DNA source, including degraded or low-quality DNA. Disadvantages are short read lengths, which may be problematic for sequence assembly particularly in areas associated with sequence repeats, the need for expensive biotinylated primers, and the inability to accurately detect variants within long (~ 5 or 6 bp) homopolymer stretches. In addition, multiplexing, while possible, is difficult to design.

The pyrosequencing technology has not been widely applied for the control of fungal plant diseases yet. However, Nunes et al. (2011) applied 454 sequencing technology to elucidate and characterize the small RNA transcriptome (15 - 40 nt) of mycelia and appressoria of *Magnaporthe oryzae*. Thus, they propose that a better understanding of key small RNA players in *M. oryzae* pathogenesis-related processes may illuminate alternative strategies to engineer plants capable of modifying the *M. oryzae* small transcriptome, and suppress disease development in an effective manner. Another application of this new sequencing technology is the rapid generation of genomic information to identify putative singlenucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. Thus, Broders et al. (2011) described the sequencing, assembly and discovery of SNPs from the plant fungal pathogen *Ophiognomonia clavigignenti-juglandacearum*, for which virtually no sequence information was previously available. Moreover, Malausa et al. (2011) described a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and 454 pyrosequencing in different organisms, such as *Phytophthora alni* subsp. *uniformis*.

The principle of the Illumina/Solexa system is also based on sequencing-by-synthesis chemistry, with novel reversible terminator nucleotides for the four bases each labeled with a different fluorescent dye, and a special DNA polymerase enzyme able to incorporate them (Ansorge, 2010). In the ABI SOLiD (Sequencing by Oligo Ligation and Detection), the sequence extension reaction is not carried out by polymerases but rather by ligases. In the sequencing-by-ligation process, a sequencing primer is hybridized to single-stranded copies of the library molecules to be sequenced. (Kircher & Kelso, 2010). These two above mentioned systems have not been currently used in studies on plant pathogenic fungi.

#### **2.5.2 DNA barcoding**

172 Plant Pathology

chemiluminescence and a fifth protein, SSB, which can be included to enhance the quality of the obtained sequences and thereby prolong the read length. The detection system is based on the pyrophosphate released when a nucleotide is introduced in the DNA strand. Thereby, the signal can be quantitatively connected to the number of bases added (Ahmadian et al., 2006). The pyrosequencing principle is used by the 454 platform, the rst next-generation sequencing technology released to the market by Roche Applied Science. 454 technology is based in emulsion PCR (Tawfik et al., 1998), which uses fixing adapterligated-DNA fragments to streptavidin beads in water-in-oil emulsion droplets. In each droplet the DNA fixed to these beads is then amplified by PCR producing about 107 copies of a unique DNA template per bead. Each DNA-bound bead is placed into a ~29 μm well on a PicoTiterPlate, a fiber optic chip, and analyzed using a pyrosequencing reaction. The use of the picotiter plate allows hundreds of thousands of pyrosequencing reactions to be carried out in parallel, massively increasing the sequencing throughput. 454 platform is capable of generating 80–120 Mb of sequence in 200 to 300 bp reads in a 4 h run. This technology enables a rapid and accurate quantification of sequence variation, including mutation detection, SNP genotyping, estimation of allele frequency and gene copy number, allelic imbalance and methylation status. Pyrosequencing can be applied to any DNA source, including degraded or low-quality DNA. Disadvantages are short read lengths, which may be problematic for sequence assembly particularly in areas associated with sequence repeats, the need for expensive biotinylated primers, and the inability to accurately detect variants within long (~ 5 or 6 bp) homopolymer stretches. In addition,

The pyrosequencing technology has not been widely applied for the control of fungal plant diseases yet. However, Nunes et al. (2011) applied 454 sequencing technology to elucidate and characterize the small RNA transcriptome (15 - 40 nt) of mycelia and appressoria of *Magnaporthe oryzae*. Thus, they propose that a better understanding of key small RNA players in *M. oryzae* pathogenesis-related processes may illuminate alternative strategies to engineer plants capable of modifying the *M. oryzae* small transcriptome, and suppress disease development in an effective manner. Another application of this new sequencing technology is the rapid generation of genomic information to identify putative singlenucleotide polymorphisms (SNPs) to be used for population genetic, evolutionary, and phylogeographic studies on non-model organisms. Thus, Broders et al. (2011) described the sequencing, assembly and discovery of SNPs from the plant fungal pathogen *Ophiognomonia clavigignenti-juglandacearum*, for which virtually no sequence information was previously available. Moreover, Malausa et al. (2011) described a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and 454

pyrosequencing in different organisms, such as *Phytophthora alni* subsp. *uniformis*.

The principle of the Illumina/Solexa system is also based on sequencing-by-synthesis chemistry, with novel reversible terminator nucleotides for the four bases each labeled with a different fluorescent dye, and a special DNA polymerase enzyme able to incorporate them (Ansorge, 2010). In the ABI SOLiD (Sequencing by Oligo Ligation and Detection), the sequence extension reaction is not carried out by polymerases but rather by ligases. In the sequencing-by-ligation process, a sequencing primer is hybridized to single-stranded copies of the library molecules to be sequenced. (Kircher & Kelso, 2010). These two above mentioned systems have not been currently used in studies on plant pathogenic fungi.

multiplexing, while possible, is difficult to design.

DNA barcoding is a taxonomic method that uses a short genetic marker in the DNA to identify an organism as belonging to a particular species. It has facilitated the description of numerous new species and the characterisation of species complexes. Current fungal species identification platforms are available: *Fusarium*-ID was created as a simple, web-accessible BLAST server that consisted of sequences of the *TEF 1* gene from representative species of *Fusarium* (Geiser et al., 2004). Sequences of multiple marker loci from almost all known *Fusarium* species have been progressively included for supporting strain identification and phylogenetic analyses. Two additional platforms have been constructed: the Fusarium Comparative Genomics Platform (FCGP), which keeps five genomes from four species, supports genome browsing and analysis, and shows computed characteristics of multiple gene families and functional groups; and the Fusarium Community Platform (FCP), an online research and education forum. All together, these platforms form the Cyber infrastructure for *Fusarium* (CiF; http://www.fusariumdb.org/) (Park, B. et al., 2011).

For *Phytophthora* identification two web-based databases have been created: (i) *Phytophthora* Database (http://www.phytophthoradb.org/) based in nine loci sequences including the ITS region and the 5' portion of the large subunit of rRNA genes; nuclear genes encoding 60S ribosomal protein L10, β-tubulin, enolase, heat shock protein 90, TigA fusion protein, and TEF 1; the mitochondrial gene *cox* II and spacer region between *cox* I and *cox* II genes (Park, J. et al., 2008); and (ii) Phytophthora-ID (http://phytophthora-id.org/) based on sequences of the ITS and the *cox* I and *cox* II spacer regions (Grunwald et al., 2011). Additional web-based databases are available including UNITE (http://unite.ut.ee/index.php), an ITS database supporting the identification of ectomycorrhizal fungi (Koljalg et al., 2005); TrichOKey (http://www.isth.info/tools/molkey/index.php), a database supporting the identification of *Hypocrea* and *Trichoderma* species (Druzhinina et al., 2005); and BOLD (http://www.boldsystems.org/) containing ITS and *cox* I databases from oomycetes (Ratnasingham et al., 2007; Robideau et al., 2011). Consortium for the Barcode of Life (CBOL) is an international collaborative effort which aims to use DNA barcoding to generate a unique genetic barcode for every species of life on earth. The *cox* I mitochondrial gene is emerging as the standard barcode region for eukariotes.

#### **3. Fungicide resistance**

Despite extensive fungicide use in the previous 90 years, resistance emerged as a practical problem as recently as 1970. The incidence of resistance has been restricted largely to systemic fungicides that operate to biochemical targets (single-site inhibitors). These included several of major groups of fungicides: sterol demethylation, bezimidazoles, pyrimidines, phenylamines, dicarboximides, carboxanilides and morpholines.

The resistance to toxic compounds is a genetic adaptation of the fungus to one or more fungicides that leads to a reduction in sensitivity to these compounds. This phenomenon, described as genotypic or acquired resistance, is found in numerous fungi that have been sensitive to the fungicide prior to exposure. Fungicide resistance can be acquired by single mutations in genes of the pathogen or by increasing the frequency of subpopulations that are naturally less sensitive. We have to differentiate between resistance and natural or intrinsic insensitivity of species that are not sensitive to the action of these compounds (Delp & Bekker,

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 175

The overexpression of ABC and MFS genes plays an essential role in the resistance of chemically unrelated phenomenon described as multidrug resistance or MDR drugs (Del Sorbo et al., 2000; White, 1997). This phenomenon has been observed in a wide variety of organisms and can be a real threat to the effective control of fungal pathogens (Fling et al., 1991). In phytopathogenic fungi, these transporters can also be a virulence factor in providing protection against defense compounds produced by the plant or mediating the secretion of host-specific toxins. Also play a major role in determining the baseline sensitivity to fungicides and other antifungal agents. (De Waard, 1997; Stergiopoulos et al.,

In *P. digitatum*, causal agent of citrus green rotten, four ABC transporters have been identified so far. ABC transporters PMR1 (Hamamoto et al., 2001b; Nakaune et al., 1998) and PMR5 (Nakaune, 2001; Nakaune et al., 2002) have been studied previously. Disruption of PMR1 in sensitive and resistant strains results in an increased sensitivity to DMIs and other compounds (Nakaune et al., 1998; Nakaune et al., 2002). However, the introduction of resistant strains from PMR1 restores the resistance while the introduction of PMR1 from sensitive strains does not have the same effect and does not restore the resistance. This suggests that although PMR1 plays an important role in the sensitivity of *P. digitatum* against DMIs alone does not explain the differences between sensitive and resistant strains (Hamamoto et al., 2001). Another of the genes studied is PMR5. This gene has highly homologous to PMR1, and also to *atrB* from *Aspergillus nidulans* and *BcAtrB* from *Botrytis cinerea* (Schoonbeek et al., 2003), however, is strongly induced by benzimidazoles, resveratrol and other compounds, but not for DMIs. This shows the different substrate specificity of both proteins and may play an important role in providing protection against

Sequence analysis in all four ABC transporter genes in several sensitive and resistant strains revealed no mutations in PMR1, PMR3 and PMR4, and point mutations only were observed in both the promoter and coding regions of PMR5 in multiple resistant strains (TBZ- and DMI-resistant) (Sánchez-Torres & Tuset, 2011). But no explanation was ascertained for the absence of sequence changes relating to fungicide resistance in the other ABC transporters, particularly in the PMR1 gene given that transcription of PMR1 has proven to be strongly activated in the presence of different fungicides (Hamamoto et al., 2001b; Nakaune, 2001;

To date, MFS transporters have been described in several fungi, e.g. *Botrytis cinerea* shows a broad spectrum of resistance to different fungicides and their expression has been induced by many of them, particularly noteworthy *Bcmfs4* induction in the presence of strobilurin (trifloxiestrobin) (Hayashi et al., 2002a, 2002b, 2003; Schoonbeek et al., 2003; Vermeulen et al., 2001). All this means that these transporters are potential candidates for the study of factors involved in resistance based on active eflux of these toxic compounds since they have remarkably broad substrate specificity although they can also transport specific compounds. Recently, five different MFS transporters have been identified and characterized in the postharvest phytopathogenic fungus *Penicillium digitatum* (PdMFS1-PdMFS5). Sequence analysis of these five genes revealed different genomic structure and although all genes seem to be implicated in pathogenicity, only 2 out of five MFS transporters confirmed to be involved in fungicide resistance (Sánchez-Torres et al., submitted). Therefore, the most recent thought for fungicide resistance based on active efflux of these toxic compounds is

natural or synthetic toxic compounds (Nakaune et al., 2002).

2003a, 2003b).

Nakaune et al., 1998).

1985; Brent, 1995). The term "lower sensitivity" is used in practical situations where there is a decreased sensitivity to a fungicide without an effect on field performance. The term "field resistance" is used when both the level of resistance and frequency of resistant strains are high and coincident, resulting in noticeable decline of field performance (Hewit, 1998).

It is to be expected that the evolutionary and dynamic progress of selection should eventually produce fungi that are resistant to fungicides. Therefore fungicide resistance is variable, with fungi developing resistance to some fungicides more rapidly than to others and in some cases no resistance had been reported after long periods of fungicide use.

In fungicide resistance, we should consider how pathogens have the ability to evolve resistant and how fungicides varies their susceptibility to resistance. Hence, resistance risk is determined by the target pathogen and selected fungicide.

Populations of fungi are so diverse that mechanisms of resistance may be present within the population before the fungicide is applied. The rate of resistance development in a population depends on the resultant mechanism of resistance such as how resistance characters are inherited, the epidemiology of the fungus, the environment, and the persistence of selective pressure.

#### **3.1. Mechanisms of fungicide resistance**

Fungicide resistance can be conferred by various mechanisms including: (I) an altered target site, which reduces the binding of the fungicide; (II) the synthesis of an alternative enzyme capable of substituting the target enzyme; (III) the overproduction of the fungicide target; (IV) an active efflux or reduced uptake of the fungicide; and (V) a metabolic breakdown of the fungicide. In addition, some unrecognized mechanisms could also be responsible for fungicide resistance.

#### **3.1.1 The reduction of intracellular concentration of antifungal compound**

Currently, the most widespread hypothesis to explain the reduced levels of toxic products in the cell is based on active efflux of these compounds by ABC transporters (ATP binding cassette) and MFS (Major Facilitator Superfamily) (Hayashi et al., 2001, 2002a, 2002b, 2003; Stergiopoulos et al., 2002a, 2002b; Stergiopoulos & de Waard, 2002, Vermeulen et al., 2001). ABC and MFS transporters are the most studied so far and have been described in many fungi such as *Aspergillus nidulans, Botrytis cinerea, Mycosphaerella graminicola, Magnaporthe grisea, Penicillium digitatum***,** etc (Andrade et al., 2000a, 2000b; del Sorbo et al., 2000; Nakaune, 2001; Schoonbeek et al., 2003; Stergiopoulos et al., 2002a**;** Stergiopoulos & de Waard, 2002; Vermeulen et al., 2001; Yoder and Turgeon 2001; Zwiers et al., 2003). Its function is to prevent or reduce the accumulation of compounds and therefore to avoid or minimize their toxic action (Bauer et al., 1999, Pao et al., 1998). ABC transporters comprise a large family of proteins and are located outside the plasma membrane or within the cell in intracellular compartments as vacuoles, endoplasmic reticulum, peroxisomes and mitochondria. They can carry a wide variety of toxic against the gradient (Del Sorbo et al., 2000; Theodoulou, 2000). ABC transporters include systems both capture and removal, generally showing activity on a wide range of substrates (fungicides, drugs, alkaloids, lipids, peptides, sterols, flavonoids, sugars, etc), but there are specific transporters for substrates (Bauer et al., 1999, Del Sorbo et al., 2000).

1985; Brent, 1995). The term "lower sensitivity" is used in practical situations where there is a decreased sensitivity to a fungicide without an effect on field performance. The term "field resistance" is used when both the level of resistance and frequency of resistant strains are high

It is to be expected that the evolutionary and dynamic progress of selection should eventually produce fungi that are resistant to fungicides. Therefore fungicide resistance is variable, with fungi developing resistance to some fungicides more rapidly than to others and in some cases no resistance had been reported after long periods of fungicide use.

In fungicide resistance, we should consider how pathogens have the ability to evolve resistant and how fungicides varies their susceptibility to resistance. Hence, resistance risk is

Populations of fungi are so diverse that mechanisms of resistance may be present within the population before the fungicide is applied. The rate of resistance development in a population depends on the resultant mechanism of resistance such as how resistance characters are inherited, the epidemiology of the fungus, the environment, and the

Fungicide resistance can be conferred by various mechanisms including: (I) an altered target site, which reduces the binding of the fungicide; (II) the synthesis of an alternative enzyme capable of substituting the target enzyme; (III) the overproduction of the fungicide target; (IV) an active efflux or reduced uptake of the fungicide; and (V) a metabolic breakdown of the fungicide. In addition, some unrecognized mechanisms could also be responsible for

Currently, the most widespread hypothesis to explain the reduced levels of toxic products in the cell is based on active efflux of these compounds by ABC transporters (ATP binding cassette) and MFS (Major Facilitator Superfamily) (Hayashi et al., 2001, 2002a, 2002b, 2003; Stergiopoulos et al., 2002a, 2002b; Stergiopoulos & de Waard, 2002, Vermeulen et al., 2001). ABC and MFS transporters are the most studied so far and have been described in many fungi such as *Aspergillus nidulans, Botrytis cinerea, Mycosphaerella graminicola, Magnaporthe grisea, Penicillium digitatum***,** etc (Andrade et al., 2000a, 2000b; del Sorbo et al., 2000; Nakaune, 2001; Schoonbeek et al., 2003; Stergiopoulos et al., 2002a**;** Stergiopoulos & de Waard, 2002; Vermeulen et al., 2001; Yoder and Turgeon 2001; Zwiers et al., 2003). Its function is to prevent or reduce the accumulation of compounds and therefore to avoid or minimize their toxic action (Bauer et al., 1999, Pao et al., 1998). ABC transporters comprise a large family of proteins and are located outside the plasma membrane or within the cell in intracellular compartments as vacuoles, endoplasmic reticulum, peroxisomes and mitochondria. They can carry a wide variety of toxic against the gradient (Del Sorbo et al., 2000; Theodoulou, 2000). ABC transporters include systems both capture and removal, generally showing activity on a wide range of substrates (fungicides, drugs, alkaloids, lipids, peptides, sterols, flavonoids, sugars, etc), but there are specific transporters for substrates (Bauer et al., 1999,

**3.1.1 The reduction of intracellular concentration of antifungal compound** 

and coincident, resulting in noticeable decline of field performance (Hewit, 1998).

determined by the target pathogen and selected fungicide.

persistence of selective pressure.

fungicide resistance.

Del Sorbo et al., 2000).

**3.1. Mechanisms of fungicide resistance** 

The overexpression of ABC and MFS genes plays an essential role in the resistance of chemically unrelated phenomenon described as multidrug resistance or MDR drugs (Del Sorbo et al., 2000; White, 1997). This phenomenon has been observed in a wide variety of organisms and can be a real threat to the effective control of fungal pathogens (Fling et al., 1991). In phytopathogenic fungi, these transporters can also be a virulence factor in providing protection against defense compounds produced by the plant or mediating the secretion of host-specific toxins. Also play a major role in determining the baseline sensitivity to fungicides and other antifungal agents. (De Waard, 1997; Stergiopoulos et al., 2003a, 2003b).

In *P. digitatum*, causal agent of citrus green rotten, four ABC transporters have been identified so far. ABC transporters PMR1 (Hamamoto et al., 2001b; Nakaune et al., 1998) and PMR5 (Nakaune, 2001; Nakaune et al., 2002) have been studied previously. Disruption of PMR1 in sensitive and resistant strains results in an increased sensitivity to DMIs and other compounds (Nakaune et al., 1998; Nakaune et al., 2002). However, the introduction of resistant strains from PMR1 restores the resistance while the introduction of PMR1 from sensitive strains does not have the same effect and does not restore the resistance. This suggests that although PMR1 plays an important role in the sensitivity of *P. digitatum* against DMIs alone does not explain the differences between sensitive and resistant strains (Hamamoto et al., 2001). Another of the genes studied is PMR5. This gene has highly homologous to PMR1, and also to *atrB* from *Aspergillus nidulans* and *BcAtrB* from *Botrytis cinerea* (Schoonbeek et al., 2003), however, is strongly induced by benzimidazoles, resveratrol and other compounds, but not for DMIs. This shows the different substrate specificity of both proteins and may play an important role in providing protection against natural or synthetic toxic compounds (Nakaune et al., 2002).

Sequence analysis in all four ABC transporter genes in several sensitive and resistant strains revealed no mutations in PMR1, PMR3 and PMR4, and point mutations only were observed in both the promoter and coding regions of PMR5 in multiple resistant strains (TBZ- and DMI-resistant) (Sánchez-Torres & Tuset, 2011). But no explanation was ascertained for the absence of sequence changes relating to fungicide resistance in the other ABC transporters, particularly in the PMR1 gene given that transcription of PMR1 has proven to be strongly activated in the presence of different fungicides (Hamamoto et al., 2001b; Nakaune, 2001; Nakaune et al., 1998).

To date, MFS transporters have been described in several fungi, e.g. *Botrytis cinerea* shows a broad spectrum of resistance to different fungicides and their expression has been induced by many of them, particularly noteworthy *Bcmfs4* induction in the presence of strobilurin (trifloxiestrobin) (Hayashi et al., 2002a, 2002b, 2003; Schoonbeek et al., 2003; Vermeulen et al., 2001). All this means that these transporters are potential candidates for the study of factors involved in resistance based on active eflux of these toxic compounds since they have remarkably broad substrate specificity although they can also transport specific compounds.

Recently, five different MFS transporters have been identified and characterized in the postharvest phytopathogenic fungus *Penicillium digitatum* (PdMFS1-PdMFS5). Sequence analysis of these five genes revealed different genomic structure and although all genes seem to be implicated in pathogenicity, only 2 out of five MFS transporters confirmed to be involved in fungicide resistance (Sánchez-Torres et al., submitted). Therefore, the most recent thought for fungicide resistance based on active efflux of these toxic compounds is

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 177

alternative route in mitochondrial respiration (Gisi et al., 2000; Schnabel et al., 2001; Wood &

The procedures for detecting fungicide resistance using conventional methods are laborintensive and time-consuming if large numbers of isolates have to be tested. Advances in molecular biology have provided new opportunities for rapidly detecting fungicide resistant genotype once the mechanisms of resistance have been elucidated at a molecular level. Several molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), allele specific PCR, allele-specific real-time PCR and massive sequencing techniques (all above described) have been used successfully to detect fungicideresistant genotypes of several plant pathogens. Many examples of these techniques have

PCR has revolutionized molecular biology and diagnostics and has become a fast tool for detecting fungicide-resistant pathogens. In *Penicillium digitatum*, the DMI resistance resulted from over-expression of the *PdCYP51* genes driven by a tandem repeat of five copies of a 126 transcriptional enhancer (Hamamoto et al., 2000), or insertion of 199-bp in the promoter region of *PdCYP51A* (Gosoph et al., 2007) or by the presence of 199-bp enhancer in the promoter region of *PdCYP51B* (Sun et al., 2011). Based on the DNA sequence of the *PdCYP51*-genes, PCR primers have been developed which are able to distinguish not only between sensitive and resistant DMI strains but allow identification of molecular mechanism that takes place (Hamamoto et al., 2001; Gosoph et al., 2007; Sánchez-Torres &

**3.2.2 PCR-RFLP and primer-introduced restriction analysis PCR (PIRA-PCR)** 

PCR amplification followed by restriction enzyme analysis (PCR-RFLP) is a general technique to detect a point mutation that alters a restriction enzyme site. This method has been used to rapidly detect benzimidazole-resistant isolates of *Monilinia laxa* from stone fruit and almond crops in California (Ma & Michailides, 2005) and can also detect azoxystrobinresistant isolates of *Alternaria alternata, A. tenuissima*, and *A. arborescens* within a few hours (Ma et al., 2003b). Since the PCR-RFLP is an easy and rapid technique, these assays have been developed for detecting benzimidazole resistance in *Botrytis cinerea* (Luck & Gillings, 1995), *Cladobotryum dendroides* (McKay et al., 1998), *Helminthosporium solani* (Cunha & Rizzo, 2003; McKay & Cooke, 1997), and for detecting strobilurin resistance in *Blumeria graminis*  f.sp. *hordei* (Baumler et al., 2003), *Erysiphe graminis* f. sp. *tritici* (Sierotzki et al., 2000), and

Although PCR-RFLP is a simple method for detection of point mutations, some target DNA fragments may not contain a restriction endonuclease recognition sequence at the site of point mutation. Thus, primer-introduced restriction analysis PCR (PIRA-PCR) has become a useful method to create diagnostic artificial RFLPs (Haliassos et al., 1989). A PIRA-PCR assay was developed for detecting carpropamid resistant *Magnaporthe grisea* (Kaku et al.,

**3.2 Molecular detection of fungicide resistance in phytopathogenic fungi** 

Hollomon, 2003).

been reported.

Tuset, 2011; Sun et al., 2011).

*Podosphaera fusca* (Ishii et al., 2001).

2003).

**3.2.1 PCR detection of DMI-resistant isolates** 

now discussed. These results suggest that many genes could be involved in the mechanisms conferring fungicide resistance to phytopathogenic fungi and some are fungicidedependent.

From a practical standpoint, the fact that ABC and MFS transporters determine the baseline sensitivity to fungicides, are responsible for MDR and can act as virulence factors implies that these carriers are an attractive target for chemical control. In this context, inhibitors of these transporters could improve the effectiveness of control and reduce the virulence of fungal pathogens.

#### **3.1.2 Changes in binding target that causes a reduced affinity of the compound fungicide**

The most extent mechanism to confer DMI resistance involved mutations of *CYP51* gene, the target enzyme of DMIs fungicides and has been described for a large number of pathogens such as *Botrytis cinerea* (Albertini et al., 2002, Albertini & Leroux, 2004), a substitution of Phe for Tyr at position 136 (Y136F) was found in *Uncinula necator* (Délye et al., 1997) and also in *Erisiphe graminis* f. sp. *hordei* (Délye et al., 1998). Two single nucleotide mutations of *CYP51*  resulting in amino acid substitutions Y136F and K147Q in *Blumeria graminis* were also found (Wyand & Brown, 2005). Different mutations were also found in *Tapesia* sp (Albertini et al., 2003), *Penicillium italicum* (Joseph-Horne & Hollomon, 1997), *Ustilago maydis* (Butters et al., 2000) and *Blumeriella jaapii* (Ma et al., 2006)

Similarly, mutations have been described in the cytochrome b gene that lead to change of its corresponding protein G143A, conferring resistance to strobilurin (Avila-Adame & Koller, 2003a, 2003b; Gisi et al., 2000, Zheng et al., 2000, Zhang, Z. et al., 2009) or the amino acid substitution in the -tubulin target protein involved in development of resistance to benzimidazoles in *Botrytis cinerea* (Banno, 2008), *Venturia inaequalis* and *Penicillium italicum* (Koenraadt et al., 1992)*, Monilinia fructicola* (Ma et al., 2003a), *M. laxa* (Ma & Michailides, 2005), *P. expansum* (Baraldi et al., 2003) and *P. digitatum* (Sánchez-Torres & Tuset, 2011).

#### **3.1.3 Over-expression of the target of union of fungicide**

P45014DM increased by over-expression of the *CYP51* gene has been described as a mechanism of resistance to azoles. In *Penicillium digitatum* a unique 126-bp sequence in the promoter region of *CYP51* was tandem repeated five times in resistant isolates and was present only one in sensitive isolates. This provided a quick and easy method to detect DMIresistant strains of *P. digitatum* (Hamamoto et al., 2001). Insertions in the promoter were also found in *Blumeriella jaapii* (Ma et al., 2006), *Venturia inaequalis* (Schnabel & Jones 2001) and in *Monilinia fructicola* (Luo et al., 2008) and recently another insertion of 199-bp was found in *P. digitatum CYP51A* gene (Ghosoph et al., 2007) and *PdCYP51B* gene (Sun et al., 2011; Sánchez-Torres et al., submitted) leading to resistant phenotypes.

#### **3.1.4 Compensation of the toxic effects of the fungicide by altered biosynthetic or metabolic pathway**

This phenomenon has been described both in the case of DMIs because they exert their toxic effect by depletion of ergosterol and the accumulation of C14 methylated precursors and in the case of strobilurins since the presence of AOX (alternative oxidase) allows the use an alternative route in mitochondrial respiration (Gisi et al., 2000; Schnabel et al., 2001; Wood & Hollomon, 2003).

#### **3.2 Molecular detection of fungicide resistance in phytopathogenic fungi**

The procedures for detecting fungicide resistance using conventional methods are laborintensive and time-consuming if large numbers of isolates have to be tested. Advances in molecular biology have provided new opportunities for rapidly detecting fungicide resistant genotype once the mechanisms of resistance have been elucidated at a molecular level. Several molecular techniques, such as PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), allele specific PCR, allele-specific real-time PCR and massive sequencing techniques (all above described) have been used successfully to detect fungicideresistant genotypes of several plant pathogens. Many examples of these techniques have been reported.

#### **3.2.1 PCR detection of DMI-resistant isolates**

176 Plant Pathology

now discussed. These results suggest that many genes could be involved in the mechanisms conferring fungicide resistance to phytopathogenic fungi and some are fungicide-

From a practical standpoint, the fact that ABC and MFS transporters determine the baseline sensitivity to fungicides, are responsible for MDR and can act as virulence factors implies that these carriers are an attractive target for chemical control. In this context, inhibitors of these transporters could improve the effectiveness of control and reduce the virulence of

The most extent mechanism to confer DMI resistance involved mutations of *CYP51* gene, the target enzyme of DMIs fungicides and has been described for a large number of pathogens such as *Botrytis cinerea* (Albertini et al., 2002, Albertini & Leroux, 2004), a substitution of Phe for Tyr at position 136 (Y136F) was found in *Uncinula necator* (Délye et al., 1997) and also in *Erisiphe graminis* f. sp. *hordei* (Délye et al., 1998). Two single nucleotide mutations of *CYP51*  resulting in amino acid substitutions Y136F and K147Q in *Blumeria graminis* were also found (Wyand & Brown, 2005). Different mutations were also found in *Tapesia* sp (Albertini et al., 2003), *Penicillium italicum* (Joseph-Horne & Hollomon, 1997), *Ustilago maydis* (Butters et al.,

Similarly, mutations have been described in the cytochrome b gene that lead to change of its corresponding protein G143A, conferring resistance to strobilurin (Avila-Adame & Koller, 2003a, 2003b; Gisi et al., 2000, Zheng et al., 2000, Zhang, Z. et al., 2009) or the amino acid substitution in the -tubulin target protein involved in development of resistance to benzimidazoles in *Botrytis cinerea* (Banno, 2008), *Venturia inaequalis* and *Penicillium italicum* (Koenraadt et al., 1992)*, Monilinia fructicola* (Ma et al., 2003a), *M. laxa* (Ma & Michailides, 2005), *P. expansum* (Baraldi et al., 2003) and *P. digitatum* (Sánchez-Torres & Tuset, 2011).

P45014DM increased by over-expression of the *CYP51* gene has been described as a mechanism of resistance to azoles. In *Penicillium digitatum* a unique 126-bp sequence in the promoter region of *CYP51* was tandem repeated five times in resistant isolates and was present only one in sensitive isolates. This provided a quick and easy method to detect DMIresistant strains of *P. digitatum* (Hamamoto et al., 2001). Insertions in the promoter were also found in *Blumeriella jaapii* (Ma et al., 2006), *Venturia inaequalis* (Schnabel & Jones 2001) and in *Monilinia fructicola* (Luo et al., 2008) and recently another insertion of 199-bp was found in *P. digitatum CYP51A* gene (Ghosoph et al., 2007) and *PdCYP51B* gene (Sun et al., 2011; Sánchez-

**3.1.4 Compensation of the toxic effects of the fungicide by altered biosynthetic or** 

This phenomenon has been described both in the case of DMIs because they exert their toxic effect by depletion of ergosterol and the accumulation of C14 methylated precursors and in the case of strobilurins since the presence of AOX (alternative oxidase) allows the use an

**3.1.2 Changes in binding target that causes a reduced affinity of the compound** 

dependent.

**fungicide** 

fungal pathogens.

2000) and *Blumeriella jaapii* (Ma et al., 2006)

**3.1.3 Over-expression of the target of union of fungicide** 

Torres et al., submitted) leading to resistant phenotypes.

**metabolic pathway** 

PCR has revolutionized molecular biology and diagnostics and has become a fast tool for detecting fungicide-resistant pathogens. In *Penicillium digitatum*, the DMI resistance resulted from over-expression of the *PdCYP51* genes driven by a tandem repeat of five copies of a 126 transcriptional enhancer (Hamamoto et al., 2000), or insertion of 199-bp in the promoter region of *PdCYP51A* (Gosoph et al., 2007) or by the presence of 199-bp enhancer in the promoter region of *PdCYP51B* (Sun et al., 2011). Based on the DNA sequence of the *PdCYP51*-genes, PCR primers have been developed which are able to distinguish not only between sensitive and resistant DMI strains but allow identification of molecular mechanism that takes place (Hamamoto et al., 2001; Gosoph et al., 2007; Sánchez-Torres & Tuset, 2011; Sun et al., 2011).

#### **3.2.2 PCR-RFLP and primer-introduced restriction analysis PCR (PIRA-PCR)**

PCR amplification followed by restriction enzyme analysis (PCR-RFLP) is a general technique to detect a point mutation that alters a restriction enzyme site. This method has been used to rapidly detect benzimidazole-resistant isolates of *Monilinia laxa* from stone fruit and almond crops in California (Ma & Michailides, 2005) and can also detect azoxystrobinresistant isolates of *Alternaria alternata, A. tenuissima*, and *A. arborescens* within a few hours (Ma et al., 2003b). Since the PCR-RFLP is an easy and rapid technique, these assays have been developed for detecting benzimidazole resistance in *Botrytis cinerea* (Luck & Gillings, 1995), *Cladobotryum dendroides* (McKay et al., 1998), *Helminthosporium solani* (Cunha & Rizzo, 2003; McKay & Cooke, 1997), and for detecting strobilurin resistance in *Blumeria graminis*  f.sp. *hordei* (Baumler et al., 2003), *Erysiphe graminis* f. sp. *tritici* (Sierotzki et al., 2000), and *Podosphaera fusca* (Ishii et al., 2001).

Although PCR-RFLP is a simple method for detection of point mutations, some target DNA fragments may not contain a restriction endonuclease recognition sequence at the site of point mutation. Thus, primer-introduced restriction analysis PCR (PIRA-PCR) has become a useful method to create diagnostic artificial RFLPs (Haliassos et al., 1989). A PIRA-PCR assay was developed for detecting carpropamid resistant *Magnaporthe grisea* (Kaku et al., 2003).

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 179

improve our ability of studying the evolution of fungicide resistance at the population level. Molecular techniques can be also developed based on the different fungicide mechanisms to rapidly detect resistant isolates. Furthermore, a timely detection of resistance levels in populations of phytopathogenic fungi in a field would help growers make proper decisions

Authors want to thank Dr. J.M. Colmenero for critical reading of the manuscript. A.M. Pastrana is recipient of an IFAPA fellowship from the Consejería de Agricultura y Pesca,

Abad, Z.G.; Abad, J.A.; Coffey, M.D.; Oudemans, P.V.; Man, W.A.; de Gruyter, H.;

Abbott, C.L.; Gilmore, S.R.; Lewis, C.T.; Chapados, J.T.; Peters, R.D.; Platt, H.W.; Coffey,

Abd-Elsalam, K.; Bahkali, A.; Moslem, M.; Amin, O.E. & Niessen, L. (2011). An optimized

Abd-Elsalam, K.A.; Asran-Amal, A.; Schnieder, F.; Migheli, Q. & Verreet, J.-A. (2006).

Ahmadian, A.; Ehn, M. & Hober, S. (2006). Pyrosequencing: History, biochemistry and future. *Clinica Chimica Acta*, Vol.363, No.1-2, pp. 83-94, ISSN 0009-8981 Alaei, H.; Baeyen, S.; Maes, M.; Hofte, M. & Heungens, K. (2009). Molecular detection of

Albertini, C. & Leroux, P. (2004). A *Botrytis cinerea* putative 3-keto reductase gene (ERG27)

Albertini, C.; Thebaud G.; Fournier, E. & Leroux P. (2002). Eburicol 14*α*-demethylase gene

*Journal of Plant Pathology,* Vol.109, pp. 117–128, ISSN 0929-1873

continents. *Mycologia*, Vol.100, No.1, pp. 99-110, ISSN 0027-5514

*Journal of Plant Pathology,* Vol.4, No.32, pp. 440-457, ISSN 0706-0661

Cunnington, J. & Louws, F.J. (2008). *Phytophthora bishetia* sp nov., a new species identified in isolates from the Rosaceous raspberry, rose and strawberry in three

M.D. & Lévesque, C.A. (2010). Development of a SNP genetic marker system based on variation in microsatellite flanking regions of *Phytophthora infestans*. *Canadian* 

protocol for DNA extraction from wheat seeds and Loop-Mediated Isothermal Amplification (LAMP) to detect *Fusarium graminearum* contamination of wheat grain. *International Journal of Molecular Sciences,* Vol.12, No.6, pp. 3459-3472, ISSN

Molecular detection of *Fusarium oxysporum* f. sp. *vasinfectum* in cotton roots by PCR and real-time PCR assay. *Journal of Plant Diseases and Protection*, Vol.113, No.1, pp.

*Puccinia horiana* in *Chrysanthemum* x *morifolium* through conventional and real-time PCR. *Journal of Microbiological Methods,* Vol.76, No.2, pp. 136-145, ISSN 0167-7012 Albertini, C.; Gredt, M. & Leroux, P. (2003). Polymorphism of the 14\_a demethylase gene

(CYP51) in the cereal eyespot fungi *Tapesia acuformis* and *Tapesia yallundae*. *European* 

that is homologous to the mammalian 17 beta-hydroxysteroid dehydrogenase type 7 gene (17 beta-HSD7). European Journal of Plant Pathology Vol.110, No.7, pp.723-

(CYP51) polymorphism and speciation in *Botrytis cinerea*. *Mycological Research,*

on resistance management programs to control plant diseases.

**5. Acknowledgements** 

Junta de Andalucía, Spain.

1422-0067

14–19, ISSN 1861-3829

733, ISSN: 0929-1873

Vol.106, pp. 1171-1178, ISSN 0953-7562

**6. References** 

#### **3.2.3 Allele-specific PCR and quantitative allele-specific real-time PCR**

Allele-specific PCR is another simple and rapid method for detecting point mutations. Usually, one of two PCR primers used in an allele specific amplification is designed to amplify preferentially one allele by matching the desired allele and mismatching the other allele at the 3' end of primer.

Allele-specific PCR assays have been developed for detecting benzimidazole-low resistant isolates of *Monilinia laxa* (Ma & Michailides, 2005), and azoxystrobin-resistant isolates of *Alternaria alternata, A. tenuissima*, and *A. arborascens* (Ma & Michailides, 2004). Additionally, allele-specific PCR assays have been developed for the rapid detection of strobilurinresistant isolates of *Blumeria graminis* f. sp. *tritici* (Fraaije et al., 2002) and *Mycosphaerella fijiensis* (Gisi et al., 2002), and DMI-resistant isolates of *Erysiphe graminis* f. sp. *hordei* (Dèlye et al., 1997).

Real-time PCR technique can be used to quantitatively determine the amount of target DNA in a sample. An allele-specific real-time PCR assay has been used to follow the dynamics of *QoI* resistant allele *A143* in field populations of *Blumeria graminis* f. sp. *tritici* before and after fungicide application (Fraaije et al., 2002). A real-time PCR assay to rapidly detect azoxystrobin-resistant *Alternaria* has been developed in California pistachio orchards (Ma & Michailides, 2005). Additionally, a real-time PCR using the *Alternaria* specific PCR primer pair can quantify both resistant and sensitive alleles in the same tested samples, thereby enabling a rapid determination of frequencies of the azoxystrobin-resistant allele in *Alternaria* populations.

#### **4. Conclusion**

Advances in the development of molecular methods, especially PCR technology have provided diagnostic laboratories with powerful tools for detection and identification of phytopathogenic fungi. Molecular techniques have also contributed to elucidate the phenotypic and genetic structure within species and the complexity of plant and environment fungal populations. New technologies and improved methods with reduced cost and improved speed, throughput, multiplexing, accuracy and sensitivity have emerged as an essential strategy for the control of plant fungal diseases. These advances have been complemented by the development of new nucleic acids extraction methods, increased automation, reliable internal controls, multiplexing assays, *online* information and *on site* molecular diagnostics. Nevertheless, molecular diagnostic tools should be complemented with other techniques, either traditional culture-based methods or the newly emerged proteomic, a promising tool for providing information about pathogenicity and virulence factors that will open up new possibilities for crop disease diagnosis and crop protection.

On the other hand, fungicides continue to play a key role in strategies for the control of diseases in crops, and the development of resistance in the target pathogens is a continuing risk. This fact leads to many losses as control systems are not longer effective. Therefore, the better understanding on mechanisms developed during fungicide resistance is essential for a better management of chemical control, environment and human health.

Great advances have been made in the development of molecular methods to identify and monitor resistance of plant pathogens to fungicides. The highly sensitive methods can improve our ability of studying the evolution of fungicide resistance at the population level. Molecular techniques can be also developed based on the different fungicide mechanisms to rapidly detect resistant isolates. Furthermore, a timely detection of resistance levels in populations of phytopathogenic fungi in a field would help growers make proper decisions on resistance management programs to control plant diseases.

#### **5. Acknowledgements**

Authors want to thank Dr. J.M. Colmenero for critical reading of the manuscript. A.M. Pastrana is recipient of an IFAPA fellowship from the Consejería de Agricultura y Pesca, Junta de Andalucía, Spain.

#### **6. References**

178 Plant Pathology

Allele-specific PCR is another simple and rapid method for detecting point mutations. Usually, one of two PCR primers used in an allele specific amplification is designed to amplify preferentially one allele by matching the desired allele and mismatching the other

Allele-specific PCR assays have been developed for detecting benzimidazole-low resistant isolates of *Monilinia laxa* (Ma & Michailides, 2005), and azoxystrobin-resistant isolates of *Alternaria alternata, A. tenuissima*, and *A. arborascens* (Ma & Michailides, 2004). Additionally, allele-specific PCR assays have been developed for the rapid detection of strobilurinresistant isolates of *Blumeria graminis* f. sp. *tritici* (Fraaije et al., 2002) and *Mycosphaerella fijiensis* (Gisi et al., 2002), and DMI-resistant isolates of *Erysiphe graminis* f. sp. *hordei* (Dèlye et

Real-time PCR technique can be used to quantitatively determine the amount of target DNA in a sample. An allele-specific real-time PCR assay has been used to follow the dynamics of *QoI* resistant allele *A143* in field populations of *Blumeria graminis* f. sp. *tritici* before and after fungicide application (Fraaije et al., 2002). A real-time PCR assay to rapidly detect azoxystrobin-resistant *Alternaria* has been developed in California pistachio orchards (Ma & Michailides, 2005). Additionally, a real-time PCR using the *Alternaria* specific PCR primer pair can quantify both resistant and sensitive alleles in the same tested samples, thereby enabling a rapid determination of frequencies of the azoxystrobin-resistant allele in

Advances in the development of molecular methods, especially PCR technology have provided diagnostic laboratories with powerful tools for detection and identification of phytopathogenic fungi. Molecular techniques have also contributed to elucidate the phenotypic and genetic structure within species and the complexity of plant and environment fungal populations. New technologies and improved methods with reduced cost and improved speed, throughput, multiplexing, accuracy and sensitivity have emerged as an essential strategy for the control of plant fungal diseases. These advances have been complemented by the development of new nucleic acids extraction methods, increased automation, reliable internal controls, multiplexing assays, *online* information and *on site* molecular diagnostics. Nevertheless, molecular diagnostic tools should be complemented with other techniques, either traditional culture-based methods or the newly emerged proteomic, a promising tool for providing information about pathogenicity and virulence factors that will open up new possibilities for crop disease diagnosis and crop protection.

On the other hand, fungicides continue to play a key role in strategies for the control of diseases in crops, and the development of resistance in the target pathogens is a continuing risk. This fact leads to many losses as control systems are not longer effective. Therefore, the better understanding on mechanisms developed during fungicide resistance is essential for

Great advances have been made in the development of molecular methods to identify and monitor resistance of plant pathogens to fungicides. The highly sensitive methods can

a better management of chemical control, environment and human health.

**3.2.3 Allele-specific PCR and quantitative allele-specific real-time PCR** 

allele at the 3' end of primer.

al., 1997).

*Alternaria* populations.

**4. Conclusion** 


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 181

Baayen, R.P.; O'Donnell, K.; Bonants, P.J.M.; Cigelnik, E.; Kroon, L.P.N.M.; Roebroeck, E.J.A.

Babu, B.K..; Mesapogu, S.; Sharma A.; Somasani, S.R. & Arora, D.K. (2011). Quantitative

Bailey, A.M.; Mitchell, D.J.; Manjunath, K.L.; Nolasco, G. & Niblett, C.L. (2002).

Baraldi, E.; Mari, M.; Chierici, E.; Pondrelli, M.; Bertollini, P. & Pratella, G.C. (2003). Studies

Bauer, B.E.; Wolfger, H. & Kuchler, K. (1999). Inventory and function of yeast ABC proteins:

Baumler, S.; Felsenstein, F.G. & Schwarz, G. (2003). CAPS and DHPLC analysis of a single

Bayraktar, H.; Dolar, F.S. & Tor, M. (2007). Determination of genetic diversity within

Bearchell, S.J.; Fraaije, B.A.; Shaw, M.W. & Fitt, B.D.L. (2005). Wheat archive links long-term

Bilodeau, G.J.; Pelletier, G.; Pelletier, F.; Lévesque, C.A. & Hamelin, R.C. (2009). Multiplex

Bindslev, L.; Oliver, R.P. & Johansen, B. (2002). *In situ* PCR for detection and identication of fungal species. *Mycological Research*, Vol.106, No.3, pp. 277–279, ISSN 0953-7562 Biswas, K. & Biswas, R.A. (2011). Modified method to isolate genomic DNA from plants

*Journal of Plant Pathology,* Vol.89, No.3, pp. 341-347, ISSN 1125-4653

assays. *Phytopathology,* Vol.98, pp. 397–404, ISSN 0031-949X

*Phytopathology,* Vol.97, No.6, pp. 717-727, ISSN 0031-949X

*Biophysic Acta,* Vol.1461, No.2, pp. 217-236, ISSN 0005-2736

Vol.151, pp. 149–152, ISSN 0931-1785

No.2, pp. 195-210, ISSN 0706-0661

ISSN 0027-8424

3891

949X

No.3, pp.466-473, ISSN: 0027-5514

& Waalwijk, C. (2000). Gene genealogies and AFLP analyses in the *Fusarium oxysporum* complex identify monophyletic and nonmonophyletic formae speciales causing wilt and rot disease. *Phytopathology*, Vol.90, No.8, pp. 891-900, ISSN 0031-

real-time PCR assay for rapid detection of plant and human pathogenic *Macrophomina phaseolina* from field and environmental samples. *Mycologia* Vol.103,

Identification to the species level of the plant pathogens *Phytophthora* and *Pythium* by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR Elisa. *Fems Microbiology Letters,* Vol.207, No.2, pp. 153-158, ISSN 0378-1097 Banno, S.; Fukumori, F.; Ichiishi, A.; Okada, K.; Uekusa, H.; Kimura, M. & Fujimura, M.

(2008). Genotyping of benzimidazole-resistant and dicarboximideresistantmutations in *Botrytis cinerea* using real-time polymerase chain reaction

of thiabendazole resistance of *Penicillium expansum* of pears: pathogenic fitness and genetic characterization. *Plant Pathology,* Vol.52, pp. 362-370, ISSN 0032-0862 Barnes, C.W. & Szabo, L.J. (2007). Detection and identification of four common rust

pathogens of cereals and grasses using real-time polymerase chain reaction.

about sex, stress, pleiotropic drug and heavy metal resistance. *Biochimestry* 

nucleotide polymorphism in the cytochrome b gene conferring resistance to strobilurin in field isolates of *Blumeria graminis* f. sp*. hordei. Journal of Phytopathology,*

*Ascochyta rabiei* (pass.) labr., the cause of ascochyta blight of chickpea in Turkey.

fungal pathogen population dynamics to air pollution. *Proceedings of the National Academy of sciences of the United States of America,* Vol.102, No.15, pp. 5438-5442,

real-time polymerase chain reaction (PCR) for detection of *Phytophthora ramorum*, the causal agent of sudden oak death. *Canadian Journal of Plant Pathology,* Vol.31,

without liquid nitrogen. *Current Science*, Vol.100, No.11, pp. 1622-1624 , ISSN 0011-


Altschul, S.F.; Madden, T.L.; Schaffer, A.A.; Zhang, J.H.; Zhang, Z.; Miller, W. & Lipman,

Anderson, I.C. & Cairney, J.W.G. (2004). Diversity and ecology of soil fungal communities:

Anderson, I.C. & Parkin, P.I. (2007). Detection of active soil fungi by RT-PCR amplification

Anderson, N.; Szemes, M.; O'Brien, P.; De Weerdt, M.; Schoen, C.; Boender, P. & Bonants, P.

Andrade, A.C.; Van Nistelrooy, J.G.M.; Peery, R.B.; Skatrud, P.L. & de Waard M.A. (2000b).

Ansorge, W.J. (2010). Novel Next-Generation DNA Sequencing Techniques for Ultra High-

Arici, S.E. & Koc N.K. (2010). RAPD–PCR analysis of genetic variation among isolates of

Aroca, A.; Raposo, R. & Lunello, P. (2008). A biomarker for the identification of four

Ashizawa, T.; Takahashi, M.; Moriwaki, J. & Hirayae, K. (2010). Quantification of the rice

Avila-Adame, C. & Koller, W. (2003a). Characterization of spontaneous mutants of

Avila-Adame, C. & Koller, W. (2003b). Impact of alternative respiration and target-site

azoxystrobin. *Current Genet*ics, Vol.42, pp. 332-338, ISSN 0172-8083

*Microbiology,* Vol.73, No.9, pp. 2911–2918, ISSN 0099-2240

*Microbiology and Biotechnology,* Vol.80, No.6, pp. 1131-1140

search programs. *Nucleic Acids Research*, Vol.25, pp. 3389-3402

*Environmental Microbiology,* Vol.6, No. 8, pp. 769–779

1987-1997, ISSN 1350-0872

966-977, ISSN 0026-8925

374537-8

865–872

D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database

increased understanding through the application of molecular techniques.

of precursor rRNA molecules. *Journal of Microbiological Methods*, Vol.68, pp. 248-253

(2006). Use of hybridization melting kinetics for detecting *Phytophthora* species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets. *Mycological Research,* Vol.110, pp. 664-671 Andrade, A.C.; Del Sorbo, G.; Van Nistelrooy, J.G.M. & de Waard, M.A. (2000a). The ABC

transporter AtrB from *Aspergillus nidulans* is involved in resistance to all major classes of fungicides and natural toxic compouns. *Microbiology U.K*., Vol.146, pp.

ABC transporters from *Aspergillus nidulans* are involved in protection against cytotoxic agents and antibiotic production. *Molecular General Genet*ics, Vol.263, pp.

Throughput Applications in Bio-Medicine, In: *Molecular Diagnostics* (Second Edition), G.P. Patrinos and W.J. Ansorge, pp. 365-378, Elsevier, ISBN 978-0-12-

*Fusarium graminearum* and *Fusarium culmorum* from wheat in Adana Turkey. *Pakistan Journal of Biological Sciences*, Vol.13, No. 3, pp. 138–142, ISSN 1028-8880 Aroca, A. & Raposo, R. (2007). PCR-based strategy to detect and identify species of

*Phaeoacremonium* causing grapevine diseases. *Applied and Environmental* 

*Phaeoacremonium* species using the beta-tubulin gene as the target sequence. *Applied* 

false smut pathogen *Ustilaginoidea virens* from soil in Japan using real-time PCR. E*uropean Journal of Plant Pathology,* Vol.128, No.2, pp. 221-232, ISSN 0929-1873 Attallah, Z.K.; Bae, J.; Jansky, S.H.; Rouse, D.I. & Stevenson, W.R. (2007). Multiplex real-time

quantitative PCR to detect and quantify *Verticillium dahliae* colonization in potato lines that differ in response to *Verticillium* wilt. *Phytopathology,* Vol.97, No.7, pp.

*Magnaporthe grisea* expressing stable resistance to the Qo-inhibiting fungicide

mutations on responses of germinating conidia of *Magnaporthe grisea* to Qoinhibiting fungicides. *Pest Managment Science,* Vol.59, pp. 303-309, ISSN 1526-498X


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 183

Chi, M.-H.; Park, S.-Y. & Lee, Y.-H. (2009). A quick and safe method for fungal DNA extraction. *Plant Pathology Journal,* Vol.25, No.1, pp. 108-111, ISSN 1598-2254 Chimento, A.; Scibetta, S.; Schena, L.; Cacciola, S.O. & Cooke, D.E.L. (2005). A new method

Cipriani, M.G.; Stea, G.; Moretti, A.; Altomare, C.; Mulè, G. & Vurro, M. (2009).

Collado-Romero, M.; Mercado-Blanco, J.; Olivares-Garcia, C. & Jimenez-Diaz, R. M. (2008).

Cruz-Perez, P.; Buttner, M.P. & Stetzenbach, L. D. 2001. Detection and quantitation of

Cunha, M.G. & Rizzo, D.M. (2003). Development of fungicide cross resistance in

Daval, S.; Lebreton L.; Gazengel K.; Guillerm-Erckelboudt A.-Y. & Sarniguet A. (2010).

major groups. *Plant Pathology,* Vol.59, No. 1, pp. 165–178, ISSN 0032-0862 de Cock, A.W.A.M. & Lévesque, A. (2004). New species of *Pythium* and *Phytophthora*. *Studies* 

De Waard, M.A. (1997). Significance of ABC transporters in fungicide sensitivity and

de Weerdt, M.; Zijlstra, C.; van Brouwershaven, I.R.; van Leeuwen, G.C.M.; de Gruyter, J. &

Del Sorbo, G.; Schoonbeek, H. & de Waard, M.A. (2000). Fungal transporters involved in

Delp, C.J. & Dekker, J. (1985). Fungicide resistance: definitions and use of terms. *EPPO* 

Dèlye, C.; Laigret, F. & Coster, M.F. (1997). A mutation in 14 demethylase gene of *Uncinula* 

Demeke, T. & Jenkins, G.R. (2010). Influence of DNA extraction methods, PCR inhibitors

Demeke, T.; Gräfenhan, T.; Clear, R.M.; Phan, A.; Ratnayaka, I.; Chapados, J.T.; Patrick, S.K.;

*Environmental Microbiology,* Vol.63, pp. 2966-2970, ISSN 0099-2240

*in Mycology*, Vol.50, Special Issue, pp. 481–487, ISSN 0166-0616

resistance. *Pesticide Science,* Vol.51, pp. 271-275, ISSN 0031-613X

*Phytopathology,* Vol.154, No.11-12, pp. 694-700, ISSN 0931-1785

CiF, Cyber infrastructure for *Fusarium* : ( http://www.fusariumdb.org)

*Phytopathology*, Vol.98, No.9, pp. 1019-1028, ISSN 0031-949X

*Cellular Probes,* Vol.15, No.2, pp. 81-88, ISSN 0890-8508

1755-098X

*Pathology*, Vol.87, pp. 290

pp. 78–84, ISSN 1049-9644

798–803, ISSN 0191-2917

pp. 1-15, ISSN 1087-1845

*Bulletin*, Vol.15, pp. 333-335

*subgenus Penicillium*. *Molecular Ecology Resources*, Vol.9 (Suppl. 1), pp. 114-129, ISSN

for the monitoring of *Phytophthora* diversity in soil and water. *Journal of Plant* 

Development of a PCR-based assay for the detection of *Fusarium oxysporum* strain FT2, a potential mycoherbicide of *Orobanche ramosa*. *Biological Control*, Vol.50, No.1,

Phylogenetic analysis of *Verticillium dahliae* vegetative compatibility groups.

*Aspergillus fumigatus* in pure culture using polymerase chain reaction. *Molecular and* 

*Helminthosporium solani* population from California. *Plant Disease,* Vol.87, No.7, pp.

Genetic evidence for differentiation of *Gaeumannomyces graminis* var. *tritici* into two

Kox, L.F.F. (2006). Molecular detection of *Fusarium foetens* in Begonia. *Journal of* 

efflux of natural toxic compounds and fungicides. *Fungal Genetics Biology,* Vol.30,

*necator* that correlates with resistance to a sterol biosynthesis inhibitor. *Applied* 

and quantification methods on real-time PCR assay of biotechnology-derived traits. *Analytical and Bioanalytical Chemistry,* Vol.396, No.6, pp. 1977–1990, ISSN 1618-2642

Gaba, D.; Seifert, K.A. & Lévesque, C.A. (2010). Development of a specific TaqMan® real-time PCR assay for quantification of *Fusarium graminearum* clade 7 and comparison of fungal biomass determined by PCR with deoxynivalenol

BOLD, Barcode of Life Database Systems: (http://www.boldsystems.org)


Børja, I.; Solheim, H.; Hietala, A.M. & Fossdal, C.G. (2006). Etiology and real-time

Braasch, D.A. & Corey, D.R. (2001) Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. *Chemistry & Biology*, Vol.8, No.1, pp. 1-7, ISSN 1074-5521 Brandfass, C. & Karlovsky, P. (2006). Simultaneous detection of *Fusarium culmorum* and *F*-

Brent, K.J. (1995). *Fungicide resistance in crop pathogen: How can it be managed?*. FRAC Monograph Nº 1 Global Crop Protection Federation, ISBN 90-72398-07-6, Brussels Broders, K.D.; Woeste, K.E.; SanMiguel, P.J.; Westerman, R.P. & Boland, G.J. (2011).

Bruns T.D., White T.J. & Taylor J.W. (1991). Fungal molecular systematics. *Annual Review of* 

Budge, G.E.; Shaw, M. W.; Colyer, A.; Pietravalle, S. & Boonham, N. Molecular tools to

Burgess, T.I.; Webster, J.L.; Ciampini, J.A.; White, D.; Hardy, G.E.S. & Stukely, M.J.C. (2009).

Butters, J.A.; Zhou, M. & Hollomon, D.W. (2000). The mechanism of resistance tosterol 14\_-

Campanile, G.; Schena L. & Luisi, N. (2008). Real-time PCR identification and detection of

Carisse, O.; Tremblay, D.M.; Lévesque, C.A.; Gindro, K.; Ward, P. & Houde, A. (2009).

Causin, R.; Scopel, C.; Grendene, A. & Montecchio L. (2005). An improved method for the

Chen, W.; Seifert, K.A. & Lévesque, C.A. (2009). A high density COX1 barcode

*Phytopathology*, Vol.99, No.11, pp. 1273-1280, ISSN 0031-949X

polymerase chain reaction-based detection of *Gremmeniella*- and *Phomosis*associated disease in Norway spruce seedlings. *Phytopathology*, Vol.96, No.12, pp.

*graminearum* in plant material by duplex PCR with melting curve analysis. *BMC* 

Discovery of single-nucleotide polymorphisms (SNPs) in the uncharacterized genome of the ascomycete *Ophiognomonia clavigignenti-juglandacearum* from 454 sequence data. *Molecular Ecology Resources*, Vol.11, No.4, pp. 693-702, ISSN 1755-

investigate *Rhizoctonia solani* distribution in soil. *Plant Pathology*, Vol.58, No.6, pp.

Re-evaluation of *Phytophthora* species isolated during 30 years of vegetation health surveys in western Australia using molecular techniques. *Plant Disease*, Vol.93,

demethylation inhibitors in amutant (Erg40) of *Ustilago maydis*. *Pest Managment* 

*Fuscoporia torulosa* in *Quercus ilex*. *Plant Pathology,* Vol.57, No.1, pp. 76-83, ISSN

Development of a TaqMan real-time PCR assay for quantification of airborne conidia of *Botrytis squamosa* and management of Botrytis leaf blight of onion.

detection of *Phytophthora cactorum* (L.C.) Schröeter in infected plant tissues using SCAR markers. *Journal of Plant Pathology,* Vol.87, No.1, pp. 25-35, ISSN 1125-4653 Chen, R.S.; Chu, C.; Cheng, C.W.; Chen W.Y. & Tsay J.G. (2008). Differentiation of two

powdery mildews of sunflower (Helianthus annuus) by a PCR-mediated method based on ITS sequences. *European Journal of Plant Pathology*, Vol.121, No.1, pp. 1-8,

oligonucleotide array for identification and detection of species of *Penicillium* 

BOLD, Barcode of Life Database Systems: (http://www.boldsystems.org)

1305-1314, ISSN 0031-949X

1071-1080, ISSN 0032-0862

No.3, pp. 215-223, ISSN 0191-2917

098X

0032-0862

ISSN 0929-1873

*Microbiology*, Vol.6, No.4, ISSN 1471-2180

*Ecology and Systematics*, Vol.22, pp. 525-564.

*Science,* Vol.56, pp. 257–263, ISSN 1526-498X

*subgenus Penicillium*. *Molecular Ecology Resources*, Vol.9 (Suppl. 1), pp. 114-129, ISSN 1755-098X


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 185

Fraaije, B.A.; Butters, J.A.; Coelho, J.M.; Johes, D.R. & Hollomon, D.W. (2002). Following the

Fraaije, B.A.; Lovell, D.J.; Coelho, J.M.; Baldwin, S. & Hollomon, D.W. (2001). PCR-based

Garrido, C.; Carbú, M.; Fernández-Acero, F.J.; Boonham, N.; Colyer, A.; Cantoral, J.M. &

Gayoso, C.; de Ilárduya, O.M.; Pomar, F. & de Cáceres, F.M. (2007). Assessment of real-time

Geiser, D. M.; Jiménez-Gasco, M.; Kang, S.; Makalowska, I.; Veeraraghavan, N.; Ward, T.J.;

Ghosoph, J.M.; Schmidt, L.S.; Margosan, D.A. & Smilanick, J.L. (2007). Imazalil resistance

Gisi, U.; Chin, K.M.; Knapova, G.; Küng Färber, R.; Mohr, U.; Parisi, S.; Sierotzki, H, &

Gisi, U.; Sierotzki, H.; Cook, A. & McCaffery, A. (2002). Mechanisms influencing the

Glienke, C.; Pereira, O.L.; Stringari, D.; Fabris, J.; Kava-Cordeiro, V.; Galli-Terasawa, L.;

Godoy-Lutz, G.; Kuninaga, S.; Steadman, J. R. & Powers, K. (2008). Phylogenetic analysis of

Black Spot. *Persoonia*, Vol.26, pp. 47-56, ISSN 0031-5850

*Journal of Plant Pathology*, Vol.107, No.9, pp. 905-917, ISSN 0929-1873 Fraaije, B.A., Lovell, D.J., Rohel, E.A. & Hollomon, D.W. (1999). Rapid detection and

*Plant Pathology,* Vol.51, pp. 45–54, ISSN 0032-0862

Vol.58, No.1, pp. 43–51, ISSN 0032-0862

Vol.110, No.5-6, pp. 473-479, ISSN 0929-1873

542, ISSN 1567-1348

1364-5072

ISSN 0929-1873

ISSN 0261-2194

pp. 859–867, ISSN 1526-498X

32-40, ISSN 1345-2630

5214

*Fusarium oxysporum* complex. *Infection Genetics and Evolution*, Vol.11, No.3, pp. 533-

dynamics of strobilurin resistance in *Blumeria graminis f. sp. tritici* using quantitative allele-specific realtime PCR measurements with the fluorescent dye SYBR green I.

assays to assess wheat varietal resistance to blotch (*Septoria tritici* and *Stagonospora nodorum*) and rust (*Puccinia striiformis* and *Puccinia recondita*) diseases. *European* 

diagnosis of *Septoria tritici* epidemics in wheat using a polymerase chain reaction PicoGreen assay. *Journal of Applied Microbiology*, Vol.86, No.4, pp. 701-708, ISSN

Budge G. (2009). Development of protocols for detection of *Colletotrichum acutatum* and monitoring of strawberry anthracnose using real-time PCR. *Plant Pathology*,

PCR as a method for determining the presence of *Verticillium dahliae* in different *Solanaceae* cultivars. *European Journal of Plant Pathology*, Vol.118, No.3, pp. 199–209,

Zhang, N.; Kuldau, G.A. & O'Donnell, K. (2004). *Fusarium*-ID v.1.0: A DNA sequence database for identifying *Fusarium*. *European Journal of Plant Pathology*,

linked to a unique insertion sequence in the PdCYP51 promoter region of *Penicillium digitatum*. *Postharvest Biology Technology,* Vol.44, pp. 9–18, ISSN 0925-

Steinfeld, U. (2000). Recent development in elucidating modes of resistance to phenylamide, DMI and strobilurin fungicides. *Crop Protection,* Vol.19, pp. 863-872,

evolution of resistance to Qo inhibitor fungicides. *Pest Managment Science,* Vol.58,

Cunnington, J.; Shivas, R.G.; Groenewald, J.Z. & Crous, P.W. (2011). Endophytic and pathogenic *Phyllosticta* species, with reference to those associated with Citrus

*Rhizoctonia solani* subgroups associated with web blight symptoms on common bean based on ITS-5.8S rDNA. *Journal of General Plant Pathology*, Vol.74, No.1, pp.

content in wheat and barley. *International Journal of Food Microbiology*, Vol.141, No.1- 2, pp. 45-50, ISSN 0168-1605

Demontis, M.A.; Cacciola, S.O.; Orru, M.; Balmas, V.; Chessa, V.; Maserti, B.E.; Mascia, L.; Raudino, F.; Quirico, G.M.D.S.L. & Migheli, Q. (2008). Development of real-time PCR systems based on SYBR (R) Green I and TaqMan (R) technologies for specific quantitative detection of *Phoma tracheiphila* in infected Citrus. *European Journal of Plant Pathology,* Vol.120, No.4, pp. 339-351, ISSN 0929-1873

DNA Multiscan®: (http://www.DNAmultiscan.com)


Demontis, M.A.; Cacciola, S.O.; Orru, M.; Balmas, V.; Chessa, V.; Maserti, B.E.; Mascia, L.;

Diguta, C. F.; Rousseaux, S.; Weidmann, S.; Bretin, N.; Vincent, B.; Guilloux-Benatier, M. &

Dixon, L.J.; Schlub, R.L.; Pernezny, K. & Datnoff, L.E. (2009). Host specialization and

Drenth, A.; Wagals, G.; Smith, B.; Sendall, B.; O'Dwyer, C.; Irvine, G. & Irwin, J.A.G. (2006).

Druzhinina, I. S.; Kopchinskiya, A. G.; Komoja, M.; Bissettb, J.; Szakacs, G. & Kubiceka, C.P.

*pinifolia* in Chile. *Fungal Biology*, Vol.114, No.9, pp. 746-752, ISSN 1878-6146 Dyer, P.S.; Furneaux, P.A.; Douhan, G. & Murray, T.D. (2001). A multiplex PCR test for

Feng, J.; Hwang, R.; Chang, K. F.; Hwang, S. F.; Strelkov, S. E.; Gossen, B. D. & Zhou, Q.

Fling, M.E.; Kopf, J.; Tamarkin, A.; Gorman, J.A.; Smith, H.A. & Koltin, Y. (1991). Analysis of

Fountaine, J.A.; Shaw, M.W.; Napier, B.; Ward, E. & Fraaije, B. A. (2007). Application of real-

Fourie, G.; Steenkamp, E.T.; Ploetz, R.C.; Gordon, T.R. & Viljoen, A. (2011). Current status of

in barley. *Phytopathology*, Vol.97, No.3, pp. 297-303, ISSN 0031-949X

Vol.108, No.4, pp. 379-383, ISSN 0929-1873

*Plant Pathology,* Vol.120, No.4, pp. 339-351, ISSN 0929-1873

2, pp. 45-50, ISSN 0168-1605

1015-1027, ISSN 0031-949X

0378-1097

0815-3191

1845

0661

DNA Multiscan®: (http://www.DNAmultiscan.com)

content in wheat and barley. *International Journal of Food Microbiology*, Vol.141, No.1-

Raudino, F.; Quirico, G.M.D.S.L. & Migheli, Q. (2008). Development of real-time PCR systems based on SYBR (R) Green I and TaqMan (R) technologies for specific quantitative detection of *Phoma tracheiphila* in infected Citrus. *European Journal of* 

Alexandre, H. (2010). Development of a qPCR assay for specific quantification of *Botrytis cinerea* on grapes. *Fems Microbiology Letters,* Vol.313, No.1, pp. 81-87, ISSN

phylogenetic diversity of *Corynespora cassiicola*. *Phytopathology*, Vol.99, No.9, pp.

Development of a DNA-based method for the detection and identification of *Phytophthora species*. *Australasian Plant Pathology* , Vol.35, No.2, pp. 147–159, ISSN

(2005). An oligonucleotide barcode for species identification in *Trichoderma* and *Hypocrea*. *Fungal Genetics and Biology,* Vol.42, No.10, pp. 813-828, ISSN 1087-1845 Durán, A.; Gryzenhout, M; Drenth, A; Slippers, B; Ahumada, R; Wingfield, B.D. &

Wingfield, M.J. (2010). AFLP analysis reveals a clonal population of *Phytophthora* 

determination of mating type applied to the plant pathogens *Tapesia yallundae* and *Tapesia acuformis*. *Fungal Genetics and Biology*, Vol.33, No.3, pp. 173-180, ISSN 1087-

(2010). An inexpensive method for extraction of genomic DNA from fungal mycelia. *Canadian Journal of Plant Pathology,* Vol.32, No.3, pp. 396-401, ISSN 0706-

a *Candida albicans* gene encodes a novel mechanism for resistance to benomyl and methotrexate. *Molecular General Genetics*, Vol.227, pp. 318-329, ISSN 0026-8925 Foster, S.J.; Ashby, A.M. & Fitt, B.D.L. (2002). Improved PCR-based assays for pre-

symptomatic diagnosis of light leaf spot and determination of mating type of *Pyrenopeziza brassicae* on winter oilseed rape. *European Journal of Plant Pathology,*

time and multiplex polymerase chain reaction assays to study leaf blotch epidemics

the taxonomic position of *Fusarium oxysporum formae specialis cubense* within the

*Fusarium oxysporum* complex. *Infection Genetics and Evolution*, Vol.11, No.3, pp. 533- 542, ISSN 1567-1348


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 187

Hayashi, K.; Schoonbek, H.J. & de Waard, M.A. (2002a). *Bcmfs1*. A novel major facilitator

*Applied Environmental Microbiology,* Vol.68, pp. 4996-5004, ISSN 0099-2240 Hayashi, K.; Schoonbeek, H.J. & de Waard, M.A. (2002b). Expression of the ABC transporter

fungicides. *Pest Biochemical Physiology,* Vol.73, pp. 110-121, ISSN 0048-3575 Hayashi, K.; Schoonbeek, H.J. & de Waard MA. (2003). Modulators of membrane drug

*Botrytis cinerea*. *Pest Managment Science,* Vol.59, pp. 294-302, ISSN 1526-498X Hayashi, K.; Schoonbeek, H.J.; Sugiura, H. & de Waard, M.A. (2001). Multidrug resistance in

Heid, C.A.; Stevens, J.; Livak, K.J. & Williams, P.M. (1996). Real time quantitative PCR.

Hewit, H. G. (1998). *Fungicides in crop protection*. CAB Intenational*.* University Press, ISBN 0-

Hirsch, P.R.; Mauchline, T.H. & Clark, I.M. (2010). Culture-independent molecular

Hong, S.Y.; Kang, M.R.; Cho, E.J.; Kim, H.K. & Yun, S.H. (2010). Specific PCR detection of

Huang, J. & Kang, Z. (2010). Detection of *Thielaviopsis basicola* in soil with real-time

Hughes, K.J.D.; Giltrap, P.M.; Barton, V.C.; Hobden E.; Tomilson, J.A. & Barber, P. (2006).

*persica* in the UK. *Plant Pathology,* Vol.55, No.6, pp. 813-813, ISSN 0032-0862 Hyakumachi, M.; Priyatmojo, A.; Kubota, M. & Fukui, H. (2005). New anastomosis groups,

Hyun, J. W.; Yi, S. H.; MacKenzie, S. J.; Timmer, L. W.; Kim, K. S.; Kang, S. K.; Kwon, H. M.

Inami, K.; Yoshioka, C.; Hirano, Y.; Kawabe, M.; Tsushima, S.; Teraoka, T. & Arie, T. (2010).

techniques for soil microbial ecology. *Soil Biology & Biochemistry*, Vol.42, pp. 878-

four quarantine *Fusarium Species* in Korea. *Plant Pathology Journal*, Vol.26, No.4, pp.

quantitative PCR assays. *MicrobiologicaL Research* , Vol.165, No.5, pp. 411-417, ISSN

On-site real-time PCR detection of *Phytophthora ramorum* causing dieback of *Parrotia* 

AG-T and AG-U, of binucleate *Rhizoctonia* spp. causing root and stem rot of cutflower and miniature roses. *Phytopathology,* Vol.95, No.7, pp. 784-792, ISSN 0031-

& Lim, H. C. (2009). Pathotypes and genetic relationship of worldwide collections of *Elsinoe* spp. causing scab diseases of citrus. *Phytopathology,* Vol.99, No.6, pp. 721-

Real-time PCR for differential determination of the tomato wilt fungus, *Fusarium oxysporum f. sp lycopersici*, and its races. *Journal of General Plant Pathology*, Vol.76,

*Biochemical Physiology,* Vol.70, pp. 168-179, ISSN 0048-3575

*Genome Research,* Vol.6, pp. 986–994

85199-201-3, Cambridge. UK.

887, ISSN 0038-0717

0944-5013

949X

409-416, ISSN 1598-2254

728, ISSN 0031-949X

No.2, pp.116-121, ISSN 1345-2630

19-26, ISSN 0048-3575

inhibitor resistance in *Penicillium digitatum. Pest Biochemical Physiology,* Vol.70, pp.

superfamily transporter from *Botrytis cinerea* provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides.

*BcatrD* from *Botrytis cinerea* reduces sensitivity to sterol demethylation inhibitor

transporters potentiate the activity of the DMI fungicide oxpoconazole against

*Botrytis cinerea* associated with decreased accumulation of the azole fungicide oxpoconazole and increased transcription of the ABC transporter gene *BcatrD*. *Pest* 


Gómez-Alpizar, L.; Saalau, E.; Picado I.; Tambong, J.T. & Saborio, F. (2011). A PCR-RFLP

González, C.; Noda, J.; Espino, J.J & Brito, N. (2008). Drill-assisted genomic DNA extraction

González-Mendoza, D.; Argumedo-Delira, R.; Morales-Trejo, A.; Pulido-Herrera, A.;

Goud, J.C. & Termorshuizen A.J. (2003) Quality of methods to quantify microsclerotia of

Gril, T.; Celar, F.; Munda, A.; Javornik, B. & Jakse, J. (2008). AFLP analysis of intraspecific

Groenewald, S.; Van Den Berg, N.; Marasas, W.F.O. & Viljoen, A. (2006). The application of

Grote, D.; Olmos, A.; Kofoet, J.J.; Tuset, E.; Bertolini, E. & Cambra, M. (2002). Specific and

Grünwald, N.J.; Martin, F.N.; Larsen, M.M.; Sullivan, C.M.; Press, C.M.; Coffey, M.D.;

Guglielmo, F.; Bergemann, S.E.; Gonthier, P.; Nicolotti, G. & Garbelotto, M. (2007). A

Guo, J.R.; Schnieder, F.; Beyer, M. & Verreet, J.A. (2005). Rapid detection of *Mycosphaerella* 

Haase, A.T.; Retzel, E.F. & Staskus, K.A. (1990). Amplification and detection of lentiviral

Hamamoto, H.; Hasegawa, K.; Nakaune, R.; Lee, Y.J.; Makizumi, Y.; Akutsu, K. & Hibi T.

Hamamoto, H.; Nawata, O.; Hasegawa, K.; Nakaune, R.; Lee, Y.J.; Makizumi, Y.; Akutsu, K.

*Phytopathology,* Vol.153, No.11-12, pp. 674-679, ISSN 0931-1785

*America*, Vol.87, No.13, pp. 4971-4975, ISSN 0027-8424

*Microbiology,* Vol.66, pp. 3421-3426, ISSN 099-2240

*cubense*. *Mycological Research*, Vol.110, pp. 297-305, ISSN 0953-7562

*Journal of Plant Pathology,* Vol.108, No.3, pp. 197-207, ISSN 0929-1873 Grund, E.; Darissa, O. & Adam, G. (2010). Application of FTA (R) cards to sample microbial

*Molecular Research,* Vol.9, No.1, pp. 162-166, ISSN 1676-5680

ISSN 0266-8254

534, ISSN 0929-1873

No.12, pp.1616-1624, ISSN 0191-2917

pp. 750-757, ISSN 0931-1785

1507, ISSN 1364-5072

5492

2917

assay for identification and detection of *Pythium myriotylum*, causal agent of the cocoyam root rot disease. *Letters in Applied Microbiology,* Vol.52, No.3, pp. 185-192,

from *Botrytis cinerea*. *Biotechnology Letters,* Vol.30, No.11, pp. 1989-1992, ISSN 0141-

Cervantes-Díaz, L.; Grimaldo-Juarez, O. & Alarcon, A. (2010). A rapid method for isolation of total DNA from pathogenic filamentous plant fungi. *Genetics and* 

*Verticillium dahliae* in soil. *European Journal of Plant Pathology*, Vol.109, No.6, pp. 523-

variation between *Monilinia laxa* isolates from different hosts. *Plant Disease*, Vol.92,

high-throughput AFLPs in assessing genetic diversity in *Fusarium oxysporum* f.sp.

sensitive detection of *Phytophthora nicotianae* by simple and nested-PCR. *European* 

plant pathogens for PCR and RT-PCR. *Journal of Phytopathology*, Vol.158, No.11-12,

Hansen, E.M. & Parke, JL. (2011). *Phytophthora*-ID.org: A sequence-based *Phytophthora* identification tool. *Plant Disease*, Vol.95, No.3, pp. 337-342, ISSN 0191-

multiplex PCR-based method for the detection and early identication of wood rotting fungi in standing trees. *Journal of Applied Microbiology,* Vol.103, pp. 1490-

*graminicola* in wheat using reverse transcription-PCR assay. *Journal of* 

DNA inside cells. *Proceedings of the National Academy of Sciences of the United States of* 

(2000). Tandem repeat of a transcription enhancer upstream of the sterol 14 demethylase gene (*CYP51*) in *Penicillium digitatum. Applied Environmental* 

& Hibi T. (2001). The role of the ABC transporter gene *PMR1* in demethylation

inhibitor resistance in *Penicillium digitatum. Pest Biochemical Physiology,* Vol.70, pp. 19-26, ISSN 0048-3575


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 189

Kang, S.; Mansfield, M.A.; Park, B.; Geiser, D.M.; Ivors, K.L.; Coffey, M.D.; Grünwald, N.J.;

Keiper, F.J.; Capio, E.; Grcic, M. & Wallwork, H. (2007). Development of sequence tagged

Keiper, F.J.; Hayden, M.J. & Wallwork, H. (2006) Development of sequence tagged

Kim, J.T.; Park, S.Y.; Choi, W.; Lee, Y.H. & Kim H.T. (2008). Characterization of

Kim, Y.T.; Cho, M.; Jeong, J.Y.; Lee, H.B. & Kim, S.B. (2010). Application of Terminal

Knutsen, A.K.; Torp, M. & Holst-Jensen, A. (2004). Phylogenetic analyses of the *Fusarium* 

*Journal of Food Microbiology*, Vol.95, No.3, pp. 287– 295, ISSN 0168-1605 Koenraadt, H.; Somerville, S.C.; Jones, A.L. (1992). Characterization of mutations in the beta-

pathogenic fungi. *Phytopathology,* Vol.82, pp. 1348-1354, ISSN 0031-949X Koljalg, U.; Larsson, K.-H.; Abarenkov, K.; Nilsson, R.H.; Alexander, I.J.; Eberhardt, U.;

Kristensen, R.; Torp, M.; Kosiak, B. & Holst-Jensen, A. (2005). Phylogeny and toxigenic

Kroon, L.P.N.M.; Verstappen, E.C.P.; Kox, L.F.F.; Flier, W.G. & Bonants, P. (2004). A rapid

Kurtz, B.; Karlovsky, P. & Vidal, S. (2010). Interaction between western corn rootworm

*Environmental Entomology,* Vol.39, No.5, pp. 1532-1538, ISSN 0046-225X

*Phytologist*. Vol.166, No.3, pp. 1063-1068, ISSN 0028-646X

*Phytopathology*, Vol.100, No.8, pp. 732-737, ISSN 0031-949X

*Ecology Notes,* Vol.7, No.4, pp. 664–666, ISSN 1471-8278

*Ecology Notes,* Vol.6, No.4, pp. 543–546, ISSN 1471-8278

*Journal*, Vol.24, No.1, pp. 17-23, ISSN 1598-2254

*Bioessays*, Vol.32, No. 6, pp. 524-536

173–186, ISSN 0953-7562

56, ISSN 0378-1097

Martin, F.N.; Lévesque, C.A. & Blair, J.E. (2010). The promise and pitfalls of sequence-based identification of plant-pathogenic fungi and oomycetes.

microsatellites for the barley net blotch pathogen *Pyrenophora teres*. *Molecular* 

microsatellites for the barley scald pathogen *Rhynchosporium secalis*. *Molecular* 

*Colletotrichum* isolates causing anthracnose of pepper in Korea. *Plant Pathology* 

Restriction Fragment Length Polymorphism (T-RFLP) analysis to monitor effect of biocontrol agents on rhizosphere microbial community of hot pepper (*Capsicum annuum* L.). *Journal of Microbiology*, Vol.48, No.5, pp. 566-572, ISSN 1225-8873 Kircher, M. & Kelso, J. (2010). High-throughput DNA sequencing - concepts and limitations.

*poae*, *Fusarium sporotrichioides* and *Fusarium langsethiae* species complex based on partial sequences of the translation elongation factor-1 alpha gene. *International* 

tubulin gene of benomyl-resistant field strains of *Venturia inaequalis* and other plant

Erland, S.; Hoiland, K.; Kjoller, R.; Larsson, E.; Pennanen, T.; Sen, R.; Taylor, A.F.S., Tedersoo, L., Vralstad, T., & Ursing, B.M. (2005). UNITE: A database providing web-based methods for the molecular identification of ectomycorrhizal fungi. *New* 

potential is correlated in *Fusarium* species as revealed by partial translation elongation factor 1 alpha gene sequences. *Mycological Research*, Vol.109, No.2, pp.

diagnostic test to distinguish between American and European populations of *Phytophthora ramorum Phytopathology,* Vol.94, No.6, pp. 613-620, ISSN 0031-949X Kulik, T.; Jestoi, M. & Okorski, A. (2011). Development of TaqMan assays for the

quantitative detection of *Fusarium avenaceum*/*Fusarium tricinctum* and *Fusarium poae* esyn1 genotypes from cereal grain. *FEMS Microbiology Letters*, Vol.314, No.1, pp. 49-

(Coleoptera: Chrysomelidae) larvae and root-infecting *Fusarium verticillioides.* 


Ioos, R.; Kowalski, T.; Husson, C. & Holdenrieder, O. (2009). Rapid in planta detection of

Ishii, H.; Fraaije, B.A.; Sugiyama, T.; Noguchi, K.; Nishimura, K.; Takeda, T.; Amano, T. &

Izzo, A.D. & Mazzola, M. (2009). Hybridization of an ITS-based macroarray with ITS

Jana, T.; Sharma, T.R. & Singh, N.K. (2005). SSR-based detection of genetic variability in the

Jeeva, M.L.; Mishra, A.K.; Vidyadharan, P.; Misra, R.S. & Hegde, V. (2010). A species-specific

*rolfsii*. *Australasian Plant Pathology*, Vol.39, No.6, pp. 517-523, ISSN 0815-3191 Jiang, Y.X.; Wu, J.G.; Yu, K.Q.; Ai, CX; Zou, F. & Zhou, H.W. (2011) Integrated lysis

Jiménez-Fernández D.; Montes-Borrego M.; Jimenez-Diaz R.M.; Navas-Cortes, J.A. & Landa,

Joseph-Horne, T. & Hollomon, D.W. (1997). Molecular mechanisms of azole resistance.

Justesen, A.F.; Hansen, H.J.; Pinnschmidt, H.O. (2008). Quantification of *Pyrenophora* 

Kageyama, K.; Senda, M.; Asano, T.; Suga, H. & Ishiguro, K. (2007). Intra-isolate

Kaku, K.; Takagaki, M.; Shimizu, T. & Nagayama, K. (2003). Diagnosis of dehydratase

*Journal of Plant Pathology,* Vol.125, No.2, pp. 329-335, ISSN 0929-1873 Ippolito, A.; Schena, L. & Nigro, F. (2002). Detection of *Phytophthora nicotianae* and *P.* 

*Pathology*, Vol.108, No.9, pp. 855-868, ISSN 0929-1873

Vol.91, pp. 1166–1171, ISSN 0031-949

629-636, ISSN 0191-2917

250-262, ISSN: 0031-949X

Part 1, pp. 81-86, ISSN 0953-7562

*Ecology*, Vol.46, No.3, pp. 372–382, ISSN 0929-1393

*FEMS Microbiology Letters,* Vol.149, pp. 141–149

Vol.122, No.2, pp. 253-263, ISSN 0929-1873

Vol.111, pp. 416-423, ISSN 0953-7562

*Chalara fraxinea* by a real-time PCR assay using a dual-labelled probe. *European* 

*citrophthora* in citrus roots and soils by nested PCR. *European Journal of Plant* 

Hollomon, D.W. (2001). Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. *Phytopathology,*

community probes for characterization of complex communities of fungi and fungal-like protists. *Mycological Research*, Vol.113, pp. 802-812, ISSN 0953-7562 Jackson, E.W.; Avant, J.B.; Overturf, K.E. & Bonman, J.M. (2006). A quantitative assay of

*Puccinia coronata* f. sp *avenae* DNA in *Avena sativa*. *Plant Disease,* Vol.90, No.5, pp.

charcoal root rot pathogen *Macrophomina phaseolina*. *Mycological Research*, Vol.109,

polymerase chain reaction assay for rapid and sensitive detection of *Sclerotium* 

procedures reduce extraction biases of microbial DNA from mangrove sediment. *Journal of Bioscience and Bioengineering,* Vol.111, No.2, pp. 153-157, ISSN 1389-1723 Jiménez-Fernández, D.; Montes-Borrego, M.; Navas-Cortés, J.A.; Jiménez-Díaz, R.M. &

Landa, B.B. (2010). Identication and quantication of *Fusarium oxysporum* in planta and soil by means of an improved specic and quantitative PCR assay. *Applied Soil* 

B.B. (2011). In planta and soil quantification of *Fusarium oxysporum* f. sp. *ciceris* and evaluation of Fusarium wilt resistance in chickpea with a newly developed quantitative polymerase chain reaction assay. *Phytopathology* Vol. 101, No.2, pp.

*graminea* in barley seed using real-time PCR. *European Journal of Plant Pathology,* 

heterogeneity of the ITS region of rDNA in *Pythium helicoides*. *Mycological Research*,

inhibitors in melanin biosynthesis inhibitor (MBID) resistance by primerintroduced restriction enzyme analysis in scytalonedehydratase gene of *Magnaporthe grisea*. *Pest Managment Science,* Vol.59, pp. 843–846, ISSN 1526-498X


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 191

Lievens, B.; Grauwet, T.J.M.A.; Cammue, B.P.A. & Thomma B.P.H.J. (2005a). Recent

Lievens, B.; Rep, M. & Thomma, B.P.H.J. (2008). Recent developments in the molecular

Lievens, B.; Van Baarlen P.; Verreth C.; Van Kerckhove, S.; Rep, M. & Thomma, B.P.H.J.

Liew, E.C.Y.; MacLean, D.J. & Irwin, J.A.G. (1998). Specific PCR based detection of

Liu, J.H.; Gao L.; Liu T.G. & Chen W.Q. (2009). Development of a sequence-characterized

Long, A.A. (1998). In-situ polymerase chain reaction: foundation of the technology and

Luchi, N.; Capretti, P.; Pinzani, P.; Orlando, C. & Pazzagli, M. (2005). Real-time PCR

Luck, J.E. & Gillings, M.R. (1995). Rapid identification of benomyl resistant strains of *Botrytis* 

Luo, C.; Cox, K.D.; Amiri, A. & Schnabel, G. (2008). Occurrence and detection of the

Ma, Z. & Michailides, T.J. (2004). An allele-specific PCR assay fordetecting strobilurin-

Ma, Z. & Michialides, T.J. (2005). Advances in undertanding molecular mechanisms of

phytopathogenic fungi. *Crop Protection,* Vol.24, pp. 853-863, ISSN 0261-2194 Ma, Z.; Felts, D. & Michailides, T.J. (2003b). Resistance to azoxystrobin in *Alternaria* isolates

Ma, Z.; Proffer, T.J.; Jacobs J.L. & Sundin G.W. (2006). Overexpression of the 14α-

*Applied Environmental Microbiology,* Vol.72, pp. 2581–2585, ISSN 0099-2240 Ma, Z.; Yoshimura, M. & Michailides, T.J. (2003a). Identification and characterization of

*Developments in Microbiology,* Vol.9, pp. 57-79

Vol.64, No.8, pp. 781–788, ISSN 1526-498X

pp. 1181-1191, Part: 10, ISSN 0953-7562

ISSN 0266-8254

1488, ISSN 0953-7562

ISSN 0048-3575

2240

760X

*Mycological Research*, Vol.102, pp. 73-80, ISSN 0953-7562

*Microbiology,* Vol.41, No.1, pp. 61-68, ISSN 0266-8254

*Disease,* Vol.92, pp. 1099–1103, ISSN 0191-2917

Vol.152, pp. 118–121, ISSN 0931-1785

developments in diagnostics of plant pathogens: a review. *Recent Research* 

discrimination of *formae speciales* of *Fusarium oxysporum*. *Pest Management Science*,

(2009). Evolutionary relationships between *Fusarium oxysporum* f. sp *lycopersici* and *F. oxysporum* f. sp *radicis-lycopersici* isolates inferred from mating type, elongation factor-1 alpha and exopolygalacturonase sequences. *Mycological Research*, Vol.113,

*Phytophthora medicaginis* using the intergenic spacer region of the ribosomal DNA.

amplied region marker for diagnosis of dwarf bunt of wheat and detection of *Tilletia controversa* Kühn. *Letters in Applied Microbiology*, Vol.49, No.2, pp. 235–240,

today's options. *European Journal of Histochemistry*, Vol.42, pp. 101–109, ISSN 1121-

detection of *Biscogniauxia mediterranea* in symptomless oak tissue. *Letters in Applied* 

*cinerea* using the polymerase chain reaction. *Mycological Research,* Vol.99, pp. 1483–

DMIresistance-associated genetic element 'Mona' in *Monilinia fructicola*. *Plant* 

resistant *Alternaria* isolates from pistachio in California. *Journal of Phytopathology,*

fungicide resistance and molecular detection of resistant genotypes in

from pistachio in California. *Pesticide Biochemical Physiology,* Vol.77, No.2, pp. 66–74,

demethylase target gene (CYP51) mediates fungicide resistance in *Blumeriella jaapii*.

benzimidazole resistance in *Monilinia fructicola* from stone fruit orchards in California. *Applied Environmental Microbiology,* Vol.69, pp. 7145–7152, ISSN 0099-


Landa, B.B.; Montes-Borrego, M.; Munoz-Ledesma, F.J. & Jimenez-Diaz, R.M. (2007).

Landgraf, A., Reckmann, B. & Pingoud, A. (1991). Direct analysis of polymerase chain-

Langrell, S.R.H. & Barbara, D.J. (2001). Magnetic capture hybridization for improved PCR

Langrell, S.R.H.; Glen, M. & Alfenas, A.C. (2008). Molecular diagnosis of *Puccinia psidii*

Lee, M.S.; LeMaistre, A.; Kantarjian, H.M.; Talpaz, M.; Freireich, E.J.; Trujillo, J.M. & Stass,

Leisova, L.; Minarikova, V.; Kucera, L. & Ovesna, J. (2006). Quantification of *Pyrenophora* 

Lévesque, C.A.; Harlton, C.E. & de Cock, A.W.A.M. (1998). Identification of some oomycetes

Licciardello, G.; Grasso, F. M.; Bella, P.; Cirvilleri, G.; Grimaldi, V. & Catara, V. (2006).

Lievens, B. & Thomma B.P.H.J., (2005). Recent developments in pathogen detection arrays:

Lievens, B.; Brouwer, M.; Vanachter, A.C.R.C.; Levesque, C.A.; Cammue, B.P.A. & Thomma

Lievens, B.; Claes, L.; Vakalounakis, D.J., Vanachter, A.C.R.C. & Thomma, B.P.H.J. (2007). A

Lievens, B.; Claes, L.; Vanachter, A.C.R.C.; Cammue, B.P.A. & Thomma, B.P.H.J. (2006).

*Microbiology*, Vol.9, No.9, pp. 2145-2161, ISSN 1462-2912

biodiversity. *Plant Pathology*, Vol.57, No.4, pp. 687-701, ISSN 0032-0862 Larsen, J.E.; Hollingsworth, C.R.; Flor, J.; Dornbusch, M.R.; Simpson, N.L. & Samac, D.A.

*Analytical Biochemistry*, Vol.198, pp. 86-91, ISSN 0003-2697

*Reporter*, Vol.19, No.1, pp. 5–11, ISSN 0735-9640

Vol.67, No.3, pp. 446-455, ISSN 0167-7012

No.11, pp. 1380-1390, ISSN 0031-949X

0006-4971

0031-949X

1523-1530, ISSN 0191-2917

1698-1710, ISSN 1462-2912

No.12, pp. 1374-1380, ISSN 0031-949X

Phylogenetic analysis of downy mildew pathogens of opium poppy and PCR-Based in planta and seed detection of *Peronospora arborescens*. *Phytopathology*, Vol.97,

reaction products using enzyme-linked-immunosorbent-assay techniques.

detection of *Nectria galligena* from lignified apple extracts*. Plant Molecular Biology* 

(guava rust) - a quarantine threat to Australian eucalypt and *Myrtaceae*

(2007). Distribution of *Phoma sclerotioides* on alfalfa and winter wheat crops in the north central United States. *Plant Disease*, Vol.91, No.5, pp. 551-558, ISSN 0191-2917

S.A. (1989). Detection of two alternative bcr/abl mRNA junctions and minimal residual disease in Philadelphia chromosome positive chronic myelogenous leukemia by polymerase chain reaction. *Blood,* Vol.73, No.8, pp. 2165–2170, ISSN

*teres* in infected barley leaves using real-time PCR*. Journal of Microbiological Methods,* 

by reverse dot-blot hybridization. *Phytopatholgy*, Vol.88, No.3, pp. 213-222, ISSN

Identification and detection of *Phoma tracheiphila*, causal agent of citrus mal secco disease, by real-time polymerase chain reaction. *Plant Disease,* Vol.90, No.12, pp.

implications for fungal plant pathogens and use in practice. *Phytopathology,* Vol.95,

B.P.H.J. (2005b). Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray. *Environmental Microbiology,* Vol.7, No.11, pp.

robust identification and detection assay to discriminate the cucumber pathogens *Fusarium oxysporum* f. sp *cucumerinum* and f. sp *radicis-cucumerinum. Environmental* 

Detecting single nucleotide polymorphisms using DNA arrays for plant pathogen diagnosis. *FEMS Microbiology Letters* Vol.255, No.1, pp.129-139, ISSN 0378-1097


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 193

McKay, G.J. & Cooke, L.R. (1997). A PCR-based method to characterize and identify

McKay, G.J.; Egan, D.; Morris, E. & Brown, A.E. (1998). Identification of benzimidazole

Meng, J. & Wang, Y. (2010). Rapid detection of *Phytophthora nicotianae* in infected tobacco

Mercado-Blanco, J.; Rodríguez-Jurado, D.; Pérez-Artés, E. & Jiménez-Díaz, R.M. (2001).

Meudt, H.M. & Clarke, A.C. (2007). Almost forgotten or latest practice? AFLP applications,

Midorikawa, G.E.O.; Pinheiro, M.R.R.; Vidigal, B.S.; Arruda, M.C.; Costa, F.F.; Pappas, G.J.

Moradi, M.; Oerke, E.-C.; Steiner, U.; Tesfaye, D.; Schellander, K. & Dehne, H.-W. (2010).

Mostert, L.; Groenewald, J.Z.; Summerbell, R.C.; Gams, W. & Crous, P.W. (2006). Taxonomy

Mulè, G.; Susca, A.; Stea, G. & Moretti, A. (2004). Specific detection of the toxigenic species

Mullis, K.B. & Faloona, F.A. (1987). Specific synthesis of DNA in vitro via a polymerasecatalyzed chain-reaction. *Methods in Enzymology*, Vol.155, pp. 335-350 Mumford, R.; Boonham, N.; Tomlinson, J. & Barker, I. (2006). Advances in molecular

Nakaune, R. (2001). ABC transporter genes involved in multidrug resistance in *Pencillium* 

Nakaune, R.; Adachi, K.; Nawata, O.; Tomiyama, M.; Akutsu, K. & Hibi, T. (1998). A novel

*digitatum*. *Journal of General Plant Pathol*ogy, Vol.67, No.3, pp. 251

*Studies in Mycology*, Vol.54, pp. 1–113, ISSN 0166-0616

*Pathology,* Vol.116, pp. 1-19, ISSN 0929-1873

Vol.64, pp. 3983-3988, ISSN 0099-2240

plants by nested PCR. *Plant Pathology*, Vol.50, No. 5, pp. 609-619

Vol.152, pp. 371–378, ISSN 0378-1097

No.1, pp. 1–7, ISSN 0931-1785

227-237, ISSN 0929-1873

1360-1385

2617

240

*Research,* Vol.102, pp. 671–676, ISSN 0953-7562

benzimidazole resistance in *Helminthosporium solani*. *FEMS Microbiology Letters,*

resistance in *Cladobotryum dendroides* using a PCR-based method. *Mycological* 

tissue and soil samples based on its *Ypt1* gene. *Journal of Phytopathology*, Vol.158,

Detection of the nondefoliating pathotype of *Verticillium dahliae* in infected olive

analyses and advances. *Trends in Plant Science*. Vol.12, No.3, pp. 106–117, ISSN

Jr.; Ribeiro, S. G.; Freire, F. & Miller, R.N.G. (2008). Characterization of *Aspergillus flavus* strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis. *Letters in Applied Microbiology,* Vol.47, No.1, pp. 12-18, ISSN 0266-8254 Minerdi, D.; Moretti M.; Li Y.; Gaggero, L.; Garibaldi, A. & Gullino, M.L. (2008).

Conventional PCR and real time quantitative PCR detection of *Phytophthora cryptogea* on *Gerbera jamesonii*. *European Journal of Plant Pathology*, Vol.122, No.2, pp.

Microbiological and Sybr®Green Real-Time PCR detection of major *Fusarium* Head Blight pathogens on wheat ears. *Microbiology*, Vol.79, No.5, pp. 646–654, ISSN 0026-

and pathology of *Togninia* (Diaporthales) and its *Phaeoacremonium* anamorphs.

*Fusarium proliferatum* and *F. oxysporum* from asparagus plants using primers based on calmodulin gene sequences. *FEMS Microbiology Letters*. Vol.230, No.2, pp. 235-

phytodiagnostics-new solutions for old problems. *European Journal of Plant* 

ATP-binding cassette transporter involved in multidrug resistance in phytopathogenic fungus *Pencillium digitatum*. *Applied Environmental Microbiology,*


Macia-Vicente, J.G.; Jansson, H.B.; Talbot, N.J. & Lopez-Llorca, L.V. (2009). Real-time PCR

Malausa, T.; Andre, G.; Meglecz, E.; Blanquart, H.; Duthoy, S.; Costedoat, C.; Dubut, V.;

Malvick, D.K. & Impullitti, A.E. (2007). Detection and quantification of *Phialophora gregata* in

Markakis, E.A.; Tjamos, S.E.; Antoniou, P.P.; Paplomatas, E.J. & Tjamos, E.C. (2009).

Martínez-Espinoza, A.D.; León-Ramírez, C.G.; Singh, N. & Ruiz-Herrera, J. (2003). Use of

Martínez-García, L.B.; Armas, C.; Miranda, J.D.; Padilla, F.M. & Pugnaire, F.I. (2011) .Shrubs

Massart, S.; De Clercq, D.; Salmon, M.; Dickburt, C. & Jijakli, M.H. (2005). Development of

Matsuda, Y.; Sameshima, T.; Moriura, N.; Inoue, K.; Nonomura, T.; Kakutani, K.; Nishimura,

McCartney, H.A.; Foster, S.J.; Fraaije, B.A. & Ward, E. (2003). Molecular diagnostics for

McDonald, B.A. (1997). The population genetics of fungi: tools and techniques.

*Phytopathology,* Vol.87, No. pp. 448–453, ISSN 0031-949X

*Soil Biology & Biochemistry*, Vol.43, No.3, pp. 682-689, ISSN 0038-0717 Martos, S.; Torres, E.; El Bakali, M.A.; Raposo, R.; Gramaje, D.; Armengol, J. & Luque, J.

and II genes. *Mycologia*, Vol.95, No.2, pp. 269-284, ISSN 0027-5514

Vol.182, No.1, pp. 213-228, ISSN 0028-646X

Vol.11, No.4, pp. 638-644, ISSN 1755-098X

Vol.91, No.6, pp. 736-742, ISSN 0191-2917

Vol.159, No.4, pp. 247-254, ISSN 0931-1785

120, ISSN 1139-6709

73-82, ISSN 0167-7012

1137–1143, ISSN 0031-949X

1526-498X

quantification and live-cell imaging of endophytic colonization of barley (*Hordeum vulgare*) roots by *Fusarium equiseti* and *Pochonia chlamydosporia. New Phytologist,*

Pech, N.; Castagnone-Sereno, P.; Delye, C.; Feau, N.; Frey, P.; Gauthier, P.; Guillemaud, T.; Hazard, L.; Le Corre, V.; Lung-Escarmant, B.; Male, P.J.G.; Ferreira, S.; Martin, J.F. (2011). High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries. *Molecular Ecology Resources*,

soybean and soil samples with a quantitative, real-time PCR assay. *Plant Disease*,

Symptom development, pathogen isolation and real-time QPCR quantification as factors for evaluating the resistance of olive cultivars to *Verticillium* pathotypes. *European Journal of Plant Pathology*, Vol.124, No.4, pp. 603–611, ISSN 0929-1873 Martin, F.N. & Tooley, P.W. (2003). Phylogenetic relationships among *Phytophthora* species

inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I

PCR to detect infection of differentially susceptible maize cultivars using *Ustilago maydis* strains of variable virulence. *International Microbiology*, Vol.6, No.2, pp. 117-

influence arbuscular mycorrhizal fungi communities in a semi-arid environment.

(2011) Co-operational PCR coupled with Dot Blot Hybridization for the detection of *Phaeomoniella chlamydospora* on infected grapevine wood. *Journal of Phytopathology*,

real-time PCR using Minor Groove Binding probe to monitor the biological control agent *Candida oleophila* (strain O). *Journal of Microbiological Methods,* Vol.60, No.1, pp.

H.; Kusakari, S.; Tamamatsu, S. & Toyoda, H. (2005). Identification of individual powdery mildew fungi infecting leaves and direct detection of gene expression of single conidium by polymerase chain reaction. *Phytopathology*, Vol.95, No.10, pp.

fungal plant pathogens. *Pest Management Science*, Vol.59, No.2, pp. 129–142, ISSN


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 195

Olmos, A.; Bertolini, E. & Cambra, M. (2002). Simultaneous and co-operational amplification

Omirou, M.; Rousidou, C.; Bekris, F.; Papadopoulou, K.K.; Menkissoglou-Spiroudi, U.;

Ozakman, M. & Schaad, N.W. (2003). A real-time BIO-PCR assay for detection of *Ralstonia* 

Pan, L.; Ash, G.J.; Ahn, B. & Watson, A.K. (2010). Development of strain specific molecular

Pannecoucque, J. & Hofte M. (2009). Detection of rDNA ITS polymorphism in *Rhizoctonia solani* AG 2-1 isolates. *Mycologia,* Vol.101, No. 1, pp. 26-33, ISSN 0027-5514 Pao, S.S.; Paulsen, I.T. & Saier MH Jr. (1998). Major facilitator superfamily. *Microbiology* 

Park, B.; Park, J.; Cheon,g K-C.; Choi, J.; Jung, K.; Kim, D.; Lee, Y.H.; Ward, T.J.; O'Donnell,

Park, J.; Park, B.; Veeraraghavan, N.; Blair, J.E.; Geiser, D.M.; Isard, S.; Mansfield, M.A.;

Paul, B. (2006). *Pythium apiculatum* sp. nov. isolated from Burgundian vineyards:

Paul, B., Bala, K., Gognies, S. & Belarbi, A. (2005). Morphological and molecular taxonomy

*FEMS Microbiology Letters,* Vol.246, No.2, pp. 207–212, ISSN 0378-1097 Pereira, V.J.; Fernandes, D.; Carvalho, G.; Benoliel, M.J.; Romao, M.V.S. & Crespo, M.T.B.

Special Issue: SI, pp. 4850-4859, ISSN 0043-1354 Phytophthora Database: ( http://www.phytophthoradb.org)

*Methods,* Vol.106, No.1, pp. 51-59, ISSN 0166-0934

*Microbial Ecology,* Vol.61, No.1, pp. 201–213, ISSN 0095-3628

*Plant Pathology,* Vol.25, No.3, pp. 232-239, ISSN 0706-0661

*Molecular Biology Review,* Vol.62, pp. 1-34, ISSN 1092-2172

online

D640-D646, ISSN 0305-1048

No.6, pp. 966-972, ISSN 0191-2917

257, ISSN 0378-1097

(Co-PCR): a new concept for detection of plant viruses. *Journal of Virological* 

Ehaliotis, C. & Karpouzas, D.G. (2011). The impact of biofumigation and chemical fumigation methods on the structure and function of the soil microbial community.

*solanacearum* race 3, biovar 2, in asymptomatic potato tubers. *Canadian Journal of* 

markers for the *Sclerotinia minor* bioherbicide strain IMI 344141. *Biocontrol Science and Technology*, Vol.20, No.9, pp. 939-959, ISSN 0958-3157 print/ISSN 1360-0478

K.; Geiser, D.M. & Kang, S. (2011). Cyber infrastructure for Fusarium: three integrated platforms supporting strain identification, phylogenetics, comparative genomics and knowledge sharing. *Nucleic Acids Research* Vol.39, Supplement. 1, pp.

Nikolaeva, E.; Park, S.-Y.; Russo, J.; Kim, S.H.; Greene, M.; Ivors, K.L.; Balci, Y.; Peiman, M.; Erwin, D.C.; Coffey, M.D.; Jung, K.; Lee, Y.-H.; Rossman, A.; Farr, D.; Cline, E.; Grünwald, N.J.; Luster, D.G.; Schrandt, J.; Martin, F.; Ribeiro, O.K.; Makalowska, I. & Kang, S. (2008). *Phytophthora* database: forensic database supporting the identification and monitoring of *Phytophthora*. *Plant Disease*, Vol.92,

morphology, taxonomy, ITS region of its rRNA, and comparison with related species. *FEMS Microbiology Letters*, Vol.263, No.2, pp. 194–199, ISSN 0378-1097 Paul, B. (2009). *Pythium burgundicum* sp. nov. isolated from soil samples taken in French vineyards. *FEMS Microbiology Letters*, Vol.301, No. , pp. 109-114, ISSN 0378-1097 Paul, B. & Bala, K. (2008). A new species of *Pythium* with inated sporangia and coiled

antheridia, isolated from India. *FEMS Microbiology Letters*, Vol.282, No.2, pp. 251–

of *Pythium longisporangium* sp. nov. isolated from the Burgundian region of France.

(2010). Assessment of the presence and dynamics of fungi in drinking water sources using cultural and molecular methods. *Water Research*, Vol.44, No.17,


Nakaune, R.; Hamamoto, H.; Imada, J. & Akutsu, K. (2002). A novel ABC transporter gene,

Nanayakkara, U.N.; Singh, M.; Al-Mugharabi, K.I. & Peters, R.D. (2009). Detection of

Nayaka, S.C.; Wulff, E.G.; Udayashankar, A.C.; Nandini, B.P.; Niranjana, S.R.; Mortensen,

Nechwatal, J. & Mendgen, K. (2006). *Pythium litorale* sp nov., a new species from the littoral

Nguyen, H.D.T. & Seifert, K.A. (2008). Description and DNA barcoding of three new species

Nicolaisen, M.; Justesen, A.F.; Thrane, U.; Skouboe, P. & Holmstrom, K. (2005). An

Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N. & Hase,

Nunes, C.C.; Gowda, M.; Sailsbery, J.; Xue, M.F.; Chen, F.; Brown, D.E.; Oh, Y.; Mitchell, T.K.

Nuovo, G.J. (1992). *PCR in situ hybridization: Protocols and applications.* Raven Press, ISBN

Nuovo, G.J.; MacConnell, P. & Gallery, F. (1994). Analysis of nonspecific DNA synthesis during in situ PCR and solution-phase PCR. *Genome Research*, Vol.4, pp. 89-96 Nuovo, G.J., MacConnell, P.; Forde, A. & Delvenne, P. (1991). Detection of human

Okubara, P.A.; Schroeder, K.L. & Paulitz, T.C. (2008). Identification and quantification of

Vol.28, No. 12, Article Number: e63, ISSN 0305-1048

Vol.139, No.4, pp. 847-854, ISSN 0002-9440

*Phytopathology,* Vol.98, No.7, pp. 837-847, ISSN 0031-949X

1617-4615

0175-7598

ISSN 0378-1097

ISSN 0031-5850

2822, ISSN 1684-5315

288, ISSN 1471-2164

0881679402, New York

No.3, pp. 239-245, ISSN 1099-209X

NCBI, National Center for Biotechnology Information: (http://www.ncbi.nlm.nih.gov/Genbank/)

*PMR5*, is involved in multidrug resistance in the phytopathogenic fungus *Pencillium digitatum*. *Molecular Genetics and Genomics,* Vol.267, pp. 179-185, ISSN

*Phytophthora erythroseptica* in above-ground potato tissues, progeny tubers, stolons and crop debris using PCR techniques. *American Journal of Potato Research*, Vol.86,

C.N. & Prakash, H.S. (2011). Prospects of molecular markers in *Fusarium* species diversity. *Applied Microbiology and Biotechnology*. Vol.90, No.5, pp. 1625-1639, ISSN

of Lake Constance, Germany *Fems Microbiology Letters*, Vol.255, No.1, pp. 96-101,

of *Leohumicola* from South Africa and the United States. *Persoonia*, Vol.21, pp. 57–69,

oligonucleotide microarray for the identification and differentiation of trichothecene producing and nonproducing *Fusarium* species occurring on cereal grain. *Journal of Microbiological Methods*, Vol.62, No. 1, pp. 57–69, ISSN 0167-7012 Niu, C.; Kebede, H.; Auld, D.L.; Woodward, J.E.; Burow, G. & Wright R.J. (2008). A safe

inexpensive method to isolate high quality plant and fungal DNA in an open laboratory environment. *African Journal of Biotechnology,* Vol.7, No.16, pp. 2818-

T. (2000). Loop-mediated isothermal amplification of DNA. *Nucleic Acids Research*,

& Dean, R.A. (2011). Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, *Magnaporthe oryzae*. *BMC Genomics*, Vol.12, Article Number:

papillomavirus DNA in formalin fixed tissues by in situ hybridization after amplification by the polymerase chain reaction. *American Journal of Pathology*,

*Rhizoctonia solani* and *R. oryzae* using real-time polymerase chain reaction.


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 197

Schoonbeek, H-J.; van Nistelrooy, J.G.M. & de Waard, M.A. (2003). Functional analysis of

Schroeder, K.L.; Okubara, P.A.; Tambong, J.T.; Levesque, C.A. & Paulitz, T.C. (2006).

Seifert, K.A.; Samson, R.A.; deWaard, J.R.; Houbraken, J.; Lévesque C.A.; Moncalvo, J-M.;

Sheridan, G.E.C.; Masters, C.I.; Shallcross, J.A. & Mackey, B.M. (1998). Detection of mRNA

Sierotzki, H.; Wullschleger, J. & Gisi, U. (2000). Point mutation in cytochrome b gene

isolates. *Pesticide Biochemical Physiology,* Vol.68, pp. 107–112, ISSN 0048-3575 Silvar, C.; Díaz, J. & Merino, F. (2005). Real-time polymerase chain reaction quantification of

Singh, M. & Singh, R.P. (1997). Potato virus Y detection: Sensitivity of RT-PCR depends on

Smith, L.M. & Burgoyne, L.A. (2004). Collecting, archiving and processing DNA from

Somai, B.M.; Keinath, A.P. & Dean, R.A. (2002). Development of PCR-ELISA detection and

Stergiopoulos, I. & de Waard, M.A. (2002). Activity of azole fungicides and ABC

Stergiopoulos, I.; Gielkens, M.M.C.; Goodall, S.D.; Venema, K. & de Waard, M.A. (2002b).

Stergiopoulos, I.; van Nistelrooy, J.G.; Kema, G.H. & de Waard, M.A. (2003b). Multiple

Stergiopoulos, I.; Zwiers, L-H. & de Waard, M.A. (2002a). Secretion of natural and synthetic

wildlife samples using FTA® databasing paper. *BMC Ecology*, 4:4

in Japan. *Plant Pathology,* Vol.60, No.2, pp. 253-260, ISSN 0032-0862

*De Phytopathologie*, Vol.19, pp. 149-155, ISSN 0706-0661

Vol.86, No.7, pp. 710–716, ISSN 0191-2917

Vol.150, pp. 313-320, ISSN 0931-1785

Vol.289, pp. 141-149, ISSN 0378-1119

ISSN 1526-498X

No.6, pp. 637-647, ISSN 0031-949X

pp. 1423–1429, ISSN 0031-949X

ISSN 0027-8424

ABC transporter genes from *Botrytis cinerea* identifies *BcatrB* as a transporter of eugenol. *European Journal of Plant Pathology,* Vol.109, pp. 1003-1011, ISSN 0929-1873

Identification and quantification of pathogenic *Pythium* spp. from soils in eastern Washington using real-time polymerase chain reaction. *Phytopathology,* Vol.96,

Louis-Seize, G. & Hebert P.D.N. (2007). Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case. *Proceedings of the National Academy of Sciences of the United States of America*, Vol.104, No.10, pp. 3901-3906,

by reverse transcription-PCR as an indicator of viability in *Escherichia coli* cells. *Applied Environmental Microbiology,* Vol.64, No.4, pp. 1313–1318, ISSN 0099-2240 Shimomoto, Y.; Sato, T.; Hojo, H.; Morita, Y.; Takeuchi, S.; Mizumoto, H.; Kiba, A. & Hikichi,

Y. (2011). Pathogenic and genetic variation among isolates of *Corynespora cassiicola* 

conferring resistance to strobilurin fungicides in *Erysiphe graminis* f. sp. *tritici* field

*Phytophthora capsici* in different pepper genotypes. *Phytopathology*, Vol.95, No.12,

the size of fragment amplified. *Canadian Journal of Plant Pathology-Revue Canadienne* 

differentiation of *Didymella bryoniae* from related *Phoma* species. *Plant Disease*,

transporters modulators on *Mycosphaerella graminicola. Journal of Phytopathology,*

Molecular cloning and charaterization of three new ATP-binding cassette transporter genes from the wheat pathogen *Mycosphaerella graminicola*. *Gene,*

mechanisms account for variation in base-line sensitivity to azole fungicides in field isolates of *Mycosphaerella graminicola*. *Pest Managment Science,* Vol.59, pp. 1333-1343,

toxic compounds from filamentous fungi by membrane transporters of the ATP-

Phytophthora-ID: (http://phytophthora-id.org)


Porterjordan, K.; Rosenberg, E.I.; Keiser, J.F.; Gross, J.D.; Ross, A.M.; Nasim, S. & Garrett,

Portillo, M.C.; Villahermosa, D.; Corzo, A. & González, J.M. (2011). Microbial community

Prospero, S.; Black, J.A. & Winton, L.M. (2004). Isolation and characterization of

Saldanha, R.L.; García, J.E.; Dekker, R.F.H.; Vilas-Boas, L.A. & Barbosa, A.M. (2007). Genetic

Samuelian, S.K.; Greer, L.A.; Savocchia, S. & Steel, C.C. (2011). Detection and monitoring of

Sánchez-Torres, P. & Tuset, J.J. (2011). Molecular insights into fungicide resistance in

Schena, L. & Ippolito, A. (2003). Rapid and sensitive detection of *Rosellinia necatrix* in roots

Schena, L.; Hughes K.J.D. & Cooke D.E.L. (2006). Detection and quantification of *P. ramorum,* 

Schnabel, G. & Jones, A.L. (2001). The 14-demethylase (*CYP51A1*) gene is overexpressed in

Schnabel, G.; Dait, Q. & Paradkar, M.R. (2001). Cloning and expression analysis of the ATP-

PCR. *Molecular Plant Pathology,* Vol.7, No.5, pp. 365-379, ISSN 1464-6722 Schena, L., Nigro, F., Ippolito, A. & Gallitelli, D. (2004). Real-time quantitative PCR: a new

*Journal of Plant Pathology,* Vol.110, No.9, pp. 893-908, ISSN 0929-1873 Schmidt, H.; Taniwaki, M.H.; Vogel, R.F. & Niessen, L. (2004) Utilization of AFLP markers

death. *Molecular Ecology Notes*, Vol.4, No.4, pp. 672-674, ISSN 1471-8278 Qin, L.; Fu, Y.; Xie, J.; Cheng, J.; Jiang, D.; Li, G. & Huang, J. (2011). A nested-PCR method

*napus*). *Plant Pathology,* Vol.60, No.2, pp. 271-277, ISSN 0032-0862

time PCR. *Plant Disease*, Vol.95, No.3, pp. 298-303, ISSN 0191-2917

*Biology and Technology,* Vol.59, No.2, pp. 159–165, ISSN 0925-5214

C.T. (1990). Nested polymerase chain reaction assay for the detection of cytomegalovirus overcomes false positives caused by contamination with fragmented DNA. *Journal of Medical Virology,* Vol.30, No.4, pp. 85-91, ISSN 0146-

fingerprinting by differential display-denaturing gradient gel electrophoresis. *Applied and Environmental Microbiology*, Vol.77, No.1, pp. 351–354, ISSN 0099-2240

microsatellite markers in *Phytophthora ramorum*, the causal agent of sudden oak

for rapid detection of *Sclerotinia sclerotiorum* on petals of oilseed rape (*Brassica* 

diversity among *Botryosphaeria* isolates and their correlation with cell wall-lytic enzyme production. *Brazilian Journal of Microbiology,* Vol.38, No.2, pp. 259-264, ISSN

*Greeneria uvicola* and *Colletotrichum acutatum* development on grapevines by real-

sensitive and resistant *Penicillium digitatum* strains infecting citrus. *Postharvest* 

and soils by real time Scorpion-PCR. *Journal of Plant Pathology,* Vol.85, No.1, pp. 15-

*P. kernoviae, P. citricola* and *P. quercina* in symptomatic leaves by multiplex realtime

technology to detect and study phytopathogenic and antagonistic fungi. *European*

for PCR-based identification of *Aspergillus carbonarius* and indication of its presence in green coffee samples. Journal of Applied Microbiology Vol.97, No.5, pp.899-909,

*Venturia inaequalis* strains resistant to myclobutanil. *Phytopathology,* Vol.91, pp. 102–

binding cassette transporter gene *MFABC1* and the alternative oxidase gene *MfAOX1* from *Monilinia fructicola*. *Pest Managment Scence,* Vol.59, pp. 1143-1151,

Phytophthora-ID: (http://phytophthora-id.org)

Primer3: ( http://frodo.wi.mit.edu/primer3)

6615

1517-8382259

25, ISSN 1125-4653

ISSN 1364-5072

ISSN 1526-498X

110, ISSN 0031-949X


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 199

Tomlinson, J.A.; Boonham, N.; Hughes, K.J.D.; Griffen, R.L. & Barker, I. (2005). On-site DNA

Tomlinson J.A.; Dickinson M.J. & Boonham N. (2010). Rapid detection of *Phytophthora* 

Tooley, P.W.; Martin, F.N.; Carras, M.M. & Frederick, R.D. (2006). Real-time fluorescent

Torres-Calzada, C.; Tapia-Tussell, R.; Quijano-Ramayo, A.; Martin-Mex, R.; Rojas-Herrera,

Tyagi, S. & Kramer, F.R. (1996). Molecular beacons: Probes that fluoresce upon hybridization. *Nature Biotechnology*, Vol.14, No.3, pp. 303-308, ISSN 1087-0156 UNITE, A molecular database for identification of fungi: (http://unite.ut.ee/index.php) Validov, S.Z.; Kamilova F.D. & Lugtenberg B.J.J. (2011). Monitoring of pathogenic and non-

Valsesia, G.; Gobbin, D.; Patocchi, A.; Vecchione, A.; Pertot, I. & Gessler, C. (2005).

van Doorn, R.; Slawiak, M.; Szemes, M.; Dullemans, A.M.; Bonants, P.; Kowalchuk, G.A. &

van Gent-Pelzer, M.P.E.; van Brouwershaven, I.R.; Kox, L.F.F. & Bonants, P.J.M. (2007). A

VanderLee, T.; DeWitte, I.; Drenth, A.; Alfonso, C. & Govers, F. (1997). AFLP linkage map of

Vermeulen, T.; Schoonbeek, H. & de Waard, M.A. (2001). The ABC transporter BcatrB from

fludiioxonil. *Pest Managment Science,* Vol.57, pp. 393-402, ISSN 1526-498X Vos, P.; Hogers, R.; Bleeker, M.; Reijans, M.; Vandelee, T.; Hornes, M.; Frijters, A.; Pot, J.;

reaction. *Phytopathology*, Vol.95, No.6, pp. 672-678, ISSN 0031-949X

Vol.100, No.2, pp, 143-149, ISSN 0031-949X

*Biotechnology,* Vol.49, No.1, pp. 48-55, ISSN 1073-6085

*Biotechnology,* Vol.4, No.1, pp. 82-88, ISSN 1751-7907

TrichOKey, Molecular Barcode: (http://www.isth.info/tools/molkey/index.php)

336–345, ISSN 0031-949X

4185-4193, ISSN 0099-2240

278–291, ISSN 1087-1845

0931-1785

extraction and real-time PCR for detection of *Phytophthora ramorum* in the field. *Applied and Environmental Microbiology* Vol.71, No.11, pp. 6702-6710, ISSN 0099-2240

*ramorum* and *P. kernoviae* by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device. *Phytopathology*

polymerase chain reaction detection of *Phytophthora ramorum* and *Phytophthora pseudosyringae* using mitochondrial gene regions. *Phytopathology,* Vol.96, No.4, pp.

R.; Higuera-Ciapara, I.; Perez-Brito, D. (2011). A species-specific polymerase chain reaction assay for rapid and sensitive detection of *Colletotrichum capsici*. *Molecular* 

pathogenic *Fusarium oxysporum* strains during tomato plant infection. *Microbial* 

Development of a high-throughput method for quantification of *Plasmopara viticola* DNA in grapevine leaves by means of quantitative real-time polymerase chain

Schoen, C.D. (2009). Robust detection and identification of multiple oomycetes and fungi in environmental samples by using a novel cleavable padlock probe-based ligation detection assay. *Applied and Environmental Microbiology,* Vol.75, No.12, pp.

TaqMan PCR method for routine diagnosis of the quarantine fungus *Guignardia citricarpa* on citrus fruit. *Journal of Phytopathology,* Vol.155, No.6, pp. 357-363, ISSN

the oomycete *Phytophthora infestans*. *Fungal Genetics and Biology,* Vol.21, No.3, pp.

*Botrytis cinerea* is a determinant of the activity of the phenylpyrrole fungicide

Peleman, J.; Kuiper, M. & Zabeau, M. (1995). AFLP: a new technique for DNA fingerprinting. *Nucleic Acids Research*, Vol.23, No.21, pp. 4407–14, ISSN 0305-1048

binding cassette and major facilitator superfamily*. European Journal of Plant Pathology,* Vol.108, pp. 719-734, ISSN 0929-1873


Stergiopoulos, I.; Zwiers, L-H. & de Waard, M.A. (2003a). The ABC transporter MgAtr4 is a

Stewart, J.E.; Kim, M.-S.; James, R.L.; Dumroese, R.K. & Klopfenstein, N.B. (2006). Molecular

Sun, X.; Wang, J.; Feng, D.; Ma, Z. & Li, H. (2011). PdCYP51B, a new putative sterol 14α-

Sundelin, T.; Christensen, C.B.; Larsen, J.; Møller, K.; Lübeck, M.; Bødker, L. & Jensen, B.

Takamatsu, S.; Nakano, M.; Yokota, H. & Kunoh, H. (1998). Detection of *Rhizoctonia solani* 

Tewoldemedhin, Y.T.; Mazzola, M.; Botha W.J.; Spies, C.F.J. & McLeod, A. (2011).

Theodoulou, F.L. (2000). Plant ABC transporters. *Biochimica Biophysica Acta,* Vol.1465, pp. 79-

Thies, J.E. (2007). Soil microbial community analysis using terminal restriction fragment

Tomlinson J. A.; Barker I. & Boonham N. (2007). Faster, simpler, more-specific methods for

*Environmental Microbiology* Vol.73, No.12, pp. 4040-4047, ISSN 0099-2240

*Plant Pathology,* Vol.130, No 2, pp. 215-229, ISSN 0929-1873

conifer nursery. *Phytophathology*, Vol.96, pp. 1124-1133, ISSN 0031-949X Stringari, D.; Glienke, C.; de Christo, D.; Maccheroni, W. & de Azevedo, J.L. (2009). High

*biology and technology*, Vol.52, No.5, pp. 1063-1073, ISSN 1516-8913

*Biotechnology*, Vol.91, No.4, pp. 1107-1119, ISSN 0175-7598

*Pathology,* Vol.108, pp. 719-734, ISSN 0929-1873

689-698, ISSN 0894-0282

572–574, ISSN 1389-1723

0099-2240

103, ISSN 0005-2736

591, ISSN 0361-5995

binding cassette and major facilitator superfamily*. European Journal of Plant* 

virulence factor of *Mycosphaerella graminicola* that effects colonisation of substomatal cavities in wheat leaves. *Molecular Plant-Microbe Interaction,* Vol.16, pp.

characterization of *Fusarium oxysporum* and *Fusarium commune* isolates from a

molecular diversity of the fungus *Guignardia citricarpa* and *Guignardia mangiferae* and new primers for the diagnosis of the Citrus Black Spot. *Brazilian archives of* 

demethylase gene of *Penicillium digitatum* involved in resistance to imazalil and other fungicides inhibiting ergosterol synthesis. *Applied Microbioly and* 

(2010). In planta quantification of *Plasmodiophora brassicae* using signature fatty acids and real-time PCR. *Plant Disease,* Vol.94, No.4, pp. 432-438, ISSN 0191-2917 Suzuki, S.; Taketani, H.; Kusumoto, K.I. & Kashiwagi, Y. (2006). High-throughput

genotyping of filamentous fungus *Aspergillus oryzae* based on colony direct polymerase chain reaction. *Journal of Bioscience and Bioengineering,* Vol.102, No.6, pp.

AG-2-2-IV, the causal agent of large patch of Zoysiagrass, using plasmid DNA as probe. *Annals of the Phytopathological Society of Japan*, Vol.64, No.5, pp. 451–457 Tawfik, D.S. & Griffiths, A.D. (1998). Man-made cell-like compartments for molecular evolution, *Nature Biotechnology*, Vol.16, No.7, pp. 652–656, ISSN 1087-0156 Tebbe, C.C. & Vahjen, W. (1993). Interference of humic acids and dna extracted directly from

soil in detection and transformation of recombinant-DNA from bacteria and a yeast. *Applied and Environmental Microbiology* Vol.59, No.8, pp. 2657-2665, ISSN

Characterization of fungi (*Fusarium* and *Rhizoctonia*) and oomycetes (*Phytophthora* and *Pythium*) associated with apple orchards in South Africa. *European Journal of* 

length polymorphisms. *Soil Science Society of America Journal*, Vol.71, No.2, pp. 579-

improved molecular detection of *Phytophthora ramorum* in the field. *Applied and* 


Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance 201

Yan, L.; Zhang, C.; Ding, L. & Ma, Z. (2008). Development of a real-time PCR assay for the

Yang F.; Jensen J. D.; Svensson B.; Jorgensen, H. J. L.; Collinge, D.B. & Finnie, C. (2010).

Yin, Y.; Ding, L.; Liu, X.; Yang, J. & Ma, Z. (2009). Detection of *Sclerotinia sclerotiorum* in

Yoder, O.C. & Turgeon, B. (2001). Fungal genomics and pathogenicity. *Current Opinion in* 

Zaccaro, R. P.; Carareto-Alves, L.M.; Travensolo, R.F.; Wickert, E. & Lemos, E.G.M. (2007).

Zelaya-Molina L.X.; Ortega M.A. & Dorrance A.E. (2011). Easy and efficient protocol for

Zhang, N.; Tantardini, A.; Miller, S.; Eng, A. & Salvatore, N. (2011). TaqMan real-time PCR

Zhang, Y.J.; Zhang, S.; Liu, X.Z.; Wen, H.A. & Wang, M. (2010). A simple method of genomic

Zhang, Z.; Zhu, Z.; Ma, Z., & Li, H. (2009). A molecular mechanism of azoxystrobin

Zhao, J.; Wang, X. J.; Chen, C. Q.; Huang, L. L. & Kang, Z.S. (2007). A PCR-Based assay for

Zheng, D.; Olaya, G. & Koller, W. (2000). Characterization of laboratory mutants of *Venturia* 

Zheng, P.H.; Meng, X.Y.; Yu, Z.X.; Wu, Y; Bao, Y.L.; Yu, C.L. & Li, Y.X. (2009). Genetic and

Zheng, Y.; Zhang, G.; Lin, F.C.; Wang, Z.H.; Jin, G.L.; Yang, L.; Wang, Y.; Chen, X.; Xu, Z.H.;

*Journal of Food Microbiology,* Vol.131, pp. 157–161, ISSN 0168-1605

Vol.104, No.5, pp. 1417-1424, ISSN 1364-5072

*Plant Biology*, Vol.4, pp. 315-321, ISSN 1369-5266

*Letters*, Vol.33, No.4, pp. 715-720, ISSN 0141-5492

*Microbiology,* Vol.51, No.1, pp. 114-118, ISSN 0266-8254

3748-3755, ISSN 1615-9853

469, ISSN 0931-1785

570, ISSN 0100-2945

551-558, ISSN 0929-1873

1969-1674, ISSN 0191-2917

1087-1845

*Genetics,* Vol.38, pp. 148-155, ISSN 0172-8083

*Letters*, Vol.42, No.10, pp. 1479-1494, ISSN 0003-2719

detection of *Cladosporium fulvum* in tomato leaves. *Journal of Applied Microbiology,*

Analysis of early events in the interaction between *Fusarium graminearum* and the susceptible barley (*Hordeum vulgare*) cultivar Scarlett. *Proteomics,* Vol.10, No.21, pp.

planta by a real-time PCR assay. *Journal of Phytopathology*, Vol.157, No.7-8, pp. 465–

Use of molecular marker SCAR in the identification of *Fusarium subglutinans*, causal agent of mango malformation. *Revista brasileira de fruticultura,* Vol.29, No.3, pp. 563-

oomycete DNA extraction suitable for population genetic analysis. *Biotechnology* 

method for detection of *Discula destructiva* that causes dogwood anthracnose in Europe and North America. *European Journal of Plant Pathology*, Vol.130, No.4, pp.

DNA extraction suitable for analysis of bulk fungal strains. *Letters in Applied* 

resistancein *Penicillium digitatum* UV mutants and a PCR-based assay for detection of azoxystrobin-resistant strains in packing- or store-house isolates. *Interantional* 

detection of *Puccinia striiformis* f. sp *tritici* in wheat. *Plant Disease*, Vol.91, No.12, pp.

*inaequalis* resistant to the strobilurin-related fungicide kresoxim-methyl. *Current* 

phytochemical analysis of *Armillaria mellea* by RAPD, ISSR, and HPLC. *Analytical* 

Zhao, X.Q.; Wang, H.K.; Lu, J.P.; Lu, G.D. & Wu, W.R. (2008). Development of microsatellite markers and construction of genetic map in rice blast pathogen *Magnaporthe grisea*. *Fungal Genetics and Biology*, Vol.45, No.10, pp. 1340-1347, ISSN


Wakelin S.A.; Warren, R.A.; Kong, L. & Harvey, P.R. (2008). Management factors affecting

Welsh, J. & McClelland, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. *Nucleic Acids Research*, Vol.18, No.24, pp. 7213–7218, ISSN 0305-1048 Whitcombe, D.; Kelly, S.; Mann, J.; Theaker, J.; Jones, C. & Little, S. (1999). Scorpions (TM)

White, T. (1997). Increased mRNA levels of *ERG16*, *CDR*, and *MDR1* correlate with increases

Williams, J.G.K.; Kubelik, A.R.; Livak, K.J.; Rafalski, J.A. & Tingey, S.V. (1990). DNA

Winton, L.M.; Krohn, A.L. & Leiner, R.H. (2007). Microsatellite markers for *Sclerotinia* 

Wittwer, C.T.; Herrmann, M.G.; Moss, A.A. & Rasmussen, R.P. (1997). Continuous

Wood, P.M. & Hollomon, D.W. (2003). A critical evaluation of the role of alternative oxidase

Wyand, R.A. & Brown, J.K.M. (2005). Sequence variation in the *CYP51* gene of *Blumeria* 

Xu, M.L.; Melchinger, A.E. & Lübberstedt, T. (1999). Species-specific detection of the maize

PCR-based assays. *Plant Disease*, Vol.83, No.4, pp. 390–395, ISSN 0191-2917 Xue, B; Goodwin, P.H. & Annis, S.L. (1992). Pathotype identification of *Leptosphaeria-*

complex III. *Pest Managment Science,* Vol.59, pp. 499-511, ISSN 1526-498X Wu, C.P.; Chen, G.Y.; Li, B.; Su, H.; An, Y.L.; Zhen, S.Z. & Ye, J.R. (2011). Rapid and accurate

time PCR. *Forest Pathology,* Vol.41, No.1, pp. 15-21, ISSN 1437-4781

*Fungal Genetics Biol*ogy, Vol.42, pp. 726–765, ISSN 1087-1845

*Nucleic Acids Research*, Vol.18, No.22, pp. 6531–6535, ISSN 0305-1048 Wilson, I.G. (1997) Inhibition and facilitation of nucleic acid amplification. Applied and Environmental Microbiology Vol.63, No.10, pp. 3741–3751, ISSN 0099-2240 Winton, L.M. & Hansen, E.M. (2001). Molecular diagnosis of *Phytophthora lateralis* in trees,

*Applied Soil Ecology*, Vol.39, pp. 201-209, ISSN 0929-1393

Academic Press, Inc., New York. pp. 315-322

Vol.31, No.5, pp. 275-283, ISSN 1437-4781

No.1, pp. 130–138, ISSN 0736-6205

No.3, pp. 179-188, ISSN 0885-5765

*Ecology Notes,* Vol.7, No.6, pp. 1077-1079, ISSN 1471-8278

9297

size and structure of soil *Fusarium* communities under irrigated maize in Australia.

primers - a novel method for use in single-tube genotyping. *American Journal of Human Genetics,* Vol.65, No.4, pp. A412-A412, Meeting Abstract: 2333, ISSN 0002-

in azole resistance in *Candida albicans* isolates from a patient infected with human immunodeficiency virus. *Antimicrobe Agents Chemotherapy,* Vol.41, pp. 1482-1487 White, T.J.; Bruns, T.; Lee, S. & Taylor, J.W. (1990). Amplification and direct sequencing of

fungal ribosomal RNA genes for phylogenetics. In: *PCR Protocols: A Guide to Methods and Applications*, Innis, M.A.; Gelfand, D.H.; Sninsky, J.J. & White, T.J.

polymorphisms amplied by arbitrary primers are useful as genetic markers.

water, and foliage baits using multiplex polymerase chain reaction. *Forest Pathology*,

*subarctica* nom. prov., a new vegetable pathogen of the High North. *Molecular* 

fluorescence monitoring of rapid cycle DNA amplification. *Biotechniques,* Vol.22,

in the performance of strobilurin and related fungicides acting at the Qo site of

detection of *Ceratocystis fagacearum* from stained wood and soil by nested and real-

*graminis* associated with resistance to sterol demethylase inhibiting fungicides.

pathogens *Sporisorium reiliana* and *Ustilago maydis* by dot-blot hybridization and

*maculans* with PCR and oligonucleotide primers from ribosomal internal transcribed spacer sequences. *Physiological and Molecular Plant Pathology*, Vol.41,


**8** 

*Austria* 

**Novel Methods for the Quantification** 

**Quantitative PCR and ELISA Accurately** 

*Vienna University of Technology, Institute of Chemical Engineering, Vienna* 

Fungi of the genus *Fusarium* are worldwide occurring plant pathogens which cause severe damages to numerous cultivable plants (Weiland et al., 2000; Mirete et al., 2004; Youssef et al., 2007; Li et al., 2008) with the highest economical losses upon infection of maize, wheat and barley (Windels et al., 2000; Nganje et al., 2004). *Fusarium* caused diseases can destroy crops within several weeks and the infection leads to quality losses in two different aspects: besides the reduced yield due to reduced kernel size, the fungus produces various toxic metabolites while colonizing the plant. These mycotoxins heavily impair the quality of the harvest (McMullen et al., 1997). The acute or chronic toxicity of *Fusarium* released compounds led to the introduction of national or international limits to regulate mycotoxin

Most *Fusarium* species are widely distributed in substrates such as soil, on subterranean and aerial plant parts, plant debris, and on dead organic matter. Many *Fusarium* species have active or passive means to disperse spores or conidia in the atmosphere. The ability to grow on a broad range of substrates combined with their efficient dispersal mechanisms enables a widespread distribution of these fungi (Burgess, 1981). *Fusarium* species are well adapted to grass hosts (Leonard & Bushnell, 2003) and can colonize on many agricultural commodities such as rice, bean and soybean. According to several studies *F. graminearum* and *F. culmorum* are among the most aggressive plant pathogenic fungi known. *F. graminearum* mainly occurs in temperate and warmer regions of the USA, China and the southern hemisphere (Osborne & Stein, 2007). It is regarded as one of the most vigorous toxin producers and has therefore become the most intensively studied plant pathogen (Goswami & Kistler, 2004). In contrast, *F. culmorum* predominates in the cooler regions including the U.K., Northern Europe and Canada (Osborne & Stein, 2007) and is also associated with the occurrence of many

A worldwide problem of agricultural industry producing wheat, barley and maize is the fungal disease *Fusarium* head blight (FHB), with maize and wheat as the economically most

levels in food and feed (e.g. limits of the European Community since 2006).

**1. Introduction** 

mycotoxins (Desjardins, 2006).

**2. Toxigenicity and impacts** 

**of Pathogenic Fungi in Crop Plants:** 

Kurt Brunner, Andreas Farnleitner and Robert L. Mach

**Determine** *Fusarium* **Biomass** 

Zwiers, L-H.; Stergiopoulos, I.; Gielkens, M.M.C.; Goodall, S.D. & de Waard, M.A. (2003). ABC transporters of the wheat pathogen *Mycosphaerella graminicola* function as protectants against biotic and xenobiotic toxic compounds. *Molecular Genetics and Genomics,* Vol.269, pp. 499-507, ISSN 1617-4615

### **Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants: Quantitative PCR and ELISA Accurately Determine** *Fusarium* **Biomass**

Kurt Brunner, Andreas Farnleitner and Robert L. Mach *Vienna University of Technology, Institute of Chemical Engineering, Vienna Austria* 

#### **1. Introduction**

202 Plant Pathology

Zwiers, L-H.; Stergiopoulos, I.; Gielkens, M.M.C.; Goodall, S.D. & de Waard, M.A. (2003).

*Genomics,* Vol.269, pp. 499-507, ISSN 1617-4615

ABC transporters of the wheat pathogen *Mycosphaerella graminicola* function as protectants against biotic and xenobiotic toxic compounds. *Molecular Genetics and* 

> Fungi of the genus *Fusarium* are worldwide occurring plant pathogens which cause severe damages to numerous cultivable plants (Weiland et al., 2000; Mirete et al., 2004; Youssef et al., 2007; Li et al., 2008) with the highest economical losses upon infection of maize, wheat and barley (Windels et al., 2000; Nganje et al., 2004). *Fusarium* caused diseases can destroy crops within several weeks and the infection leads to quality losses in two different aspects: besides the reduced yield due to reduced kernel size, the fungus produces various toxic metabolites while colonizing the plant. These mycotoxins heavily impair the quality of the harvest (McMullen et al., 1997). The acute or chronic toxicity of *Fusarium* released compounds led to the introduction of national or international limits to regulate mycotoxin levels in food and feed (e.g. limits of the European Community since 2006).

> Most *Fusarium* species are widely distributed in substrates such as soil, on subterranean and aerial plant parts, plant debris, and on dead organic matter. Many *Fusarium* species have active or passive means to disperse spores or conidia in the atmosphere. The ability to grow on a broad range of substrates combined with their efficient dispersal mechanisms enables a widespread distribution of these fungi (Burgess, 1981). *Fusarium* species are well adapted to grass hosts (Leonard & Bushnell, 2003) and can colonize on many agricultural commodities such as rice, bean and soybean. According to several studies *F. graminearum* and *F. culmorum* are among the most aggressive plant pathogenic fungi known. *F. graminearum* mainly occurs in temperate and warmer regions of the USA, China and the southern hemisphere (Osborne & Stein, 2007). It is regarded as one of the most vigorous toxin producers and has therefore become the most intensively studied plant pathogen (Goswami & Kistler, 2004). In contrast, *F. culmorum* predominates in the cooler regions including the U.K., Northern Europe and Canada (Osborne & Stein, 2007) and is also associated with the occurrence of many mycotoxins (Desjardins, 2006).

#### **2. Toxigenicity and impacts**

A worldwide problem of agricultural industry producing wheat, barley and maize is the fungal disease *Fusarium* head blight (FHB), with maize and wheat as the economically most

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants:

with high yield and *Fusarium* resistance.

Quantitative PCR and ELISA Accurately Determine *Fusarium* Biomass 205

Christensen (Schroeder & Christensen, 1963) described in their work two phenotypic measures of disease resistance; type I resistance operates against initial infection and type II resistance describes the resistance to spreading within infected tissues. A third type of *Fusarium* resistance (type III) was described by Wang and Miller (1988) as the insensitivity of

Integrated control strategies are essential to prevent *Fusarium* diseases in modern agriculture. The use of resistant cultivars is considered necessary for numerous reasons. Firstly, modern agronomic practices such as reduced tillage and cereal and maize rich crop rotations have the tendency to increase the amount of *Fusarium* related diseases. In addition to this, chemical control measures are only partly effective as only few fungicides are sufficiently active against *Fusarium* and their application must be performed within a time frame of a few days around the flowering stage of the plants. For the protection of maize cultures no fungicides are commercially available up to now. Understanding *Fusarium* lifecycle and infection pathways is the first step in preventing disease. Nevertheless, uncontrollable factors such as weather conditions during as well as economic or ecologic interests prevent sustainable success in the reduction of these pathogens. Plant breeders have made significant progress in the development of *Fusarium* resistant maize and wheat varieties by identifying genetic regions, which are linked to the resistance of plants (Buerstmayer et al., 2003; Draeger et al., 2007). Resistance against *Fusarium* is a quantitative trait, which is governed by many independent genes distributed on several distinct genetic regions (quantitative trait loci, QTLs) in the plant genome. Therefore an effective approach to investigate FHB resistance is the identification of the QTLs and their mapping (Buerstmayer et al., 2009). The mapping of QTLs is the basis for further efforts towards a straightforward marker assisted resistance breeding strategy. Interestingly, often the best regionally adapted and highly productive crop varieties are susceptible to FHB. For this reason, breeders are faced with the difficult task to combine adaption to certain locations

The production of mycotoxins has been identified as a crucial factor for successful infection . Deoxynivalenol is considered the most common occurring mycotoxin involving *Fusarium* infection. However, DON levels in infected grain vary significantly amongst wheat cultivars (Bai et al., 2001) infected with the same *Fusarium* isolate. The evaluation of *Fusarium* resistance of either the parents in current breeding programs or the control of resistance levels of novel lines can be carried out by chemical analysis of the DON accumulation in the seeds. In general DON levels in resistant plants are much lower than those found in more susceptible cultivars. Artificial infection of wheat-ears with *Fusarium* conidia followed by determination of DON content and visual scoring of the head blight symptoms are the currently established methods for resistance assessment. Nevertheless, the determinations of disease symptoms in combination with the mycotoxin content of grain are both indirect methods to evaluate plant resistance. Furthermore, both methods observe the same effect, as visual disease symptoms are mainly caused by mycotoxin intoxication and not by the growth of mycelium. Studies have shown that the amount of fungal mycelia formed during infection not always correlates well with these parameters (Waalwijk et al, 2004; Hill et al., 2006; Brunner et al., 2009; Kulik et al. 2011). Asymptomatic kernels may contain significant

amounts of mycotoxins while symptomatic kernels within the same sample may not.

wheat lines to toxins or the ability of the resistant cultivar to degrade mycotoxins.

important host plants. The disease is associated with *F. graminearum*, *F. culmorum*, *F. poae*, *F. avenaceum*, *F. sporotrichoides* and *Microdochium nivale*. Each fungus contributing to FHB has particular biological and environmental requirements, which in part explains the frequency of occurrence in specific locations.

This destructive disease was initially described in 1884 in England and was considered a major threat during the early years of the twentieth century (Goswami & Kistler, 2004). Nowadays, *Fusarium* head blight is a disease of massive economic impact worldwide and has been ranked by the United States Department of Agriculture (USDA) as the worst plant disease to hit the nation (Windels, 2000). The losses due to direct and secondary economic impact of FHB on wheat and barley were estimated at \$2.7 billion for the period from 1998 – 2000 in the US (Nganje et al., 2002). Epidemics of FHB are strongly influenced by various factors such as local and regional environment, the physiological state and genetic background of the host, as well as pathogen related factors including host adaptation and virulence.

Infected plant debris serves as primary substrate for common FHB pathogens, which are able to survive the winter period as saprophytic mycelium or thick walled chlamydospores. Warm, moist conditions in spring are favorable for the development and maturation of conidia and perithecia, which produce ascospores (Markell & Frankl, 2002). These sticky spores are discharged from the surface of crop debris and dispersed by wind, rain or even insects to the host plants. Infected wheat or maize presents brown, dark purple to black lesions on the exterior. The head of cereals has a characteristically bleached appearance, hence the name *Fusarium* head blight. During prolonged wet periods infected spikelets, glumes and kernels present pink to salmon-orange spore masses of the fungus. Maize infections occur at the ear apex following colonization of the kernels with white mycelium, which turns pink to red with time.

Fungal colonization is affected by numerous variables such as spike morphology, canopy density, plant height, rainfall, relative humidity, temperature and host plant resistance (Kolb et al., 2001; Rudd et al., 2001). *Fusarium* infections are increased due to the lack of completely resistant plant material and a frequent application of unsuitable cropping systems. Pathogen survival is favored in reduced tillage systems as residue burial speeds up the decomposition and reduces pathogen reproduction and survival (Khonga et al., 1988; Pereyra et al., 2004). Petcu et al., (1998) investigated the influence of crop rotations on the severity of FHB. Studies show that the economically most lucrative cultivation of alternating maize and wheat cultures turned out to be problematic. The increased production of these *Fusarium* favorable crops and the dramatic increase in their residues remaining on the soil surface provide a large increase in the amount of niche available to the pathogen. *Fusarium* non-host plants used as preceding crops or intertillage of wheat and maize are often less profitable and therefore of low interest for farmers. In North and South America erosion causes a dramatic loss of fertile topsoil. To avoid this loss, no-till systems have been established to overcome the drawback of conventional farming. However, *Fusarium* inoculum density increases in soil in reduced tillage systems compared to plough treated fields (Steinkellner & Langer, 2004).

#### **3. Plant resistance to** *Fusarium*

Plant resistance can be defined as the relative amount of heritable qualities possessed by a plant that reduces the degree of damage to the plant caused by pathogens. Schroeder and

important host plants. The disease is associated with *F. graminearum*, *F. culmorum*, *F. poae*, *F. avenaceum*, *F. sporotrichoides* and *Microdochium nivale*. Each fungus contributing to FHB has particular biological and environmental requirements, which in part explains the frequency

This destructive disease was initially described in 1884 in England and was considered a major threat during the early years of the twentieth century (Goswami & Kistler, 2004). Nowadays, *Fusarium* head blight is a disease of massive economic impact worldwide and has been ranked by the United States Department of Agriculture (USDA) as the worst plant disease to hit the nation (Windels, 2000). The losses due to direct and secondary economic impact of FHB on wheat and barley were estimated at \$2.7 billion for the period from 1998 – 2000 in the US (Nganje et al., 2002). Epidemics of FHB are strongly influenced by various factors such as local and regional environment, the physiological state and genetic background of the host, as well

Infected plant debris serves as primary substrate for common FHB pathogens, which are able to survive the winter period as saprophytic mycelium or thick walled chlamydospores. Warm, moist conditions in spring are favorable for the development and maturation of conidia and perithecia, which produce ascospores (Markell & Frankl, 2002). These sticky spores are discharged from the surface of crop debris and dispersed by wind, rain or even insects to the host plants. Infected wheat or maize presents brown, dark purple to black lesions on the exterior. The head of cereals has a characteristically bleached appearance, hence the name *Fusarium* head blight. During prolonged wet periods infected spikelets, glumes and kernels present pink to salmon-orange spore masses of the fungus. Maize infections occur at the ear apex following colonization of the kernels with white mycelium,

Fungal colonization is affected by numerous variables such as spike morphology, canopy density, plant height, rainfall, relative humidity, temperature and host plant resistance (Kolb et al., 2001; Rudd et al., 2001). *Fusarium* infections are increased due to the lack of completely resistant plant material and a frequent application of unsuitable cropping systems. Pathogen survival is favored in reduced tillage systems as residue burial speeds up the decomposition and reduces pathogen reproduction and survival (Khonga et al., 1988; Pereyra et al., 2004). Petcu et al., (1998) investigated the influence of crop rotations on the severity of FHB. Studies show that the economically most lucrative cultivation of alternating maize and wheat cultures turned out to be problematic. The increased production of these *Fusarium* favorable crops and the dramatic increase in their residues remaining on the soil surface provide a large increase in the amount of niche available to the pathogen. *Fusarium* non-host plants used as preceding crops or intertillage of wheat and maize are often less profitable and therefore of low interest for farmers. In North and South America erosion causes a dramatic loss of fertile topsoil. To avoid this loss, no-till systems have been established to overcome the drawback of conventional farming. However, *Fusarium* inoculum density increases in soil in reduced tillage

Plant resistance can be defined as the relative amount of heritable qualities possessed by a plant that reduces the degree of damage to the plant caused by pathogens. Schroeder and

as pathogen related factors including host adaptation and virulence.

systems compared to plough treated fields (Steinkellner & Langer, 2004).

of occurrence in specific locations.

which turns pink to red with time.

**3. Plant resistance to** *Fusarium*

Christensen (Schroeder & Christensen, 1963) described in their work two phenotypic measures of disease resistance; type I resistance operates against initial infection and type II resistance describes the resistance to spreading within infected tissues. A third type of *Fusarium* resistance (type III) was described by Wang and Miller (1988) as the insensitivity of wheat lines to toxins or the ability of the resistant cultivar to degrade mycotoxins.

Integrated control strategies are essential to prevent *Fusarium* diseases in modern agriculture. The use of resistant cultivars is considered necessary for numerous reasons. Firstly, modern agronomic practices such as reduced tillage and cereal and maize rich crop rotations have the tendency to increase the amount of *Fusarium* related diseases. In addition to this, chemical control measures are only partly effective as only few fungicides are sufficiently active against *Fusarium* and their application must be performed within a time frame of a few days around the flowering stage of the plants. For the protection of maize cultures no fungicides are commercially available up to now. Understanding *Fusarium* lifecycle and infection pathways is the first step in preventing disease. Nevertheless, uncontrollable factors such as weather conditions during as well as economic or ecologic interests prevent sustainable success in the reduction of these pathogens. Plant breeders have made significant progress in the development of *Fusarium* resistant maize and wheat varieties by identifying genetic regions, which are linked to the resistance of plants (Buerstmayer et al., 2003; Draeger et al., 2007). Resistance against *Fusarium* is a quantitative trait, which is governed by many independent genes distributed on several distinct genetic regions (quantitative trait loci, QTLs) in the plant genome. Therefore an effective approach to investigate FHB resistance is the identification of the QTLs and their mapping (Buerstmayer et al., 2009). The mapping of QTLs is the basis for further efforts towards a straightforward marker assisted resistance breeding strategy. Interestingly, often the best regionally adapted and highly productive crop varieties are susceptible to FHB. For this reason, breeders are faced with the difficult task to combine adaption to certain locations with high yield and *Fusarium* resistance.

The production of mycotoxins has been identified as a crucial factor for successful infection . Deoxynivalenol is considered the most common occurring mycotoxin involving *Fusarium* infection. However, DON levels in infected grain vary significantly amongst wheat cultivars (Bai et al., 2001) infected with the same *Fusarium* isolate. The evaluation of *Fusarium* resistance of either the parents in current breeding programs or the control of resistance levels of novel lines can be carried out by chemical analysis of the DON accumulation in the seeds. In general DON levels in resistant plants are much lower than those found in more susceptible cultivars. Artificial infection of wheat-ears with *Fusarium* conidia followed by determination of DON content and visual scoring of the head blight symptoms are the currently established methods for resistance assessment. Nevertheless, the determinations of disease symptoms in combination with the mycotoxin content of grain are both indirect methods to evaluate plant resistance. Furthermore, both methods observe the same effect, as visual disease symptoms are mainly caused by mycotoxin intoxication and not by the growth of mycelium. Studies have shown that the amount of fungal mycelia formed during infection not always correlates well with these parameters (Waalwijk et al, 2004; Hill et al., 2006; Brunner et al., 2009; Kulik et al. 2011). Asymptomatic kernels may contain significant amounts of mycotoxins while symptomatic kernels within the same sample may not.

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants:

developed to detect plant pathogens belonging to different genera.

developed tests.

the fungal biomass.

Quantitative PCR and ELISA Accurately Determine *Fusarium* Biomass 207

scientists working on agricultural and plant pathology related topics recognized the potential of this analytical tool. Within only a few years the number of annual ELISA based publications in the AGRICOLA database rose from zero to more than 150 and numerous antibodies were

The first immuno assay for *Fusarium* was developed at the University of Göttingen in 1989 by J.G. Unger and published in his PhD thesis. The author succeeded in the recognition of *F. culmorum* exoantigens (compounds secreted by *Fusarium* during growth) by an antibody. However, after this first success of the ELISA in *Fusarium* detection almost 15 years passed by until this *F. culmorum* ELISA was applied to practical applications (Chala et al., 2003). During these field tests the authors found out that the developed antibody is not specific to *F. culmorum* but binds to antigens from various *Fusarium* species. Over the years numerous studies on the development of anti-*Fusarium* antibodies have been published. Although most authors used *F. graminearum* and/or *F. culmorum* for immunization, no group succeeded in the production of highly specific antibodies. Gan et al., (1997) immunized chicken with soluble exoantigens and with soluble frations from homogenized mycelia of F. graminarum, *F. poae* and *F. sporotrichioides*. The *F. graminearum* and *F. sporotrichioides* exoantigen antibodies showed cross-reactivities with almost all common *Fusarium* species. The antibody against *F. poae* exoantigens showed absolutely no cross-reactivity against other *Fusarium* species or against other filamentous fungi. Up to now this is the only speciesspecific antibody for a *Fusarium* spp. Hill et al., (2006) probably produced the bestcharacterized antibody so far by isolating monoclonal antibodies produced by IF8 cell lines for the detection of *Fusarium*. These antibodies detected species from different phylogenetic clades and the authors also demonstrated that no cross-reaction – even with closely related ascomycetes – could be observed. Unfortunately, no publication describes the essential analytical parameters like limit of detection, limit of quantification or a linear range of the

In general, antibodies detect many different *Fusarium* species but the specificity of PCR based assays has not yet been obtained by immunoassays. Neither the detection of particular species – except one example for *F. poae* – nor the detection of a particular toxigenic group (e.g. trichothecene producers or fumonisin producers) is possible with ELISA methods. Furthermore, most antibodies were raised against unknown compounds secreted by *Fusarium* during growth in liquid medium (exoantigens). As usually the secretion is not a constant attribute but is subjected to complex regulation, the amount of exoantigens secreted during the infection of grains might not always be constant. For this reason, the amount of *Fusarium* antigen detected in a sample may not always correlate with

Many of the developed PCR based tests for the determination of *Fusarium* focus on the screening of harvested grain and frequently the correlation of *Fusarium* DNA content and toxin accumulation is the central topic. Only few (references) of the many *Fusarium* PCR publications deal with plant pathology related questions whereas all developed ELISA methods were extensively used in field applications to monitor the efficacy of fungicide

**4.1.2 Applications of** *Fusarium* **ELISAs for agriculture and plant pathology** 

**4.1.1 ELISAs based methods for the detection and quantification of** *Fusarium*

#### **4. Methods for the identification of** *Fusarium* **and quantification of the infection**

Quantification and identification of *Fusarium* species in agriculture or plant pathology is traditionally carried out using culture based methods. Usually single Kernels are spread on petri dishes with appropriate medium and the number of infected kernels is counted after several days. For a further classification of the species a morphological investigation of the grown mycelium is necessary. This kind of species determination is time consuming and requires experience and specific expertise. Furthermore, culture based methods rely on living propagules and the obtained results not always reflects the biological situation. The pathogen detection is only reliable at late stages of the infection when a spread of the disease can no longer be controlled by fungicides (McCArtney et al., 2003). Besides these drawbacks the results are only semi-quantitative as only the number of infected kernels can be determined but not the grade of infection of each kernel. Other conventional methods include instrumental analysis such as ergosterol quantification. Ergosterol is a characteristic compound of fungal cell walls. However this approach is not specific to pathogens or a certain fungal species and the ergosterol amount relative to the fungal mycelia varies within *Fusarium* species (Pasanen et al., 1999). Finally, the analytical determination of ergosterol is not easier than that of DON, resulting in an alternative method, which is not commercially applicable to determine the *Fusarium* resistance of plants.

Recent studies aim at developing more direct techniques to quantify *Fusarium* related diseases which can i) reduce the number of analyses to accurately assess *Fusarium* infection, ii) eliminate pleiotropy between disease symptomology and mycotoxin production, iii) reduce the error associated with environmental effects and iv) reduce errors related with asymptomatic expression of FHB (Hill et al., 2008). In general, two distinct approaches have become accepted within the last two decades: immunoassays and (quantitative) PCR. The enzyme linked immunosorbent assay (ELISA) has been developed as a direct measure of *Fusarium* spp. biomass in infested grain samples or plant tissue. ELISA provides at least genus specificity through specific fungal antigens and its ease of sample preparation and low costs make it an interesting novel method. Another approach to overcome the drawbacks of conventional identifications is the development of screening techniques based on DNA identification which are nowadays well established for *Fusarium* species and a broad range of available literature guarantees to find a suitable PCR assay for many applications. Unlike conventional detection methods, samples can be tested directly with ELISA or PCR – no elaborate isolation and cultivation steps are necessary for a suitable detection or quantification.

#### **4.1 Enzyme linked immunosorbent assays (ELISAs)**

Enzyme linked immunosorbent assays are based on the specific recognition capabilities of antibodies. These antibodies are usually derived from the immunization of animals (usually rabbits, mice, chicken or goat) with certain immunogens such as culture filtrates or mycelial compounds. After repeated injection of the immunogen blood samples are taken and the serum is used either as a whole or it is applied after certain clean-up steps for the ELISA tests. For the production of the well-defined monoclonal antibodies lymphocytes are isolated from immunized mice and are fused to myeloma cells. The resultant clonal hybridoma cells can be maintained in culture. Soon after the development of the ELISA method in the early 1970s

Quantification and identification of *Fusarium* species in agriculture or plant pathology is traditionally carried out using culture based methods. Usually single Kernels are spread on petri dishes with appropriate medium and the number of infected kernels is counted after several days. For a further classification of the species a morphological investigation of the grown mycelium is necessary. This kind of species determination is time consuming and requires experience and specific expertise. Furthermore, culture based methods rely on living propagules and the obtained results not always reflects the biological situation. The pathogen detection is only reliable at late stages of the infection when a spread of the disease can no longer be controlled by fungicides (McCArtney et al., 2003). Besides these drawbacks the results are only semi-quantitative as only the number of infected kernels can be determined but not the grade of infection of each kernel. Other conventional methods include instrumental analysis such as ergosterol quantification. Ergosterol is a characteristic compound of fungal cell walls. However this approach is not specific to pathogens or a certain fungal species and the ergosterol amount relative to the fungal mycelia varies within *Fusarium* species (Pasanen et al., 1999). Finally, the analytical determination of ergosterol is not easier than that of DON, resulting in an alternative method, which is not commercially

Recent studies aim at developing more direct techniques to quantify *Fusarium* related diseases which can i) reduce the number of analyses to accurately assess *Fusarium* infection, ii) eliminate pleiotropy between disease symptomology and mycotoxin production, iii) reduce the error associated with environmental effects and iv) reduce errors related with asymptomatic expression of FHB (Hill et al., 2008). In general, two distinct approaches have become accepted within the last two decades: immunoassays and (quantitative) PCR. The enzyme linked immunosorbent assay (ELISA) has been developed as a direct measure of *Fusarium* spp. biomass in infested grain samples or plant tissue. ELISA provides at least genus specificity through specific fungal antigens and its ease of sample preparation and low costs make it an interesting novel method. Another approach to overcome the drawbacks of conventional identifications is the development of screening techniques based on DNA identification which are nowadays well established for *Fusarium* species and a broad range of available literature guarantees to find a suitable PCR assay for many applications. Unlike conventional detection methods, samples can be tested directly with ELISA or PCR – no elaborate isolation and cultivation steps are necessary for a suitable

Enzyme linked immunosorbent assays are based on the specific recognition capabilities of antibodies. These antibodies are usually derived from the immunization of animals (usually rabbits, mice, chicken or goat) with certain immunogens such as culture filtrates or mycelial compounds. After repeated injection of the immunogen blood samples are taken and the serum is used either as a whole or it is applied after certain clean-up steps for the ELISA tests. For the production of the well-defined monoclonal antibodies lymphocytes are isolated from immunized mice and are fused to myeloma cells. The resultant clonal hybridoma cells can be maintained in culture. Soon after the development of the ELISA method in the early 1970s

**4. Methods for the identification of** *Fusarium* **and quantification of the** 

applicable to determine the *Fusarium* resistance of plants.

**4.1 Enzyme linked immunosorbent assays (ELISAs)** 

detection or quantification.

**infection** 

scientists working on agricultural and plant pathology related topics recognized the potential of this analytical tool. Within only a few years the number of annual ELISA based publications in the AGRICOLA database rose from zero to more than 150 and numerous antibodies were developed to detect plant pathogens belonging to different genera.

#### **4.1.1 ELISAs based methods for the detection and quantification of** *Fusarium*

The first immuno assay for *Fusarium* was developed at the University of Göttingen in 1989 by J.G. Unger and published in his PhD thesis. The author succeeded in the recognition of *F. culmorum* exoantigens (compounds secreted by *Fusarium* during growth) by an antibody. However, after this first success of the ELISA in *Fusarium* detection almost 15 years passed by until this *F. culmorum* ELISA was applied to practical applications (Chala et al., 2003). During these field tests the authors found out that the developed antibody is not specific to *F. culmorum* but binds to antigens from various *Fusarium* species. Over the years numerous studies on the development of anti-*Fusarium* antibodies have been published. Although most authors used *F. graminearum* and/or *F. culmorum* for immunization, no group succeeded in the production of highly specific antibodies. Gan et al., (1997) immunized chicken with soluble exoantigens and with soluble frations from homogenized mycelia of F. graminarum, *F. poae* and *F. sporotrichioides*. The *F. graminearum* and *F. sporotrichioides* exoantigen antibodies showed cross-reactivities with almost all common *Fusarium* species. The antibody against *F. poae* exoantigens showed absolutely no cross-reactivity against other *Fusarium* species or against other filamentous fungi. Up to now this is the only speciesspecific antibody for a *Fusarium* spp. Hill et al., (2006) probably produced the bestcharacterized antibody so far by isolating monoclonal antibodies produced by IF8 cell lines for the detection of *Fusarium*. These antibodies detected species from different phylogenetic clades and the authors also demonstrated that no cross-reaction – even with closely related ascomycetes – could be observed. Unfortunately, no publication describes the essential analytical parameters like limit of detection, limit of quantification or a linear range of the developed tests.

In general, antibodies detect many different *Fusarium* species but the specificity of PCR based assays has not yet been obtained by immunoassays. Neither the detection of particular species – except one example for *F. poae* – nor the detection of a particular toxigenic group (e.g. trichothecene producers or fumonisin producers) is possible with ELISA methods. Furthermore, most antibodies were raised against unknown compounds secreted by *Fusarium* during growth in liquid medium (exoantigens). As usually the secretion is not a constant attribute but is subjected to complex regulation, the amount of exoantigens secreted during the infection of grains might not always be constant. For this reason, the amount of *Fusarium* antigen detected in a sample may not always correlate with the fungal biomass.

#### **4.1.2 Applications of** *Fusarium* **ELISAs for agriculture and plant pathology**

Many of the developed PCR based tests for the determination of *Fusarium* focus on the screening of harvested grain and frequently the correlation of *Fusarium* DNA content and toxin accumulation is the central topic. Only few (references) of the many *Fusarium* PCR publications deal with plant pathology related questions whereas all developed ELISA methods were extensively used in field applications to monitor the efficacy of fungicide

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants:

resistance than visual disease assessment or toxin measurements.

**4.2 Detection and quantification of** *Fusarium* **by PCR methods** 

with the two other methods.

target organisms (McCartney et al., 2003).

to different genera.

Quantitative PCR and ELISA Accurately Determine *Fusarium* Biomass 209

and disease data were collected by visual disease assessment, determination of the DON accumulation and the *Fusarium* ELISA. The obtained data were subjected to statistical analyses and the authors calculated a model for the prediction of different replications and different environments necessary to certainly identify differences in *Fusarium* resistances. Interestingly, the determination of DON was identified as the factor that is least feasible for resistance studies. The quantification of the *Fusarium* biomass by ELISA required less different locations and fewer field replicates per location to classify barley varieties according to their *Fusarium*

Often the correlation between visual disease scoring and DON content is better than between ELISA results and DON content. This fact has also been demonstrated in a Slovakian study (Slikova et al., 2009) for the infection of winter wheat with *F. culmorum*. As visual assessment just records the damage caused by the fungal toxins the correlation between symptoms and the DON content is obvious. The fact that visual symptoms and DON content are not independent features makes the combined use of only these two factors for resistance determination questionable. Therefore, Slikova et al. (2009) recommend the application of the *Fusarium* ELSIA to get more reliable results for plant resistance than

The extensive application of DNA based identification technologies has increased the knowledge on suitable diagnostic DNA fragments of *Fusarium* species such as ITS (internal transcribes spacer) or IGS (intergenic spacer) sequences (Gagkaeva & Yli-Mattila, 2004, Jurado et al., 2006, Konstantinova & Yli-Mattila, 2004, Kulik 2008, Yli-Mattila, 2004), mitochondrial DNA (Laday et al., 2004), the β-tubulin encoding gene (Yli-Mattila, 2004, Mach et al., 2004, Reischer et al., 2004), the translation elongation factor gene (Knutsen et al., 2004) and the calmodulin gene (Mule et al., 2004) which were sequenced from numerous *Fusarium* spp. As a result highly specific PCR primers could be developed for *Fusarium* detection. In order to distinguish between producers and non-producers of certain toxins the genes from biosynthesis pathways for mycotoxins were accurately studied. The sequence information gained throughout numerous molecular taxonomic investigations allows not only qualitative applications to detect particular species but also quantitative measurement of fungal biomass. Different techniques like DGGE, RFLP and AFLP can be applied for the identification of adequate PCR targets for a review see Brunner & Mach, (2010), for quantitative detection methods however, only the real-time PCR has been shown to be applicable. Molecular diagnosis of plant pathogenic fungi has been proven to be highly specific, very sensitive and fast and relatively insensitive to microbial backgrounds and non-

Within the last decade numerous PCR based assays have been published for the detection of most agriculturally important *Fusarium* species. An intensive literature study reveals that all such publications can be separated into two distinct groups. Either the authors focus on highly specific test systems to allow the differentiation of *Fusarium* species or the focus is the comprehensive detection of a whole group sharing a common feature. The latter assays usually use a key-gene for a biosynthetic pathway – e.g. for mycotoxin synthesis – as a target for PCR. This system allows the detection or even the quantification of all isolates, which are able to produce a certain class of toxins even if they belong to different species or

application (Chala et al., 2003) or to assess the *Fusarium* resistance of crop lines (Miedaner et al., 1994, Miedaner et al., 2004, Hill et al., 2006, Slikova 2009, Rohde & Rabenstein 2005).

#### **4.1.2.1 Evaluation of fungicide efficacy with a** *Fusarium* **ELISA**

Chala et al., (2003) presented a comprehensive study on the evaluation of fungicide application to reduce *Fusarium* infection and DON content in wheat. Winter wheat was planted in four field replications and artificially infected with *F. culmorum* conidia at flowering stage by spray inoculation. Five different fungicides were tested at different application times alone and in various combinations. The efficiency of the treatment was determined by visual scoring of disease symptoms, *Fusarium* ELISA, yield a 1000-grain weight. The combination of these different methods allows the evaluation of the results obtained by the immunoassay. The results of the field trials reveal clearly that only one fungicide showed a high efficiency in the reduction of fungal biomass and DON content. All applied methods led to the same conclusion. Interestingly, the application of some fungicides at a later growth stage after flowering did not affect the fungal biomass but slightly increased the production of DON. In general the study demonstrates clearly the potential of the developed *Fusarium* ELISA to obtain a deeper insight into biomass formation. The main advantage of the ELISA based determination of infection is the enhanced failure-safety.

#### **4.1.2.2 Evaluation of** *Fusarium* **resistance with ELISA tests**

The comparison of the resistance of different plant varieties is often a challenge for breeders. The increase in *Fusarium* resistance of novel crop lines is just marginal and for some plants like durum or triticale disease symptoms are usually not obvious. Furthermore, other fungal diseases like *Microdochium nivale* or *Septoria nodurum* also lead to similar symptoms as *Fusarium* head blight and the result of the visual assessment might be misinterpreted. Besides the PCR based methods described above the ELISA tests represent the only available tool to determine fungal biomass and so the application for resistance tests is self-evident.

The first field test for *Fusarium* resistance based on ELISA analysis was performed in the early 1990s (Miedaner et al., 1994). Winter rye was infected with *F. culmorum* colonized wheat flour in November when the plants have reached the three-leaf status. The colonization of the host plant started early after inoculation and increased continuously till full maturity. Interestingly, different genotypes showed the highest variance in *Fusarium* protein content during medium growth states. In adult states almost no difference in *Fusarium* infection between resistant and susceptible plants was found. The disappearance of the discrepancy results in saprophytic growth of *F. culmorum* on rye during ripening and therefore the medium growth stages are the optimal date to discriminate resistant from susceptible varieties. The same group also investigated the *F. culmorum* resistance of wheat and triticale cultivars in six different environments and demonstrated the performance of ELISA biomass determination for two more cereal crops (Miedaner et al., 2004). However, the correlation of the ELISA based *Fusarium* protein content to the DON content in a sample was not better than the correlation between DON and visual disease symptom assessment. Another ELISA test based on monoclonal antibodies was optimized for the quantification of *Fusarium* in barley (Hill et al., 2006) and was used to study the influence of the applied analytical method on the results of resistance tests (Hill et al., 2008). A mapping population was grown in two environments and breeding lines were grown at four different locations

application (Chala et al., 2003) or to assess the *Fusarium* resistance of crop lines (Miedaner et al., 1994, Miedaner et al., 2004, Hill et al., 2006, Slikova 2009, Rohde & Rabenstein 2005).

Chala et al., (2003) presented a comprehensive study on the evaluation of fungicide application to reduce *Fusarium* infection and DON content in wheat. Winter wheat was planted in four field replications and artificially infected with *F. culmorum* conidia at flowering stage by spray inoculation. Five different fungicides were tested at different application times alone and in various combinations. The efficiency of the treatment was determined by visual scoring of disease symptoms, *Fusarium* ELISA, yield a 1000-grain weight. The combination of these different methods allows the evaluation of the results obtained by the immunoassay. The results of the field trials reveal clearly that only one fungicide showed a high efficiency in the reduction of fungal biomass and DON content. All applied methods led to the same conclusion. Interestingly, the application of some fungicides at a later growth stage after flowering did not affect the fungal biomass but slightly increased the production of DON. In general the study demonstrates clearly the potential of the developed *Fusarium* ELISA to obtain a deeper insight into biomass formation. The main advantage of the ELISA based determination of infection is the

The comparison of the resistance of different plant varieties is often a challenge for breeders. The increase in *Fusarium* resistance of novel crop lines is just marginal and for some plants like durum or triticale disease symptoms are usually not obvious. Furthermore, other fungal diseases like *Microdochium nivale* or *Septoria nodurum* also lead to similar symptoms as *Fusarium* head blight and the result of the visual assessment might be misinterpreted. Besides the PCR based methods described above the ELISA tests represent the only available tool to

The first field test for *Fusarium* resistance based on ELISA analysis was performed in the early 1990s (Miedaner et al., 1994). Winter rye was infected with *F. culmorum* colonized wheat flour in November when the plants have reached the three-leaf status. The colonization of the host plant started early after inoculation and increased continuously till full maturity. Interestingly, different genotypes showed the highest variance in *Fusarium* protein content during medium growth states. In adult states almost no difference in *Fusarium* infection between resistant and susceptible plants was found. The disappearance of the discrepancy results in saprophytic growth of *F. culmorum* on rye during ripening and therefore the medium growth stages are the optimal date to discriminate resistant from susceptible varieties. The same group also investigated the *F. culmorum* resistance of wheat and triticale cultivars in six different environments and demonstrated the performance of ELISA biomass determination for two more cereal crops (Miedaner et al., 2004). However, the correlation of the ELISA based *Fusarium* protein content to the DON content in a sample was not better than the correlation between DON and visual disease symptom assessment. Another ELISA test based on monoclonal antibodies was optimized for the quantification of *Fusarium* in barley (Hill et al., 2006) and was used to study the influence of the applied analytical method on the results of resistance tests (Hill et al., 2008). A mapping population was grown in two environments and breeding lines were grown at four different locations

determine fungal biomass and so the application for resistance tests is self-evident.

**4.1.2.1 Evaluation of fungicide efficacy with a** *Fusarium* **ELISA** 

**4.1.2.2 Evaluation of** *Fusarium* **resistance with ELISA tests** 

enhanced failure-safety.

and disease data were collected by visual disease assessment, determination of the DON accumulation and the *Fusarium* ELISA. The obtained data were subjected to statistical analyses and the authors calculated a model for the prediction of different replications and different environments necessary to certainly identify differences in *Fusarium* resistances. Interestingly, the determination of DON was identified as the factor that is least feasible for resistance studies. The quantification of the *Fusarium* biomass by ELISA required less different locations and fewer field replicates per location to classify barley varieties according to their *Fusarium* resistance than visual disease assessment or toxin measurements.

Often the correlation between visual disease scoring and DON content is better than between ELISA results and DON content. This fact has also been demonstrated in a Slovakian study (Slikova et al., 2009) for the infection of winter wheat with *F. culmorum*. As visual assessment just records the damage caused by the fungal toxins the correlation between symptoms and the DON content is obvious. The fact that visual symptoms and DON content are not independent features makes the combined use of only these two factors for resistance determination questionable. Therefore, Slikova et al. (2009) recommend the application of the *Fusarium* ELSIA to get more reliable results for plant resistance than with the two other methods.

#### **4.2 Detection and quantification of** *Fusarium* **by PCR methods**

The extensive application of DNA based identification technologies has increased the knowledge on suitable diagnostic DNA fragments of *Fusarium* species such as ITS (internal transcribes spacer) or IGS (intergenic spacer) sequences (Gagkaeva & Yli-Mattila, 2004, Jurado et al., 2006, Konstantinova & Yli-Mattila, 2004, Kulik 2008, Yli-Mattila, 2004), mitochondrial DNA (Laday et al., 2004), the β-tubulin encoding gene (Yli-Mattila, 2004, Mach et al., 2004, Reischer et al., 2004), the translation elongation factor gene (Knutsen et al., 2004) and the calmodulin gene (Mule et al., 2004) which were sequenced from numerous *Fusarium* spp. As a result highly specific PCR primers could be developed for *Fusarium* detection. In order to distinguish between producers and non-producers of certain toxins the genes from biosynthesis pathways for mycotoxins were accurately studied. The sequence information gained throughout numerous molecular taxonomic investigations allows not only qualitative applications to detect particular species but also quantitative measurement of fungal biomass. Different techniques like DGGE, RFLP and AFLP can be applied for the identification of adequate PCR targets for a review see Brunner & Mach, (2010), for quantitative detection methods however, only the real-time PCR has been shown to be applicable. Molecular diagnosis of plant pathogenic fungi has been proven to be highly specific, very sensitive and fast and relatively insensitive to microbial backgrounds and nontarget organisms (McCartney et al., 2003).

Within the last decade numerous PCR based assays have been published for the detection of most agriculturally important *Fusarium* species. An intensive literature study reveals that all such publications can be separated into two distinct groups. Either the authors focus on highly specific test systems to allow the differentiation of *Fusarium* species or the focus is the comprehensive detection of a whole group sharing a common feature. The latter assays usually use a key-gene for a biosynthetic pathway – e.g. for mycotoxin synthesis – as a target for PCR. This system allows the detection or even the quantification of all isolates, which are able to produce a certain class of toxins even if they belong to different species or to different genera.

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants:

2008;).

compounds.

Quantitative PCR and ELISA Accurately Determine *Fusarium* Biomass 211

In contrast to the practical applications of the developed ELISA tests, the PCR assays are

Some methods were developed to identify the chemotype of different *Fusarium* strains on a molecular basis. These assays are designed to differentiate between nivalenol, 3- and 15 acetyl-deoxynivalenol chemotypes of *F. graminearum*, *F. culmorum*, and F. cerealis. The biosynthetic pathway of trichothecene production is highly conserved between different *Fusarium* species and the genes within the trichothecene gene cluster are well investigated. Based on this knowledge three genes were chosen in all studies to identify the chemotypes: tri3, tri5, tri7 and tri13 or regions between these genes. The developed assays were applied to *Fusarium* monitoring programs in numerous countries of Europe and Nortehrn America (Chandler et al., 2003; Jennings et al. 2004; Lia et al., 2005; Quarta et al., 2006; Stepien et al.,

A tri5 based assay was used to evaluate the efficiency of fungicides against *F. graminearum* and *F. culmorum* (Edwards et al., 2001. Three chemical fungicides were applied alone in various concentrations and in combinations. Interestingly, the authors could demonstrate that different pathogenic fungi require different fungicides for the optimal control of the disease. The study revealed a good correlation between the DON content of samples and the *Fusarium* DNA. Furthermore, the ration between DON and the fungal DNA was not altered by the application of fungicides. This fact is of particular interest as previous in vitro studies postulated the enhanced toxin production of *Fusarium* after the treatment with antifungal

Most of the published studies focus on correlations between accumulated toxins and the PCR determined fungal DNA concentration in harvested grain and the PCR is postulated as a screening method for applications in food and feed safety. For some of these studies species specific assays were used to assess the biomass of various *Fusarium* species (Schnerr et al., 2000; Waalwijk et al., 2004; Brandfass & Karlovsky, 2006; Yli-Mattila et al. 2008) or group specific assays were used to identify of quantify certain toxin producers (Schnerr et

Although the sensitive PCR methods represent an optimal tool to monitor even minor amounts of *Fusarium* during colonialization of a host these applications are rare. Reischer et al. (2004) developed a TaqMan based quantitative PCR assay to quantify *F. graminearum* directly from infected plant material. A central spikelet of wheat heads was artificially infected with conidia and the increase of fungal biomass was monitored by real-time PCR and the disease symptoms were also assessed visually. Due to the superior sensitivity of the PCR assay the disease could be detected and quantified the first day after inoculation, whereas the first visual symptoms became obvious five days later. Nicholson et al. (1998) used PCR tests for *F. culmorum* and *F. graminearum* to address the fungal spread in wheat. The authors found out that the severity of *Fusarium* stem rot caused by *F. culmorum* correlates not only with the inoculums load but also with the time point the inoculums is applied. The earlier in the season the conidia are applied the more fungal DNA could be measured in the stem. The influence of trichothecene production on successful infection was tested in the same study with toxigenic and atoxigenic *F. graminearum* isolates. The toxin producers colonized grain better than non-producers, supporting the theory of trichothecens

al. 2001, 2002; Bluhm et al. 2004; Waalwijk et al., 2008; Kulik et al., 2011).

**4.2.3 Applications of the** *Fusarium* **PCR in agriculture and plant pathology** 

only rarely used to address typical questions related to plant pathology.

#### **4.2.1 Species-specific PCR assays**

Species-specific assays allow the determination of one particular *Fusarium* species. This method focuses on high selectivity in order to quantify a target species even in a background of highly similar isolates. Several TaqMan based PCR assays have been developed for the quantification of some of the predominant species associated with head blight in Europe including *F. graminearum*, *F. poae*, *F. culmorum*, F. sporotrichioides, F. verticillioides or *F. avenaceum* . The authors clearly demonstrated the advantage of quantitative real time PCR systems combined with a high-throughput DNA extraction protocol over morphologic based methods. Unlike conventional agar-plating techniques, the quantification of different fungal species is possible directly from infested material (grain or plant tissue) and the entire procedure is less time consuming than microbiological methods (for a review see Brunner & Mach, 2010).

#### **4.2.2 Group specific assays**

Group specific assays were designed for the simultaneous quantification of all strains, which produce certain toxins such as trichothecenes, fumonisins or enniatin, based on key-genes involved in the mycotoxin biosynthesis. In contrast to the species-specific assays, a whole group of *Fusarium* spp., regardless of taxonomic origin, that is able to produce a certain class of mycotoxins can be quantified in a single run. A correlation between qPCR determined fungal biomass and DON, fumonisin or enniatin content in cereals could be shown using these group specific assays.


Table 1. Overview of frequently applied PCR assays. This table cites the original method development papers (qualitative and quantitative) but not potentially following publications demonstrating the practical application of these assays.

Species-specific assays allow the determination of one particular *Fusarium* species. This method focuses on high selectivity in order to quantify a target species even in a background of highly similar isolates. Several TaqMan based PCR assays have been developed for the quantification of some of the predominant species associated with head blight in Europe including *F. graminearum*, *F. poae*, *F. culmorum*, F. sporotrichioides, F. verticillioides or *F. avenaceum* . The authors clearly demonstrated the advantage of quantitative real time PCR systems combined with a high-throughput DNA extraction protocol over morphologic based methods. Unlike conventional agar-plating techniques, the quantification of different fungal species is possible directly from infested material (grain or plant tissue) and the entire procedure is less time consuming than microbiological methods

Group specific assays were designed for the simultaneous quantification of all strains, which produce certain toxins such as trichothecenes, fumonisins or enniatin, based on key-genes involved in the mycotoxin biosynthesis. In contrast to the species-specific assays, a whole group of *Fusarium* spp., regardless of taxonomic origin, that is able to produce a certain class of mycotoxins can be quantified in a single run. A correlation between qPCR determined fungal biomass and DON, fumonisin or enniatin content in cereals could be shown using

RAPD derived qualitative Schilling et al., 1996

RAPD derived qualitative Nicholson et al., 1998

RAPD derived quantitative Waalwijk et al. 2004

quantitative Li et al., 2008

Detectable species PCR target Type of assay Reference

trichothecene producers *tri5* qualitative Niessen et al., 1998

trichothecene producers *tri5* quantitative Schnerr et al., 2001 trichothecene producers *tri5* qualitative Edwards et al., 2001 *F. graminearum, F. poae* IGS quantitative Yli-Mattila et al., 2008 fumonisin producers fum1 quantitative Bluhm et al., 2004 *F. graminearum tub1* quantitative Reischer et al., 2004

fumonisin producers *fum1* quantitative Waalwijk et al. 2008 enniatin producers *esyn1* quantitative Kulik et al. 2011 Table 1. Overview of frequently applied PCR assays. This table cites the original method

development papers (qualitative and quantitative) but not potentially following

**4.2.1 Species-specific PCR assays** 

(for a review see Brunner & Mach, 2010).

**4.2.2 Group specific assays** 

these group specific assays.

*F. graminearum, F. culmorum, F. avenaceum*

*F. graminearum*, *F.* 

*F. graminearum, F. culmorum, F. avenaceum* 

*F. solani* rRNA, small

subunit

publications demonstrating the practical application of these assays.

*culmorum*

#### **4.2.3 Applications of the** *Fusarium* **PCR in agriculture and plant pathology**

In contrast to the practical applications of the developed ELISA tests, the PCR assays are only rarely used to address typical questions related to plant pathology.

Some methods were developed to identify the chemotype of different *Fusarium* strains on a molecular basis. These assays are designed to differentiate between nivalenol, 3- and 15 acetyl-deoxynivalenol chemotypes of *F. graminearum*, *F. culmorum*, and F. cerealis. The biosynthetic pathway of trichothecene production is highly conserved between different *Fusarium* species and the genes within the trichothecene gene cluster are well investigated. Based on this knowledge three genes were chosen in all studies to identify the chemotypes: tri3, tri5, tri7 and tri13 or regions between these genes. The developed assays were applied to *Fusarium* monitoring programs in numerous countries of Europe and Nortehrn America (Chandler et al., 2003; Jennings et al. 2004; Lia et al., 2005; Quarta et al., 2006; Stepien et al., 2008;).

A tri5 based assay was used to evaluate the efficiency of fungicides against *F. graminearum* and *F. culmorum* (Edwards et al., 2001. Three chemical fungicides were applied alone in various concentrations and in combinations. Interestingly, the authors could demonstrate that different pathogenic fungi require different fungicides for the optimal control of the disease. The study revealed a good correlation between the DON content of samples and the *Fusarium* DNA. Furthermore, the ration between DON and the fungal DNA was not altered by the application of fungicides. This fact is of particular interest as previous in vitro studies postulated the enhanced toxin production of *Fusarium* after the treatment with antifungal compounds.

Most of the published studies focus on correlations between accumulated toxins and the PCR determined fungal DNA concentration in harvested grain and the PCR is postulated as a screening method for applications in food and feed safety. For some of these studies species specific assays were used to assess the biomass of various *Fusarium* species (Schnerr et al., 2000; Waalwijk et al., 2004; Brandfass & Karlovsky, 2006; Yli-Mattila et al. 2008) or group specific assays were used to identify of quantify certain toxin producers (Schnerr et al. 2001, 2002; Bluhm et al. 2004; Waalwijk et al., 2008; Kulik et al., 2011).

Although the sensitive PCR methods represent an optimal tool to monitor even minor amounts of *Fusarium* during colonialization of a host these applications are rare. Reischer et al. (2004) developed a TaqMan based quantitative PCR assay to quantify *F. graminearum* directly from infected plant material. A central spikelet of wheat heads was artificially infected with conidia and the increase of fungal biomass was monitored by real-time PCR and the disease symptoms were also assessed visually. Due to the superior sensitivity of the PCR assay the disease could be detected and quantified the first day after inoculation, whereas the first visual symptoms became obvious five days later. Nicholson et al. (1998) used PCR tests for *F. culmorum* and *F. graminearum* to address the fungal spread in wheat. The authors found out that the severity of *Fusarium* stem rot caused by *F. culmorum* correlates not only with the inoculums load but also with the time point the inoculums is applied. The earlier in the season the conidia are applied the more fungal DNA could be measured in the stem. The influence of trichothecene production on successful infection was tested in the same study with toxigenic and atoxigenic *F. graminearum* isolates. The toxin producers colonized grain better than non-producers, supporting the theory of trichothecens

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants:

pathologist might still be high to enter a technical field like quantitative PCR.

Development Fund (ERDF) of the European Union for financial support.

applications.

**7. References** 

**6. Acknowledgements** 

An investigation of the NCBI pubmed database reveals that the published studies applying *Fusarium* PCR methods in field-trials increase continuously. Although less novel assays are developed, more and more previously published methods are included in current studies, which might indicate the changeover from PCR based method development to PCR

The authors thank the Federal Country Lower Austria and the European Regional

Bai, G.H., Plattner, R., Desjardins, A. & Kolb, F. (2001). Resistance to *Fusarium* head blight and deoxynivalenol accumulation in wheat. *Plant Breeding*, 120(1), pp 1-6. Baird, R., Abbas, H.K., Windham, G., Williams, P., Baird, S., Ma , P., Kelley, R., Hawkins L.

& Scruggs M. (2008). Identification of select fumonisin forming *Fusarium* species using PCR applications of the polyketide synthase gene and its relationship to fumonisin production in vitro. *International Journal of Molecular Sciences*, 9: 554-570.

Quantitative PCR and ELISA Accurately Determine *Fusarium* Biomass 213

always the case as long dry periods during ripening in combination with intense UV radiation can reduce the survival of fungal mycelium or even of conidia. Furthermore, the morphologic discrimination of grown mycelium is difficult and experienced personal is indispensible. Novel methods for the detection and quantification of fungal biomass based on marker molecules (e.g. antigens or DNA) have the potential to revolutionize the field of pathogen monitoring in plant pathology. Within the last twenty years immunoassays and PCR methods found their way into this scientific field. ELISA test for different *Fusarium* species have been developed intensely in the 1990s and were applied in numerous field studies. However, the specificity of the produced antibodies leaded a lot to desire. Although particular species were used for the immunization of animals the resulting antibodies did detect many different *Fusarium* species. Only one group succeeded in the production of polyclonal antibodies which were specific to a single species, namely to *F. poae* . Furthermore, the re-production of antibodies is difficult as immunization is a complex procedure. The resulting immunoglobulins can vary in their features and therefore a careful characterization must be performed each production cycle. Interestingly with the availability of affordable real-time PCR cyclers on the market published studies using *Fusarium* ELISA almost disappeared. Most probably this tendency can be ascribed to two crucial advantages of PCR methods: i) these methods can be performed in high throughput and are easily transferred to other laboratories. Primers are synthesized commercially, are cheap and they are available within a few days. ii) PCR assays are highly specific and can be used to detect either genera or species and even distinctive isolates can be differentiated. Although quantitative PCR provides numerous advantages to study *Fusarium* in its environment only a few studies have been published to address biological problems. This might be due to the fact that nowadays food safety related questions are of high relevance and so the comparison between fungal DNA and mycotoxins is estimated as more important than other applications. On the other hand, the barrier for traditional plant

as a virulence factor. However, non-producing strains could also infect the wheat heads but the amount of detected fungal DNA was only 1 to 10% of the DNA amount found in plants infected with toxin producing strains. The infected wheat heads were also subjected to visual assessment of disease symptoms. Although the conservative method led to the same results the difference between toxigenic and atoxigenis strains was less pronounced. The above described assay for *F. culmorum* was also used to monitor natural stem rot in wheat in the U.K. (Nicholson et a., 2002), The authors found out that usually other pathogens than *F. culmorum* were the causal agent of this disease as almost no quantifiable DNA of *Fusarium* was present in the samples. The same *F. culmorum* assay was again used in combination with a *F. poae* assay to evaluate fungicide efficiency for *Fusarium* head blight control in a greenhouse experiment (Doohan et al., 1999). Under all tested conditions the applied fungicides reduced the disease severity between 20% and 80% and in general the results could be confirmed by visual monitoring of the disease symptoms.

A Canadian *Fusarium* monitoring program integrated PCR tests for *F. avenaceum*, *F. culmorum*, F. crookwellense, *F. poae*, *F. sporotrichioides*, *F. equiseti*, *F. pseudograminearum*, and *F. graminearum* to compare results with the traditional agar plating method (Demeke et al., 2005). For 83% of the tested grain samples the two methods led to the same results. However, the agar plate method not always gave positive results when DNA was found in samples. This leads to the assumption that PCR based methods provide enhanced sensitivity. Additionally, *F. graminearum*, *F. pseudograminearum* and *F. crookwellense* could not be distinguished by morphological analysis whereas PCR could clearly differentiate these species. This comprehensive study clearly demonstrated the power of DNA based methods if integrated into *Fusarium* monitoring programs. PCR allows the quantification (if applied as real-time PCR) and classification of numerous strains within a few hours of analysis. Classical methods need some days to weeks to give the same results and quantification is difficult as only the number of infected kernels can be determined.

An LNA-TaqMan based assays was used to investigate the differences in *Fusarium* resistance of twenty novel wheat lines ranging from highly susceptible to highly resistant (Brunner et al., 2009). The wheat-ears were inoculated with *F. graminearum* and *F. culmorum* in two consecutive years and the formation of mycotoxins, the accumulation of *Fusarium* DNA and the visual disease symptoms were recorded. This study demonstrated a certain discrepancy between visual scoring and quantitative PCR results. In accordance to other studies the visible symptoms matched perfectly the DNA content in medium to low resistant lines. In contrast, highly resistant lines with low *Fusarium* biomass – but nevertheless high amount of toxins – did sometimes not show any disease symptoms. The authors addressed this effect to the detoxification mechanism of high resistant plants as some toxins can be "masked" by the plant. So they are converted to less plant-toxic metabololites which do not damage the wheat-ears.

#### **5. Conclusions**

Visual assessment of disease caused by *Fusarium* species is frequently insufficient to identify the causal agent or to quantify the plant pathogen. In natural systems pathogenic fungi often occur in a combination of species, which can induce similar symptoms and a visual discrimination might be impossible. Other methods like agar plate assays rely on the fact that viable propagules are present in a sample. Especially in harvested grain this is not always the case as long dry periods during ripening in combination with intense UV radiation can reduce the survival of fungal mycelium or even of conidia. Furthermore, the morphologic discrimination of grown mycelium is difficult and experienced personal is indispensible. Novel methods for the detection and quantification of fungal biomass based on marker molecules (e.g. antigens or DNA) have the potential to revolutionize the field of pathogen monitoring in plant pathology. Within the last twenty years immunoassays and PCR methods found their way into this scientific field. ELISA test for different *Fusarium* species have been developed intensely in the 1990s and were applied in numerous field studies. However, the specificity of the produced antibodies leaded a lot to desire. Although particular species were used for the immunization of animals the resulting antibodies did detect many different *Fusarium* species. Only one group succeeded in the production of polyclonal antibodies which were specific to a single species, namely to *F. poae* . Furthermore, the re-production of antibodies is difficult as immunization is a complex procedure. The resulting immunoglobulins can vary in their features and therefore a careful characterization must be performed each production cycle. Interestingly with the availability of affordable real-time PCR cyclers on the market published studies using *Fusarium* ELISA almost disappeared. Most probably this tendency can be ascribed to two crucial advantages of PCR methods: i) these methods can be performed in high throughput and are easily transferred to other laboratories. Primers are synthesized commercially, are cheap and they are available within a few days. ii) PCR assays are highly specific and can be used to detect either genera or species and even distinctive isolates can be differentiated. Although quantitative PCR provides numerous advantages to study *Fusarium* in its environment only a few studies have been published to address biological problems. This might be due to the fact that nowadays food safety related questions are of high relevance and so the comparison between fungal DNA and mycotoxins is estimated as more important than other applications. On the other hand, the barrier for traditional plant pathologist might still be high to enter a technical field like quantitative PCR.

An investigation of the NCBI pubmed database reveals that the published studies applying *Fusarium* PCR methods in field-trials increase continuously. Although less novel assays are developed, more and more previously published methods are included in current studies, which might indicate the changeover from PCR based method development to PCR applications.

#### **6. Acknowledgements**

The authors thank the Federal Country Lower Austria and the European Regional Development Fund (ERDF) of the European Union for financial support.

#### **7. References**

212 Plant Pathology

as a virulence factor. However, non-producing strains could also infect the wheat heads but the amount of detected fungal DNA was only 1 to 10% of the DNA amount found in plants infected with toxin producing strains. The infected wheat heads were also subjected to visual assessment of disease symptoms. Although the conservative method led to the same results the difference between toxigenic and atoxigenis strains was less pronounced. The above described assay for *F. culmorum* was also used to monitor natural stem rot in wheat in the U.K. (Nicholson et a., 2002), The authors found out that usually other pathogens than *F. culmorum* were the causal agent of this disease as almost no quantifiable DNA of *Fusarium* was present in the samples. The same *F. culmorum* assay was again used in combination with a *F. poae* assay to evaluate fungicide efficiency for *Fusarium* head blight control in a greenhouse experiment (Doohan et al., 1999). Under all tested conditions the applied fungicides reduced the disease severity between 20% and 80% and in general the results

A Canadian *Fusarium* monitoring program integrated PCR tests for *F. avenaceum*, *F. culmorum*, F. crookwellense, *F. poae*, *F. sporotrichioides*, *F. equiseti*, *F. pseudograminearum*, and *F. graminearum* to compare results with the traditional agar plating method (Demeke et al., 2005). For 83% of the tested grain samples the two methods led to the same results. However, the agar plate method not always gave positive results when DNA was found in samples. This leads to the assumption that PCR based methods provide enhanced sensitivity. Additionally, *F. graminearum*, *F. pseudograminearum* and *F. crookwellense* could not be distinguished by morphological analysis whereas PCR could clearly differentiate these species. This comprehensive study clearly demonstrated the power of DNA based methods if integrated into *Fusarium* monitoring programs. PCR allows the quantification (if applied as real-time PCR) and classification of numerous strains within a few hours of analysis. Classical methods need some days to weeks to give the same results and quantification is

An LNA-TaqMan based assays was used to investigate the differences in *Fusarium* resistance of twenty novel wheat lines ranging from highly susceptible to highly resistant (Brunner et al., 2009). The wheat-ears were inoculated with *F. graminearum* and *F. culmorum* in two consecutive years and the formation of mycotoxins, the accumulation of *Fusarium* DNA and the visual disease symptoms were recorded. This study demonstrated a certain discrepancy between visual scoring and quantitative PCR results. In accordance to other studies the visible symptoms matched perfectly the DNA content in medium to low resistant lines. In contrast, highly resistant lines with low *Fusarium* biomass – but nevertheless high amount of toxins – did sometimes not show any disease symptoms. The authors addressed this effect to the detoxification mechanism of high resistant plants as some toxins can be "masked" by the plant. So they are converted to less plant-toxic

Visual assessment of disease caused by *Fusarium* species is frequently insufficient to identify the causal agent or to quantify the plant pathogen. In natural systems pathogenic fungi often occur in a combination of species, which can induce similar symptoms and a visual discrimination might be impossible. Other methods like agar plate assays rely on the fact that viable propagules are present in a sample. Especially in harvested grain this is not

could be confirmed by visual monitoring of the disease symptoms.

difficult as only the number of infected kernels can be determined.

metabololites which do not damage the wheat-ears.

**5. Conclusions** 

Bai, G.H., Plattner, R., Desjardins, A. & Kolb, F. (2001). Resistance to *Fusarium* head blight and deoxynivalenol accumulation in wheat. *Plant Breeding*, 120(1), pp 1-6.

Baird, R., Abbas, H.K., Windham, G., Williams, P., Baird, S., Ma , P., Kelley, R., Hawkins L. & Scruggs M. (2008). Identification of select fumonisin forming *Fusarium* species using PCR applications of the polyketide synthase gene and its relationship to fumonisin production in vitro. *International Journal of Molecular Sciences*, 9: 554-570.

Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants:

crops. *Molecular Plant Pathology*, 2004. 5(6): p. 515-525.

Nurseries. *Crop Science*, 48, pp 1389-1398.

*Applied Microbiology*, 29: 681-689.

*Journal of Food Protection*, 95: 287-295.

*of Applied Genetics*, 49(3): 305–311.

Minn, USA: APS Press, 2003.

95: 321-331.

200.

*Science*, 46: p. 2636 - 2642.

Quantitative PCR and ELISA Accurately Determine *Fusarium* Biomass 215

Gagkaeva, T.Y. & Yli-Mattila, T. (2004). Genetic diversity of *Fusarium* graminearum in

Gan, Z., Marquardt, R.R., Abramson, D. & Clear R.M. (1997). The characterization of chicken

Goswami, R.S. & Kistler, H.C. 2004). Heading for disaster: *Fusarium* graminearum on cereal

Hill, N.S., Hiatt, E.E. & Chanh, T.C. (2006). ELISA analysis for *Fusarium* in barley. *Crop* 

Hill, N.S., Neate, S.M., Cooper, B., Horsley, R., Schwarz, P., Dahleen, L.S., Smith, K.P.,

Jennings, P., Coates, M.E., Turner, J.A., Chandler, E.A. & Nicholson P. (2004). Determination

Jurado, M., Vázquez, C., Sanchis, V. & González-Jaén, M.T. (2006). PCR-based strategy to

Khonga, E.B. & Sutton, J.C. (1988). Inoculum production and survival of Gibberella zeae in maize and wheat residues. Canadian Journal of Plant Pathology 10: p. 232-240. Knutsen, A.K. & Holst-Jensen, A. (2004). Phylogenetic analyses of the *Fusarium* poae,

Kolb, F.L., Baib, G-H., Muehlbauer, G.J., Andersonc, J.A., Smithc K.P. & Fedak G. (2001).

Konstantinova, P. & Yli-Mattila, T. (2004). IGS-RFLPanalysis and development of molecular

Kulik, T. (2008). Detection of *Fusarium* tricinctum from cereal grain using PCR assay. *Journal* 

Kulik, T., Jestoi, M. & Okorski, A. (2011). Development of TaqMan assays fort he

Leonard, K.J. & Bushnell, W. (2003). *Fusarium* head blight of wheat and barley. St.Paul,

Li, S., Hartman, G. L., Domier L.L. & Boykin, D. (2008). Quantification of *Fusarium solani* f.

quantitative PCR. *Theoretical and Applied Genetics*, 117: 343-352.

with Molecular Markers. *Crop Science*, 41(3), pp 611-619.

*European Journal of Plant Pathology*, 110, pp 563–571

England and Wales by PCR assay. *Plant Pathology,* 53, pp 182–190.

antibodies raised against *Fusarium* spp. by enzyme-linked immunosorbent assay and immunoblotting. *International Journal of Food Microbiology*, 16, 38(2-3), pp 191-

O'Donnell, K. & Reeves, J. (2008). Comparison of ELISA for *Fusarium*, Visual Screening, and Deoxynivalenol Analysis of *Fusarium* Head Blight for Barley Field

of deoxynivalenol and nivalenol chemotypes of *Fusarium* culmorum isolates from

detect contamination with mycotoxigenic *Fusarium* species in maize. *Systematic and* 

*Fusarium* sporotrichioides and *Fusarium* langsethiae species complex passed on partial sequences of the translation elongation factor-1 alpha gene. *International* 

Host Plant Resistance Genes for *Fusarium* Head Blight: Mapping and Manipulation

markers for identification of *Fusarium* poae, *Fusarium* langsethiae, *Fusarium* sporotrichioides and *Fusarium* kyushuense. *International Journal of Food Microbiology*,

quantitative detection of *Fusarium* avenaceum/*Fusarium* tritinctum and *Fusarium* poae esyn1 genotypes from cereal grain. *FEMS Microbiology Letters*, 314, pp49-56. Laday, M., Mule, G., Moretti, A., Hamari, Z., Juhasz, A., Szecsi, A., Logrieco, A., (2004).

Mitochondrial DNA variability in *Fusarium* proliferatum (Gibberella intermedia).

sp. glycines isolates in soybean roots by colony-forming unit assays and real-time

Europe and Asia. *European Journal of Plant Pathology*, 110: 551-562.


Bluhm, B.H., Cousin, M.A. & Woloshuk, C.P. (2004). Multiplex real-time PCR detection of

Brandfass, C. & Karlovsky, P. (2006). Simultaneous detection of *F. culmorum* and *F.* 

Brunner, K., Kovalsky Paris, M.P., Paolino, G., Bürstmayr, H., Lemmens, M., Berthiller, F.,

Brunner, K. & Mach R.L. (2010). Quantitative Detection of Fungi by Molecular Methods: A

Buerstmayr, H., Steiner, B., Hartl, L., Griesser, M., Angerer, N., Lengauer, D., Miedaner, T.,

Buerstmayr, H., Ban, T. & Anderson, J.A. (2009). QTL mapping and marker-assisted selection

Burgess, L.W. (1981). General ecology of the Fusaria. P. E. Nelson, T. A. Toussoun, & R. J.

Chala, A., Weinert J., Wolf G.A. (2003). An Integrated Approach to the Evaluation of the

Chandler, E.A., Simpson, D.R., Thomsett,M.A. & Nicholson, P. (2003). Development of PCR

Demeke, T., Clear, R.M., Patrick, S.K. & Gaba, D. (2005). Species-specific PCR-based assays

Desjardins, A.E. (2006). *Fusarium* mycotoxins. Chemistry, Genetics, and Biology. APS Press,

Doohan, F.M., Parry, D.W. & Nicholson, P. (1999). *Fusarium* ear blight of wheat: the use of

Draeger, R., Gosman, N., Steed, A., Chandler, E., Thomsett, M., Srinivasachary, J.

Edwards, S.G., Progozliev, S.R., Hare, M.C. & Jenkinson, P. (2001). Quantification of

*Applied and Environmental Microbiology*, 67(4): 1575-1580.

*Analytical and Bioanalytical Chemistry*, 395( 5), pp 1385 - 1394.

spread. *Theoretical and Applied Genetics*, 107(3), pp 503-508.

*Physiological and Molecular Plant Pathology* , 62 (2003) 355–367.

Springer-Verlag, Berlin Heidelberg, pp 93-105.

Press, University Park, 1981: p. 225-235.

Wheat. *Journal of Phytopathology*, 151, 673-678.

*Journal of Food Protection*, 67(3): 536-543.

*Microbiology*, 6:4

103, pp 271-284.

St. Paul, MN, 2006.

*Pathology*, 48, pp 209-217

*Genetics*, 115(5), pp 617-625.

fumonisin-producing and trichothecene-producing groups of *Fusarium* species.

*graminearum* in plant material by duplex PCR with melting curve analysis. *BMC* 

Schuhmacher, R., Krska, R. & Mach R.L. (2009). A reference-gene-based quantitative PCR method as a tool to determine *Fusarium* resistance in wheat.

Case Study on *Fusarium*. In: *Molecular Identification of Fungi*. Gherbawy, Y.,

Schneider, B. & Lemmens, M. (2003). Molecular mapping of QTLs for *Fusarium* head blight resistance in spring wheat. II. Resistance to fungal penetration and

for *Fusarium* head blight resistance in wheat: a review. *Plant Breeding*, 128(1), pp 1-26.

Cook, In: *Fusarium: diseases, biology, and taxonomy*. Pennsylvania State University

Efficacy of Fungicides Against *Fusarium culmorum*, the Cause of Head Blight of

assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of *Fusarium graminearum*, *Fusarium culmorum* and *Fusarium cerealis*.

for the detection of *Fusarium* species and comparison with the whole seed agar plate method and trichothecene analysis. *International Journal of Food Microbiology*,

quantitative PCR and visual disease assessment in studies of disease control. *Plant* 

Schondelmaier, H., Buerstmayr, H., Lemmens, M. & Schmolke, M.. (2007). Identification of QTLs for resistance to *Fusarium* head blight, DON accumulation and associated traits in the winter wheat variety Arina. *Theoretical and Applied* 

trichothecene producing *Fusarium* species in harvested grain by competitive PCR to determine efficiacies of fungicides against *Fusarium* head blight of winter wheat.


Novel Methods for the Quantification of Pathogenic Fungi in Crop Plants:

Breeding Systems*. Crop Science*, 41(3), pp 620-627.

*Molecular Plant Patholology*, 86(5):515-522

*Journal of Food Microbiology*, 71, pp 53-61.

*Journal of Plant Patholohy*, 124, pp 163-170.

*Plant and Soil*, 267(1-2): pp 13-22.

*Applied Genetics*, 49(4), pp 433-441

*of Plant Pathology*. 110: 481-494.

oft he rRNA gene. *Plant Disease*, 84(4), pp 475-482.

39-47.

pp 118-125.

caused by *Gibberella zeae*. *Phyto*pathology 53: pp 831-839.

83-91.

Quantitative PCR and ELISA Accurately Determine *Fusarium* Biomass 217

Petcu, G. & Ioniþã, S. (1998). Influence of crop rotation on weed infestion and *Fusarium* spp.

Quarta, A., Mita, G., Haidukowski, M., Logrieco, A., Mulè, G., & Visconti, A. (2006).

Reischer G.H., Lemmens, M., Farnleitner, A.H., Adler, A. & Mach, R.L. (2004).

Rhode, S. & Rabenstein, F. (2005). Standardization of an indirect PTA-ELISA for detection of *Fusarium* spp. In infected grains. *Mycotoxin Research,* 21(2), pp 100-104. Rudd, J.C., Horsley, R.D., McKendry, A.L. & Elias, E.M.. (2001). Host Plant Resistance Genes

Schilling, A.G., Möller, E.M. & Geiger H.H. (1996). Polymerase Chain Reaction-Based Assay

Schnerr, H., Niessen, L. & Vogel, R.F. (2001). Real-time detection of the tri5 gene in *Fusarium*

Šliková, S., Šudyová, V., Martinek, P., Polišenská, I., Gregová, E. & Mihálik D. (2009).

Schroeder, H.W. & Christensen, J.J. (1963). Factors affecting resistance of wheat to scab

Steinkellner, S. & Langer, I. (2004). Impact of tillage on the incidence of *Fusarium* spp. in soil.

Stepień, L., Popiel, D., Koczyk, G. & Chełkowski, J. (2008). Wheat-infecting *Fusarium* species

Waalwijk, C., Koch, S.H., Ncube, E., Allwood, J., Flett, B., de Vries, I. & Kema, G.H.J. (2008).

Waalwijk, C., van der Heide, R., de Vries, I., van der Lee, T., Schoen, C., Corainville, G.C.,

Wang, Y.Z. & Miller, J.D. (1988). Effects of *Fusarium* graminearum metabolites on wheat

Weiland, J.J. & Sundsbak, J.L. (2000). Differentiation and detection of sugar beet fungal

attack, yield and quality of winter wheat. Romanian Agricultural Research, 9-10:

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyldeoxynivalenol chemotypes in *Fusarium*. *FEMS Microbiology Letters*, 259(1), pp 7-13

Quantification of *Fusarium graminearum* in infected wheat by species specific realtime PCR applying a TaqMan probe. *Journal of Microbiology Methods*, 59, pp 141-146.

for *Fusarium* Head Blight: Sources, Mechanisms, and Utility in Conventional

for Species-Specific Detection of *F. culmorum*, *F. graminearum* , and *F. avenaceum*.

by LightCycler-PCR using SYBR Green I for continous fluorescence monitoring. Int.

Assessment of infection in wheat by *Fusarium* protein equivalent levels. *European* 

in Poland--their chemotypes and frequencies revealed by PCR assay. *Journal of* 

Quantitative detection of *Fusarium* spp. and its correlation with fumonisin content in maize from South African subsistence farmers. *World Mycotoxin Journal*, 1(1), pp

Häusler-Hahn, I., Kastelein, P., Köhl, J., Lonnet, P., Demarquet, T. & Kema, G.H.J. (2004). Quantitative detection of *Fusarium* in wheat using TaqMan. *European Journal* 

tissue in relation to *Fusarium* head blight resistance. *Journal of Phytopathology*, 122,

pathogens using PCR amplification of actin coding sequences and the ITS region


Lia, H., Wua, A., Zhaoa, C., Scholten, O., Löffler H., & Yu-Cai Liaoa (2005).Development of a

Mach, R.L., Kullnig-Gradinger, C.M., Farnleitner, A.H., Reischer, G. Adler, A. & Kubicek,

Markell, S.G. & Francl, L.J. (2003). *Fusarium* head blight inoculum: species prevalence and

McCartney, H.A., Foster, S.J., Fraaije, B.A. & Ward E. (2003). Molecular diagnostics for

McMullen, M., Jones, R., & Gallenberg, D. (1997). Scab of Wheat and Barley: A Re-emerging

Miedaner, T., Beyer, W., Höxter, H. & Geiger, H.H. (1994). Growth Stage Specific Resistance

Miedaner, T., Heinrich, N., Schneider, B., Oettler, G., Rohde, S. & Rabenstein, F. (2004).

Mirete, S., Vázquez, C., Mulè, G., Jurado M. & González-Jaén M.T. (2004). Differentiation of

Mulè, G., Susca, A. Stea, G. & Moretti, A. (2004). A species specific PCR assay based on the

Nganje, W.A., Bangsund, D.A., Leistritz, F.L., Wilson,W.W., & Tiapo, N.M. (2004). Regional

Nicholson, P. Simpson, D.R., Wilson, A.H., Chandler, E. & Thomsett A. (2004). Detection

Osborne, L.E. and Stein, J.M. (2007). Epidemiology of *Fusarium* head blight on small-grain

Pasanen A.L., Yli-Pietilä, K., Pasanen, P., Kalliokoski, P. & Tarhanen J. (1999). Ergosterol

Pereyra, S.A., Dill-Macky, R. & A.L. (2004). Sims, Survival and inoculum production of

small-grain cereals. *European Journal of Plant Pathology*. 110: 503-514. Nicholson, P., Simpson, D.R., Weston, G., Rezanoor, H.N., Lees, A.K., Parry, D.W., & Joyce,

cereals. *International Journal of Food Microbiology*, 119(1-2): p. 103-8.

D. (1998). *Physiological and Molecular Plant Pathology*, 53: 17-37

*Applied and Environmental Microbiology*, 65(1), pp 138-142.

*Gibberella zeae* in wheat residue. *Plant Disease*, 88, pp 724-730.

and *F. subglutinans*. *European Journal of Plant Pathology*, 110: 495-502. Mulfinger, S., Niessen, L. & Vogel, R. (2000). PCR based quality control of toxigenic

of Winter Rye to *Microdochium nivale* and *Fusarium* spp. In the Field Assessed by

Estimation of deoxynivalenol (DON) grain content by symptom rating and exoantigen content for resistance selection in wheat and triticale. *Euphytica*, 139(2),

*Fusarium verticillioides* from Banana Fruits by IGS and EF-1α Sequence Analyses.

calmodulin partial gene for identification of *Fusarium verticillioides*, *F. roliferatum*

*Fusarium* spp.in brewing malt using ultrasonication for rapid sample preparation.

Economic Impacts of *Fusarium* Head Blight in Wheat and Barley. *Review of* 

and differentiation of trichothecene and enniatin-producing *Fusarium* species on

Content in Various Fungal Species and Biocontaminated Building Materials.

graminearum. *FEMS Microbiology Letters*, 243(2), pp 505-511

*Gibberella zeae* spore type. *Plant Disease*, 87, pp 814-820.

fungal plant pathogens. *Pest Management Sciences*, 59, pp129-142

Disease of Devastating Impact. *Plant Disease* 81(12): 1340-1348.

Immunological Methods. *Phytopathology*, 85, pp 416-421.

*European Journal of Plant Pathology*, 110(5-6), pp 515-523.

*Advances in Food Science*, 22(1/2): p. 38 - 46.

*Agricultural Economics*, 26(3), pp 332-337.

*Protection*, 95, pp 333-339.

pp 123-132

generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of *Fusarium*

C.P. (2004). Specific detection of *Fusarium* langsethiae and related species by DGGE and ARMS-PCR of a β-tubulin (*tub1*) gene fragment. *International Journal of Food* 


**1. Introduction** 

**2. Gray mold of castor** 

**2.1 Historic and economic importance** 

**9** 

*Brazil* 

Dartanhã José Soares

**Gray Mold of Castor: A Review** 

Castor plant (*Ricinus communis* L.) is a non-edible oilseed crop with unique oil features for the chemistry industry. The crop was very important in the mid and late nineteenth century and also during WWI. After that the crop lost its importance in developed countries (Godfrey, 1923), but in India and Brazil it has remained as the most important non-edible oilseed crop of the arid and semi-arid regions (Dange et al., 2005; Santos et al., 2007). Nowadays, due the constant pressure for renewable fuels, castor has been investigated as a potential source of biofuel, mainly in Brazil due to governmental stimulus, and this has raised the crop importance once again. Regardless of the lack of a well established crop system, castor hosts several pests and diseases which cause heavy losses in the crop yield. One of the most destructive diseases of castor is gray mold, caused by the fungus *Botryotinia ricini* (Godfrey) Whetzel. Actually, it is the anamorphic phase of *B. ricini*, known as *Amphobotrys ricini* (N.F. Buchw.) Hennebert, that is responsible for disease epidemics and heavy yield losses frequently observed in castor crops. The first epidemic outbreak caused by this fungus was reported by H.E. Stevens of the Florida Experiment Station, Gainesville, Florida (Godfrey, 1919, 1923). At that time, a meticulous study was conducted and much of our knowledge regarding the disease and its causal agent was published in the classic work of Godfrey (1923). Subsequently, only sporadic works were conducted by other scientists around the world, consequently few advances have been made on management of gray mold. Breeding programs have failed in developing varieties with satisfactory resistance levels (Kolte, 1995), and chemical control is still ineffective and economically prohibitive, mainly due to the lack of basal information about the causal organism and its biology. In

this chapter, the major aspects of castor gray mold will be reviewed.

Castor gray mold was first reported in the USA in 1918, following pioneering investigations by H.E. Stevens and F. W. Patterson, who promptly suggested that the causal organism of castor gray mold was an unknown *Botrytis* species (Godfrey, 1919, 1923). This fungus had caused serious losses of castor crop in the summer of 1918 mainly in Florida and others southern States, where it was responsible for losses up to 100% of castor yield (Godfrey,

*Empresa Brasileira de Pesquisa Agropecuária,* 

*Embrapa Algodão, Campina Grande* 


### **Gray Mold of Castor: A Review**

#### Dartanhã José Soares

*Empresa Brasileira de Pesquisa Agropecuária, Embrapa Algodão, Campina Grande Brazil* 

#### **1. Introduction**

218 Plant Pathology

Windels, C.E. (2000). Economic and Social Impacts of *Fusarium* Head Blight: Changing

Yli-Mattila, T., Mach, R.L., Alekhina, I.A., Bulat, S.A., Koskinen, S., Kullnig-Gradinger, C.M.,

Yli-Mattila, T., Paavanen-Huhtala, S., Jestoi, M., Parikka, P., Hietanieme, V., Gagkaeva, T.,

Youssef, S.A., Maymon, M., Zveibil, A., Klein-Gueta, D., Sztejnberg, A., Shalaby, A.A. &

pp 17-21.

95, pp 267-285.

Plant Protection, 41(4), pp 243-260.

Egypt. *Plant Pathology*, 56, pp 257–263.

Farms and Rural Communities in the Northern Great Plains. *Phytopathology*, 90(1),

Kubicek, C.P. & Klemsdal, S.S. (2004). Phylogenetic relationship of *Fusarium* langsethiae to *Fusarium* poae and *Fusarium* sporotrichioides as inferred by IGS, ITS, β-tubulin sequences and UP-PCR analysis*. International Journal of Food Microbiology*,

Sarlin, T., Haikara, A., Laaksonen, S. & Rizzo, A. (2008). Real-time PCR detection and quantification of *Fusarium poae*, *F. graminearum*, *F. sporotrichioides* and *F. langsethiae* in cereal grains in Finland and Russia. Archives of Phytopathology and

Freeman S. (2007). Epidemiological aspects of mango malformation disease caused by *Fusarium mangiferae* and source of infection in seedlings cultivated in orchards in

Castor plant (*Ricinus communis* L.) is a non-edible oilseed crop with unique oil features for the chemistry industry. The crop was very important in the mid and late nineteenth century and also during WWI. After that the crop lost its importance in developed countries (Godfrey, 1923), but in India and Brazil it has remained as the most important non-edible oilseed crop of the arid and semi-arid regions (Dange et al., 2005; Santos et al., 2007). Nowadays, due the constant pressure for renewable fuels, castor has been investigated as a potential source of biofuel, mainly in Brazil due to governmental stimulus, and this has raised the crop importance once again. Regardless of the lack of a well established crop system, castor hosts several pests and diseases which cause heavy losses in the crop yield. One of the most destructive diseases of castor is gray mold, caused by the fungus *Botryotinia ricini* (Godfrey) Whetzel. Actually, it is the anamorphic phase of *B. ricini*, known as *Amphobotrys ricini* (N.F. Buchw.) Hennebert, that is responsible for disease epidemics and heavy yield losses frequently observed in castor crops. The first epidemic outbreak caused by this fungus was reported by H.E. Stevens of the Florida Experiment Station, Gainesville, Florida (Godfrey, 1919, 1923). At that time, a meticulous study was conducted and much of our knowledge regarding the disease and its causal agent was published in the classic work of Godfrey (1923). Subsequently, only sporadic works were conducted by other scientists around the world, consequently few advances have been made on management of gray mold. Breeding programs have failed in developing varieties with satisfactory resistance levels (Kolte, 1995), and chemical control is still ineffective and economically prohibitive, mainly due to the lack of basal information about the causal organism and its biology. In this chapter, the major aspects of castor gray mold will be reviewed.

#### **2. Gray mold of castor**

#### **2.1 Historic and economic importance**

Castor gray mold was first reported in the USA in 1918, following pioneering investigations by H.E. Stevens and F. W. Patterson, who promptly suggested that the causal organism of castor gray mold was an unknown *Botrytis* species (Godfrey, 1919, 1923). This fungus had caused serious losses of castor crop in the summer of 1918 mainly in Florida and others southern States, where it was responsible for losses up to 100% of castor yield (Godfrey,

Gray Mold of Castor: A Review 221

*Botryotinia ricini* belongs to Sclerotiniaceae (Helotiales, Ascomycota) and is characterized by its dark, plane-convex, elongated sclerotia (Fig.1), which give rise to cinnamon brown to chestnut brown, long stipitate apothecia, with cylindrical to cylindro-clavate asci, apex slightly thickened, 8-spored; ascopores ellipsoidal, often sub-fusoid, one-celled, bi-guttulate and hyaline; paraphyses hyaline, filiform, septate (Godfrey, 1919). Its anamorphic phase is characterized by cylindrical, straight, dichotomously branched, pale brown conidiophores, with conidiogeneous cell not inflated, thin-walled; conidia globose, maturing synchronically, on short denticles, smooth, one-celled, sub-hyaline to pale brown (Fig.1) (Godfrey, 1919; Hennebert, 1973; Lima et al., 2008). A synanamorph (*Myrioconium* sp. – spermatial state) may sometimes be present on culture media (Godfrey, 1923; Hennebert, 1973; Seifert et al., 2011). According to Kirk et al. (2008), the genus *Amphobotrys* remains

*Botryotinia ricini* is regarded as a homothallic species (Beveer & Weeds, 2007), so sexual reproduction will readily take place. If sexual reproduction had taken place, a high degree of diversity would be expected within the *B. ricini* population; however, Bezerra (2007) shows evidence that, in the state of Paraíba (Northeast Brazil) populations of *B. ricini* are clonal, which means that sexual reproduction had not taken place within those populations. Nonetheless, this conclusion must be viewed with caution because the population sampled was relatively small. Unfortunately, there is no other work on this subject and, therefore, the

Fig. 1. Dark sclerotia on culture medium (A); close-up view of the sclerotia (B); transversal section through a sclerotium to show its plane-convex form (D); dichotomous branch of the conidiophores (E); conidiogenous cells showing the synchronic conidiogenesis (C); and close-up the conidiogenous cell to show the denticles and globose conidia (F). Photos: D.J.

monotypic.

Soares.

population structure of *B. ricini* remains unknown.

1923). Later, the disease was reported in almost all countries where castor has been cultivated (Kolte, 1995), having nowadays a worldwide distribution.

The first occurrence of this disease in USA was directly linked to seeds imported from Bombay (now Mumbai), India, even though until that time, such disease had not been described in that country (Godfrey, 1923). In his work, Godfrey (1923) did a detailed account of the destructive potential of the gray mold of castor under favourable condition. By attacking mainly reproductive organs of the castor plant, gray mold disease is implicated in direct losses of yield whatever the level of infection.

In India, today the major castor producer, gray mold is found in few states and is regarded as troublesome only in Andhra Pradesh and Tamil Nadu, in the South, where the weather conditions are more favourable for disease development where in 1987, an epidemic outbreak of gray mold occurred (Dange et al., 2005).

In Brazil, the disease was first reported in the São Paulo state in 1932. However, it was only in 1936 that any attention was given to the disease due to the serious losses which occurred that year (Gonçalves, 1936). Currently, gray mold is present in almost all Brazilian states and its importance has grown at the same time that the crop cultivation has been intensified, mainly in those regions where the weather conditions are favourable for disease development, including the Southern and South-eastern Brazilian states (Araújo et al., 2007; Freire et al., 2007). In the region of the "Brejo Paraibano", where the recommended sowing period is between mid-April to early-May (Amorim Neto et al., 2000), the flowering period (mid-June to early-August) usually coincides with highly favourable conditions for disease development (Moraes et al., 2009). Yield losses of up to 100% are quite frequent when highly susceptible cultivars are planted. Conversely, in Bahia the major castor producer in Brazil gray mold is not a problem because the weather conditions are usually not favourable for disease development.

#### **2.2 Etiology, taxonomy and population structure**

The causal agent of gray mold of castor was originally described by Godfrey (1919) as *Sclerotinia ricini* Godfrey, based on the holomorph. Later, Whetzel (1945) transferred the species *S. ricini* to the genus *Botryotinia*, which since then has been known as *Botryotinia ricini* (Godfrey) Whetzel. Subsequently, the anamorphic state of *Botryotinia ricini* was named as *Botrytis ricini* N.F. Buchw. (Buchwald, 1949). This led to general confusion between the non-mycologist communities, which adopted the name *Botrytis ricini* N.F. Buchw., instead of *Botryotinia ricini* (Godfrey) Whetzel. In 1973, Hennebert erected the genus *Amphobotrys* to accommodate the anamorphic state of *B. ricini*, based mainly on the distinctive pattern of conidiophore ramification, and since then the anamorphic state became known as *Amphobotrys ricini* (N.F. Buchw.) Hennebert (Hennebert, 1973). Even so, several authors used, and still use, the erroneous name "*Botrytis ricini*" attributing its authority to Godfrey (Barreto & Evans, 1998; Batista et al., 1998; Dange et al., 2005; Lima & Soares, 1990).

Although the correct name to be applied to the causal agent of gray mold of castor is *Botryotinia ricini*, only the anamorphic state is observed in the field by most authors, and thus the name of the anamorph, at the expense of the name of the holomorph, is preferred (Holcomb et al., 1989; Lima et al., 2008).

1923). Later, the disease was reported in almost all countries where castor has been

The first occurrence of this disease in USA was directly linked to seeds imported from Bombay (now Mumbai), India, even though until that time, such disease had not been described in that country (Godfrey, 1923). In his work, Godfrey (1923) did a detailed account of the destructive potential of the gray mold of castor under favourable condition. By attacking mainly reproductive organs of the castor plant, gray mold disease is implicated in

In India, today the major castor producer, gray mold is found in few states and is regarded as troublesome only in Andhra Pradesh and Tamil Nadu, in the South, where the weather conditions are more favourable for disease development where in 1987, an epidemic

In Brazil, the disease was first reported in the São Paulo state in 1932. However, it was only in 1936 that any attention was given to the disease due to the serious losses which occurred that year (Gonçalves, 1936). Currently, gray mold is present in almost all Brazilian states and its importance has grown at the same time that the crop cultivation has been intensified, mainly in those regions where the weather conditions are favourable for disease development, including the Southern and South-eastern Brazilian states (Araújo et al., 2007; Freire et al., 2007). In the region of the "Brejo Paraibano", where the recommended sowing period is between mid-April to early-May (Amorim Neto et al., 2000), the flowering period (mid-June to early-August) usually coincides with highly favourable conditions for disease development (Moraes et al., 2009). Yield losses of up to 100% are quite frequent when highly susceptible cultivars are planted. Conversely, in Bahia the major castor producer in Brazil gray mold is not a problem because the weather conditions are usually not favourable for

The causal agent of gray mold of castor was originally described by Godfrey (1919) as *Sclerotinia ricini* Godfrey, based on the holomorph. Later, Whetzel (1945) transferred the species *S. ricini* to the genus *Botryotinia*, which since then has been known as *Botryotinia ricini* (Godfrey) Whetzel. Subsequently, the anamorphic state of *Botryotinia ricini* was named as *Botrytis ricini* N.F. Buchw. (Buchwald, 1949). This led to general confusion between the non-mycologist communities, which adopted the name *Botrytis ricini* N.F. Buchw., instead of *Botryotinia ricini* (Godfrey) Whetzel. In 1973, Hennebert erected the genus *Amphobotrys* to accommodate the anamorphic state of *B. ricini*, based mainly on the distinctive pattern of conidiophore ramification, and since then the anamorphic state became known as *Amphobotrys ricini* (N.F. Buchw.) Hennebert (Hennebert, 1973). Even so, several authors used, and still use, the erroneous name "*Botrytis ricini*" attributing its authority to Godfrey

(Barreto & Evans, 1998; Batista et al., 1998; Dange et al., 2005; Lima & Soares, 1990).

Although the correct name to be applied to the causal agent of gray mold of castor is *Botryotinia ricini*, only the anamorphic state is observed in the field by most authors, and thus the name of the anamorph, at the expense of the name of the holomorph, is preferred

cultivated (Kolte, 1995), having nowadays a worldwide distribution.

direct losses of yield whatever the level of infection.

outbreak of gray mold occurred (Dange et al., 2005).

**2.2 Etiology, taxonomy and population structure** 

(Holcomb et al., 1989; Lima et al., 2008).

disease development.

*Botryotinia ricini* belongs to Sclerotiniaceae (Helotiales, Ascomycota) and is characterized by its dark, plane-convex, elongated sclerotia (Fig.1), which give rise to cinnamon brown to chestnut brown, long stipitate apothecia, with cylindrical to cylindro-clavate asci, apex slightly thickened, 8-spored; ascopores ellipsoidal, often sub-fusoid, one-celled, bi-guttulate and hyaline; paraphyses hyaline, filiform, septate (Godfrey, 1919). Its anamorphic phase is characterized by cylindrical, straight, dichotomously branched, pale brown conidiophores, with conidiogeneous cell not inflated, thin-walled; conidia globose, maturing synchronically, on short denticles, smooth, one-celled, sub-hyaline to pale brown (Fig.1) (Godfrey, 1919; Hennebert, 1973; Lima et al., 2008). A synanamorph (*Myrioconium* sp. – spermatial state) may sometimes be present on culture media (Godfrey, 1923; Hennebert, 1973; Seifert et al., 2011). According to Kirk et al. (2008), the genus *Amphobotrys* remains monotypic.

*Botryotinia ricini* is regarded as a homothallic species (Beveer & Weeds, 2007), so sexual reproduction will readily take place. If sexual reproduction had taken place, a high degree of diversity would be expected within the *B. ricini* population; however, Bezerra (2007) shows evidence that, in the state of Paraíba (Northeast Brazil) populations of *B. ricini* are clonal, which means that sexual reproduction had not taken place within those populations. Nonetheless, this conclusion must be viewed with caution because the population sampled was relatively small. Unfortunately, there is no other work on this subject and, therefore, the population structure of *B. ricini* remains unknown.

Fig. 1. Dark sclerotia on culture medium (A); close-up view of the sclerotia (B); transversal section through a sclerotium to show its plane-convex form (D); dichotomous branch of the conidiophores (E); conidiogenous cells showing the synchronic conidiogenesis (C); and close-up the conidiogenous cell to show the denticles and globose conidia (F). Photos: D.J. Soares.

Gray Mold of Castor: A Review 223

came from the fact that the male flowers are the first to be exposed, at the earlier stage of inflorescence formation; consequently such flowers are exposed longer to the infection units of the fungus. However, as soon as the male flowers suffer anthesis they are no longer a target and are hardly infected, contradicting the statement of Drumond & Coelho (1981) that "the fungus attacks first the male flowers because the anthers, being soaked with the rain

Fig. 2. Symptoms of gray mold attack on castor inflorescence and raceme. A-B. Symptoms on young inflorescences, before fertilization of female flowers. C-H. Symptoms on capsules

The first symptoms are visible as bluish spots on the inflorescences, on both female and male (before anthesis) flowers, and on developing fruits. On fruits, the symptoms can evolve to circular or elliptic, sunken, dark coloured spots that can result in rupture of the capsule (Fig.3 A-B) (Araújo et al., 2007). These symptoms are usually more frequent when a period of low relative humidity unfavourable to fungal sporulation occurs soon after the fungus

Depending on weather conditions (e.g. long periods with high relative humidity soon after the fungus penetrates the host), the occurrence of yellow ooze at the point of infection is frequent (Fig. 3 C-D) (Batista et al., 1996; Dange et al., 2005) as a result of the rapid enzymatic tissue degradation. The symptoms on the male flowers, before anthesis, are small, pale brown, necrotic spots, which can evolve to larger brown spots with a darker edge (Fig.3 E). The infected flowers and young capsules became softened due the fungal colonization and mycelial growth is, at first, pale gray and later dark olivaceous. A profuse sporulation is usually observed in such stage (Fig.3 F). When the infection starts on immature capsules, they become rotten; if the infection starts later, with fully developed capsules, the seeds usually became hollow, with coat discoloration and weight loss (Dange et al., 2005). On the

at distinct development stages. Photos: D.J. Soares.

penetrates the host tissues.

water or dew, easily retain the fungus spores carried by the wind".

#### **2.3 Host penetration and colonization**

In his classical work, G.H. Godfrey also investigated the infection process of *B. ricini* on leaves of the castor plant and concluded that penetration occurs directly through the host cuticle, in a process similar to *Botrytis cinerea* (Godfrey, 1923). After penetrating the cuticle, the fungus quickly spreads over the host tissues leading to a complete disorganization and breakdown. Although Godfrey had made mention as to the possible role of an enzymatic action in the penetration process, his conclusion pointed out to a mechanical penetration of the germ-tube, without tissues dissolving, prior to infection. On the other hand, Thomas & Orellana (1963b) found that it was not possible to verify the direct germ-tube penetration of *B. ricini*, through the cuticle or stomata, on castor capsules, before tissue maceration by pectic enzymes action, suggesting that the fungus first degraded the cuticle and later penetrated the host tissues (Orellana & Thomas, 1962). Probably *B. ricini* uses both mechanical and chemical processes to penetrate undamaged host tissue, however, no further studies have been done to clarify these questions.

Although the infection process of *B. ricini* needs to be better understood, it is likely that enzymes, such as lipases and cutinases, play an important role in the infection process similar to several other *Botrytis*-host interactions (Kars & van Kan, 2007). Additionally, *Botrytis* species can also affect the 'redox' process in the host plants, during the colonization of host tissues, through the production of enzymes like superoxide dismutase (Lyon et al., 2007). Due the great biological similarity of *Botrytis* spp. and *B. ricini*, probably, such enzymes also have an important role in the infection process of the causal agent of castor gray mold, as already evidenced by Orellana & Thomas (1962). Hoffmann et al. (2004) had, for example, extracted alpha and beta esterase and superoxide dismutase from *B. ricini,* however they did not perform a study to determine the role of such enzymes in the infection process of this fungus.

It is important to highlight the great distinction between the penetration process of a fungus under controlled and highly favourable condition in contrast with the natural process in the field. In the latter case, all aerial parts of the host are potential targets for deposition and penetration of *B. ricini*, because not only the conidia, regarded as the major propagative unit, usually responsible for the epidemic outbreak, but also the ascospores, sclerotia and mycelia fragments can give rise to infection, as observed in *Botrytis* spp. (Jarvis, 1978). So, besides direct penetration, probably natural openings and wounds also serve as a point of entrance for the fungus. Growth of the fungus on the host surface and consequently its penetration in the host tissues will depend on factors such as inoculum type, free water and nutrient availability, cuticle features, presence of exudates on floral organs and other glands, besides the abundance of natural openings and the size and age of wounds, as pointed out by Holz et al., (2007) for *Botrytis* species.

#### **2.4 Symptoms**

The primary targets of the fungus are the inflorescence and the capsules, in any development stage (Fig.2) (Araújo et al., 2007; Dange et al., 2005; Gonçalves, 1936; Lima et al., 2001). Some authors (Drumond & Coelho, 1981; Batista et al., 1996) claim that the male flowers are the first to be infected, but it is not always the case because any part of the inflorescence can be infected, the female flowers being the preferential target. That claim

In his classical work, G.H. Godfrey also investigated the infection process of *B. ricini* on leaves of the castor plant and concluded that penetration occurs directly through the host cuticle, in a process similar to *Botrytis cinerea* (Godfrey, 1923). After penetrating the cuticle, the fungus quickly spreads over the host tissues leading to a complete disorganization and breakdown. Although Godfrey had made mention as to the possible role of an enzymatic action in the penetration process, his conclusion pointed out to a mechanical penetration of the germ-tube, without tissues dissolving, prior to infection. On the other hand, Thomas & Orellana (1963b) found that it was not possible to verify the direct germ-tube penetration of *B. ricini*, through the cuticle or stomata, on castor capsules, before tissue maceration by pectic enzymes action, suggesting that the fungus first degraded the cuticle and later penetrated the host tissues (Orellana & Thomas, 1962). Probably *B. ricini* uses both mechanical and chemical processes to penetrate undamaged host tissue, however, no

Although the infection process of *B. ricini* needs to be better understood, it is likely that enzymes, such as lipases and cutinases, play an important role in the infection process similar to several other *Botrytis*-host interactions (Kars & van Kan, 2007). Additionally, *Botrytis* species can also affect the 'redox' process in the host plants, during the colonization of host tissues, through the production of enzymes like superoxide dismutase (Lyon et al., 2007). Due the great biological similarity of *Botrytis* spp. and *B. ricini*, probably, such enzymes also have an important role in the infection process of the causal agent of castor gray mold, as already evidenced by Orellana & Thomas (1962). Hoffmann et al. (2004) had, for example, extracted alpha and beta esterase and superoxide dismutase from *B. ricini,* however they did not perform a study to determine the role of such enzymes in the infection

It is important to highlight the great distinction between the penetration process of a fungus under controlled and highly favourable condition in contrast with the natural process in the field. In the latter case, all aerial parts of the host are potential targets for deposition and penetration of *B. ricini*, because not only the conidia, regarded as the major propagative unit, usually responsible for the epidemic outbreak, but also the ascospores, sclerotia and mycelia fragments can give rise to infection, as observed in *Botrytis* spp. (Jarvis, 1978). So, besides direct penetration, probably natural openings and wounds also serve as a point of entrance for the fungus. Growth of the fungus on the host surface and consequently its penetration in the host tissues will depend on factors such as inoculum type, free water and nutrient availability, cuticle features, presence of exudates on floral organs and other glands, besides the abundance of natural openings and the size and age of wounds, as pointed out by Holz

The primary targets of the fungus are the inflorescence and the capsules, in any development stage (Fig.2) (Araújo et al., 2007; Dange et al., 2005; Gonçalves, 1936; Lima et al., 2001). Some authors (Drumond & Coelho, 1981; Batista et al., 1996) claim that the male flowers are the first to be infected, but it is not always the case because any part of the inflorescence can be infected, the female flowers being the preferential target. That claim

**2.3 Host penetration and colonization** 

process of this fungus.

et al., (2007) for *Botrytis* species.

**2.4 Symptoms** 

further studies have been done to clarify these questions.

came from the fact that the male flowers are the first to be exposed, at the earlier stage of inflorescence formation; consequently such flowers are exposed longer to the infection units of the fungus. However, as soon as the male flowers suffer anthesis they are no longer a target and are hardly infected, contradicting the statement of Drumond & Coelho (1981) that "the fungus attacks first the male flowers because the anthers, being soaked with the rain water or dew, easily retain the fungus spores carried by the wind".

Fig. 2. Symptoms of gray mold attack on castor inflorescence and raceme. A-B. Symptoms on young inflorescences, before fertilization of female flowers. C-H. Symptoms on capsules at distinct development stages. Photos: D.J. Soares.

The first symptoms are visible as bluish spots on the inflorescences, on both female and male (before anthesis) flowers, and on developing fruits. On fruits, the symptoms can evolve to circular or elliptic, sunken, dark coloured spots that can result in rupture of the capsule (Fig.3 A-B) (Araújo et al., 2007). These symptoms are usually more frequent when a period of low relative humidity unfavourable to fungal sporulation occurs soon after the fungus penetrates the host tissues.

Depending on weather conditions (e.g. long periods with high relative humidity soon after the fungus penetrates the host), the occurrence of yellow ooze at the point of infection is frequent (Fig. 3 C-D) (Batista et al., 1996; Dange et al., 2005) as a result of the rapid enzymatic tissue degradation. The symptoms on the male flowers, before anthesis, are small, pale brown, necrotic spots, which can evolve to larger brown spots with a darker edge (Fig.3 E). The infected flowers and young capsules became softened due the fungal colonization and mycelial growth is, at first, pale gray and later dark olivaceous. A profuse sporulation is usually observed in such stage (Fig.3 F). When the infection starts on immature capsules, they become rotten; if the infection starts later, with fully developed capsules, the seeds usually became hollow, with coat discoloration and weight loss (Dange et al., 2005). On the

Gray Mold of Castor: A Review 225

There are few studies of the epidemiological aspects of gray mold on castor. Godfrey (1923) mentioned that temperatures around 25ºC and high relative humidity are highly favourable to disease development. Such statements have been exhaustively repeated in almost all publications about the subject over the last decades (Araújo et al., 2007; Batista et al., 1998; Gonçalves, 1936; Kimati, 1980; Lima & Soares, 1990; Massola JR & Bedendo, 1997; Melhorança & Staut, 2005;). The minimum and maximum temperature for mycelial growth

Some complementary studies have confirmed that temperatures around 25ºC are favourable to fungal growth and disease development (Araújo et al., 2003; Suassuna et al., 2003; Sussel, 2008). At temperatures below 20ºC, the disease is little expressed and highly dependent on long periods of high relative humidity (Sussel, 2008). According to Sussel et al. (2011), there is a high correlation between the temperature and duration of leaf wetness with the disease incidence and severity. The same authors also concluded that the disease was more intense with a temperature of 28ºC and 72 hours of leaf wetness, and that for temperatures below 15º the fungus needs more than 6 hours of leaf wetness, otherwise the disease does not occur (Sussel et al., 2011). Even when under optimal temperatures (near 25ºC), the fungus appears to be highly dependent on periods of high humidity, since in a study developed by Esuruoso (1966) with 39 castor varieties in two distinct places, Ibadan and Ilora (West Nigeria), that share similar temperatures (24.4 to 27.9 and 24.2 to 27.2°C, respectively), but that rainfall was twofold higher in Ibadan than Ilora, the disease level in Ibadan was 1.5 fold higher than in Ilora. This dependence on high rainfall, and not only on high relative humidity, was also observed in a study conducted in the Paraiba state (Northeast Brazil) where a high correlation between the disease progress and the accumulated rainfall between the seventh and fifth day before the evaluation was observed (D.J. Soares, unpublished data). The correlation, however, was inversely proportional to the rainfall intensity which means that long periods of low-intensity rains

Sussel (2008) concluded that the disease shows a random distribution pattern, matching its airborne nature. However, under high rainfall, the disease assumes an aggregate pattern, typical of those dispersed by water splash. With reference to aerobiology, Sussel (2008) obtained a positive correlation between the average number of conidia collected daily in air and the weather variables: minimum temperature, mean relative humidity, mean precipitation and leaf wetness. There was an increase in the number of conidia collected with the elevation of the minimum temperature from 14.6 to 18.1ºC; the same occurred when the mean relative humidity was raised from 42 to 95% and when the daily

Under controlled conditions, at 25ºC and relative humidity near saturation, Soares et al. (2010) had determined that the incubation period of *B. ricini* can vary from 44 to 88 hours (average 72 h) and the latent period from 72 to 144 hours (average 96 h), depending on the

Although in the recent years, there have been some advances in knowledge about the epidemiology of gray mold of castor, several issues remains unresolved. Further studies are needed to understand the role of each potential dispersal unit; how the fungus survives

was established by Godfrey as 12 and 35ºC, respectively (Godfrey, 1923).

are more favourable than short periods of high-intensity rains.

precipitation increased from 1 to 20 hours (Sussel, 2008, 2009a).

**2.5 Epidemiology** 

genotype.

inflorescence, the male flowers can be infected first, but the fungus has a clear preference for the female flowers (Fig.3 G). Infection can lead to complete destruction of the raceme (Fig.3 H), particularly if it reaches the main stem and the weather conditions are favourable for the disease. Several other plant parts, e.g. leaves, petioles and stem can also be infected, mainly due to the deposition or fall of infected material from the inflorescence or racemes. On leaves, the lesions are usually irregular, but can assume an elliptic or circular pattern, the size is very variable, sometimes coalescing and resulting in a foliar blight (Fig.3 I-J). On petioles and stems, necrotic, sunken lesions usually are formed which can cause the strangulation and consequently death of the parts above the infection point (Fig. K-N) (Batista et al., 1996; Dange et al., 2005).

Fig. 3. Symptoms of gray mold on castor plants. Dark-bluish spot (A) and capsule rupture (B) under unfavourable conditions; Gray-bluish spot with yellowish-brown ooze (C-D) under favourable conditions; Dark brown spot on male flowers before anthesis (E); Profuse sporulation on infected capsule (F); Infected inflorescence showing the fungus preference for female flowers (G); Completely destroyed inflorescence (H); Leaf spot (I) and blight (J); Apical infection (K); Petiole infection (L); Stem (M) and rachis infection (N). Photos: D.J. Soares.

#### **2.5 Epidemiology**

224 Plant Pathology

inflorescence, the male flowers can be infected first, but the fungus has a clear preference for the female flowers (Fig.3 G). Infection can lead to complete destruction of the raceme (Fig.3 H), particularly if it reaches the main stem and the weather conditions are favourable for the disease. Several other plant parts, e.g. leaves, petioles and stem can also be infected, mainly due to the deposition or fall of infected material from the inflorescence or racemes. On leaves, the lesions are usually irregular, but can assume an elliptic or circular pattern, the size is very variable, sometimes coalescing and resulting in a foliar blight (Fig.3 I-J). On petioles and stems, necrotic, sunken lesions usually are formed which can cause the strangulation and consequently death of the parts above the infection point (Fig. K-N)

Fig. 3. Symptoms of gray mold on castor plants. Dark-bluish spot (A) and capsule rupture (B) under unfavourable conditions; Gray-bluish spot with yellowish-brown ooze (C-D) under favourable conditions; Dark brown spot on male flowers before anthesis (E); Profuse sporulation on infected capsule (F); Infected inflorescence showing the fungus preference for female flowers (G); Completely destroyed inflorescence (H); Leaf spot (I) and blight (J); Apical infection (K); Petiole infection (L); Stem (M) and rachis infection (N). Photos: D.J.

(Batista et al., 1996; Dange et al., 2005).

Soares.

There are few studies of the epidemiological aspects of gray mold on castor. Godfrey (1923) mentioned that temperatures around 25ºC and high relative humidity are highly favourable to disease development. Such statements have been exhaustively repeated in almost all publications about the subject over the last decades (Araújo et al., 2007; Batista et al., 1998; Gonçalves, 1936; Kimati, 1980; Lima & Soares, 1990; Massola JR & Bedendo, 1997; Melhorança & Staut, 2005;). The minimum and maximum temperature for mycelial growth was established by Godfrey as 12 and 35ºC, respectively (Godfrey, 1923).

Some complementary studies have confirmed that temperatures around 25ºC are favourable to fungal growth and disease development (Araújo et al., 2003; Suassuna et al., 2003; Sussel, 2008). At temperatures below 20ºC, the disease is little expressed and highly dependent on long periods of high relative humidity (Sussel, 2008). According to Sussel et al. (2011), there is a high correlation between the temperature and duration of leaf wetness with the disease incidence and severity. The same authors also concluded that the disease was more intense with a temperature of 28ºC and 72 hours of leaf wetness, and that for temperatures below 15º the fungus needs more than 6 hours of leaf wetness, otherwise the disease does not occur (Sussel et al., 2011). Even when under optimal temperatures (near 25ºC), the fungus appears to be highly dependent on periods of high humidity, since in a study developed by Esuruoso (1966) with 39 castor varieties in two distinct places, Ibadan and Ilora (West Nigeria), that share similar temperatures (24.4 to 27.9 and 24.2 to 27.2°C, respectively), but that rainfall was twofold higher in Ibadan than Ilora, the disease level in Ibadan was 1.5 fold higher than in Ilora. This dependence on high rainfall, and not only on high relative humidity, was also observed in a study conducted in the Paraiba state (Northeast Brazil) where a high correlation between the disease progress and the accumulated rainfall between the seventh and fifth day before the evaluation was observed (D.J. Soares, unpublished data). The correlation, however, was inversely proportional to the rainfall intensity which means that long periods of low-intensity rains are more favourable than short periods of high-intensity rains.

Sussel (2008) concluded that the disease shows a random distribution pattern, matching its airborne nature. However, under high rainfall, the disease assumes an aggregate pattern, typical of those dispersed by water splash. With reference to aerobiology, Sussel (2008) obtained a positive correlation between the average number of conidia collected daily in air and the weather variables: minimum temperature, mean relative humidity, mean precipitation and leaf wetness. There was an increase in the number of conidia collected with the elevation of the minimum temperature from 14.6 to 18.1ºC; the same occurred when the mean relative humidity was raised from 42 to 95% and when the daily precipitation increased from 1 to 20 hours (Sussel, 2008, 2009a).

Under controlled conditions, at 25ºC and relative humidity near saturation, Soares et al. (2010) had determined that the incubation period of *B. ricini* can vary from 44 to 88 hours (average 72 h) and the latent period from 72 to 144 hours (average 96 h), depending on the genotype.

Although in the recent years, there have been some advances in knowledge about the epidemiology of gray mold of castor, several issues remains unresolved. Further studies are needed to understand the role of each potential dispersal unit; how the fungus survives

Gray Mold of Castor: A Review 227

(Holcomb et al., 1989; Russo & Rossman, 1991), *Euphorbia milli* (Sanoamuang, 1990), *Euphorbia pulcherrima* (Holcomb & Brown, 1990), *Euphorbia heterophylla* (Barreto & Evans, 1998), *Euphorbia inarticulata* (Alwadie & Baka, 2003), *Acalypha hispida* and *Jatropha podagrica* (Lima et al., 2008). Besides these reports of natural infection, artificial inoculations tests have shown that this pathogen has a wide host range within the Euphorbiaceae, including species of economic interest, e.g. cassava (*Manihot utilissima*) (Holcomb et al., 1989; Kumar et al., 2007; Lima et al., 2008). The sole report of natural infection by *B. ricini* outside the Euphorbiaceae family was made by Hansen & Bega (1955) on *Caladium bicolor* (Araceae)

Fig. 4. Visitor insects (flies and stingless bees) on castor inflorescence infected by *B. ricini* (A-C). Seeds with profuse gray mold growth and sporulation (D-E). Photos: D.J. Soares.

Hence, it is possible that the fungus can survive in the field on several other plant species belonging to Euphorbiaceae, and these can be an inoculum reservoir of the fungus in such a way that it would be ready to infect its preferential host, the castor plant, as soon as

susceptible tissues become available.

and, it is quite likely a case of fungus misidentification.

between the growth seasons, for example, as sclerotia, on secondary hosts, on spontaneous (volunteer) castor plants. Additionally, it is also crucial to determine whether it is possible to predict disease development based on environmental variables, like precipitation or surface wetness.

#### **2.5.1 Live cycle and host range**

The disease starts with spore deposition on the host surface, followed by penetration and colonization of the host tissues. Soon after colonization, the fungus, under favourable conditions, sporulates profusely on the dead tissues, and then the conidia became the main inoculum source for new infection sites. Although most authors recognized the major role of conidia after the disease had been established in the field, there is much speculation about the primary inoculum source of *B. ricini*.

Godfrey (1923) claims that the fungus survives on soil or crop residues as sclerotia and, under the right conditions, these can produce sexual structures, which will be responsible for the initial infection. However, there is no report of sexual reproduction under natural conditions, other than the original reports of Godfrey (1919, 1923), so the purported role of ascospores as the initial inoculum source remains unclear, despite the fact that apothecia are easily overlooked in the field.

Under tropical climate, the initial inoculum source are probably the conidia from wild castor plants which grows spontaneously near the crop areas all year (Gonçalves, 1936). Wild castor plants can produce flowers throughout the year and consequently new susceptible tissue will be available for the fungus to self perpetuate in its anamorphic state through the year. By infecting the first inflorescence under favourable conditions, the fungus produces abundant sporulation, thus allowing multiple rounds of re-infection, since this pathogen is easily spread by wind, rain splash and, probably, by insects (Fig. 4 A-C) (Dange et al., 2005).

It was also mentioned by Godfrey (1923), that the fungus is seed-borne, the seeds being regarded as the primary inoculum source (Fig. 4 D-E). However, the role of seeds as a primary inoculum source requires further study. Probably the seeds have no essential role at the beginning of the epidemic because there is usually an interval of almost two months between sowing and flowering, so the inoculum originating from the seeds will not be available to infect the flowers. This means that, although *B. ricini* is a seed-borne fungus, it is probably not seed transmitted, because it could hardly infect the crops which grow from them, as conceived by Maude (1996). Thus, the most important role of the seeds is to carry the pathogen to new areas, rather than to act as a primary inoculum source for epidemic on the crop season.

Initially, it was suspected that *B. ricini* was host-specific and that it has a very narrow host range (Godfrey, 1923). Through artificial inoculation, Godfrey (1923) showed that the fungus is extremely dependent on high humidity and it was not able to cause disease, at same levels observed on castor plant, when inoculated on several other hosts, including members of Euphorbiaceae. In field inspections, the same author did not detect any other plant species showing symptoms of natural infection in the surroundings of castor-growing areas which were severely affected by the disease. Nevertheless, since the 1980s several reports of natural infection of *B. ricini* on members of Euphorbiaceae have been made, including both weeds and ornamentals: such as *Caperonia palustris* (Whitney & Taber,1986), *Euphorbia supina*

between the growth seasons, for example, as sclerotia, on secondary hosts, on spontaneous (volunteer) castor plants. Additionally, it is also crucial to determine whether it is possible to predict disease development based on environmental variables, like precipitation or surface

The disease starts with spore deposition on the host surface, followed by penetration and colonization of the host tissues. Soon after colonization, the fungus, under favourable conditions, sporulates profusely on the dead tissues, and then the conidia became the main inoculum source for new infection sites. Although most authors recognized the major role of conidia after the disease had been established in the field, there is much speculation about

Godfrey (1923) claims that the fungus survives on soil or crop residues as sclerotia and, under the right conditions, these can produce sexual structures, which will be responsible for the initial infection. However, there is no report of sexual reproduction under natural conditions, other than the original reports of Godfrey (1919, 1923), so the purported role of ascospores as the initial inoculum source remains unclear, despite the fact that apothecia are

Under tropical climate, the initial inoculum source are probably the conidia from wild castor plants which grows spontaneously near the crop areas all year (Gonçalves, 1936). Wild castor plants can produce flowers throughout the year and consequently new susceptible tissue will be available for the fungus to self perpetuate in its anamorphic state through the year. By infecting the first inflorescence under favourable conditions, the fungus produces abundant sporulation, thus allowing multiple rounds of re-infection, since this pathogen is easily spread by wind, rain splash and, probably, by insects (Fig. 4

It was also mentioned by Godfrey (1923), that the fungus is seed-borne, the seeds being regarded as the primary inoculum source (Fig. 4 D-E). However, the role of seeds as a primary inoculum source requires further study. Probably the seeds have no essential role at the beginning of the epidemic because there is usually an interval of almost two months between sowing and flowering, so the inoculum originating from the seeds will not be available to infect the flowers. This means that, although *B. ricini* is a seed-borne fungus, it is probably not seed transmitted, because it could hardly infect the crops which grow from them, as conceived by Maude (1996). Thus, the most important role of the seeds is to carry the pathogen to new

areas, rather than to act as a primary inoculum source for epidemic on the crop season.

Initially, it was suspected that *B. ricini* was host-specific and that it has a very narrow host range (Godfrey, 1923). Through artificial inoculation, Godfrey (1923) showed that the fungus is extremely dependent on high humidity and it was not able to cause disease, at same levels observed on castor plant, when inoculated on several other hosts, including members of Euphorbiaceae. In field inspections, the same author did not detect any other plant species showing symptoms of natural infection in the surroundings of castor-growing areas which were severely affected by the disease. Nevertheless, since the 1980s several reports of natural infection of *B. ricini* on members of Euphorbiaceae have been made, including both weeds and ornamentals: such as *Caperonia palustris* (Whitney & Taber,1986), *Euphorbia supina*

wetness.

**2.5.1 Live cycle and host range** 

the primary inoculum source of *B. ricini*.

easily overlooked in the field.

A-C) (Dange et al., 2005).

(Holcomb et al., 1989; Russo & Rossman, 1991), *Euphorbia milli* (Sanoamuang, 1990), *Euphorbia pulcherrima* (Holcomb & Brown, 1990), *Euphorbia heterophylla* (Barreto & Evans, 1998), *Euphorbia inarticulata* (Alwadie & Baka, 2003), *Acalypha hispida* and *Jatropha podagrica* (Lima et al., 2008). Besides these reports of natural infection, artificial inoculations tests have shown that this pathogen has a wide host range within the Euphorbiaceae, including species of economic interest, e.g. cassava (*Manihot utilissima*) (Holcomb et al., 1989; Kumar et al., 2007; Lima et al., 2008). The sole report of natural infection by *B. ricini* outside the Euphorbiaceae family was made by Hansen & Bega (1955) on *Caladium bicolor* (Araceae) and, it is quite likely a case of fungus misidentification.

Fig. 4. Visitor insects (flies and stingless bees) on castor inflorescence infected by *B. ricini* (A-C). Seeds with profuse gray mold growth and sporulation (D-E). Photos: D.J. Soares.

Hence, it is possible that the fungus can survive in the field on several other plant species belonging to Euphorbiaceae, and these can be an inoculum reservoir of the fungus in such a way that it would be ready to infect its preferential host, the castor plant, as soon as susceptible tissues become available.

Gray Mold of Castor: A Review 229

usually is conferred by numerous and complex mechanisms and can include traits such as tolerance and disease-escape which are not strictly resistance mechanisms *per se* (Robinson, 1976). It is also important to highlight the fact that horizontal resistance usually is an efficient way to achieve disease control, and that in the recent years selection for horizontal resistance has become easier with the use of marker-assisted breeding

In Brazil, several studies have been carried out aiming to select for a resistance source to gray mold. However, what has become clear is that whilst there are differences in susceptibility among the assayed genotypes, none has the desired resistance level (Batista et al., 1998; Costa et al., 2004; Lima & Soares, 1990; Milani et al., 2005; Rego Filho et al., 2007). Among the genotypes assessed in different countries, thus far, several distinct levels of susceptibility have been observed, but not immunity (Anjani et al. 2004; Esuruoso, 1996; Zarzycka, 1958). The way that the breeding programs are being conducted, using only information generated by field investigations, and which are usually affected by uncontrollable factors, is much more likely to select for "field resistance" rather than for genetic resistance, i.e., the plant and raceme architecture, together with the weather conditions, play a bigger role in the disease development, by inducing a micro-climate formation, which might be more or less favourable to the pathogen. This means that unless we redirect our thinking to incorporate horizontal resistance, through a careful marker-assisted breeding program, whether it be genetic or morphological, it will probably never be possible to obtain a variety with satisfactory

Some castor plant varieties have a thick outer wax layer which can act as a constitutive barrier to pathogen infection. However, there is no information about the role of this wax layer in the infection process of *B. ricini* and, although the adhesion of the conidia and subsequently their penetration into the host tissue could be difficult, the wax could also act as an elicitor; being responsible for the initial recognition of the host-pathogen interaction. Thus, the wax layer could actually favour the pathogen rather than inhibit its development. Field observations lead us to hypothesize that the latter, is the most probable scenario in the

As commented previously, most of the research involving the assessment of genotypes resistance has been conducted under field conditions and without any standard to quantify the disease and, worst still, sometimes using very subjective assessment methods, like that adopted by Lima & Soares (1990). This has been reflected by the fact that it is almost impossible perform a comparison among the different already studies undertaken. However, a diagrammatic scale to assess gray mold severity in castor- was published recently (Fig.5) (Sussel et al., 2009; Sussel, 2009b). This was developed in order to standardize disease assessment in field experiments, and was constructed based on the Weber-Fechner law and divided into 10 levels. Although useful, such a scale faces two crucial factors which might constitute an impediment to its wider adoption: first, there is great variation in raceme architecture among the castor genotypes, and; secondly, it only considers the fully developed raceme. In the first case, as the diagrammatic scale was drawn based on long conical racemes, its use to assess disease severity in genotypes with more or

(Keane, 2012).

levels of resistance.

present pathosystem.

**2.6.1 Host resistance assessment** 

#### **2.6 Host resistance**

The search for resistance to gray mold has been investigated ever since the disease was described. In his classic work, Godfrey (1923) pointed out general conclusions that clearly have been overlooked over the decades by plant breeders and plant pathologists alike. Among these, three are worth mentioning: "(1) Plants of more ornamental type, with stalk, foliage, and sometimes pods in different shades of red or reddish green were more resistant; (2) All smaller, many branched plants, which by their yield indicated commercial possibilities, showed high susceptibility to the disease; and (3) Cross pollination in castorbean fields probably occurs very extensively. It would require years of work to develop pure strains and then to select and breed for desirable qualities combined with resistance before permanent results could be secured" (Godfrey, 1923). Based on this information, it is clear that only minor progress has been achieved toward the development of resistant cultivars during the last century. As a result, breeding programs have failed to develop a resistant cultivar or hybrid until today.

Gonçalves (1936) reported that "spontaneous varieties" are highly resistant to the disease, since the fungus attacks only few capsules, while most of the capsules of the same raceme or from others raceme remain healthy. Although this behaviour is frequently observed in wild types, and even in some commercial cultivars, this statement must be viewed with caution since it has no scientific basis and probably such behaviour is a result of the wide genetic variation within castor plants.

According to several authors, varieties with more compact racemes, shorter internodes, and male flowers distributed all long the inflorescence are considered to be more susceptible to the pathogen (Batista et al., 1998; Costa et al., 2004; Dange et al., 2005; Milani et al., 2005; Thomas & Orellana, 1963b; Ueno et al., 2006; Zarzycka, 1958): a fact already noted by Godfrey (1923), and also by Esuruoso (1966), who concluded that the disease severity was more intense on short-stalked varieties than on those with longer stalks. Another relevant aspect is that, apparently, the presence of spines in the capsule predisposes them to pathogen attack (Alcântara et al., 2008; Cook, 1981; Lima & Soares, 1990).

Plants with capsules containing high soluble sugar concentrations are more susceptible to fungal development than plants with low soluble sugar concentrations (Orellana & Thomas, 1962). According to these authors, capsule resistance is intimately associated with its capacity of inactivation of pectic, cellulolytic and others hydrolytic enzymes through the products of the phenol oxidation (Thomas & Orellana, 1963a), as well as lower content of water-soluble pectin, higher content of calcium and magnesium and lower sodium and potassium contents (Thomas & Orellana, 1964).

Although several studies have been conducted to assess the resistance of castor bean genotypes, none of them deals with the inheritance of resistance to *B. ricini*, nor how it is governed. Probably, the resistance to gray mold is quantitative and possibly governed by several minor genes.

Quantitative resistance, also called horizontal resistance, is, according to Robinson (1976), universal and occurs in all plants against all parasites; it is also permanent and permits cumulative plant breeding. Horizontal resistance can be either passive or active, and

The search for resistance to gray mold has been investigated ever since the disease was described. In his classic work, Godfrey (1923) pointed out general conclusions that clearly have been overlooked over the decades by plant breeders and plant pathologists alike. Among these, three are worth mentioning: "(1) Plants of more ornamental type, with stalk, foliage, and sometimes pods in different shades of red or reddish green were more resistant; (2) All smaller, many branched plants, which by their yield indicated commercial possibilities, showed high susceptibility to the disease; and (3) Cross pollination in castorbean fields probably occurs very extensively. It would require years of work to develop pure strains and then to select and breed for desirable qualities combined with resistance before permanent results could be secured" (Godfrey, 1923). Based on this information, it is clear that only minor progress has been achieved toward the development of resistant cultivars during the last century. As a result, breeding programs have failed to develop a

Gonçalves (1936) reported that "spontaneous varieties" are highly resistant to the disease, since the fungus attacks only few capsules, while most of the capsules of the same raceme or from others raceme remain healthy. Although this behaviour is frequently observed in wild types, and even in some commercial cultivars, this statement must be viewed with caution since it has no scientific basis and probably such behaviour is a result of the wide genetic

According to several authors, varieties with more compact racemes, shorter internodes, and male flowers distributed all long the inflorescence are considered to be more susceptible to the pathogen (Batista et al., 1998; Costa et al., 2004; Dange et al., 2005; Milani et al., 2005; Thomas & Orellana, 1963b; Ueno et al., 2006; Zarzycka, 1958): a fact already noted by Godfrey (1923), and also by Esuruoso (1966), who concluded that the disease severity was more intense on short-stalked varieties than on those with longer stalks. Another relevant aspect is that, apparently, the presence of spines in the capsule predisposes them to pathogen attack (Alcântara et al., 2008; Cook, 1981; Lima & Soares,

Plants with capsules containing high soluble sugar concentrations are more susceptible to fungal development than plants with low soluble sugar concentrations (Orellana & Thomas, 1962). According to these authors, capsule resistance is intimately associated with its capacity of inactivation of pectic, cellulolytic and others hydrolytic enzymes through the products of the phenol oxidation (Thomas & Orellana, 1963a), as well as lower content of water-soluble pectin, higher content of calcium and magnesium and lower sodium and

Although several studies have been conducted to assess the resistance of castor bean genotypes, none of them deals with the inheritance of resistance to *B. ricini*, nor how it is governed. Probably, the resistance to gray mold is quantitative and possibly governed by

Quantitative resistance, also called horizontal resistance, is, according to Robinson (1976), universal and occurs in all plants against all parasites; it is also permanent and permits cumulative plant breeding. Horizontal resistance can be either passive or active, and

**2.6 Host resistance** 

resistant cultivar or hybrid until today.

potassium contents (Thomas & Orellana, 1964).

variation within castor plants.

1990).

several minor genes.

usually is conferred by numerous and complex mechanisms and can include traits such as tolerance and disease-escape which are not strictly resistance mechanisms *per se* (Robinson, 1976). It is also important to highlight the fact that horizontal resistance usually is an efficient way to achieve disease control, and that in the recent years selection for horizontal resistance has become easier with the use of marker-assisted breeding (Keane, 2012).

In Brazil, several studies have been carried out aiming to select for a resistance source to gray mold. However, what has become clear is that whilst there are differences in susceptibility among the assayed genotypes, none has the desired resistance level (Batista et al., 1998; Costa et al., 2004; Lima & Soares, 1990; Milani et al., 2005; Rego Filho et al., 2007). Among the genotypes assessed in different countries, thus far, several distinct levels of susceptibility have been observed, but not immunity (Anjani et al. 2004; Esuruoso, 1996; Zarzycka, 1958). The way that the breeding programs are being conducted, using only information generated by field investigations, and which are usually affected by uncontrollable factors, is much more likely to select for "field resistance" rather than for genetic resistance, i.e., the plant and raceme architecture, together with the weather conditions, play a bigger role in the disease development, by inducing a micro-climate formation, which might be more or less favourable to the pathogen. This means that unless we redirect our thinking to incorporate horizontal resistance, through a careful marker-assisted breeding program, whether it be genetic or morphological, it will probably never be possible to obtain a variety with satisfactory levels of resistance.

Some castor plant varieties have a thick outer wax layer which can act as a constitutive barrier to pathogen infection. However, there is no information about the role of this wax layer in the infection process of *B. ricini* and, although the adhesion of the conidia and subsequently their penetration into the host tissue could be difficult, the wax could also act as an elicitor; being responsible for the initial recognition of the host-pathogen interaction. Thus, the wax layer could actually favour the pathogen rather than inhibit its development. Field observations lead us to hypothesize that the latter, is the most probable scenario in the present pathosystem.

#### **2.6.1 Host resistance assessment**

As commented previously, most of the research involving the assessment of genotypes resistance has been conducted under field conditions and without any standard to quantify the disease and, worst still, sometimes using very subjective assessment methods, like that adopted by Lima & Soares (1990). This has been reflected by the fact that it is almost impossible perform a comparison among the different already studies undertaken. However, a diagrammatic scale to assess gray mold severity in castor- was published recently (Fig.5) (Sussel et al., 2009; Sussel, 2009b). This was developed in order to standardize disease assessment in field experiments, and was constructed based on the Weber-Fechner law and divided into 10 levels. Although useful, such a scale faces two crucial factors which might constitute an impediment to its wider adoption: first, there is great variation in raceme architecture among the castor genotypes, and; secondly, it only considers the fully developed raceme. In the first case, as the diagrammatic scale was drawn based on long conical racemes, its use to assess disease severity in genotypes with more or

Gray Mold of Castor: A Review 231

the disease (Esuruoso 1969). At the Embrapa Algodão Station - a branch of the Brazilian Agricultural Research Agency, responsible for developing research on castor - we have developed a controlled method to assess castor resistance to gray mold. The principal advantage of this method is to eliminate the unpredictable changes usually present under field evaluation, and then to select the castor plants based solely on their genetic background rather than select a phenotype. Based on such studies, it has been possible to obtain a clear difference among the so-called susceptible and resistant genotypes, which had been previously screened under field conditions. It has also been possible to stratify the genotypes within at least three ranks: highly susceptible, moderately susceptible and less susceptible (Silva et al., 2008; Soares et al., 2010), although none of the screened

Without doubt, protection of the inflorescences and immature capsules is crucial to avoid heavy yield losses when castor is cultivated under favourable disease conditions. Fully developed capsules are less susceptible to pathogen attack and the severity levels are usually lower when compared with infection in young capsules or on the inflorescence. However, it is important to note that under highly favourable conditions with high inoculum pressure, losses of 100% are relatively frequent (Anjani et al., 2004). There is no single measure to keep the disease under acceptable levels; as well as there is no knowledge about any acceptable disease level. As the pathogen has a very short incubation period, and is easily wind-dispersed, its destructive potential is very high and usually the growers do not want to take their chances and wait passively, they usually prefer to act before or at the

Cultural practices are usually applied at aiming to prevent the introduction of inoculum into the field, reducing its survival, spread or build-up, or rendering the host less prone to disease attack (Palti & Rotem, 1983; Termorshuizen, 2001). The use of varietal resistance is regarded as the better method for disease management. However, as highlighted previously, there are no varieties with satisfactory resistance levels to gray mold (Anjani et al., 2003; Araújo et al., 2007; Cook, 1981; Dange et al., 2005; Kolte, 1995; Milani et al.,

Several authors have recommended the use of healthy seeds, removal of plant debris, adequate choice of planting area and growing season, and use of less susceptible cultivars (Galli et al., 1968; Massola Jr. & Bedendo, 2005; Sussel, 2009a). It is also recommended to use plant spacing adjusted for maximum aeration (Kolte, 1995; Lima et al., 2005). The use of healthy seeds, including seed treatment with fungicides, is always a desirable practice, however, its practical benefits for the management gray mold are questionable, because as mentioned previously, it is unlikely that such seeds will serve as an inoculum source for that crop season, so this practice has much more value in promoting vigorous plant growth and avoiding the introduction of the pathogen into new areas. Elimination of alternate and reservoir hosts (euphorbiaceous hosts), as well as removal and destruction of inoculum persisting in plant residues, are welcome practices for management of gray mold, and

genotypes were regarded as resistant.

**2.7 Disease management** 

first signs of the disease.

**2.7.1 Cultural** 

2005).

less globose raceme will be difficult. In the second case, the scale does not take into account disease severity in inflorescences or even in immature racemes, where the disease is usually more severe and more difficult to estimate. Therefore, it is possible that the scale will underestimate disease severity. Nonetheless, the simple fact that such a diagrammatic scale is now available is a huge advance towards more reliable disease assessment and the possibility of comparing assays conducted by different researchers in different localities. Chagas et al. (2010) also developed a diagrammatic scale, with six levels, to assess the disease severity of gray mold on castor, but similar to the scale developed by Sussel et al. (2009), this scale was also developed based on long conical, full developed, racemes, and thus, will face the same problem mentioned above.

Fig. 5. Diagrammatic scale to assess the gray mold severity on castor (Reprinted from: Sussel et al. *Tropical Plant Pathology*, Vol. 34, No.3, pp.186-191, 2009, by permission). Numbers represents the percent area affected by the disease.

The first attempt to evaluate disease resistance under controlled conditions was made by C.A. Thomas & R.G. Orellana in 1963 using a biochemical test (Thomas & Orellana, 1963b). This methodology was reproduced by O.F. Esuruoso in Nigeria, a few years later (Esuruoso, 1969). This last author concluded that the laboratory results were similar to previous observations from field tests, and none of the tested varieties were resistant to

less globose raceme will be difficult. In the second case, the scale does not take into account disease severity in inflorescences or even in immature racemes, where the disease is usually more severe and more difficult to estimate. Therefore, it is possible that the scale will underestimate disease severity. Nonetheless, the simple fact that such a diagrammatic scale is now available is a huge advance towards more reliable disease assessment and the possibility of comparing assays conducted by different researchers in different localities. Chagas et al. (2010) also developed a diagrammatic scale, with six levels, to assess the disease severity of gray mold on castor, but similar to the scale developed by Sussel et al. (2009), this scale was also developed based on long conical, full developed, racemes, and

Fig. 5. Diagrammatic scale to assess the gray mold severity on castor (Reprinted from: Sussel et al. *Tropical Plant Pathology*, Vol. 34, No.3, pp.186-191, 2009, by permission). Numbers

The first attempt to evaluate disease resistance under controlled conditions was made by C.A. Thomas & R.G. Orellana in 1963 using a biochemical test (Thomas & Orellana, 1963b). This methodology was reproduced by O.F. Esuruoso in Nigeria, a few years later (Esuruoso, 1969). This last author concluded that the laboratory results were similar to previous observations from field tests, and none of the tested varieties were resistant to

thus, will face the same problem mentioned above.

represents the percent area affected by the disease.

the disease (Esuruoso 1969). At the Embrapa Algodão Station - a branch of the Brazilian Agricultural Research Agency, responsible for developing research on castor - we have developed a controlled method to assess castor resistance to gray mold. The principal advantage of this method is to eliminate the unpredictable changes usually present under field evaluation, and then to select the castor plants based solely on their genetic background rather than select a phenotype. Based on such studies, it has been possible to obtain a clear difference among the so-called susceptible and resistant genotypes, which had been previously screened under field conditions. It has also been possible to stratify the genotypes within at least three ranks: highly susceptible, moderately susceptible and less susceptible (Silva et al., 2008; Soares et al., 2010), although none of the screened genotypes were regarded as resistant.

#### **2.7 Disease management**

Without doubt, protection of the inflorescences and immature capsules is crucial to avoid heavy yield losses when castor is cultivated under favourable disease conditions. Fully developed capsules are less susceptible to pathogen attack and the severity levels are usually lower when compared with infection in young capsules or on the inflorescence. However, it is important to note that under highly favourable conditions with high inoculum pressure, losses of 100% are relatively frequent (Anjani et al., 2004). There is no single measure to keep the disease under acceptable levels; as well as there is no knowledge about any acceptable disease level. As the pathogen has a very short incubation period, and is easily wind-dispersed, its destructive potential is very high and usually the growers do not want to take their chances and wait passively, they usually prefer to act before or at the first signs of the disease.

#### **2.7.1 Cultural**

Cultural practices are usually applied at aiming to prevent the introduction of inoculum into the field, reducing its survival, spread or build-up, or rendering the host less prone to disease attack (Palti & Rotem, 1983; Termorshuizen, 2001). The use of varietal resistance is regarded as the better method for disease management. However, as highlighted previously, there are no varieties with satisfactory resistance levels to gray mold (Anjani et al., 2003; Araújo et al., 2007; Cook, 1981; Dange et al., 2005; Kolte, 1995; Milani et al., 2005).

Several authors have recommended the use of healthy seeds, removal of plant debris, adequate choice of planting area and growing season, and use of less susceptible cultivars (Galli et al., 1968; Massola Jr. & Bedendo, 2005; Sussel, 2009a). It is also recommended to use plant spacing adjusted for maximum aeration (Kolte, 1995; Lima et al., 2005). The use of healthy seeds, including seed treatment with fungicides, is always a desirable practice, however, its practical benefits for the management gray mold are questionable, because as mentioned previously, it is unlikely that such seeds will serve as an inoculum source for that crop season, so this practice has much more value in promoting vigorous plant growth and avoiding the introduction of the pathogen into new areas. Elimination of alternate and reservoir hosts (euphorbiaceous hosts), as well as removal and destruction of inoculum persisting in plant residues, are welcome practices for management of gray mold, and

Gray Mold of Castor: A Review 233

days), the use of the same fungicides could not stop the disease and the losses reached 100%

Despite the limited studies dealing with chemical control of gray mold of castor, there are several fungicides known as "botryoticides" which are effective in protecting crops against *Botrytis* spp. (Leroux, 2007), and probably we can assume that these fungicides also are effective against *B. ricini.* In several *Botrytis* pathosystems, there is a high concern about the use of fungicides just before, or even after the harvest because of the toxicological risks of their residues (Leroux, 2007). In contrast, in castor crops, such a restriction is not a concern and fungicides can be applied just before harvest. However, if used indiscriminately, there must be concern about resistance phenomena associated with several major botryticide families, including benzimidazoles, phenylcarbamates and dicarboxymides (Leroux, 2007). Hence, control of gray mold of castor must consider several, rather than just one, management practices, as is currently recommended for the management of other *Botrytis*

There are several studies dealing with the use of biological control agents, mainly *Trichoderma* spp. and *Clonostachys rosea* to control diseases caused by *Botrytis* spp. (Elad & Stewart, 2007). If we consider the fact that the genera *Botrytis* and *Amphobotrys* are biologically similar and, probably, phylogenetically related, we could expect that the use of such biological control agents could be applied as an effective strategy in the pathosystem *B. ricini* x *R. communis*. Actually, there are some studies conducted with *Trichoderma* and *Clonostachys rosea* for the control of gray mold of castor and promising results have been obtained (Bhattiprolu & Bhattiprolu, 2006; Chagas, 2009; Demant et al., 2006; Raoof et al., 2003; Tirupathi et al., 2006). However, it is clear that, although promising, these results are still experimental and much work needs to be done before permanent recommendations regarding the use of biological control agents can be

The major problem about castor gray mold remains the lack of basic knowledge about its causal agent, how the disease develops, which factors are conducive to epidemics, and how we can manage it. It is necessary to elucidate, for example, what is the role of the climatic variables over the monocyclic components of the disease and how they affect the development of epidemics in the field. A better understanding of such relationships will determine which areas are suitable to grow castor. It is also imperative to know whether sexual reproduction is occurring within *B. ricini* populations in order to prevent fungicide resistance developing, perhaps by recommending permutations of fungicides with distinct active molecules and to determine the role of ascospores during the beginning of epidemics. However, we must first determine which fungicides are effective against gray mold, as well as their timing and frequency of application. We must also determine how resistance to the disease is inherited and if there are any phenotypic or genetic markers associated with it, so that breeders can more effectively generate resistant varieties using marker-assisted programs. Additionally, economic and cost-benefit analyses should be conducted to

(D.J. Soares unpublished data).

diseases (Leroux, 2007).

secured for gray mold management.

**3. Future challenges** 

**2.7.3 Biological** 

usually result in lower disease levels. Nevertheless, this practice must be followed by rigorous field inspection since the fungus is easily wind-dispersed, and once established in an area, such practice will become unfeasible. Perhaps, among the recognized cultural practices, the choice of growing season, or sowing time in such a way that spike development and maturity occur during the dry season, consequently avoiding the long, wet periods favourable to disease development, should be the most efficient one (Dange et al., 2005; Kolte, 1995); as evidenced by the fact that in locations where dry weather prevails, the disease does not occur (Godfrey, 1923; Kolte, 1995). This situation is typically observed in the Bahia state of Brazil, as mentioned previously.

#### **2.7.2 Chemical**

Seed treatment has been the management strategy most frequently recommended (Araújo et al., 2007; Batista et al., 1996; Godfrey, 1923; Gonçalves, 1936; Massola Jr. & Bedendo, 2005; Milani et al., 2005; Sussel, 2009), mainly to avoid the introduction of the pathogen into new areas. However, as the pathogen is air-borne and since it is already reported in almost all countries where castor is cultivated, the efficacy of such measures needs to be corroborated, because doubts about the role of the seeds in this pathosystem, as a primary inoculum source, still need to be clarified.

After disease establishment, fungicide spraying is usually the only way to stop or reduce the disease progress. However, there are few studies on chemical control of gray mold, and, of the fungicides tested, none is registered for use on castor crops in Brazil. According to Araújo et al. (2007), spraying of systemic fungicides soon after the appearance of the first symptoms delayed the epidemic and reduced disease progress.

In India Dange et al. (2005) recommended two prophylactic sprays with carbendazim (0.05%): the first at 50% of flowering; and the second, when the first disease symptoms appear. Although, Anjani et al. (2004) considered that non-genetic management measures have failed to control gray mold.

In the last decades, significant progress has been achieved regarding the use of fungicides to control plant diseases. Several new active fungicides with distinct modes of action, and usually with high specificity, have provided satisfactory levels of control of many plant diseases. However, for gray mold of castor, the main issue is not the ineffectiveness of fungicidal products, but the lack of research on the most appropriate timing of fungicide application and optimum dose, including also cost-benefit analyses.

Preliminary studies under controlled conditions have shown that carbendazim and azoxystrobin are effective against the gray mold pathogen (Bezerra, 2007). According to Chagas (2009), however, azoxystrobin was ineffective against *B. ricini*, while carbendazin and several others fungicides, including tebuconazole, iprodione and procymidone, were highly effective.

A field study, conducted under highly favourable conditions for disease development and using susceptible varieties, has confirmed that procymidone and iprodione are effective in disease control, but only if applied at the beginning of the epidemic and at weekly intervals. Where the timing of the first spraying was lost, and the application intervals were longer (15

usually result in lower disease levels. Nevertheless, this practice must be followed by rigorous field inspection since the fungus is easily wind-dispersed, and once established in an area, such practice will become unfeasible. Perhaps, among the recognized cultural practices, the choice of growing season, or sowing time in such a way that spike development and maturity occur during the dry season, consequently avoiding the long, wet periods favourable to disease development, should be the most efficient one (Dange et al., 2005; Kolte, 1995); as evidenced by the fact that in locations where dry weather prevails, the disease does not occur (Godfrey, 1923; Kolte, 1995). This situation is typically observed

Seed treatment has been the management strategy most frequently recommended (Araújo et al., 2007; Batista et al., 1996; Godfrey, 1923; Gonçalves, 1936; Massola Jr. & Bedendo, 2005; Milani et al., 2005; Sussel, 2009), mainly to avoid the introduction of the pathogen into new areas. However, as the pathogen is air-borne and since it is already reported in almost all countries where castor is cultivated, the efficacy of such measures needs to be corroborated, because doubts about the role of the seeds in this pathosystem, as a primary inoculum

After disease establishment, fungicide spraying is usually the only way to stop or reduce the disease progress. However, there are few studies on chemical control of gray mold, and, of the fungicides tested, none is registered for use on castor crops in Brazil. According to Araújo et al. (2007), spraying of systemic fungicides soon after the appearance of the first

In India Dange et al. (2005) recommended two prophylactic sprays with carbendazim (0.05%): the first at 50% of flowering; and the second, when the first disease symptoms appear. Although, Anjani et al. (2004) considered that non-genetic management measures

In the last decades, significant progress has been achieved regarding the use of fungicides to control plant diseases. Several new active fungicides with distinct modes of action, and usually with high specificity, have provided satisfactory levels of control of many plant diseases. However, for gray mold of castor, the main issue is not the ineffectiveness of fungicidal products, but the lack of research on the most appropriate timing of fungicide

Preliminary studies under controlled conditions have shown that carbendazim and azoxystrobin are effective against the gray mold pathogen (Bezerra, 2007). According to Chagas (2009), however, azoxystrobin was ineffective against *B. ricini*, while carbendazin and several others fungicides, including tebuconazole, iprodione and procymidone, were

A field study, conducted under highly favourable conditions for disease development and using susceptible varieties, has confirmed that procymidone and iprodione are effective in disease control, but only if applied at the beginning of the epidemic and at weekly intervals. Where the timing of the first spraying was lost, and the application intervals were longer (15

in the Bahia state of Brazil, as mentioned previously.

symptoms delayed the epidemic and reduced disease progress.

application and optimum dose, including also cost-benefit analyses.

**2.7.2 Chemical** 

source, still need to be clarified.

have failed to control gray mold.

highly effective.

days), the use of the same fungicides could not stop the disease and the losses reached 100% (D.J. Soares unpublished data).

Despite the limited studies dealing with chemical control of gray mold of castor, there are several fungicides known as "botryoticides" which are effective in protecting crops against *Botrytis* spp. (Leroux, 2007), and probably we can assume that these fungicides also are effective against *B. ricini.* In several *Botrytis* pathosystems, there is a high concern about the use of fungicides just before, or even after the harvest because of the toxicological risks of their residues (Leroux, 2007). In contrast, in castor crops, such a restriction is not a concern and fungicides can be applied just before harvest. However, if used indiscriminately, there must be concern about resistance phenomena associated with several major botryticide families, including benzimidazoles, phenylcarbamates and dicarboxymides (Leroux, 2007). Hence, control of gray mold of castor must consider several, rather than just one, management practices, as is currently recommended for the management of other *Botrytis* diseases (Leroux, 2007).

#### **2.7.3 Biological**

There are several studies dealing with the use of biological control agents, mainly *Trichoderma* spp. and *Clonostachys rosea* to control diseases caused by *Botrytis* spp. (Elad & Stewart, 2007). If we consider the fact that the genera *Botrytis* and *Amphobotrys* are biologically similar and, probably, phylogenetically related, we could expect that the use of such biological control agents could be applied as an effective strategy in the pathosystem *B. ricini* x *R. communis*. Actually, there are some studies conducted with *Trichoderma* and *Clonostachys rosea* for the control of gray mold of castor and promising results have been obtained (Bhattiprolu & Bhattiprolu, 2006; Chagas, 2009; Demant et al., 2006; Raoof et al., 2003; Tirupathi et al., 2006). However, it is clear that, although promising, these results are still experimental and much work needs to be done before permanent recommendations regarding the use of biological control agents can be secured for gray mold management.

#### **3. Future challenges**

The major problem about castor gray mold remains the lack of basic knowledge about its causal agent, how the disease develops, which factors are conducive to epidemics, and how we can manage it. It is necessary to elucidate, for example, what is the role of the climatic variables over the monocyclic components of the disease and how they affect the development of epidemics in the field. A better understanding of such relationships will determine which areas are suitable to grow castor. It is also imperative to know whether sexual reproduction is occurring within *B. ricini* populations in order to prevent fungicide resistance developing, perhaps by recommending permutations of fungicides with distinct active molecules and to determine the role of ascospores during the beginning of epidemics. However, we must first determine which fungicides are effective against gray mold, as well as their timing and frequency of application. We must also determine how resistance to the disease is inherited and if there are any phenotypic or genetic markers associated with it, so that breeders can more effectively generate resistant varieties using marker-assisted programs. Additionally, economic and cost-benefit analyses should be conducted to

Gray Mold of Castor: A Review 235

Barreto, R.W. & Evans H.C. (1998). Fungal pathogens of *Euphorbia heterophylla* and E. *hirta* in

Batista, F.A.S.; Lima, E.F.; Moreira, J.A.N.; Azevedo, D.M.P.; Pires, V.A.; Vieira, R.M. &

Beever, R.E. & Weeds, P.L. (2007) *Taxonomy and Genetic Variation of* Botrytis *and* Botryotinia*.*

Bezerra, C.S. (2007). Estrutura genética e sensibilidade a fungicidas de *Amphobotrys ricini* 

Buchwald, N. F. (1949). Studies in the Scletoriniaceae: I. Taxonomy of the Sclerotiniaceae. *Kongelige Veterinær- og Landbohøjskole, Aarsskrift*, Vol.32, pp.1-116. ISSN 03687171 Chagas, H.A. (2009). Controle de mofo-cinzento (*Amphobotrys ricini*) da mamoneira (*Ricinus*

Chagas, H.A.; Basseto, M.A.; Rosa, D.D.; Zanotto, M.D.; & Furtado E.L. (2010). Escala

Cook, A.A. (1981). *Disease of Tropical and Subtropical Field, Fiber and Oil Plants,* Macmillan,

Costa, R. S.; Suassuna, T. M .F.; Milani, M.; Costa, M. N. & Suassuna, N. D. (2004). Avaliação

Dange, S.R.S; Desal, A.G. & Patel, S.I. (2005). *Diseases of castor*. In: G.S. Saharan; N. Mehta &

Demant, C.A.R.; Furtado, E.L.; Zanotto, M. & Chagas, A.A. Controle do mofo cinzento com

http://www.cnpa.embrapa.br/produtos/mamona/publicacoes/trabalhos\_cbm2/

Técnica 21], ISSN 0100-6460, Campina Grande, Brazil

36, ISSN 0301-486X

Netherlands

pp.101-104, ISSN 0253-4355

Filho", Botucatu, Brazil

ISBN 0-02-949300-5, New York, United States

81-7387-176-0, New Delhi, India

5405

049.pdf

043.pdf

Available from:

Brazil and their potential as weed biocontrol agents. *Mycopathologia*, Vol.141, pp.21-

Santos, J.W. (1998). Avaliação da resistência de genótipos de mamoneira, *Ricinus communis* L., ao mofo cinzento causado por *Botrytis ricini* Godfrey. Embrapa Algodão. [Comunicado Técnico 73], ISSN 0102-0099, Campina Grande, Brazil Batista, F.A.S.; Lima, E.F.; Soares, J.J. & Azevedo, D.M.P. (1996). Doenças e pragas da

mamoneira (*Ricinus communis* L.) e seu controle. Embrapa Algodão, [Circular

In: Botrytis: *Biology*, *Pathology and Control,* Y. Elad; B. Williamson; P. Tudzynski & N. Delen (Eds), 29-52, Springer, ISBN 978-1-4020-6586-6, Dordrecht, The

agente causal do mofo cinzento da mamoneira. MSc Dissertation (Genetic and Molecular Biology), Universidade Federal do Rio Grande do Norte, Natal, Brazil Bhattiprolu, S.L. & Bhattiprolu, G.R. (2006). Management of castor grey rot disease using

botanical and biological agents. *Indian Journal of Plant Protection*, Vol.34, No.1,

*communis* L.) por métodos químico, biológico e com óleos essenciais. MSc Dissertation, (Agronomy), Universidade Estadual Paulista "Júlio de Mesquita

diagramática para avaliação de mofo cinzento (*Amphobotrys ricini*) da mamoneira (*Ricinus communis* L.). *Summa Phytopathologica*, Vol.36, No.2, pp.164-167, ISSN 0100-

de resistência de genótipos de mamoneira ao mofo cinzento (*Amphobotrys ricini*), *Proccedings of 1st Congresso Brasileiro de Mamona*. 04.06.2010. Available from: http://www.cnpa.embrapa.br/produtos/mamona/publicacoes/trabalhos\_cbm2/

M.S. Sangwan (Eds), 211-234, *Diseases of Oilseed Crops*, Indus Publishing Co, ISBN

o uso de *Trichoderma*, *Proccedings of 2nd Congresso Brasileiro de Mamona.* 04.06.2010.

determine which practices for disease management are worthwhile recommending. In others words, there is still much work to be done before we can better define the best strategies to avoid economic losses due to gray mold in castor crops.

#### **4. Conclusion**

Despite being one of the most important diseases of castor worldwide, causing severe losses on castor yield for almost a century, since its first report, gray mold is still poorly studied. The recent concern about renewable energy sources has proportioned a unique opportunity to draw attention back to this pathosystem. So, researchers involved with castor cultivation must now take this opportunity to try to elucidate the many still to be answered questions about this disease in order to mitigate the constant menace of gray mold to castor crops.

#### **5. Acknowledgment**

The author wishes to thank the CNPq (Proc. 472953/2009-5) and Petrobrás (TC 0050.0064181.10.9) for the financial support on research of castor gray mold. I also wish to thanks Dr. Harry C. Evans by his priceless suggestions to improve the text.

#### **6. References**

Alcântara, M.J.; Nassur, R.C.M.R.; Sá, G.A.; Fraga, A.C. & Neto, P.C. (2008). Avaliação de materiais de mamoneira visando obtenção de resistência ao mofo cinzento, *Proccedings of 5th Congresso Brasileiro de Plantas Oleaginosas, Óleos, Gorduras e Biodiesel.* 04.06.2010. Available from

http://oleo.ufla.br/anais\_05/artigos/a5\_\_464.pdf


determine which practices for disease management are worthwhile recommending. In others words, there is still much work to be done before we can better define the best

Despite being one of the most important diseases of castor worldwide, causing severe losses on castor yield for almost a century, since its first report, gray mold is still poorly studied. The recent concern about renewable energy sources has proportioned a unique opportunity to draw attention back to this pathosystem. So, researchers involved with castor cultivation must now take this opportunity to try to elucidate the many still to be answered questions about this disease in order to mitigate the constant menace of gray

The author wishes to thank the CNPq (Proc. 472953/2009-5) and Petrobrás (TC 0050.0064181.10.9) for the financial support on research of castor gray mold. I also wish to

Alcântara, M.J.; Nassur, R.C.M.R.; Sá, G.A.; Fraga, A.C. & Neto, P.C. (2008). Avaliação de

Alwadie, H.M. & Baka, Z.A.M. (2003). New records of fungal pathogens of *Euphorbia*

Amorim Neto, M. S.; Araújo, A.E.; Beltrão, N. E.M.; Silva, L.C. & Gomes, D.C. (2000)

Araújo, A.E.; Suassuna, N.D.; Bandeira, C.M. & Agra, K.N. (2003). Efeito da temperatura na

Araújo, A.E.; Suassuna, N.D. & Coutinho, W.M. (2007). *Doenças e seu Manejo*. In: *O* 

materiais de mamoneira visando obtenção de resistência ao mofo cinzento, *Proccedings of 5th Congresso Brasileiro de Plantas Oleaginosas, Óleos, Gorduras e* 

*inarticulate* from Aseer region. *Archives of Phytopathology and Plant Protection*, Vol.36,

Zoneamento e época de plantio para a mamoneira – estado da Paraíba. Embrapa Algodão, [Comunicado Técnico 108], ISSN 0102-0099, Campina Grande, Brazil Anjani, K.; Raoof, M.A.; Ashoka Vardhana Reddy, V. & Hanumanata Rao, C. (2004). Sources

of resistance to major castor (*Ricinus communis* L.) diseases. *Plant Genetic Resources* 

germinação de esporos de *Amphobotrys ricini* (=*Botrytis ricini*). *Fitopatologia*

*Agronegócio da Mamona no Brasil,* D.M.P. Azevedo & N.E. de M. Beltrão, (Eds.), 283-303, Embrapa Informação Tecnológica, ISBN 978-85-7383-381-2, Brasília,

thanks Dr. Harry C. Evans by his priceless suggestions to improve the text.

*Newsletter*. Vol.137, (March 2004) pp.46-48, ISSN 0048-4334

*Brasileira*, Vol.28, pp.S200 [Abstract], ISSN 0100-4158

*Biodiesel.* 04.06.2010. Available from http://oleo.ufla.br/anais\_05/artigos/a5\_\_464.pdf

No.3/4, pp.195-209, ISSN 0323-5408

strategies to avoid economic losses due to gray mold in castor crops.

**4. Conclusion** 

mold to castor crops.

**5. Acknowledgment** 

Brazil

**6. References** 


 http://www.cnpa.embrapa.br/produtos/mamona/publicacoes/trabalhos\_cbm2/ 043.pdf

Gray Mold of Castor: A Review 237

Kars, I. & van Kan, J.A.L. (2007) *Extracellular Enzymes and Metabolites Involved in Pathogenesis*

Keane, P.J. (2012) *Horizontal or Generalized Resistance to Pathogens in Plants*. In: *Plant Pathology*, C.J.R. Cumagun (Ed), 317-352, InTech, ISBN 978-953-307-933-2, Viena, Austria Kimati, H. (1980). *Doenças da Mamoneira*. In: *Manual de Fitopatologia: Doenças das Plantas Cultivadas*, F. Galli, (Ed), 347-351, Editora Agronomica Ceres, São Paulo, Brazil Kirk, P.M.; Cannon, P.F.; Minter, D.W. & Stalpers, J.A. (2008). *Dictionary of the Fungi*, CABI

Kolte, J.S. (1995). *Castor: Diseases and Crop Improvement*. Shipra Publications, ISBN 81-85402-

Kumar, A.; Reddy, P.N. & Rao, T.G.N. (2007). Host range studies of *Botrytis ricini*, the causal

Leroux, P. (2007) *Chemical Control of* Botrytis *and its Resistance to Chemical Fungicides*. In:

Lima, B.V.; Soares, D.J.; Perreira, O.L. & Barreto, R.W. (2008). Natural infection of *Acalypha*

Lima, E.F.; Araújo, A.E. & Batista, F.A.S. (2001) *Doenças e seu Controle*. In: *O Agronegócio da* 

Lima, E.F. & Soares, J.J. (1990). Resistência de cultivares de mamoneira ao mofo cinzento,

Lima, V.P.T.; Graça Leite, E.A.; Botrel, E.P.; Fraga, A.C. &Castro Neto, P. (2005). Avaliação

Lyon, G.D.; Goodman, B.A. & Williamson, B. (2007). Botrytis cinerea *Perturbs Redox Processes* 

Massola JR, N. S. & Bedendo, I. P. (2005). *Doenças da Mamoneira (*Ricinus communis *L.).* In:

Maude, R.B. (1996). *Seedborne Diseases and their Control: Principles and Practice.* 978-

Melhorança, A. L. & Staut, L.A. (2005). Informações técnicas para a cultura da mamona em

*Oleaginosas, Óleos, Gorduras e Biodiesel*. 12.06.2010. Available from:

*Australasian Plant Disease Notes* Vol.3, pp.5*-*7, ISSN 1833-928X

Tecnológica, ISBN85-7383-116-2, Brasília, Brazil

http://oleo.ufla.br/anais\_02/artigos/t183.pdf

6586-6, Dordrecht, The Netherlands

Ceres, ISBN 85-318-0008-0, São Paulo, Brazil

0851989228, CABI Publishing, Kew, UK

1679-1320, Dourados, Brazil

agent of castor grey mold. *Indian Journal of Plant Protection*, Vol.35, No.1, pp.140-

Botrytis: *Biology*, *Pathology and Control,* Y. Elad; B. Williamson; P. Tudzynski & N. Delen (Eds), 195-222, Springer, ISBN 978-1-4020-6586-6, Dordrecht, The

hisp*i*da and *Jatropra podagrica* inflorescences by *Amphobotrys ricini* in brazil.

*Mamona no Brasil*, D.M.P. Azevedo & E.F.Lima (Eds), 192-212, Embrapa Informação

causado por *Botrytis ricini*. *Fitopatologia Brasileira*, Vol.15, No.1, pp.96-98, ISSN 0100-

de ataque de mofo cinzento da mamoneira, variedade Al Guarany 2002, em diferentes espaçamentos. *Proccedings of 2nd Congresso Brasileiro de Plantas* 

*as an Attack Strategy in Plants*. In: Botrytis: *Biology*, *Pathology and Control,* Y. Elad; B. Williamson; P. Tudzynski & N. Delen (Eds), 119-142, Springer, ISBN 978-1-4020-

*Manual de Fitopatologia: Doenças das Plantas Cultivadas,* H. Kimati; L. Amorim; A. Bergamin Filho; L.E.A. Camargo & J.A.M. Rezende (Eds), 497-500,. Agronomica

Mato Grosso do Sul. Embrapa Agropecuária Oeste [Sistemas de Produção 8], ISSN

Publishing, ISBN 978-0-85199-826-8, Wallingford, UK

The Netherlands

54-X, Delhi, India

141, ISSN 0253-4355

Netherlands

4158

*of* Botrytis. In: Botrytis: *Biology*, *Pathology and Control,* Y. Elad; B. Williamson; P. Tudzynski & N. Delen (Eds), 99-118, Springer, ISBN 978-1-4020-6586-6, Dordrecht,


Drumond, O. A. & Coelho S. J. (1981). Doenças da mamoneira. *Informe Agropecuário* Vol.7,

Elad, Y & Stewart, A. (2007). *Microbial Control* of Botrytis *spp*. In: Botrytis: *Biology*, *Pathology*

Esuruoso, O.F. (1966). A preliminary study on the susceptibility of certain varieties of castor

Esuruoso, O.F. (1969). The reaction of the capsules of certain varieties of castor to a bio-

Freire, E.C.; Lima, E.F.; Andrade, F.P.; Milani, M. & Nóbrega, M.B.M. (2007). *Melhoramento* 

Galli, F.; Tokeshi, H.; Carvalho, P.C.T.; Balmer, E.; Cardoso, C.O.N. & Salgado, C.L. (1968).

Godfrey, G.H. (1919). *Sclerotinia ricini* n. sp. on the castor bean (*Ricinus communis*).

Godfrey, G. H. (1923). Gray mold of castor bean. *Journal* of *Agricultural Research*, Vol.23, No.

Gonçalves, R.D. (1936). Mofo cinzento da mamoneira. *O Biológico*, Vol.2, No.7, pp.232-235,

Hansen H.N. & Bega, R.V. (1955). *Botrytis* rot of *Caladium* tubers. *Plant Disease Reporter,* 

Hennebert, G.L. (1973). *Botrytis* and *Botrytis*-like genera. *Persoonia*, Vol.7, No.2, pp.183-204,

Hoffmann, L.V.; Coutinho, T.C.; Duarte, E.A.A.; Bandeira, C.M. & Suassuna, N. D. (2004)

Holcomb, G.E. & Brown, W.L. (1990). Basal stem rot of cultivated *Poinsettia* caused by

Holcomb, G.E.; Jones, J.P. & Wells, D.W. (1989). Blight of prostate spurge and cultivated

Holz, G.; Coertze, S. & Williamson, B. (2007). *The Ecology of* Botrytis *on Plant Surfaces*. In:

*Amphobotrys ricini*. *Plant Disease*, Vol.74, pp.828, ISSN 0191-2917

Cultivo de *Amphobotrys ricini* e detecção das enzimas málica, superóxido dismutase e esterase, *Proccedings of 1st Congresso Brasileiro de Mamona*, 14.06.2010. Available from: http://www.cnpa.embrapa.br/produtos/mamona/publicacoes/trabalhos\_cbm1/

*Poinsettia* caused by *Amphobotrys ricini*. *Plant Disease*, Vol.73, pp.74-75, ISSN 0191-

Botrytis: *Biology*, *Pathology and Control,* Y. Elad; B. Williamson; P. Tudzynski & N. Delen (Eds), 9-28, Springer, ISBN 978-1-4020-6586-6, Dordrecht, The Netherlands Jarvis, W.R. (1978). Epidemiology. In: *The Biology of* Botrytis, J.R. Coley-Smith, K. Verhoeff & W.R. Jarvis (Eds), 219-250, Academic Press, ISBN 0-12-179850-X, London, UK

Springer, ISBN 978-1-4020-6586-6, Dordrecht, The Netherlands

*Agricultural Journal*, Vol.6, No.1, pp.15-17, ISSN 0300-368X

292-197, Editora Agronomica Ceres, São Paulo, Brazil

*Phytopathology*, Vol.9, pp.565-567, ISSN 0031-949X

9, pp.679-715 + 13 plates, ISSN 0095-9758

Vol.39, No.3, pp.283, ISSN 0032-0811

*and Control,* Y. Elad; B. Williamson; P. Tudzynski & N. Delen (Eds), 223-236,

(*Ricinus communis* L.) to inflorescence blight disease caused by *Sclerotinia* (*Botrytis*) *ricini* (Godfrey) Whet. *The Nigerian Agricultural Journal*, Vol.3, No.1, pp.15-17, ISSN

chemical test for susceptibility to the inflorescence blight diseases. *The Nigerian* 

*Genético*. In: *O Agronegócio da Mamona no Brasil,* D.M.P. Azevedo & N.E. de M. Beltrão, (Eds.), 171-194, Embrapa Informação Tecnológica, ISBN 978-85-7383-381-2,

*Doenças da Mamoneira*. In: *Manual de Fitopatologia: Doenças das Plantas e seu Controle*, Galli, F.; Tokeshi, H.; Carvalho, P.C.T.; Balmer, E.; Cardoso, C.O.N. & Salgado, C.L.,

No.82, pp.38-42, ISSN 0100-3364

0300-368X

Brasília, Brazil

ISSN 0366-0567

ISSN 0031-5850

126.PDF

2917


Gray Mold of Castor: A Review 239

Soares, D.J.; Fernandes J.N. & Araújo, A.E. (2010). Componentes monociclicos do mofo

Suassuna, N.D.; Araújo, A.E.; Bandeira, C.M.; Agra, K.N. (2003). Efeito de temperatura no

Sussel, A.A.B. (2008). Epidemiologia do mofo-cinzento (*Amphobotrys ricini* Buchw.) da

Sussel, A.A.B. (2009a) Epidemiologia e Manejo do Mofo-cinzento-da-mamoneira. Embrapa

Sussel, A.A.B. (2009b) Escala Diagramática para Avaliação do Mofo-cinzento-da-

Sussel, A.A.B.; Pozza, E.A. & Castro, H.A. (2009). Elaboração e validação de escala

Sussel, A.A.B.; Pozza, E.A. & Castro, H.A. & Lasmar, E.B.C. (2011). Incidência e severidade

Termorshuizen, A.J. (2001). *Cutural Control.* In: *Plant Pathologist's Pokcketbook*, J.M. Waller;

Thomas, C.A. & Orellana, R.G. (1963a) Nature of predisposition of castor beans to *Botrytis*.

Thomas, C.A. & Orellana, R.G. (1963b) Biochemical tests indicative of reaction of castor bean

Thomas, C.A. & Orellana, R.G. (1964) Phenols and pectins in relation to browning and

Tirupathi, J.; Kumar, C.P.C. & Reddy, D.R.R. (2006). *Trichoderma* as potential biocontrol

Ueno, B.; Hellwig, T.C.; Nickel, G.; Silva, S.D.A. (2006). Resistência ao mofo cinzento em 15

Whetzel, H.H. (1945). A synopsis of the genera and species of the Sclerotiniaceae, a family

to *Botrytis*. *Science*, Vol.139, pp.334-335, ISSN 0036-8075

No.4, pp.359-366, ISSN 0931-1785

Vol.34, No.2, pp.31-36

*4th Congresso Brasileiro de Mamona,* 12.07.2011. Available from

Cerrados, [Documentos 241], ISSN 1517-5111, Brasília, Brazil

*Tropical Plant Pathology*, Vol.34, No.3, pp.186-191, ISSN 1982-5676

http://www.cbmamona.com.br/pdfs/FIT-01.pdf

Lavras, Lavras, Brazil

34, ISSN0100-5405

Wallingford, UK

0031-949X

049.pdf

5514

Brazil

*Brasileira* Vol.28, pp.S232 [Abstract], ISSN0100-4158

cinzento (*Amphobotrys ricini*) em diferentes genótipos de mamoneira, *Proceedings of*

crescimento e esporulação de *Amphobotrys ricini* (=*Botrytis ricini*). *Fitopatologia*

mamoneira. PhD Thesis (Phytopathology), (June 2008)Universidade Federal de

mamoneira. Embrapa Cerrados, [Documentos 247], ISSN1517-5111, Brasília,

diagramática para avaliação da severidade do mofo cinzento da mamoneira.

do mofo-cinzento-da-mamoneira sob diferentes temperaturas, períodos de molhamento e concentração de conídios. *Summa Phytopatologica* Vol.37, No.1, pp.30-

J.M. Lenné & S.J. Waller (Eds), 318-327, CABI Publishing, ISBN 0851994598,

II. Raceme compactness, internode lenghs, position of staminate flowers and bloom in raltion to capsule susceptibility. *Phytopathology*, Vol.53, No.2, pp.249-251, ISSN

maceration of castorbean capsules by *Botrytis*. *Phytopathologische Zeitschrift*, Vol.50,

agents for the management of grey mold of castor. *Journal of Research Angrau*,

genótipos de mamoneira cultivadas na região de Pelotas, RS, na safra 2004/2005, *Proceedings of 2nd Congresso Brasileiro de Mamona*. 12.06.2010. Available from: http://www.cnpa.embrapa.br/produtos/mamona/publicacoes/trabalhos\_cbm2/

of stromatic inoperculate discomycetes. *Mycologia*, Vol.37, pp.648-714, ISSN 0027-


http://www.biodiesel.gov.br/docs/congresso2007/agricultura/3.pdf


 http://www.cnpa.embrapa.br/produtos/mamona/publicacoes/cbm3/trabalhos/ FITOSSANIDADE/F%2012.pdf

Milani M.; Nóbrega, M.B.M.; Suassuna, N.D. & Coutinho, W.M. (2005). Resistência da

Moraes, W.B.; Souza, A.F.; Tomas, M.A.; Cecilio, R.A. & Jesus Junior, W.C. (2009).

Palti, J. & Rotem, J. (1983). *Cultural Practices for the Control of Crop Diseases*. In: *Plant* 

Raoof, M.A.; Yasmeen, M. & Kausar, R. (2003). Potential of biocontrol agents for the

Rego Filho, L.M.; Bezerra Neto, F.V. & Santos, Z.M. (2007). Avaliação da incidência de mofo-

Robinson, R.A. (1976). *Horizontal Pathosystem Analysis*. In: *Plant Pathosysytem* (*Advanced Series* 

Russo, V.M. & Rossman, A.Y. (1991). Occurrence of *Amphobotrys ricini* on prostate spurge in

Santos. R.F.; Kouri, J.; Barros, M.A.L.; Firmino, P.T. & Requião. L.E.G. (2007). *Aspectos* 

Sanoamuang, N. (1996). First report of gray mold blight caused by *Amphobotrys ricini* on crown of thorns in Thailand. *Plant Disease*, Vol.80, pp.223, ISSN 0191-2917 Seifert, K.; Morgan-Jones, G.; Gams, W. & Kendrick, B. (2011). *The Genera of Hyphomycetes,* 

Silva, J.A.; Suassuna, N.D.; Coutinho, W.M. & Milani, M. (2008). Esporulação de

http://www.cnpa.embrapa.br/produtos/mamona/publicacoes/cbm3/trabalhos/

*Protection*, Vol.31, No.2, pp.124-126, ISSN 0253-4355

http://www.biodiesel.gov.br/docs/congresso2007/agricultura/3.pdf

Oklahoma. *Plant Disease*, Vol. 75, pp. 750, ISSN 0191-2917

Tecnológica, ISBN 978-85-7383-381-2, Brasília, Brazil

*Biodiesel 2006*. 27.07.2011. Available from:

Brazil.

0031-949X

460-6, Kew, England

X, Berlin, Germany

Netherlands

Available from:

FITOSSANIDADE/F%2012.pdf

mamoneira (*Ricinus communis* L.) ao mofo cinzento causado por *Amphobotrys ricini*. Embrapa Algodão [Documentos 137], ISSN 0103-0205, Campina Grande,

Zoneamento das áreas de risco da ocorrência de mofo cinzento da mamona no Brasil. *Proccedings of 13th Encontro Latino Americano de Iniciação Científica and 9th Encontro Latino Americano de Pós-Graduação*, 01.06.2010. Available from: http://www.inicepg.univap.br/cd/inic\_2009/anais/arquivos/0821\_1434\_04.pdf Orellana, R.G. & Thomas, C.A. (1962). Nature of predisposition of castor beans to *Botrytis*. I.

Relation of leachable sugar and certain other biochemical constituents of the capsule to varietal susceptibility. *Phytopathology*, Vol.52, No.6, pp.533-538, ISSN

*Pathologist's Pocketbook*, A. Johnston & C. Booth (Eds), 186-195, CMI, ISBN 0-85-198-

management of castor grey mold, *Botrytis ricini* godfrey*. Indian Journal of Plant* 

cinzento em genótipos de mamoneira no período de outono-inverno em campos dos Goytacazes-RJ. *Proccedings of the 2nd Congresso da Rede Brasileira de Tecnologia de* 

*in Agricultural Sciences 3*), R.A. Robinson, 74-89, Springer-Verlag, ISBN 3-540-07712-

*Econômicos do Agronegócio da Mamoneira*. In: *O Agronegócio da Mamona no Brasil,* D.M.P. Azevedo & N.E. de M. Beltrão, (Eds.), 22-41, Embrapa Informação

CBS-KNAW Fungal Biodiversity Centre, ISBN 978-90-70351-85-4, Utrecht, The

*Amphobotrys ricini* em frutos de mamoneira como pomponente de resistencia ao mofo cinzento, *Proccedings of the 3th Congresso Brasileiro de Mamona*. 04.06.2010.


**1. Introduction** 

barrier to disease control in tropical perennials.

**10** 

Peter McMahon

*Australia* 

**Effect of Nutrition and Soil Function** 

**on Pathogens of Tropical Tree Crops** 

Crops grown in the tropics are subject to different kinds of disease pressure from those produced in temperate regions. The greater biodiversity found in the tropics, including diversity of fungi, is reflected by the larger number of pathogen species in tropical regions (see Ploetz, 2007; Wellman**,** 1968, 1972). Perennial crops, and tropical perennials in particular, have features in common that may predispose them to pathogen infections. Pathogen inocula, such as microsclerotia, may build up from year to year in perennial crops (Pennypacker, 1989). Also, tropical conditions are usually suitable for the year-round survival and propagation of pathogen species, unlike temperate climates which have a cooler season when pathogen populations die off or are reduced. Tropical perennial crops often include susceptible genotypes on the farm and the presence of susceptible host material encourages the production of inoculum and the initiation of new infections (Ploetz, 2007). Ploetz (2007) remarks that the presence of susceptible hosts is a particularly important

Diseases in the tropics may be complicated by interactions between different pathogens, or between pathogens and insect pests (Holliday, 1980; Ploetz, 2006; Vandermeer et al., 2010; Anonymous, 2010). Disease complexes involving a number of fungal pathogens or fungi and nematodes are common in tropical situations. Interactions between pathogens and environmental stress may also occur. Crops can become more susceptible to pathogen infections when weakened by environmental stress such as drought, temperature extremes, and exposure to sunlight or wind (Agrios, 2005). Stressed plants, or plants sustaining damage caused by insects or other pathogens, may also be susceptible to attack by secondary pathogens or pathogens that infect through wounds (Palti, 1981). Nutrient deficiencies may increase the susceptibility of crops to disease. In tropical perennial crops, poor plant nutrition is likely to be a particularly important contributing factor to production losses (Schroth et al., 2000). In addition to lower production due to nutrient deficiency, low nutrition may predispose plants to diseases, increasing losses further. Nutrient deficiency causes the plant to become weakened and generally more susceptible to infection. Under such conditions, infection by weakly pathogenic species that would normally cause few problems may become more serious. The incidence and severity of particular diseases may also be linked to deficiencies of particular nutrients. However, much more research has been

*Department of Botany, La Trobe University, Bundoora Vic* 


### **Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops**

Peter McMahon

*Department of Botany, La Trobe University, Bundoora Vic Australia* 

#### **1. Introduction**

240 Plant Pathology

Whitney, N.G. & Taber, R.A. (1986). First report of *Amphobotrys ricini* infecting *Caperonia palustris* in the United States. *Plant Disease*, Vol.70, pp.892, ISSN 0191-2917 Zarzycka, H. (1958). Resistance of some varieties of castor bean (*Ricinus communis*) to the fungus *Botrytis cinerea* Pers. *Acta Agrobotanica*, Vol.7, pp.117-124, ISSN 0065-0951

> Crops grown in the tropics are subject to different kinds of disease pressure from those produced in temperate regions. The greater biodiversity found in the tropics, including diversity of fungi, is reflected by the larger number of pathogen species in tropical regions (see Ploetz, 2007; Wellman**,** 1968, 1972). Perennial crops, and tropical perennials in particular, have features in common that may predispose them to pathogen infections. Pathogen inocula, such as microsclerotia, may build up from year to year in perennial crops (Pennypacker, 1989). Also, tropical conditions are usually suitable for the year-round survival and propagation of pathogen species, unlike temperate climates which have a cooler season when pathogen populations die off or are reduced. Tropical perennial crops often include susceptible genotypes on the farm and the presence of susceptible host material encourages the production of inoculum and the initiation of new infections (Ploetz, 2007). Ploetz (2007) remarks that the presence of susceptible hosts is a particularly important barrier to disease control in tropical perennials.

> Diseases in the tropics may be complicated by interactions between different pathogens, or between pathogens and insect pests (Holliday, 1980; Ploetz, 2006; Vandermeer et al., 2010; Anonymous, 2010). Disease complexes involving a number of fungal pathogens or fungi and nematodes are common in tropical situations. Interactions between pathogens and environmental stress may also occur. Crops can become more susceptible to pathogen infections when weakened by environmental stress such as drought, temperature extremes, and exposure to sunlight or wind (Agrios, 2005). Stressed plants, or plants sustaining damage caused by insects or other pathogens, may also be susceptible to attack by secondary pathogens or pathogens that infect through wounds (Palti, 1981). Nutrient deficiencies may increase the susceptibility of crops to disease. In tropical perennial crops, poor plant nutrition is likely to be a particularly important contributing factor to production losses (Schroth et al., 2000). In addition to lower production due to nutrient deficiency, low nutrition may predispose plants to diseases, increasing losses further. Nutrient deficiency causes the plant to become weakened and generally more susceptible to infection. Under such conditions, infection by weakly pathogenic species that would normally cause few problems may become more serious. The incidence and severity of particular diseases may also be linked to deficiencies of particular nutrients. However, much more research has been

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 243

enhance disease prevention. Further studies are needed to ascertain whether nutrient

In tropical perennials crops, as in annuals, disease is often a consequence of inadequate nutrition, particularly of *nitrogen* (N). Low supplies of N may predispose plants to infections by facultative parasites such as *Fusarium* spp. However, most research on the effect of N supply on disease has been conducted on temperate annuals (Agrios, 2005; Jones et al., 1989; Palti, 1981). For example, diseases in species of the Solanaceae family including Fusarium wilt, Alternaria early blight, *Pseudomonos solanacearum* wilt, *Sclerotium rolfsii* and Pythium damping off are increased under low N conditions (see Agrios, 2005). Vascular wilts caused by *F. oxysporum* in the annual crops tomato, cotton and pea, as well as Alternaria blights of a

In contrast to cases where a low N supply predisposes crops to disease, research on annual crops has demonstrated that an excessive N supply increases disease or damage caused by some pests and pathogens (Jones et al., 1989; Palti, 1981 p.136). Pest and pathogen attack of above ground parts of the plant may be encouraged by high N in the presence of low K and P (Desaeger et al., 2004); a high N/K ratio encourages insect herbivory by increasing the content of free amino acids in plant tissues (Marschner, 1995). A number of studies with cereals and other crops have shown that obligate pathogens in particular, such as *Puccinia* spp. causing rust and other biotrophs, can be encouraged by a high N supply (see Palti, 1981). For example, an increase in rice blast disease (*Magnaporthe grisea*) was observed in upland rice, which had been treated with the green manure of an alley crop with a high N content (Maclean et al., 1992). Possibly excess N might also favour the development of infections by obligate fungal parasites in tropical perennials although there is little evidence for this. For example, a poor nutrient status in coffee plants has been reported to predispose

The form of N supplied can be a significant factor in plant disease. A supply of ammonium-N may predispose plants to certain diseases, while nitrate-N is favourable for the development of others (Palti, 1981). For example, Fusarium wilt severity in some crops is greater when N is supplied as ammonium, while Verticillium wilts are enhanced by a nitrate-N supply (see Section 4.1). Possibly this is connected to a pH effect in the rhizosphere. Fusarium wilts are favoured by acidic soils and Verticillium wilts by a higher soil pH (see Palti, 1981). Uptake of ammonium-N occurs in exchange for protons (H+ ions), causing a decrease in pH in the rhizosphere while nitrate uptake has the opposite effect on

fungi may be particularly sensitive to localised changes in the rhizosphere, such as pH

Some tropical perennial crops, notably banana, coffee, coconut and cocoa, have a high demand for *potassium* (K), suggesting that K deficiency may occur in areas that produce these crops over a long-term (see Section 3, below). This is particularly the case in areas planted with perennials that receive insufficient levels of fertiliser. In agroforestry farming systems, mulch from woody biomass (e.g. pruned branches) can be a good source of K and, conversely, as K is sequestered by woody species, this may create K-deficiency in sites which already have low levels of K in the soil (Beer et al., 1998). Potassium is a mobile

ions (Rice, 2007). Pathogenic

number of crops, may also be increased under low N conditions (Palti, 1981).

elements have similar roles in tropical perennial crops.

them to rust infection (Waller et al., 2007 p. 302).

fluctuations.

pH as OH- ions are pumped out by roots in exchange for NO3-

*Macronutrient elements* 

conducted on the relationships between particular nutrients and diseases in annual crops, than in perennial species, particularly tropical perennials.

Many tropical perennials are grown in an agroforestry situation with other crop species. Unlike the situation with annual cropping, there are fewer opportunities to include fallow periods or rotations in perennial systems during which inoculum loses its viability and the system can in general 'recover'. In tropical perennial systems, soil-borne pathogens, such as nematodes, may build up over time. An important aim in the management of these systems is to achieve a position of equilibrium between pests/diseases and the predators and parasites that keep them in check (Schroth et al., 2000). Furthermore, as perennial crops are present more or less permanently in the system, they remove nutrients on a continuous basis, without a fallow period during which soil fertility can be restored. In this light, the role of the nutrient status of tropical perennials in mitigating disease is an important topic and deserves attention from researchers.

Many diseases of tropical perennial crops are "new encounter" diseases which develop following production in new areas outside of the region of the crop's origin (Ploetz, 2007). At first, such plantings may enjoy a mainly disease-free period with high productivity. They are removed from the pressures of co-evolved pathogens and pests in their region of origin. However, such a 'honeymoon' period ends when new fungal pathogens (as well as other pathogens and pests) transfer from hosts indigenous to the region in which the crop is being produced (Keane and Putter, 1992). The indigenous hosts often remain unidentified. This is largely because the fungus causing the new disease often resides asymptomatically on its original host plant (for example, as an endophyte) (Ploetz, 2007). New encounter diseases may cause devastating losses. Unlike co-evolved pathogens in the region of the crop's origin, new encounter pathogens have few antagonists that could reduce disease incidence or severity. Poor growing conditions and poor farm management may further exacerbate the situation.

#### **2. Role of plant nutrition in mitigating disease**

Most studies on the role played by individual nutrients in preventing or reducing disease have been conducted on temperate crop species, or on tropical annuals such as rice. Little attention has been paid to the role of nutrition in alleviating diseases of tropical tree crops. However, for some diseases of tropical perennial species, a link is often observed between a deficient nutrient status caused by low soil fertility or poor plant nutrition and disease severity (Desaeger et al., 2004). Generally, plants stressed by various environmental limitations may be weakened and more vulnerable to disease and these include nutrientdeficient plants. Nutrient-deficient plants may be particularly susceptible to infection by facultative pathogens (Palti, 1981). Pathogens that are mild in normal conditions of plant growth and exist mainly as saprophytes or endophytes, such as some *Fusarium* spp. and *Alternaria* spp.*,* may cause severe disease under conditions of nutrient stress or aluminium toxicity (Desaeger et al., 2004).

Adequate nutrition helps to mitigate pest/disease damage by replacement of root and shoot tissues (Marschner, 1995). However, studies conducted on other crop species, especially temperate crops, have elucidated how particular nutrients, including micronutrients, may enhance disease prevention. Further studies are needed to ascertain whether nutrient elements have similar roles in tropical perennial crops.

#### *Macronutrient elements*

242 Plant Pathology

conducted on the relationships between particular nutrients and diseases in annual crops,

Many tropical perennials are grown in an agroforestry situation with other crop species. Unlike the situation with annual cropping, there are fewer opportunities to include fallow periods or rotations in perennial systems during which inoculum loses its viability and the system can in general 'recover'. In tropical perennial systems, soil-borne pathogens, such as nematodes, may build up over time. An important aim in the management of these systems is to achieve a position of equilibrium between pests/diseases and the predators and parasites that keep them in check (Schroth et al., 2000). Furthermore, as perennial crops are present more or less permanently in the system, they remove nutrients on a continuous basis, without a fallow period during which soil fertility can be restored. In this light, the role of the nutrient status of tropical perennials in mitigating disease is an important topic

Many diseases of tropical perennial crops are "new encounter" diseases which develop following production in new areas outside of the region of the crop's origin (Ploetz, 2007). At first, such plantings may enjoy a mainly disease-free period with high productivity. They are removed from the pressures of co-evolved pathogens and pests in their region of origin. However, such a 'honeymoon' period ends when new fungal pathogens (as well as other pathogens and pests) transfer from hosts indigenous to the region in which the crop is being produced (Keane and Putter, 1992). The indigenous hosts often remain unidentified. This is largely because the fungus causing the new disease often resides asymptomatically on its original host plant (for example, as an endophyte) (Ploetz, 2007). New encounter diseases may cause devastating losses. Unlike co-evolved pathogens in the region of the crop's origin, new encounter pathogens have few antagonists that could reduce disease incidence or severity. Poor growing conditions and poor farm management may further

Most studies on the role played by individual nutrients in preventing or reducing disease have been conducted on temperate crop species, or on tropical annuals such as rice. Little attention has been paid to the role of nutrition in alleviating diseases of tropical tree crops. However, for some diseases of tropical perennial species, a link is often observed between a deficient nutrient status caused by low soil fertility or poor plant nutrition and disease severity (Desaeger et al., 2004). Generally, plants stressed by various environmental limitations may be weakened and more vulnerable to disease and these include nutrientdeficient plants. Nutrient-deficient plants may be particularly susceptible to infection by facultative pathogens (Palti, 1981). Pathogens that are mild in normal conditions of plant growth and exist mainly as saprophytes or endophytes, such as some *Fusarium* spp. and *Alternaria* spp.*,* may cause severe disease under conditions of nutrient stress or aluminium

Adequate nutrition helps to mitigate pest/disease damage by replacement of root and shoot tissues (Marschner, 1995). However, studies conducted on other crop species, especially temperate crops, have elucidated how particular nutrients, including micronutrients, may

than in perennial species, particularly tropical perennials.

and deserves attention from researchers.

exacerbate the situation.

toxicity (Desaeger et al., 2004).

**2. Role of plant nutrition in mitigating disease** 

In tropical perennials crops, as in annuals, disease is often a consequence of inadequate nutrition, particularly of *nitrogen* (N). Low supplies of N may predispose plants to infections by facultative parasites such as *Fusarium* spp. However, most research on the effect of N supply on disease has been conducted on temperate annuals (Agrios, 2005; Jones et al., 1989; Palti, 1981). For example, diseases in species of the Solanaceae family including Fusarium wilt, Alternaria early blight, *Pseudomonos solanacearum* wilt, *Sclerotium rolfsii* and Pythium damping off are increased under low N conditions (see Agrios, 2005). Vascular wilts caused by *F. oxysporum* in the annual crops tomato, cotton and pea, as well as Alternaria blights of a number of crops, may also be increased under low N conditions (Palti, 1981).

In contrast to cases where a low N supply predisposes crops to disease, research on annual crops has demonstrated that an excessive N supply increases disease or damage caused by some pests and pathogens (Jones et al., 1989; Palti, 1981 p.136). Pest and pathogen attack of above ground parts of the plant may be encouraged by high N in the presence of low K and P (Desaeger et al., 2004); a high N/K ratio encourages insect herbivory by increasing the content of free amino acids in plant tissues (Marschner, 1995). A number of studies with cereals and other crops have shown that obligate pathogens in particular, such as *Puccinia* spp. causing rust and other biotrophs, can be encouraged by a high N supply (see Palti, 1981). For example, an increase in rice blast disease (*Magnaporthe grisea*) was observed in upland rice, which had been treated with the green manure of an alley crop with a high N content (Maclean et al., 1992). Possibly excess N might also favour the development of infections by obligate fungal parasites in tropical perennials although there is little evidence for this. For example, a poor nutrient status in coffee plants has been reported to predispose them to rust infection (Waller et al., 2007 p. 302).

The form of N supplied can be a significant factor in plant disease. A supply of ammonium-N may predispose plants to certain diseases, while nitrate-N is favourable for the development of others (Palti, 1981). For example, Fusarium wilt severity in some crops is greater when N is supplied as ammonium, while Verticillium wilts are enhanced by a nitrate-N supply (see Section 4.1). Possibly this is connected to a pH effect in the rhizosphere. Fusarium wilts are favoured by acidic soils and Verticillium wilts by a higher soil pH (see Palti, 1981). Uptake of ammonium-N occurs in exchange for protons (H+ ions), causing a decrease in pH in the rhizosphere while nitrate uptake has the opposite effect on pH as OH- ions are pumped out by roots in exchange for NO3 - ions (Rice, 2007). Pathogenic fungi may be particularly sensitive to localised changes in the rhizosphere, such as pH fluctuations.

Some tropical perennial crops, notably banana, coffee, coconut and cocoa, have a high demand for *potassium* (K), suggesting that K deficiency may occur in areas that produce these crops over a long-term (see Section 3, below). This is particularly the case in areas planted with perennials that receive insufficient levels of fertiliser. In agroforestry farming systems, mulch from woody biomass (e.g. pruned branches) can be a good source of K and, conversely, as K is sequestered by woody species, this may create K-deficiency in sites which already have low levels of K in the soil (Beer et al., 1998). Potassium is a mobile

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 245

than non-mycorrhizal plants inoculated with the nematode or mycorrhizal plants that had been inoculated with the AM fungi at a later stage. Care needs to be taken with supplying inorganic P to crops as an excessive external P supply can inhibit mycorrhizal development. This may lead to a shortage of other nutrients, such as zinc, that mycorrhizal roots are

An adequate supply of *calcium* (Ca) has been demonstrated to enhance resistance to a number of diseases in annual crop species caused by pathogens such as *Rhizoctonia solani*, *Sclerotium* spp., *Botrytis* spp., *Fusarium oxysporum* and the nematodes *Meloidogyne* spp. and *Pratylenchus* sp. (Agrios 2005; Jones et al., 1989). Resistance of lucerne to nematodes was shown to increase with supplied Ca (see Palti, 1981 p. 142). A large proportion of Ca in plants is present in the apoplast and, influences cell structural properties, especially of the cell wall (Rice, 2007). Increased levels of Ca-pectate complexes in the cell wall are likely to increase resistance to vascular wilt pathogens because this form of pectate is resistant to breakdown by endopolygalacturonase enzymes produced by fungi to degrade pectin in the xylem vessel walls. Ca-pectin complexes might also impede the progress of wilt pathogens growing within the xylem (Corden, 1965; Pennypacker, 1989; Waggoner and Dimond, 1955). However, Ca also has metabolic functions within the symplast as a secondary messenger in signalling pathways (Rice, 2007). Calcium possibly plays a significant role in mechanism(s) of disease resistance in fruits. A relatively large proportion of Ca taken up by plants is distributed to fruits and low Ca has been linked to increased incidence of fruit diseases such as brown-eye spot in coffee berries (see Section 4.2). Groundnut pods have a high Ca demand and pod rot caused by *Pythium* and *Rhizoctonia* spp. has been linked to a low Ca content. High rates of magnesium and K application can reduce the Ca content of pods,

*Sulphur* (S) is a component of defense-related peptides and proteins such as glutathione and phytoalexins. Application of S to deficient soil reduced leaf spot, caused by *Pyrenopeziza* in oil seed rape and stem canker caused by *Rhizoctonia solani* in potato (Haneklaus, 2007). The effect of S nutrition on diseases of tropical perennials is largely unknown. However, deposits of elemental sulphur (S) were observed in the xylem of cocoa plants in response to infection with *Verticillium dahliae* (Resende et al, 1996; Cooper and Williams, 2004). Similar findings were made in tomato (Williams et al., 2002 – see Haneklaus, 2007). Elemental S is toxic to some fungal pathogens and may be considered to be a phytoalexin in its own right (Resende et al., 1996). *Magnesium* (Mg) is an essential component of chlorophyll and, therefore, the photosynthetic systems of plants. However, a direct relation between Mg and plant disease has been less commonly demonstrated than with the other macronutrient elements. Magnesium, with K, plays a role in phloem-loading of sugars (Cakmak et al.,

Micronutrients have a diverse range of functions in plants: for example, as enzyme cofactors with redox roles and, in the case of elements such as boron and silicon, in tissue strengthening or structural functions. The numerous biochemical functions of micronutrients are reflected by their roles in a diverse range of mechanisms of disease resistance. *Zinc* (Zn) nutrition appears to be involved in resistance to many diseases. The mechanisms involving Zn in disease resistance are unclear but Zn acts as a co-factor for numerous enzymes (Rice, 2007). Stimulation of root growth by Zn may account for some

1994). Magnesium also activates enzymes such as glutathione synthetase.

efficient at accessing for the plant (Andrade et al., 2009).

increasing disease severity (Prabhu et al., 2007)

*Micronutrient elements* 

element with multiple functions in the plant. It acts as a counter-ion for anion transport, regulates stomatal aperture and the water potential of plant cells, affects cell wall plasticity, as well as other roles (Rice, 2007). It promotes wound healing and decreases frost injury (Palti, 1981). Potassium deficiency has been found to be linked to diseases in a number of temperate crops (see Palti, 1981) and a high K supply can improve resistance of plants to fungal and bacterial pathogens (Marscher, 1995; Perrenoud, 1977; 1990). The mechanism of resistance in some disease-resistant genotypes might be related to a greater efficiency in K uptake (Prabhu et al., 2007). The N/K ratio can affect resistance: if it is too high cells have thinner cell walls and weaker membranes and are more prone to pathogen attack (Perrenoud, 1990; Potash Institute, www.ipipotash.org). For similar reasons, cereals may become more prone to lodging. A low potassium/chlorine (K/Cl) ratio in plant tissues, which might result from the application of chloride-containing compounds such as ammonium fertilisers, may predispose plants to disease (e.g. wheat rust caused by *Puccinia* spp. or other diseases, see Prabhu et al., 2007; Jones et al., 1989). K-deficiency increases the concentration of soluble sugars in leaf tissues providing a substrate for many pathogens (Potash Institute, www.ipipotash.org). It is likely that the susceptibility of tropical perennial crops to some pathogens is also increased under conditions of K deficiency. In a study on tea plants, for example, a high K supply reduced nematode and borer damage (Muraleedharan and Chen, 1997). Another study showed that supplying K reduced Fusarium wilt in oil palm (Turner et al., 1970). However, few research studies have been conducted that could confirm a link between K nutrition and disease incidence or severity in tropical perennial crops.

*Phosphorus* (P)-deficiency is especially limiting to production of perennial crop in many tropical soils. Most P in the soil is in a fixed form (unavailable to plants) and the proportion of fixed P is increased at low soil pH levels (see Rice, 2007). Very low levels of available P are found in acid tropical soils (Mengel and Kirkby, 1982 p. 471). Woody biomass is very low in P, unlike K, and, therefore, external sources of P are often necessary in farm management (Beer et al., 1998). Phosphorus nutrition improves crop vigour and may decrease severity of diseases through new growth (Smyth and Cassell, 1995; Buresh, 1997). Improved root growth by P nutrition may allow the plant to 'escape' attack by soil-borne fungal pathogens or nematodes (Prabhu et al., 2007). Foliar application of phosphates may decrease diseases such as powdery mildew (Reuveni and Reuveni, 1998). Incidence of anthracnose (caused by *Colletotrichum lindemuthianum*) in susceptible cowpea cultivars was found to be higher in plants grown without applied P than in plants grown with P supplied at rates up to 80 kg of P fertiliser/ha (Adebiton, 1996). In the same study, disease severity in all of the cowpea cultivars tested was also decreased by P amendment and chickpea genotypes with resistance to Ascochyta blight had higher tissue concentrations of P and K than susceptible genotypes, which had a higher N content.

Mycorrhizal fungi, which form symbiotic associations with the roots of tropical perennials, such as coffee and banana, play a crucial role in accessing sources of P for their host plants. These fungi can access sources of P in the soil that are unavailable to non-mycorrhizal plant roots. As well as decreasing the impacts of plant pathogens (Azco'n-Aguilar and Barea, 1996), mycorrhizal plants have higher contents of certain nutrients, such as P. An example of this was demonstrated by greenhouse experiments conducted on coffee by Vaast et al. (1997). Coffee plants inoculated at an early stage with AM fungi had higher tissue P contents than non-mycorrhizal plants. High P tissue contents were maintained following inoculation with the nematode pathogen *Pratylenchus coffeae* and these plants also had fewer root lesions

element with multiple functions in the plant. It acts as a counter-ion for anion transport, regulates stomatal aperture and the water potential of plant cells, affects cell wall plasticity, as well as other roles (Rice, 2007). It promotes wound healing and decreases frost injury (Palti, 1981). Potassium deficiency has been found to be linked to diseases in a number of temperate crops (see Palti, 1981) and a high K supply can improve resistance of plants to fungal and bacterial pathogens (Marscher, 1995; Perrenoud, 1977; 1990). The mechanism of resistance in some disease-resistant genotypes might be related to a greater efficiency in K uptake (Prabhu et al., 2007). The N/K ratio can affect resistance: if it is too high cells have thinner cell walls and weaker membranes and are more prone to pathogen attack (Perrenoud, 1990; Potash Institute, www.ipipotash.org). For similar reasons, cereals may become more prone to lodging. A low potassium/chlorine (K/Cl) ratio in plant tissues, which might result from the application of chloride-containing compounds such as ammonium fertilisers, may predispose plants to disease (e.g. wheat rust caused by *Puccinia* spp. or other diseases, see Prabhu et al., 2007; Jones et al., 1989). K-deficiency increases the concentration of soluble sugars in leaf tissues providing a substrate for many pathogens (Potash Institute, www.ipipotash.org). It is likely that the susceptibility of tropical perennial crops to some pathogens is also increased under conditions of K deficiency. In a study on tea plants, for example, a high K supply reduced nematode and borer damage (Muraleedharan and Chen, 1997). Another study showed that supplying K reduced Fusarium wilt in oil palm (Turner et al., 1970). However, few research studies have been conducted that could confirm a link between K nutrition and disease incidence or severity in tropical perennial crops.

*Phosphorus* (P)-deficiency is especially limiting to production of perennial crop in many tropical soils. Most P in the soil is in a fixed form (unavailable to plants) and the proportion of fixed P is increased at low soil pH levels (see Rice, 2007). Very low levels of available P are found in acid tropical soils (Mengel and Kirkby, 1982 p. 471). Woody biomass is very low in P, unlike K, and, therefore, external sources of P are often necessary in farm management (Beer et al., 1998). Phosphorus nutrition improves crop vigour and may decrease severity of diseases through new growth (Smyth and Cassell, 1995; Buresh, 1997). Improved root growth by P nutrition may allow the plant to 'escape' attack by soil-borne fungal pathogens or nematodes (Prabhu et al., 2007). Foliar application of phosphates may decrease diseases such as powdery mildew (Reuveni and Reuveni, 1998). Incidence of anthracnose (caused by *Colletotrichum lindemuthianum*) in susceptible cowpea cultivars was found to be higher in plants grown without applied P than in plants grown with P supplied at rates up to 80 kg of P fertiliser/ha (Adebiton, 1996). In the same study, disease severity in all of the cowpea cultivars tested was also decreased by P amendment and chickpea genotypes with resistance to Ascochyta blight had higher tissue concentrations of P and K

Mycorrhizal fungi, which form symbiotic associations with the roots of tropical perennials, such as coffee and banana, play a crucial role in accessing sources of P for their host plants. These fungi can access sources of P in the soil that are unavailable to non-mycorrhizal plant roots. As well as decreasing the impacts of plant pathogens (Azco'n-Aguilar and Barea, 1996), mycorrhizal plants have higher contents of certain nutrients, such as P. An example of this was demonstrated by greenhouse experiments conducted on coffee by Vaast et al. (1997). Coffee plants inoculated at an early stage with AM fungi had higher tissue P contents than non-mycorrhizal plants. High P tissue contents were maintained following inoculation with the nematode pathogen *Pratylenchus coffeae* and these plants also had fewer root lesions

than susceptible genotypes, which had a higher N content.

than non-mycorrhizal plants inoculated with the nematode or mycorrhizal plants that had been inoculated with the AM fungi at a later stage. Care needs to be taken with supplying inorganic P to crops as an excessive external P supply can inhibit mycorrhizal development. This may lead to a shortage of other nutrients, such as zinc, that mycorrhizal roots are efficient at accessing for the plant (Andrade et al., 2009).

An adequate supply of *calcium* (Ca) has been demonstrated to enhance resistance to a number of diseases in annual crop species caused by pathogens such as *Rhizoctonia solani*, *Sclerotium* spp., *Botrytis* spp., *Fusarium oxysporum* and the nematodes *Meloidogyne* spp. and *Pratylenchus* sp. (Agrios 2005; Jones et al., 1989). Resistance of lucerne to nematodes was shown to increase with supplied Ca (see Palti, 1981 p. 142). A large proportion of Ca in plants is present in the apoplast and, influences cell structural properties, especially of the cell wall (Rice, 2007). Increased levels of Ca-pectate complexes in the cell wall are likely to increase resistance to vascular wilt pathogens because this form of pectate is resistant to breakdown by endopolygalacturonase enzymes produced by fungi to degrade pectin in the xylem vessel walls. Ca-pectin complexes might also impede the progress of wilt pathogens growing within the xylem (Corden, 1965; Pennypacker, 1989; Waggoner and Dimond, 1955). However, Ca also has metabolic functions within the symplast as a secondary messenger in signalling pathways (Rice, 2007). Calcium possibly plays a significant role in mechanism(s) of disease resistance in fruits. A relatively large proportion of Ca taken up by plants is distributed to fruits and low Ca has been linked to increased incidence of fruit diseases such as brown-eye spot in coffee berries (see Section 4.2). Groundnut pods have a high Ca demand and pod rot caused by *Pythium* and *Rhizoctonia* spp. has been linked to a low Ca content. High rates of magnesium and K application can reduce the Ca content of pods, increasing disease severity (Prabhu et al., 2007)

*Sulphur* (S) is a component of defense-related peptides and proteins such as glutathione and phytoalexins. Application of S to deficient soil reduced leaf spot, caused by *Pyrenopeziza* in oil seed rape and stem canker caused by *Rhizoctonia solani* in potato (Haneklaus, 2007). The effect of S nutrition on diseases of tropical perennials is largely unknown. However, deposits of elemental sulphur (S) were observed in the xylem of cocoa plants in response to infection with *Verticillium dahliae* (Resende et al, 1996; Cooper and Williams, 2004). Similar findings were made in tomato (Williams et al., 2002 – see Haneklaus, 2007). Elemental S is toxic to some fungal pathogens and may be considered to be a phytoalexin in its own right (Resende et al., 1996). *Magnesium* (Mg) is an essential component of chlorophyll and, therefore, the photosynthetic systems of plants. However, a direct relation between Mg and plant disease has been less commonly demonstrated than with the other macronutrient elements. Magnesium, with K, plays a role in phloem-loading of sugars (Cakmak et al., 1994). Magnesium also activates enzymes such as glutathione synthetase.

#### *Micronutrient elements*

Micronutrients have a diverse range of functions in plants: for example, as enzyme cofactors with redox roles and, in the case of elements such as boron and silicon, in tissue strengthening or structural functions. The numerous biochemical functions of micronutrients are reflected by their roles in a diverse range of mechanisms of disease resistance. *Zinc* (Zn) nutrition appears to be involved in resistance to many diseases. The mechanisms involving Zn in disease resistance are unclear but Zn acts as a co-factor for numerous enzymes (Rice, 2007). Stimulation of root growth by Zn may account for some

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 247

response in the host, rather than to a chemical effect. Silicon and other elements, including Ca and B, may be of particular importance in resistance to facultative pathogens, wound

Since many tropical soils are nutrient-poor and the replacement of nutrients by mineral or organic fertilisers is often inadequate, tropic perennial crops are particularly prone to nutrient stress. Growing perennial crops can lead to nutrient deficiencies if the soils in which they are grown are not adequately amended with mineral or organic fertilisers. Long-term cropping of one or a few species in tropical soils can also have other impacts on soil properties, such as the soil pH, that in turn may cause nutrient deficiencies. In addition, poor sanitation (e.g. removal of infected plant material), flooding (which may spread inoculum as well as causing plant stress) and inappropriate canopy management are common exacerbating factors leading to increases in disease incidence and severity on a farm (Kohler et al., 1997). Importantly, disease may also have the effect of reducing the plants' nutrient status or impairing water uptake. For example, coffee tree roots and the roots of other tree crops infected with nematodes or fungi may have impaired water and chemical uptake mechanisms causing wilting and nutrient

Table 1 presents data on nutrient uptake by some tropical perennial crops based on previous studies. The data refer only to the nutrient content of marketable products; the nutrients contained in waste (e.g. discarded tea leaves and coffee or cocoa beans) are not included. In the case of nutrients removed by cocoa in Nigeria (reported by Wessel, 1985), it can be seen that the pod husks, which are normally discarded, remove high amounts of K (77 kg ha-1y-1 in the husks of pods producing one ton of dry beans). Substantial amounts of Ca are also

**Yield N P K**

Cocoa pod husks 17 2 77

Table 1. Estimated removal of N, P and K from the soil by the harvested products of some tropical tree crops. Yields indicated for each crop are estimates of the quantity of produce obtained per hectare each year (sources: Krauss, 2003; Wessel, 1985). Note that where the quantities removed were given for the oxides of P and K in the original data, these figures have been converted to indicate the respective quantities of the elements removed.

Soil acidity or low pH, common in the tropics, may be increased under particular crops, especially long-term perennial crops, or by the application of some kinds of mineral fertiliser (Jones et al., 1989). Soil pH decreases when forest soils are turned over to perennial crops, such as coffee and cocoa (Beer, 1988; Beer et al., 1998; Hartemink, 2005). Lowering soil pH decreases the availability of basic cations, particularly Ca and Mg. However, increased soil

**Coffee** 1000 kg beans 40 2 42 **Rubber** Latex 6-36 1-7 5-31 **Tea** 1000 kg leaves (dry) 41 3 21 **Banana** 40 – 70 tonnes 225-450 20-40 800-1200 **Coconut** 6920 nuts 96 19 115 **Cocoa** 1000 kg beans (dry) 23 4 8

 **Nutrients removed (kg ha-1y-1)** 

**3. Management of soils supporting tropical perennial crops** 

invading pathogens and nematode infections.

deficiency (Nelson et al., 2002; Waller et al., 2007 p. 279).

removed with cocoa pods and other fruit products.

observed cases of disease resistance (Duffy, 2007). Zinc application to soils reduces attack by root pathogens of tomato, including *Fusarium solani*, *Rhizoctonia solani* and *Macrophoma phaseoli*, and also Rhizoctonia root rots of wheat, chickpea, cowpea and medicago (Duffy, 2007; Gaur and Vaidge, 1983; Kalim et al., 2003; Streeter et al., 2001). In tropical perennials, the role of Zn in disease resistance remains to be investigated. However, Zn-deficiency in rubber (*Hevea brasiliensis*) predisposes the tree to infection with *Oidium heveae* (Duffy, 2007). Zinc has been reported to alleviate Phytophthora diseases. Low Zn levels in soils and leaf tissues were associated with a high incidence of Phytophthora pod rot (or black pod) of cocoa in Papua New Guinea (Nelson et al., 2011). Supplying *Manganese* (Mn) has been shown to alleviate various diseases in a number of crop plant species (Palti, 1981; Thompson and Huber, 2007). Manganese occurs in different redox states and while it is present in healthy tissues as the Mn2+ ion, it accumulates at sites of pathogen attack in the Mn4+ form, for example in rice affected by blast (Thompson and Huber, 2007). *Iron* (Fe) has an essential role in plant cells as a co-factor in redox reactions and other functions. Fe is mainly available to plants as its reduced ion, Fe2+. Verticillium wilt in mango caused by *V. albo-atrum*, and in groundnut caused by *V. dahliae* was mitigated in both cases by the application of Fe in chelated form (see Palti, 1981 p. 142). On the other hand, control of Fusarium wilt in tomato was favoured by low Fe (Woltz and Jones, 1981). Similarly, Fusarium wilt has been shown to be lower at low levels of Mn. *F. oxysporum* has a particularly high demand for some micronutrient elements, especially Mn, Fe and Zn (Jones et al., 1989; Woltz and Jones, 1981). The supply of Mn, Fe and possibly other nutrients, to *F. oxysporum* strains causing vascular wilt may therefore increase disease incidence and/or severity (see section 4.1, below).

The availability of other micronutrients to plants has been linked to disease alleviation in particular instances. *Copper* (Cu) deficiency decreases lignification in the xylem and has been linked to lodging in cereals (Evans et al., 2007). Copper has direct toxic effects on pathogens as well. A Cu supply protects grapes and hops from Downy mildew, caused by *Plasmopara viticola* and *Pseudoperonospora humuli*, respectively (see Evans et al., 2007). *Nickel* (Ni), like Fe and Zn, is a co-factor of some enzymes, such as ureases, which break down urea into less toxic forms (Rice, 2007). Nickel application has been shown to reduce brown spot in rice (caused by *Cochliobolus miyabeanus* syn. *Helminthosporium oryzae*). Supplying *molybdenum* (Mo) reduced late blight in potato and Ascochyta blight in beans and peas (Palti, 1989 p. 143). As a co-factor of nitrate reductase, this element plays a particularly important role in the reduction of nitrate to ammonium (Rice, 2007).

*Silicon* (Si), now regarded as an essential micronutrient, has been shown to enhance disease resistance in many instances (Datnoff et al., 2007). In sugar cane, ring spot was alleviated by Si amendments (see Datnoff et al., 2007). Low Si in rice has been linked to susceptibility to a number of pathogens including *Pyricularia*, *Sclerotium oryzae*, *Cochliobolus* and *Xanthomonas oryzae* (Palti, 1981 and references within). A supply of Si enhances resistance to rice blast (Datnoff et al., 2007). Supplying Si to coffee reduced leaf disease and nematode infections in roots (see Sections 4.2 and 4.5). Possibly Si, with other nutrients such as Ca and *boron* (B), influences cell wall properties and enhances mechanical strengthening of tissues (Rice, 2007). Shen et al. (2010) tested the effect of potassium silicate on *in vitro* growth of some plant pathogens, including *Fusarium oxysporum, Rhizoctonia solani* and *Pestalotiopsis clavispora*, finding no influence if the media pH was maintained at the same level as the control. They suggested that the mechanism by which Si confers resistance may be related to provision of a physical barrier to pathogen infection or to the induction of a defense

observed cases of disease resistance (Duffy, 2007). Zinc application to soils reduces attack by root pathogens of tomato, including *Fusarium solani*, *Rhizoctonia solani* and *Macrophoma phaseoli*, and also Rhizoctonia root rots of wheat, chickpea, cowpea and medicago (Duffy, 2007; Gaur and Vaidge, 1983; Kalim et al., 2003; Streeter et al., 2001). In tropical perennials, the role of Zn in disease resistance remains to be investigated. However, Zn-deficiency in rubber (*Hevea brasiliensis*) predisposes the tree to infection with *Oidium heveae* (Duffy, 2007). Zinc has been reported to alleviate Phytophthora diseases. Low Zn levels in soils and leaf tissues were associated with a high incidence of Phytophthora pod rot (or black pod) of cocoa in Papua New Guinea (Nelson et al., 2011). Supplying *Manganese* (Mn) has been shown to alleviate various diseases in a number of crop plant species (Palti, 1981; Thompson and Huber, 2007). Manganese occurs in different redox states and while it is present in healthy tissues as the Mn2+ ion, it accumulates at sites of pathogen attack in the Mn4+ form, for example in rice affected by blast (Thompson and Huber, 2007). *Iron* (Fe) has an essential role in plant cells as a co-factor in redox reactions and other functions. Fe is mainly available to plants as its reduced ion, Fe2+. Verticillium wilt in mango caused by *V. albo-atrum*, and in groundnut caused by *V. dahliae* was mitigated in both cases by the application of Fe in chelated form (see Palti, 1981 p. 142). On the other hand, control of Fusarium wilt in tomato was favoured by low Fe (Woltz and Jones, 1981). Similarly, Fusarium wilt has been shown to be lower at low levels of Mn. *F. oxysporum* has a particularly high demand for some micronutrient elements, especially Mn, Fe and Zn (Jones et al., 1989; Woltz and Jones, 1981). The supply of Mn, Fe and possibly other nutrients, to *F. oxysporum* strains causing vascular wilt may therefore increase disease

The availability of other micronutrients to plants has been linked to disease alleviation in particular instances. *Copper* (Cu) deficiency decreases lignification in the xylem and has been linked to lodging in cereals (Evans et al., 2007). Copper has direct toxic effects on pathogens as well. A Cu supply protects grapes and hops from Downy mildew, caused by *Plasmopara viticola* and *Pseudoperonospora humuli*, respectively (see Evans et al., 2007). *Nickel* (Ni), like Fe and Zn, is a co-factor of some enzymes, such as ureases, which break down urea into less toxic forms (Rice, 2007). Nickel application has been shown to reduce brown spot in rice (caused by *Cochliobolus miyabeanus* syn. *Helminthosporium oryzae*). Supplying *molybdenum* (Mo) reduced late blight in potato and Ascochyta blight in beans and peas (Palti, 1989 p. 143). As a co-factor of nitrate reductase, this element plays a particularly important role in

*Silicon* (Si), now regarded as an essential micronutrient, has been shown to enhance disease resistance in many instances (Datnoff et al., 2007). In sugar cane, ring spot was alleviated by Si amendments (see Datnoff et al., 2007). Low Si in rice has been linked to susceptibility to a number of pathogens including *Pyricularia*, *Sclerotium oryzae*, *Cochliobolus* and *Xanthomonas oryzae* (Palti, 1981 and references within). A supply of Si enhances resistance to rice blast (Datnoff et al., 2007). Supplying Si to coffee reduced leaf disease and nematode infections in roots (see Sections 4.2 and 4.5). Possibly Si, with other nutrients such as Ca and *boron* (B), influences cell wall properties and enhances mechanical strengthening of tissues (Rice, 2007). Shen et al. (2010) tested the effect of potassium silicate on *in vitro* growth of some plant pathogens, including *Fusarium oxysporum, Rhizoctonia solani* and *Pestalotiopsis clavispora*, finding no influence if the media pH was maintained at the same level as the control. They suggested that the mechanism by which Si confers resistance may be related to provision of a physical barrier to pathogen infection or to the induction of a defense

incidence and/or severity (see section 4.1, below).

the reduction of nitrate to ammonium (Rice, 2007).

response in the host, rather than to a chemical effect. Silicon and other elements, including Ca and B, may be of particular importance in resistance to facultative pathogens, wound invading pathogens and nematode infections.

#### **3. Management of soils supporting tropical perennial crops**

Since many tropical soils are nutrient-poor and the replacement of nutrients by mineral or organic fertilisers is often inadequate, tropic perennial crops are particularly prone to nutrient stress. Growing perennial crops can lead to nutrient deficiencies if the soils in which they are grown are not adequately amended with mineral or organic fertilisers. Long-term cropping of one or a few species in tropical soils can also have other impacts on soil properties, such as the soil pH, that in turn may cause nutrient deficiencies. In addition, poor sanitation (e.g. removal of infected plant material), flooding (which may spread inoculum as well as causing plant stress) and inappropriate canopy management are common exacerbating factors leading to increases in disease incidence and severity on a farm (Kohler et al., 1997). Importantly, disease may also have the effect of reducing the plants' nutrient status or impairing water uptake. For example, coffee tree roots and the roots of other tree crops infected with nematodes or fungi may have impaired water and chemical uptake mechanisms causing wilting and nutrient deficiency (Nelson et al., 2002; Waller et al., 2007 p. 279).

Table 1 presents data on nutrient uptake by some tropical perennial crops based on previous studies. The data refer only to the nutrient content of marketable products; the nutrients contained in waste (e.g. discarded tea leaves and coffee or cocoa beans) are not included. In the case of nutrients removed by cocoa in Nigeria (reported by Wessel, 1985), it can be seen that the pod husks, which are normally discarded, remove high amounts of K (77 kg ha-1y-1 in the husks of pods producing one ton of dry beans). Substantial amounts of Ca are also removed with cocoa pods and other fruit products.


Table 1. Estimated removal of N, P and K from the soil by the harvested products of some tropical tree crops. Yields indicated for each crop are estimates of the quantity of produce obtained per hectare each year (sources: Krauss, 2003; Wessel, 1985). Note that where the quantities removed were given for the oxides of P and K in the original data, these figures have been converted to indicate the respective quantities of the elements removed.

Soil acidity or low pH, common in the tropics, may be increased under particular crops, especially long-term perennial crops, or by the application of some kinds of mineral fertiliser (Jones et al., 1989). Soil pH decreases when forest soils are turned over to perennial crops, such as coffee and cocoa (Beer, 1988; Beer et al., 1998; Hartemink, 2005). Lowering soil pH decreases the availability of basic cations, particularly Ca and Mg. However, increased soil

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 249

The main types of disease that impact production and performance of major tropical perennials and the conditions that influence disease incidence and/or severity, particularly nutrition and soil function, are outlined below. Most of the diseases described are caused by new encounter pathogens, while some such as witches' broom disease of cocoa in South America and coffee wilt disease, which has caused severe losses and tree death in East Africa, are caused by co-evolved pathogens. While each group of diseases (e.g. dieback diseases) has common features that relate to their management and control, it should be noted that pathogens from widely separate taxonomic groups may cause similar symptoms common to a particular type of disease. Therefore, control measures to reduce plant diseases need to take into account the taxon of the pathogen as well as the type of disease it causes. Diseases of tropical crops are often complex being associated with more than one pathogen or pest, or transmitted by vectors. Conversely, a particular pathogen species may cause more than one disease. Therefore, the disease groupings outlined below may overlap with

Vascular wilts can cause serious losses for a number of tropical perennial crops. Vascular wilts may be soil-borne with infections initiated in the roots, as in many wilts caused by *Fusarium oxysporum*, or else they may be initiated in the phyllosphere, especially by infections via wounds as occurs in many Ceratocystis wilts, caused by *Ceratocystis fimbriata* and closely related species on a wide range of crop hosts. Coffee wilt (tracheomycosis) attributed to *Gibberella xylarioides (anamorph: Fusarium xylarioides)* has become a serious disease problem in Robusta coffee-growing areas of East Africa (Rutherford, 2006; Flood, 2010). An adequate nutrient supply to coffee trees is recommended as part of an integrated strategy to manage this disease (e.g. see www.nyrussell.com/all-about-coffee). Wounding caused by nematodes in the roots or by insects in aerial parts may increase the possibility of infection by fungi causing soilborne diseases, including wilts. In coffee, for example, infection with nematodes can

Insects may also transmit disease by carrying spores to wounded tissues or by producing insect frass containing spores that can be dispersed by wind. For example, species of Ambrosia beetles are attracted to cocoa tissues infected with *Ceratocystis* spp. and insect frass containing their spores may be disseminated to other plants by wind. Vascular wilts have in some cases been linked to environmental factors, such as drought stress. Drought-stressed plants are more susceptible to Ceratocystis wilt (Harrington, 2004). Vascular wilt diseases are complicated by the fact that plants may be more susceptible to the causal pathogen in conditions of water or nutrient deficiency but that, additionally, the pathogen may impair transport in the vascular

Wilts caused by host-specific races of *F. oxysporum* occur in a number of crop species. Panama disease of banana caused by *F. oxysporum* f. sp. cubense is particularly devastating (Ploetz, 2006). Following infection of the root, the fungus enters the vascular system and impedes water and nutrient transport to the upper plant. The leaves of infected banana plants become yellow and dry and the plant eventually wilts. The pathogen can be spread through flooding and, therefore, drainage and prevention of over-irrigation are particularly

predispose plants to infection with *F. oxysporum* (Kohler et al., 1997; Nelson, 2002).

tissues thus causing water or nutrient deficiency in the plant tissues.

**4. Diseases of tropical perennial crops in relation to nutrient and other** 

**growing conditions** 

each other considerably.

**4.1 Vascular wilts** 

acidity increases the availability of other cations to plant roots. These include the cations of Mn and aluminium (Al) which can reach toxic levels as their uptake by plants increases. The proportion of soil P that is fixed and unavailable for plant uptake increases in acid conditions (Mengel and Kirkby, 1982). Liming can reduce the severity of a number of diseases perhaps by increasing the availability of a number of nutrients to crops, as well as providing a source of Ca, reducing Al toxicity and improving soil structure (Palti, 1981 p. 142). The increase in pH in limed soils also favours the growth of bacteria, including actinomycetes, which include species that are antagonistic to fungal pathogens (Palti, 1981 p. 29; Jones et al., 1989).

#### *Shade and nutrition*

Tropical perennials produced in agroforestry systems are affected by other species on the farm, including shade trees in the case of shade-requiring tree crops such as cocoa and coffee (Schroth et al., 2001). Importantly for such shade-requiring species, managed shade can reduce incidence and severity of some pests and diseases. Shade may also reduce stress to tree crops by preventing extremes in temperature, water loss etc. that may result from exposure (Staver et al., 2001). This, in turn, mitigates diseases that become more severe in stressed plants. Removal of shade can increase photosynthesis and, therefore, raise the productivity of tree crops such as coffee and cocoa. The removal of shade from coffee farms, for example, can provide double the yields of shaded coffee in the short-term (Waller et al., 2007). However, this may be followed by impacts from other problems, including increased susceptibility to diseases such as brown-eye spot (see Section 4.2), wind and storm damage, frost damage at higher altitudes, increased evapotranspiration (and water loss) and lower levels of soil organic matter (Waller et al., 2007 p. 313). Conditions such as overbearing dieback and sunscorch of coffee (see Section 4.4) may result from shade removal. Other twig and leaf blights, such as anthracnose caused by *Colletotrichum gloeosporioides* on cocoa grown in Indonesia become more severe following shade removal (Agus Purwantara, pers. comm.). Conversely, excessive shade and inadequate pruning can provide suitable conditions for other coffee and cocoa pathogens, such as *Corticium* spp. (causing web blight and pink disease), *Phytophthora palmivora* (causing pod rot and other diseases in cocoa) and *Mycena citricolor* (causing South American leaf spot in coffee). Shade trees may be sources of other pathogens with wide host ranges such as the root pathogens *Armillaria* and *Ganoderma* spp.

Shade trees have a mixed effect on the plant nutrition of other crops in the agroforestry system (Schroth et al., 2001). They may compete with crops for water and nutrients in the soil and sequester nutrients in their biomass (Palm, 1995). However, they also provide inputs of nutrients to the system through leaf litter or by nitrogen-fixation. In Central America, *Cordia alliodora* shade trees on each hectare of coffee produce 5.7 tons of leaf litter per year, containing 114 kg N, 7 kg P and 54 kg K (Beer, 1988). Forest trees providing shade for cocoa in West Africa produced 5 tons of leaf litter per hectare each year, containing 79 kg N and 4.5 kg P (Murray, 1975). Legume shade trees in cocoa and coffee agroforestry systems provide approximately 60 kg N ha-1y-1 by biological fixation of N2 (Beer, 1988). However, some legumes may cause decreases in soil pH. Somarriba and Beer (2011) reported that timber species grown with cocoa did not impact cocoa production. Shade trees with relatively deep roots can remobilise nutrients in the system (Schroth et al., 2001). Beer et al. (1998) cite reports of lower leaching rates of N under shaded coffee (9 kg ha-1y-1) than under unshaded coffee (24 kg ha-1y-1).

#### **4. Diseases of tropical perennial crops in relation to nutrient and other growing conditions**

The main types of disease that impact production and performance of major tropical perennials and the conditions that influence disease incidence and/or severity, particularly nutrition and soil function, are outlined below. Most of the diseases described are caused by new encounter pathogens, while some such as witches' broom disease of cocoa in South America and coffee wilt disease, which has caused severe losses and tree death in East Africa, are caused by co-evolved pathogens. While each group of diseases (e.g. dieback diseases) has common features that relate to their management and control, it should be noted that pathogens from widely separate taxonomic groups may cause similar symptoms common to a particular type of disease. Therefore, control measures to reduce plant diseases need to take into account the taxon of the pathogen as well as the type of disease it causes. Diseases of tropical crops are often complex being associated with more than one pathogen or pest, or transmitted by vectors. Conversely, a particular pathogen species may cause more than one disease. Therefore, the disease groupings outlined below may overlap with each other considerably.

#### **4.1 Vascular wilts**

248 Plant Pathology

acidity increases the availability of other cations to plant roots. These include the cations of Mn and aluminium (Al) which can reach toxic levels as their uptake by plants increases. The proportion of soil P that is fixed and unavailable for plant uptake increases in acid conditions (Mengel and Kirkby, 1982). Liming can reduce the severity of a number of diseases perhaps by increasing the availability of a number of nutrients to crops, as well as providing a source of Ca, reducing Al toxicity and improving soil structure (Palti, 1981 p. 142). The increase in pH in limed soils also favours the growth of bacteria, including actinomycetes, which include species that are antagonistic to fungal pathogens (Palti, 1981

Tropical perennials produced in agroforestry systems are affected by other species on the farm, including shade trees in the case of shade-requiring tree crops such as cocoa and coffee (Schroth et al., 2001). Importantly for such shade-requiring species, managed shade can reduce incidence and severity of some pests and diseases. Shade may also reduce stress to tree crops by preventing extremes in temperature, water loss etc. that may result from exposure (Staver et al., 2001). This, in turn, mitigates diseases that become more severe in stressed plants. Removal of shade can increase photosynthesis and, therefore, raise the productivity of tree crops such as coffee and cocoa. The removal of shade from coffee farms, for example, can provide double the yields of shaded coffee in the short-term (Waller et al., 2007). However, this may be followed by impacts from other problems, including increased susceptibility to diseases such as brown-eye spot (see Section 4.2), wind and storm damage, frost damage at higher altitudes, increased evapotranspiration (and water loss) and lower levels of soil organic matter (Waller et al., 2007 p. 313). Conditions such as overbearing dieback and sunscorch of coffee (see Section 4.4) may result from shade removal. Other twig and leaf blights, such as anthracnose caused by *Colletotrichum gloeosporioides* on cocoa grown in Indonesia become more severe following shade removal (Agus Purwantara, pers. comm.). Conversely, excessive shade and inadequate pruning can provide suitable conditions for other coffee and cocoa pathogens, such as *Corticium* spp. (causing web blight and pink disease), *Phytophthora palmivora* (causing pod rot and other diseases in cocoa) and *Mycena citricolor* (causing South American leaf spot in coffee). Shade trees may be sources of other pathogens with wide host ranges such as the root pathogens *Armillaria* and *Ganoderma* spp. Shade trees have a mixed effect on the plant nutrition of other crops in the agroforestry system (Schroth et al., 2001). They may compete with crops for water and nutrients in the soil and sequester nutrients in their biomass (Palm, 1995). However, they also provide inputs of nutrients to the system through leaf litter or by nitrogen-fixation. In Central America, *Cordia alliodora* shade trees on each hectare of coffee produce 5.7 tons of leaf litter per year, containing 114 kg N, 7 kg P and 54 kg K (Beer, 1988). Forest trees providing shade for cocoa in West Africa produced 5 tons of leaf litter per hectare each year, containing 79 kg N and 4.5 kg P (Murray, 1975). Legume shade trees in cocoa and coffee agroforestry systems provide approximately 60 kg N ha-1y-1 by biological fixation of N2 (Beer, 1988). However, some legumes may cause decreases in soil pH. Somarriba and Beer (2011) reported that timber species grown with cocoa did not impact cocoa production. Shade trees with relatively deep roots can remobilise nutrients in the system (Schroth et al., 2001). Beer et al. (1998) cite reports of lower leaching rates of N under shaded coffee (9 kg ha-1y-1) than under

p. 29; Jones et al., 1989).

unshaded coffee (24 kg ha-1y-1).

*Shade and nutrition* 

Vascular wilts can cause serious losses for a number of tropical perennial crops. Vascular wilts may be soil-borne with infections initiated in the roots, as in many wilts caused by *Fusarium oxysporum*, or else they may be initiated in the phyllosphere, especially by infections via wounds as occurs in many Ceratocystis wilts, caused by *Ceratocystis fimbriata* and closely related species on a wide range of crop hosts. Coffee wilt (tracheomycosis) attributed to *Gibberella xylarioides (anamorph: Fusarium xylarioides)* has become a serious disease problem in Robusta coffee-growing areas of East Africa (Rutherford, 2006; Flood, 2010). An adequate nutrient supply to coffee trees is recommended as part of an integrated strategy to manage this disease (e.g. see www.nyrussell.com/all-about-coffee). Wounding caused by nematodes in the roots or by insects in aerial parts may increase the possibility of infection by fungi causing soilborne diseases, including wilts. In coffee, for example, infection with nematodes can predispose plants to infection with *F. oxysporum* (Kohler et al., 1997; Nelson, 2002).

Insects may also transmit disease by carrying spores to wounded tissues or by producing insect frass containing spores that can be dispersed by wind. For example, species of Ambrosia beetles are attracted to cocoa tissues infected with *Ceratocystis* spp. and insect frass containing their spores may be disseminated to other plants by wind. Vascular wilts have in some cases been linked to environmental factors, such as drought stress. Drought-stressed plants are more susceptible to Ceratocystis wilt (Harrington, 2004). Vascular wilt diseases are complicated by the fact that plants may be more susceptible to the causal pathogen in conditions of water or nutrient deficiency but that, additionally, the pathogen may impair transport in the vascular tissues thus causing water or nutrient deficiency in the plant tissues.

Wilts caused by host-specific races of *F. oxysporum* occur in a number of crop species. Panama disease of banana caused by *F. oxysporum* f. sp. cubense is particularly devastating (Ploetz, 2006). Following infection of the root, the fungus enters the vascular system and impedes water and nutrient transport to the upper plant. The leaves of infected banana plants become yellow and dry and the plant eventually wilts. The pathogen can be spread through flooding and, therefore, drainage and prevention of over-irrigation are particularly

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 251

may be a consequence of impaired root growth and, therefore, increased exposure to microslerotia (Pennypacker, 1989). K deficiency in cotton is enhanced by infection with *V. dahliae*, perhaps due to a decrease in the ability of the roots to take up K after infection with

An increase in severity of Verticillium wilt of cotton in response to P supplied as superphosphate has been reported – possibly P promotes pathogen activity within the plant (Bell, 1989). A study on cocoa affected by Verticillium wilt in Nigeria indicated that P supply had no effect on infections (Emechebe, 1980). Calcium nutrition may enhance resistance to Verticillium wilt in some cases (Bell, 1989). This could be related to an increase in the levels of Ca-pectin complexes, which are resistant to breakdown by a pectin-degrading enzyme produced by the fungus (see Section 2). Possibly, Ca-pectin complexes are involved in

Some leaf/fruit diseases, e.g. infections by the Mycosphaerellaceae pathogen, *Cercospora mangiferae* (syn. *Stigmina mangiferae*), causing angular leaf spot in mango and other pathogens of mango are influenced by the growing conditions of the host. Similarly, brown-eye leaf spot in coffee caused by *Cercospora coffeicola* , becomes more serious under conditions of environmental stress. Brown-eye spot is encouraged if coffee is grown in unshaded conditions, particularly if the soil is nutrient-poor (see Kohler et al., 1997; Nelson, 2008a). Deficiency in N and K in particular, may accentuate brown-eye spot (Wrigley 1988). In an experiment with coffee seedlings grown in pots, Pozza et al. (2001) showed that supplying bovine manure to coffee trees, as well as other types of fertiliser reduced the severity of brown-eye leaf spot. Santos et al. (2008) compared the incidence of brown-eye spot on ten-year old coffee in neighbouring plots that were managed either conventionally using inorganic fertiliser applications, or by organic methods in which only organic amendments were applied to the soil. In a period of two consecutive years, disease incidence was higher in the plot under conventional management (28% and 29%) than in the plot managed using organic methods (9% and 12%), although higher berry yields were obtained in the conventionally managed plots. Possibly, the higher rates of disease in the conventionally-treated trees were a consequence of nutrient-deficiency, particularly of Ca and Mg. The leaf concentrations of Ca and Mg were lower in the conventionally-treated plants. Possibly this was due to the higher yields from the trees receiving conventional treatment (so that these trees had a higher nutrient

Infections in citrus-growing areas in Cameroon and other tropical countries in Africa by the Mycosphaerellaceae pathogen, *Phaeoramularia angolensis*, causing leaf and fruit lesions creates serious losses for farmers. In a survey of disease-affected areas, Ndo et al. (2010) found that disease severity was lower in citrus species grown on volcanic soils in Cameroon than in other soil-types. Altitude was also a key factor affecting disease severity. Since volcanic soils are generally nutrient-rich, this suggests that plant nutrition plays a role in mitigating this disease. In the case of *Dothiorella gregaria,* a Botryosphaeriaceae pathogen that causes fruit spot on mandarin, Ca and Zn contents of plant tissues may influence disease severity. Isolates of this pathogen were inoculated into mandarin by da Silva Moraes et al. (2007) who found that the

highest lesion rates were obtained on plants with the lowest Ca and Zn contents.

impeding growth of the fungus through the xylem (Pennypacker, 1989).

**4.2 Diseases causing lesions on leaves and fruits** 

requirement than the lower yielding organic trees).

the fungus (Bell, 1989).

*Leaf and fruit spots* 

necessary. As the pathogen can be harboured by the bunch stalks, sanitation measures should be applied (Kohler et al., 1997). Some studies on *F.oxysporum* wilts suggest that nutrient availability to both the plant and the pathogen may affect the level of disease severity. The factor(s) causing the wilt are unclear: in Fusarium wilt of tomato it has been attributed to physical blockage or by the secretion of toxins (Walker, 1972 p. 300). Reducing soil acidity by liming is used to reduce *F. oxysporum* infection of coffee plants (Kohler et al., 1997). Possibly, liming reduces the availability of some micronutrients to the fungus. As mentioned previously, *F. oxysporum* has a particularly high demand for some micronutrients, including Mn, Fe and Zn. Mn may be in particular demand by the pathogen. Jones and Woltz (1972) showed that the control of Fusarium wilt of tomato by liming could be reversed by supplying Mn in chelated form to the limed soils. Liming may also help to control Fusarium wilts by changing the soil microbial populations. Under conditions of high pH, bacteria, including actinomycete, populations increase, including those of antagonistic species (Jones et al., 1989). Increased calcium supply to the plant has been shown to reduce the severity of Fusarium wilt of tomato (Walker, 1972 p. 303). The mechanism involved may be related to the increase in resistance conferred by Ca to enzymatic breakdown of pectate compounds by the fungus, which might also account for the effect of Ca supply on reducing Verticillium wilt (see below).

As mentioned previously, the form of N that is present in the soil, whether as nitrate or ammonium ions can influence the incidence and severity of vascular wilts (see Section 2). Application of N as ammonium fertiliser can increase Fusarium wilt: this has been suggested to be an effect of increased acidity or related to a reduction in the K/Cl ratio (Jones et al., 1989). A study on Fusarium wilt of banana by Nasir et al. (2003) suggested that the effect of the form of N supplied on disease severity is not related to the activity of the pathogen. When banana plantlets were transplanted into soils infested with *F. oxysporum*, an increase of wilt disease severity and the invasion of roots by the pathogen were found to be independent of *F. oxysporum* activity in the soil (Nasir et al, 2003). Amendment of soil with chicken manure increased disease severity, but not *F. oxysporum* activity; it appeared that the increase in disease and pathogen invasion was a consequence of supplying N as the ammonium-form but that this was not connected to pathogen activity in the soil. Another study demonstrated that a lower rate of germination of *F. oxysporum* chlamydospores occurred in soils that had an adequate supply of Ca, particularly in relation to other basic cations, Mg and K (Chuang, 1988; 1991). The same study indicated that higher soil pH values and populations of actinomycetes also decreased spore germination rates. Domingues et al. (2001) compared banana field plots which differed in their capacity to suppress Fusarium wilt. Soils that suppressed disease had a lower proportion by weight of water-stable aggregates, than conducive soils. They hypothesised that the higher proportion of water-stable aggregates in the conducive soils favoured anaerobiosis, which increased availability of reduced Fe ions for the pathogen, which as mentioned previously, has a high demand for micronutrients, such as Fe.

Verticillium wilt caused by *Verticillium dahliae* also occurs in some perennial crops. While Fusarium wilt is encouraged by acidic soils, Verticillium wilt is favoured by a higher soil pH. A build-up of inocolum can occur in perennial crop species affected by *V. dahliae*; in the case of Verticillium wilt of pistachio, new infections are initiated by microsclerotia, which can survive for long periods on pistachio roots. A higher incidence of Verticillium wilt has been found in pistachio trees under conditions of K deficiency (Pennypacker, 1989). This may be a consequence of impaired root growth and, therefore, increased exposure to microslerotia (Pennypacker, 1989). K deficiency in cotton is enhanced by infection with *V. dahliae*, perhaps due to a decrease in the ability of the roots to take up K after infection with the fungus (Bell, 1989).

An increase in severity of Verticillium wilt of cotton in response to P supplied as superphosphate has been reported – possibly P promotes pathogen activity within the plant (Bell, 1989). A study on cocoa affected by Verticillium wilt in Nigeria indicated that P supply had no effect on infections (Emechebe, 1980). Calcium nutrition may enhance resistance to Verticillium wilt in some cases (Bell, 1989). This could be related to an increase in the levels of Ca-pectin complexes, which are resistant to breakdown by a pectin-degrading enzyme produced by the fungus (see Section 2). Possibly, Ca-pectin complexes are involved in impeding growth of the fungus through the xylem (Pennypacker, 1989).

#### **4.2 Diseases causing lesions on leaves and fruits**

#### *Leaf and fruit spots*

250 Plant Pathology

necessary. As the pathogen can be harboured by the bunch stalks, sanitation measures should be applied (Kohler et al., 1997). Some studies on *F.oxysporum* wilts suggest that nutrient availability to both the plant and the pathogen may affect the level of disease severity. The factor(s) causing the wilt are unclear: in Fusarium wilt of tomato it has been attributed to physical blockage or by the secretion of toxins (Walker, 1972 p. 300). Reducing soil acidity by liming is used to reduce *F. oxysporum* infection of coffee plants (Kohler et al., 1997). Possibly, liming reduces the availability of some micronutrients to the fungus. As mentioned previously, *F. oxysporum* has a particularly high demand for some micronutrients, including Mn, Fe and Zn. Mn may be in particular demand by the pathogen. Jones and Woltz (1972) showed that the control of Fusarium wilt of tomato by liming could be reversed by supplying Mn in chelated form to the limed soils. Liming may also help to control Fusarium wilts by changing the soil microbial populations. Under conditions of high pH, bacteria, including actinomycete, populations increase, including those of antagonistic species (Jones et al., 1989). Increased calcium supply to the plant has been shown to reduce the severity of Fusarium wilt of tomato (Walker, 1972 p. 303). The mechanism involved may be related to the increase in resistance conferred by Ca to enzymatic breakdown of pectate compounds by the fungus, which might also account for the effect of Ca supply on reducing

As mentioned previously, the form of N that is present in the soil, whether as nitrate or ammonium ions can influence the incidence and severity of vascular wilts (see Section 2). Application of N as ammonium fertiliser can increase Fusarium wilt: this has been suggested to be an effect of increased acidity or related to a reduction in the K/Cl ratio (Jones et al., 1989). A study on Fusarium wilt of banana by Nasir et al. (2003) suggested that the effect of the form of N supplied on disease severity is not related to the activity of the pathogen. When banana plantlets were transplanted into soils infested with *F. oxysporum*, an increase of wilt disease severity and the invasion of roots by the pathogen were found to be independent of *F. oxysporum* activity in the soil (Nasir et al, 2003). Amendment of soil with chicken manure increased disease severity, but not *F. oxysporum* activity; it appeared that the increase in disease and pathogen invasion was a consequence of supplying N as the ammonium-form but that this was not connected to pathogen activity in the soil. Another study demonstrated that a lower rate of germination of *F. oxysporum* chlamydospores occurred in soils that had an adequate supply of Ca, particularly in relation to other basic cations, Mg and K (Chuang, 1988; 1991). The same study indicated that higher soil pH values and populations of actinomycetes also decreased spore germination rates. Domingues et al. (2001) compared banana field plots which differed in their capacity to suppress Fusarium wilt. Soils that suppressed disease had a lower proportion by weight of water-stable aggregates, than conducive soils. They hypothesised that the higher proportion of water-stable aggregates in the conducive soils favoured anaerobiosis, which increased availability of reduced Fe ions for the pathogen, which as mentioned previously, has a high

Verticillium wilt caused by *Verticillium dahliae* also occurs in some perennial crops. While Fusarium wilt is encouraged by acidic soils, Verticillium wilt is favoured by a higher soil pH. A build-up of inocolum can occur in perennial crop species affected by *V. dahliae*; in the case of Verticillium wilt of pistachio, new infections are initiated by microsclerotia, which can survive for long periods on pistachio roots. A higher incidence of Verticillium wilt has been found in pistachio trees under conditions of K deficiency (Pennypacker, 1989). This

Verticillium wilt (see below).

demand for micronutrients, such as Fe.

Some leaf/fruit diseases, e.g. infections by the Mycosphaerellaceae pathogen, *Cercospora mangiferae* (syn. *Stigmina mangiferae*), causing angular leaf spot in mango and other pathogens of mango are influenced by the growing conditions of the host. Similarly, brown-eye leaf spot in coffee caused by *Cercospora coffeicola* , becomes more serious under conditions of environmental stress. Brown-eye spot is encouraged if coffee is grown in unshaded conditions, particularly if the soil is nutrient-poor (see Kohler et al., 1997; Nelson, 2008a). Deficiency in N and K in particular, may accentuate brown-eye spot (Wrigley 1988). In an experiment with coffee seedlings grown in pots, Pozza et al. (2001) showed that supplying bovine manure to coffee trees, as well as other types of fertiliser reduced the severity of brown-eye leaf spot. Santos et al. (2008) compared the incidence of brown-eye spot on ten-year old coffee in neighbouring plots that were managed either conventionally using inorganic fertiliser applications, or by organic methods in which only organic amendments were applied to the soil. In a period of two consecutive years, disease incidence was higher in the plot under conventional management (28% and 29%) than in the plot managed using organic methods (9% and 12%), although higher berry yields were obtained in the conventionally managed plots. Possibly, the higher rates of disease in the conventionally-treated trees were a consequence of nutrient-deficiency, particularly of Ca and Mg. The leaf concentrations of Ca and Mg were lower in the conventionally-treated plants. Possibly this was due to the higher yields from the trees receiving conventional treatment (so that these trees had a higher nutrient requirement than the lower yielding organic trees).

Infections in citrus-growing areas in Cameroon and other tropical countries in Africa by the Mycosphaerellaceae pathogen, *Phaeoramularia angolensis*, causing leaf and fruit lesions creates serious losses for farmers. In a survey of disease-affected areas, Ndo et al. (2010) found that disease severity was lower in citrus species grown on volcanic soils in Cameroon than in other soil-types. Altitude was also a key factor affecting disease severity. Since volcanic soils are generally nutrient-rich, this suggests that plant nutrition plays a role in mitigating this disease. In the case of *Dothiorella gregaria,* a Botryosphaeriaceae pathogen that causes fruit spot on mandarin, Ca and Zn contents of plant tissues may influence disease severity. Isolates of this pathogen were inoculated into mandarin by da Silva Moraes et al. (2007) who found that the highest lesion rates were obtained on plants with the lowest Ca and Zn contents.

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 253

such as cocoa, *Theobroma cacao* (Rubini et al., 2005), is the most common causal pathogen, infecting a number of tropical tree crops such as avocado, mango, coffee and kauri (*Agathis* sp). Symptoms include blackened and sunken lesions on fruit, and marginal necrosis of young or flush leaves. On avocado, fruit and leaves are infected by *C. gloeosporioidies*, the leaves developing large light brown lesions and, in wet weather, pink spore masses. Anthracnose is transmitted mainly by rainsplash (Kader, 2002) and heavy rainfall increases the severity of anthracnose in Robusta coffee. Rust infections can predispose Arabica coffee to anthracnose infection (Kohler et al., 1997). As an endophyte and facultative parasite, this pathogen may be asymptomatic in healthy plants of some crop species, only becoming pathogenic under conditions of stress, such as over-exposure to sun in the case of shaderequiring crops. As with other latent species (see Prakash, 2000; Kohler et al., 1997), the removal of dead twigs and branches before flowering is a crucial measure to control infection by the fungus. Possibly, nutrition plays a role in the control of anthracnose. Anthracnose in the orchid, *Cymbidium* sp., caused by *Colletototrichum orchidacearum*, for example, was reduced by applying macronutrients, especially K and P (Yi et al., 2003). Acosta-Ramos et al. (2003) took an integrated management approach, including soil and foliar application of nutrients, to mango trees in an orchard in Mexico and recorded decreases in the incidence of both anthracnose, caused by *C. gloeosporioides*, and stem end

In papaya and mango, a post-harvest problem caused by anthracnose infection of the fruit is particularly serious and can be controlled by dipping fruit in hot water or a fungicide (such as benomyl). Anthracnose infects all parts of the mango plant: new leaf flushes are particularly susceptible and, in wet weather, flowers are susceptible to blossom blight. In guava, which incurs serious losses from anthracnose disease, infections may be associated with fruit fly damage and with scab damage caused by *Sphaceloma perseae* (Ploetz, 2007). Passion fruit is also a host of anthracnose. Passion fruit produced under poor growing

Conditions predisposing perennial crops to fruit rots include high rainfall, poor drainage and poor sanitation practices, whereby infected fruits (which provided sources of new inoculum) are not disposed of correctly. This is particularly relevant in the case of *Phytophthora palmivora*, the straminopile (formerly oomycete) pathogen, which is responsible for fruit rots in a wide range of host species in the tropics including breadfruit, cocoa, black pepper, papaya and vanilla. On coconut and betel nut trees it causes bud rot, inflicting serious losses to these crops. The pathogen is dispersed mainly by motile zoospores, which require the presence of external water and infection (Guest, 2007). Hence, incidence of fruit rots caused by *P. palmivora* increases dramatically under conditions of high moisture during heavy rainfall periods in the wet season, poor drainage or slightly cooler conditions at higher altitudes that reduce evapotranspiration rates. In many host species, it also infects other parts of the plant including leaves and stems. Inoculum from one infected part of the plant can initiate infections in other tissues. On cocoa farms, insects, including ants and beetles, also transmit disease, carrying spores from infected sites to initiate new infections (Konam and Guest, 2004). Therefore, sanitation is a crucial control measure for Phytophthora diseases. A link between low levels of zinc nutrition and pod rot incidence

conditions may also be infected with *Alternaria* sp. causing brown spot.

rot, caused by *Lasiodiplodia theobromae*.

**4.3 Fruit rots** 

Particularly serious leaf diseases of banana include black sikatoga (or black leaf streak) caused by *Mycosphaerella fijiensis* (syn. *Paracercospora fijiensis*), yellow sikatoga (*Mycosphaerella musicola* syn. *Pseudocercospora musae*), freckle caused by *Guignardia musae* (syn. *Phyllosticta musarum*) and Black Cross (*Phyllachora musicola*) which may be associated with previous infections with *Cordana musae.* Black sikatoga disease is reduced under shade (Ploetz, 2003; Stover, 1972). The disease may increase under conditions of poor nutrition and, therefore, improved host nutrition is one recommended control measure (Nelson et al., 2006; Mobambo et al., 1994); adequate phosphorus nutrition may be particularly necessary for disease alleviation.

Algal leaf spot disease occurs on a number of tropical perennials including breadfruit, citrus, guava, cocoa, mango, soursop and black pepper. The causal agent is *Cephaleuros virescens* or other species of the same genus. Orange and green spots can be seen on leaves and young stems, particularly in trees weakened by stress or in periods of high rainfall (Nelson, 2008b). In avocado, poor plant nutrition, lack of soil drainage and still conditions (e.g. under dense canopies) are predisposing factors for algal infection (Nelson, 2008b).

A study in Brazil indicated a link between N nutrition and infection of citrus trees with the bacterial pathogen, *Xylella fastidiosa* causing variegated chlorosis disease (see Huber and Thompson, 2007). Nitrification increased disease severity but where a groundcover grass species was planted between rows of citrus trees, a decrease in the disease was demonstrated; this was explained by the inhibiting effect of the ground cover crop on nitrification. Nitrification decreases concentrations of ammonium ions in the soil and this, in turn, decreases Mn availability to the plant. Inhibition of nitrification caused an increase in Mn uptake by 50%. Thus in this case it appears that an increase in availability of Mn confers resistance to the host rather than favouring the pathogen, as in some Fusarium wilts.

#### *Rusts*

Rust in banana is caused by *Uredo musae* and in coffee by *Hemileia vastatrix*. In some annual crop species, rusts may be encouraged by high levels of N fertilisation (see Section 2). However, coffee rust has been reported to be more severe on plantations grown under nutrient poor conditions (Waller et. al., 2007, p. 302). Applications of silicon (Si) have been shown to decrease the level of coffee rust on coffee seedlings. Martinati et al. (2008) found that the number of rust lesions on leaves of Si-treated coffee (*C. arabica*) seedlings was decreased in proportion to the dosage of Si (as potassium silicate) supplied to the soil by up to 66% compared to the control, which received no Si.

In an epidemiological study on coffee rust in Honduras, Avelino et al. (2006) reported that the intensity of coffee rust infection was dependent on the production situation, rather than regional differences in environmental parameters, such as rainfall levels. They showed that coffee rust was associated with acid soils, soils treated with mineral fertilisers and increased yields. Possibly, an acidifying effect of the mineral fertilisers was the main factor accounting for higher levels of disease.

#### *Anthracnose*

Anthracnose diseases affect leaves, shoots and fruit of a variety of tropical perennial crops on farms and can also create severe post-harvest problems. The Ascomycotina species, *Glomerella cingulata* (anamorph: *Colletotrichum gloeosporioides)*, an endophyte on plant species such as cocoa, *Theobroma cacao* (Rubini et al., 2005), is the most common causal pathogen, infecting a number of tropical tree crops such as avocado, mango, coffee and kauri (*Agathis* sp). Symptoms include blackened and sunken lesions on fruit, and marginal necrosis of young or flush leaves. On avocado, fruit and leaves are infected by *C. gloeosporioidies*, the leaves developing large light brown lesions and, in wet weather, pink spore masses. Anthracnose is transmitted mainly by rainsplash (Kader, 2002) and heavy rainfall increases the severity of anthracnose in Robusta coffee. Rust infections can predispose Arabica coffee to anthracnose infection (Kohler et al., 1997). As an endophyte and facultative parasite, this pathogen may be asymptomatic in healthy plants of some crop species, only becoming pathogenic under conditions of stress, such as over-exposure to sun in the case of shaderequiring crops. As with other latent species (see Prakash, 2000; Kohler et al., 1997), the removal of dead twigs and branches before flowering is a crucial measure to control infection by the fungus. Possibly, nutrition plays a role in the control of anthracnose. Anthracnose in the orchid, *Cymbidium* sp., caused by *Colletototrichum orchidacearum*, for example, was reduced by applying macronutrients, especially K and P (Yi et al., 2003). Acosta-Ramos et al. (2003) took an integrated management approach, including soil and foliar application of nutrients, to mango trees in an orchard in Mexico and recorded decreases in the incidence of both anthracnose, caused by *C. gloeosporioides*, and stem end rot, caused by *Lasiodiplodia theobromae*.

In papaya and mango, a post-harvest problem caused by anthracnose infection of the fruit is particularly serious and can be controlled by dipping fruit in hot water or a fungicide (such as benomyl). Anthracnose infects all parts of the mango plant: new leaf flushes are particularly susceptible and, in wet weather, flowers are susceptible to blossom blight. In guava, which incurs serious losses from anthracnose disease, infections may be associated with fruit fly damage and with scab damage caused by *Sphaceloma perseae* (Ploetz, 2007). Passion fruit is also a host of anthracnose. Passion fruit produced under poor growing conditions may also be infected with *Alternaria* sp. causing brown spot.

#### **4.3 Fruit rots**

252 Plant Pathology

Particularly serious leaf diseases of banana include black sikatoga (or black leaf streak) caused by *Mycosphaerella fijiensis* (syn. *Paracercospora fijiensis*), yellow sikatoga (*Mycosphaerella musicola* syn. *Pseudocercospora musae*), freckle caused by *Guignardia musae* (syn. *Phyllosticta musarum*) and Black Cross (*Phyllachora musicola*) which may be associated with previous infections with *Cordana musae.* Black sikatoga disease is reduced under shade (Ploetz, 2003; Stover, 1972). The disease may increase under conditions of poor nutrition and, therefore, improved host nutrition is one recommended control measure (Nelson et al., 2006; Mobambo et al., 1994); adequate phosphorus nutrition may be particularly necessary

Algal leaf spot disease occurs on a number of tropical perennials including breadfruit, citrus, guava, cocoa, mango, soursop and black pepper. The causal agent is *Cephaleuros virescens* or other species of the same genus. Orange and green spots can be seen on leaves and young stems, particularly in trees weakened by stress or in periods of high rainfall (Nelson, 2008b). In avocado, poor plant nutrition, lack of soil drainage and still conditions (e.g. under dense canopies) are predisposing factors for algal infection (Nelson, 2008b).

A study in Brazil indicated a link between N nutrition and infection of citrus trees with the bacterial pathogen, *Xylella fastidiosa* causing variegated chlorosis disease (see Huber and Thompson, 2007). Nitrification increased disease severity but where a groundcover grass species was planted between rows of citrus trees, a decrease in the disease was demonstrated; this was explained by the inhibiting effect of the ground cover crop on nitrification. Nitrification decreases concentrations of ammonium ions in the soil and this, in turn, decreases Mn availability to the plant. Inhibition of nitrification caused an increase in Mn uptake by 50%. Thus in this case it appears that an increase in availability of Mn confers

resistance to the host rather than favouring the pathogen, as in some Fusarium wilts.

to 66% compared to the control, which received no Si.

for higher levels of disease.

*Anthracnose* 

Rust in banana is caused by *Uredo musae* and in coffee by *Hemileia vastatrix*. In some annual crop species, rusts may be encouraged by high levels of N fertilisation (see Section 2). However, coffee rust has been reported to be more severe on plantations grown under nutrient poor conditions (Waller et. al., 2007, p. 302). Applications of silicon (Si) have been shown to decrease the level of coffee rust on coffee seedlings. Martinati et al. (2008) found that the number of rust lesions on leaves of Si-treated coffee (*C. arabica*) seedlings was decreased in proportion to the dosage of Si (as potassium silicate) supplied to the soil by up

In an epidemiological study on coffee rust in Honduras, Avelino et al. (2006) reported that the intensity of coffee rust infection was dependent on the production situation, rather than regional differences in environmental parameters, such as rainfall levels. They showed that coffee rust was associated with acid soils, soils treated with mineral fertilisers and increased yields. Possibly, an acidifying effect of the mineral fertilisers was the main factor accounting

Anthracnose diseases affect leaves, shoots and fruit of a variety of tropical perennial crops on farms and can also create severe post-harvest problems. The Ascomycotina species, *Glomerella cingulata* (anamorph: *Colletotrichum gloeosporioides)*, an endophyte on plant species

for disease alleviation.

*Rusts* 

Conditions predisposing perennial crops to fruit rots include high rainfall, poor drainage and poor sanitation practices, whereby infected fruits (which provided sources of new inoculum) are not disposed of correctly. This is particularly relevant in the case of *Phytophthora palmivora*, the straminopile (formerly oomycete) pathogen, which is responsible for fruit rots in a wide range of host species in the tropics including breadfruit, cocoa, black pepper, papaya and vanilla. On coconut and betel nut trees it causes bud rot, inflicting serious losses to these crops. The pathogen is dispersed mainly by motile zoospores, which require the presence of external water and infection (Guest, 2007). Hence, incidence of fruit rots caused by *P. palmivora* increases dramatically under conditions of high moisture during heavy rainfall periods in the wet season, poor drainage or slightly cooler conditions at higher altitudes that reduce evapotranspiration rates. In many host species, it also infects other parts of the plant including leaves and stems. Inoculum from one infected part of the plant can initiate infections in other tissues. On cocoa farms, insects, including ants and beetles, also transmit disease, carrying spores from infected sites to initiate new infections (Konam and Guest, 2004). Therefore, sanitation is a crucial control measure for Phytophthora diseases. A link between low levels of zinc nutrition and pod rot incidence

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 255

Dieback diseases are caused by a range of pathogens. Some diseases may involve the interaction of more than one pathogen species and may be prevalent in ageing trees or trees weakened by environmental stress (Ploetz, 2006). This is particularly the case for infections by facultative parasites, such as some Botryosphaeriaceae species which can survive in a latent form in dead wood or are wound invaders. Similarly, the weakly aggressive pathogen, *Fusarium decemcellulare* has been linked to dieback and cushion gall of cocoa. Other tree crops are also infected with this pathogen (see Ploetz, 2006). Infection is possibly facilitated by wounds caused by insects (Holliday 1980). The pathogen can interact with other pathogens such as *Lasiodiplodia theobromae* (syn. *Botryodiplodia theobromae*) and

In coffee, overbearing dieback is linked to poor nutrition, nitrogen-deficiency in particular, which has the effect of lowering soluble sugar reserves in the stem. Exposure to the sun, resulting in plant stress and excessive cropping, poor root function (e.g. resulting from pathogen attack) and weed competition can all be factors predisposing trees to this condition. Infection by opportunistic fungi such as *Colletotrichum gloeosporioides* and *F. oxysporum* follows, causing dieback of the stem (Flood, 2010; Waller et al., 2007). The disease can be managed by supplying shade, applying N and decreasing crop density (Waller et al., 2007 p 284). Sunscorch damage is also associated with infection of berries by pathogens such

In the case of pink disease caused by the basidiomycete, *Corticium salmonicolor* (syn. *Erythricium salmonicolor)*, a lack of light caused by heavy shade and insufficient pruning, encourages infections in a number of species including citrus, cocoa, coffee, rubber, tea and black pepper. On cocoa, symptoms of the disease include a pink to creamy white crust on the bark and cracking of the bark with gum exudates. Sudden death of the whole branch may occur with the leaves remaining attached. A number of timber (forest) trees are also

The most severe dieback disease affecting cocoa in the Southeast Asian region is vascularstreak dieback (VSD) caused by another basidiomycete species, *Ceratobasidium theobromae* (syn. *Oncobasidium theobromae*). Infection is initiated in particularly wet conditions on young leaves by wind-borne spores, which are very short-lived (Keane et al., 1972; Keane, 1981; Prior, 1985; Guest and Keane, 2007). Following germination of the spores, the fungal hyphae penetrate the xylem in the leaf by a mechanism that remains unknown. The pathogen can grow via the xylem to lower parts of the branch causing leaf chlorosis and fall, and eventual dieback of the branch (Fig. 2). In susceptible varieties tree death may result, but most cocoa genotypes exhibit partial resistance to VSD. The identification of a second species, *C. ramicola*, in VSD-infected tissue (Samuels et al., 2011) raises the possibility that more than one pathogen is causing the disease. A significant and widespread change in VSD symptoms has occurred since 2004. After 2004, necrosis of infected leaves was observed (rather than only chlorosis as previously seen), with the infected leaves remaining attached to the branch longer; cracks in the midrib of the infected leaves were also observed, which allowed emergence and sporulation of the fungus from the leaves themselves, in addition to the petiolar scars on the stems as observed prior to 2004 (Purwantara et al., in process). Disease severity has also increased so that many farmers in Indonesia now identify VSD as their primary problem and the main reason given for changing their cocoa farms over to

**4.4 Dieback diseases** 

*Phytophthora palmivora* (Holliday, 1980).

as *F. stilboides* and *Cercospora coffeicola* (Waller et al., 2007 p. 285).

hosts to this pathogen. Wet conditions promote infections of tree crops.

was reported from Papua New Guinea (Nelson et al., 2011). Other *Phytophthora* species also cause fruit rots; particularly devastating losses in cocoa in West Africa are caused by *P. megakarya*, for example (Guest, 2007). Similarly devastating losses are caused in South America by the basidiomycete, *Moniliophthora roreri* that causes frosty pod. A fruit rot in cocoa is also caused by *Lasiodiplodia theobromae* in southern and Southeast Asia. On avocado, the same pathogen species causes browning of the fruit from the stem end (Kohler et al., 1997) and fruit rot in papaya (Kader, 2002). *L. theobromae* generally requires wounding to initiate infection (Kader, 2002). A possible relation to plant nutrition has been raised by studies of some Phytophthora fruit rots. For example, brown rot of citrus caused by *Phytophthora citrophthora*, is enhanced under when N fertiliser is provided as ammonium-N (Menge and Nemec, 1997).

*Verticillium theobromae* causes cigar end rot of banana. Sanitation (including removal of dead flowers), canopy aeration and exposure to light are recommended management methods for this disease (Nelson et al. 2006). A serious fruit rot of guava is caused by *Pestalotiopsis disseminate* that infects the fruit from the stalk end producing white fruiting bodies that later become brown. This disease is linked with poor nutrition; hence proper soil amendment to improve fertility is a recommended control measure. Similarly, *Pestalotiopsis* sp. infection of coconut can be reduced by improved growing conditions through the application of fertiliser (Kohler et al., 1997). *Pestaliotopsis* (Fig. 1) has been isolated from cocoa leaves that have symptoms of vascular-streak dieback – possibly it is a secondary pathogen.

Fig. 1. Conidia of *Pestalotiopsis* sp. isolated from cocoa leaves in Sulawesi, Indonesia; magnified x400 (*author's photo*).

#### **4.4 Dieback diseases**

254 Plant Pathology

was reported from Papua New Guinea (Nelson et al., 2011). Other *Phytophthora* species also cause fruit rots; particularly devastating losses in cocoa in West Africa are caused by *P. megakarya*, for example (Guest, 2007). Similarly devastating losses are caused in South America by the basidiomycete, *Moniliophthora roreri* that causes frosty pod. A fruit rot in cocoa is also caused by *Lasiodiplodia theobromae* in southern and Southeast Asia. On avocado, the same pathogen species causes browning of the fruit from the stem end (Kohler et al., 1997) and fruit rot in papaya (Kader, 2002). *L. theobromae* generally requires wounding to initiate infection (Kader, 2002). A possible relation to plant nutrition has been raised by studies of some Phytophthora fruit rots. For example, brown rot of citrus caused by *Phytophthora citrophthora*, is enhanced under when N fertiliser is provided as ammonium-N

*Verticillium theobromae* causes cigar end rot of banana. Sanitation (including removal of dead flowers), canopy aeration and exposure to light are recommended management methods for this disease (Nelson et al. 2006). A serious fruit rot of guava is caused by *Pestalotiopsis disseminate* that infects the fruit from the stalk end producing white fruiting bodies that later become brown. This disease is linked with poor nutrition; hence proper soil amendment to improve fertility is a recommended control measure. Similarly, *Pestalotiopsis* sp. infection of coconut can be reduced by improved growing conditions through the application of fertiliser (Kohler et al., 1997). *Pestaliotopsis* (Fig. 1) has been isolated from cocoa leaves that

have symptoms of vascular-streak dieback – possibly it is a secondary pathogen.

Fig. 1. Conidia of *Pestalotiopsis* sp. isolated from cocoa leaves in Sulawesi, Indonesia;

(Menge and Nemec, 1997).

magnified x400 (*author's photo*).

Dieback diseases are caused by a range of pathogens. Some diseases may involve the interaction of more than one pathogen species and may be prevalent in ageing trees or trees weakened by environmental stress (Ploetz, 2006). This is particularly the case for infections by facultative parasites, such as some Botryosphaeriaceae species which can survive in a latent form in dead wood or are wound invaders. Similarly, the weakly aggressive pathogen, *Fusarium decemcellulare* has been linked to dieback and cushion gall of cocoa. Other tree crops are also infected with this pathogen (see Ploetz, 2006). Infection is possibly facilitated by wounds caused by insects (Holliday 1980). The pathogen can interact with other pathogens such as *Lasiodiplodia theobromae* (syn. *Botryodiplodia theobromae*) and *Phytophthora palmivora* (Holliday, 1980).

In coffee, overbearing dieback is linked to poor nutrition, nitrogen-deficiency in particular, which has the effect of lowering soluble sugar reserves in the stem. Exposure to the sun, resulting in plant stress and excessive cropping, poor root function (e.g. resulting from pathogen attack) and weed competition can all be factors predisposing trees to this condition. Infection by opportunistic fungi such as *Colletotrichum gloeosporioides* and *F. oxysporum* follows, causing dieback of the stem (Flood, 2010; Waller et al., 2007). The disease can be managed by supplying shade, applying N and decreasing crop density (Waller et al., 2007 p 284). Sunscorch damage is also associated with infection of berries by pathogens such as *F. stilboides* and *Cercospora coffeicola* (Waller et al., 2007 p. 285).

In the case of pink disease caused by the basidiomycete, *Corticium salmonicolor* (syn. *Erythricium salmonicolor)*, a lack of light caused by heavy shade and insufficient pruning, encourages infections in a number of species including citrus, cocoa, coffee, rubber, tea and black pepper. On cocoa, symptoms of the disease include a pink to creamy white crust on the bark and cracking of the bark with gum exudates. Sudden death of the whole branch may occur with the leaves remaining attached. A number of timber (forest) trees are also hosts to this pathogen. Wet conditions promote infections of tree crops.

The most severe dieback disease affecting cocoa in the Southeast Asian region is vascularstreak dieback (VSD) caused by another basidiomycete species, *Ceratobasidium theobromae* (syn. *Oncobasidium theobromae*). Infection is initiated in particularly wet conditions on young leaves by wind-borne spores, which are very short-lived (Keane et al., 1972; Keane, 1981; Prior, 1985; Guest and Keane, 2007). Following germination of the spores, the fungal hyphae penetrate the xylem in the leaf by a mechanism that remains unknown. The pathogen can grow via the xylem to lower parts of the branch causing leaf chlorosis and fall, and eventual dieback of the branch (Fig. 2). In susceptible varieties tree death may result, but most cocoa genotypes exhibit partial resistance to VSD. The identification of a second species, *C. ramicola*, in VSD-infected tissue (Samuels et al., 2011) raises the possibility that more than one pathogen is causing the disease. A significant and widespread change in VSD symptoms has occurred since 2004. After 2004, necrosis of infected leaves was observed (rather than only chlorosis as previously seen), with the infected leaves remaining attached to the branch longer; cracks in the midrib of the infected leaves were also observed, which allowed emergence and sporulation of the fungus from the leaves themselves, in addition to the petiolar scars on the stems as observed prior to 2004 (Purwantara et al., in process). Disease severity has also increased so that many farmers in Indonesia now identify VSD as their primary problem and the main reason given for changing their cocoa farms over to

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 257

Various causes of mango decline have been reported. Mango decline is associated with opportunistic fungi such as *Botryosphaeria ribis*, *Physalospora* sp. and others (see Zheng et al., 2002). *L. theobromae* has also been linked to mango decline (Shahbaz et al., 2009), while a form of mango decline in Brazil is caused by *Ceratocystis fimbriata* (Ploetz, 2007). Mango decline symptoms are reported to include interveinal chlorosis in leaves, stunting and terminal and marginal necrosis with dieback of young stems, internal softening of the fruit and even tree death; these symptoms may be linked to Mn and Fe deficiency (Crane and Campbell, 1994). Both Mn and Fe may be deficient in plants growing in high pH soil. Another mango disease that has recently become a serious problems in some regions (e.g northern Australia) is mango malformation caused by *Fusarium* spp., which cause distortion

Many dieback diseases are caused by a combination of factors, including nutrient deficiency, drought and wounding or transmission of inoculum by insects. A fungus surviving in a host plant as an endophyte or as a saprophyte on dead tissue may switch to being a pathogen in stressed plants (Shulz and Boyle, 2005). Nutrient deficiency could be a key predisposing factor for this switch to occur. Generally, adequate host nutrition, as well as shade management and sanitation (such as adequate pruning and the removal of dead twigs), are

Root and collar rots are particularly severe in soils with low organic matter content, poor soil structure with a high level of compaction and poor drainage (Desaeger et al., 2004). If roots are restricted to the upper soil layers due to claypans or hardpans, this results in a greater exposure of roots to soil-borne pathogens. Similarly, old roots may leave pathogen inocula in cracks and fissures in the soil, increasing contact with new roots; an example of this is found in cases of *Eucalyptus marginate* and other native Australian species infected by

The basidiomycete, *Marasmiellus inoderma* infects all parts of the banana plant causing stem rot in particular. The pathogen has a number of hosts and is encouraged in marginal, nutrient-poor, soils with a high clay content and by poor drainage; soil improvement by organic amendments and better plant nutrition are recommended for managing the disease (Nelson et al., 2006). In breadfruit, *Lasiodiplodia theobromae* (syn. *Botryodiplodia theobromae*) causes a collar rot characterised by external white strands. As mentioned above, this is a wound invading fungus and drought-stressed trees may be particularly predisposed to infection. *Phellinus noxius* (Basidiomycota) also infects bread fruit trees at the base of the trunk creating a brown encrustation (that includes soil particles) sometimes with a white margin and gum exudates. Other hosts of this pathogen include cocoa, coffee, *Leucaena* sp., mango, oil palm and forest trees, such as *Tectonia*, *Swietenia* and laurel (Kohler et al., 1997). Diseases caused by the fungus include brown root and collar rot in cocoa: the fungus may encircle the whole trunk, causing sudden death of the tree with leaves remaining attached. As the pathogen is dispersed between roots in the soil, adjacent trees may be infected and killed. Therefore, removal of tree stumps and all large roots before planting is a necessary preventative control measure. Rigidoporus root rot (or white root rot) caused by the basidiomycete, *Rigidoporus microporus* causes serious crop losses to rubber, mango, durian

in the growth of shoots and buds.

*Root and collar rots* 

crucial preventative measure for some forms of dieback.

**4.5 Root and collar rots, cankers and nematode infections** 

the soil-borne pathogen *Phytophthora cinnamomi* (see Desaeger et al., 2004).

other crops, including maize, neelam and oil palm (pers. comm. Ade Rosmana, Hasanuddin University). Possibly the change in symptoms and increased severity of the disease is the result of an environmental factor interacting with *C. theobromae* infections or VSD-infected trees have become susceptible to infection by a secondary pathogen(s); trees weakened by stress, such as poor nutrition, might be predisposed to such a secondary infection (Mossu, 1990 cited by Schroth et al., 2000). An alternative explanation for the change in symptoms and severity of the disease is that a new strain of the pathogen, *C. theobromae*, has emerged. Further work is underway to elucidate the pathogen-environmental relationship that might lie behind the changed VSD symptoms.

A devastating disease of cocoa is witches' broom, caused by a co-evolved pathogen of cocoa, a basidiomycete species, *Moniliophthora perniciosa*. The disease causes distortion of growth in the shoots, creating a broom-like appearance. Although the pathogen co-evolved with cocoa, it has caused most damage in plantations in Bahia, Brazil away from its centre of origin in the Amazon rainforest. Improved management, including the appropriate use of fertilisers, combined with the introduction of resistant cocoa genotypes has been effective in mitigating the impact of this disease (Keane and Putter, 1992). Dieback in cocoa caused by *L. theobromae* has been observed in the Cameroons (Mbenoun et al., 2008) and in India (Kannan et al. 2010). Vascular streaking has been observed in both cases. Kannan et al. (2010) isolated the pathogen and reinoculated seedlings, which showed disease symptoms after 20 days. Infection by *L. theobromae* is facilitated by

Fig. 2. Left: Cocoa tree in Sulawesi, Indonesia infected with VSD. Right: LS of a stem of infected cocoa showing hyphae of the causal organism, *C. theobromae*, in a xylem vessel, magnified x400 (*author's photos*).

wounding or insect damage. In passion fruit, for example, infection is facilitated by tunnelling by a species of beetle. Generally, preventing insect damage or other forms of wounding may reduce infection. Removal of dead twigs or branches is also a recommended control measure since *L. theobromae* is a facultative saprophyte (Kohler et al., 1997). Thread blight or black rot in coffee is caused by a basidiomycete, *Ceratobasidium noxium* (formerly *Pellicularia koleroga* syn. *Corticium koleroga*), which infects branches of coffee trees causing blackening and drying of leaves, leading to branch dieback. The pathogen also infects cocoa, citrus and woody species. *Marasmiellus scandens* causes white thread blight of cocoa. Infection by *M. scandens* is may be associated with stem borers, especially Scolytidae beetles (observation by Asman, Hasanuddin University).

Various causes of mango decline have been reported. Mango decline is associated with opportunistic fungi such as *Botryosphaeria ribis*, *Physalospora* sp. and others (see Zheng et al., 2002). *L. theobromae* has also been linked to mango decline (Shahbaz et al., 2009), while a form of mango decline in Brazil is caused by *Ceratocystis fimbriata* (Ploetz, 2007). Mango decline symptoms are reported to include interveinal chlorosis in leaves, stunting and terminal and marginal necrosis with dieback of young stems, internal softening of the fruit and even tree death; these symptoms may be linked to Mn and Fe deficiency (Crane and Campbell, 1994). Both Mn and Fe may be deficient in plants growing in high pH soil. Another mango disease that has recently become a serious problems in some regions (e.g northern Australia) is mango malformation caused by *Fusarium* spp., which cause distortion in the growth of shoots and buds.

Many dieback diseases are caused by a combination of factors, including nutrient deficiency, drought and wounding or transmission of inoculum by insects. A fungus surviving in a host plant as an endophyte or as a saprophyte on dead tissue may switch to being a pathogen in stressed plants (Shulz and Boyle, 2005). Nutrient deficiency could be a key predisposing factor for this switch to occur. Generally, adequate host nutrition, as well as shade management and sanitation (such as adequate pruning and the removal of dead twigs), are crucial preventative measure for some forms of dieback.

#### **4.5 Root and collar rots, cankers and nematode infections**

#### *Root and collar rots*

256 Plant Pathology

other crops, including maize, neelam and oil palm (pers. comm. Ade Rosmana, Hasanuddin University). Possibly the change in symptoms and increased severity of the disease is the result of an environmental factor interacting with *C. theobromae* infections or VSD-infected trees have become susceptible to infection by a secondary pathogen(s); trees weakened by stress, such as poor nutrition, might be predisposed to such a secondary infection (Mossu, 1990 cited by Schroth et al., 2000). An alternative explanation for the change in symptoms and severity of the disease is that a new strain of the pathogen, *C. theobromae*, has emerged. Further work is underway to elucidate the pathogen-environmental relationship that might

A devastating disease of cocoa is witches' broom, caused by a co-evolved pathogen of cocoa, a basidiomycete species, *Moniliophthora perniciosa*. The disease causes distortion of growth in the shoots, creating a broom-like appearance. Although the pathogen co-evolved with cocoa, it has caused most damage in plantations in Bahia, Brazil away from its centre of origin in the Amazon rainforest. Improved management, including the appropriate use of fertilisers, combined with the introduction of resistant cocoa genotypes has been effective in mitigating the impact of this disease (Keane and Putter, 1992). Dieback in cocoa caused by *L. theobromae* has been observed in the Cameroons (Mbenoun et al., 2008) and in India (Kannan et al. 2010). Vascular streaking has been observed in both cases. Kannan et al. (2010) isolated the pathogen and reinoculated seedlings, which showed disease symptoms after 20 days.

Fig. 2. Left: Cocoa tree in Sulawesi, Indonesia infected with VSD. Right: LS of a stem of infected cocoa showing hyphae of the causal organism, *C. theobromae*, in a xylem vessel,

wounding or insect damage. In passion fruit, for example, infection is facilitated by tunnelling by a species of beetle. Generally, preventing insect damage or other forms of wounding may reduce infection. Removal of dead twigs or branches is also a recommended control measure since *L. theobromae* is a facultative saprophyte (Kohler et al., 1997). Thread blight or black rot in coffee is caused by a basidiomycete, *Ceratobasidium noxium* (formerly *Pellicularia koleroga* syn. *Corticium koleroga*), which infects branches of coffee trees causing blackening and drying of leaves, leading to branch dieback. The pathogen also infects cocoa, citrus and woody species. *Marasmiellus scandens* causes white thread blight of cocoa. Infection by *M. scandens* is may be associated with stem borers, especially Scolytidae beetles

lie behind the changed VSD symptoms.

Infection by *L. theobromae* is facilitated by

magnified x400 (*author's photos*).

(observation by Asman, Hasanuddin University).

Root and collar rots are particularly severe in soils with low organic matter content, poor soil structure with a high level of compaction and poor drainage (Desaeger et al., 2004). If roots are restricted to the upper soil layers due to claypans or hardpans, this results in a greater exposure of roots to soil-borne pathogens. Similarly, old roots may leave pathogen inocula in cracks and fissures in the soil, increasing contact with new roots; an example of this is found in cases of *Eucalyptus marginate* and other native Australian species infected by the soil-borne pathogen *Phytophthora cinnamomi* (see Desaeger et al., 2004).

The basidiomycete, *Marasmiellus inoderma* infects all parts of the banana plant causing stem rot in particular. The pathogen has a number of hosts and is encouraged in marginal, nutrient-poor, soils with a high clay content and by poor drainage; soil improvement by organic amendments and better plant nutrition are recommended for managing the disease (Nelson et al., 2006). In breadfruit, *Lasiodiplodia theobromae* (syn. *Botryodiplodia theobromae*) causes a collar rot characterised by external white strands. As mentioned above, this is a wound invading fungus and drought-stressed trees may be particularly predisposed to infection. *Phellinus noxius* (Basidiomycota) also infects bread fruit trees at the base of the trunk creating a brown encrustation (that includes soil particles) sometimes with a white margin and gum exudates. Other hosts of this pathogen include cocoa, coffee, *Leucaena* sp., mango, oil palm and forest trees, such as *Tectonia*, *Swietenia* and laurel (Kohler et al., 1997). Diseases caused by the fungus include brown root and collar rot in cocoa: the fungus may encircle the whole trunk, causing sudden death of the tree with leaves remaining attached. As the pathogen is dispersed between roots in the soil, adjacent trees may be infected and killed. Therefore, removal of tree stumps and all large roots before planting is a necessary preventative control measure. Rigidoporus root rot (or white root rot) caused by the basidiomycete, *Rigidoporus microporus* causes serious crop losses to rubber, mango, durian

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 259

nematode of guava, *M. enterolobii* (syn. *M. mayaguensis*) in naturally infested areas, were decreased by manure application to the soil (Souza et al., 2006). As mentioned above (Section 4.2), silicon (Si) supplied to coffee plants was shown to decrease the number of rust lesions on leaves; Si also has an effect on resistance to nematodes. Silva et al. (2010) found that a lower number of *M. exigua* galls and eggs occurred in the roots of coffee (*C. arabica*) plants that had been inoculated with the nematode and provided with Si, compared to

Nematode infections may predispose tree crops to infections by other pathogens creating disease complexes (Desaeger et al., 2004). Generally, nematodes may have a number of roles in facilitating disease development acting as vectors and wounding agents. They may affect the susceptibility of the host to other pathogens, or influence rhizosphere ecology (Desaeger et al., 2004). Melendez and Powell (1969) reported that nematode infection of roots of tobacco plants caused a soil-inhabiting *Trichoderma* sp. to become pathogenic. Vascular wilt pathogens are particularly encouraged by endoparasitic nematodes, while cortical root pathogens are encouraged by ectoparasitic nematodes (Hillocks and Waller, 1997). The nematode species, *Radopholus similis* causes root rot in, among other crops, banana, avocado, coconut, coffee and sugar cane. In banana, *Fusarium oxysporum* infections of the root were associated with roots infested with *R. similis* (Blake, 1966). Gomes et al. (2011) reported that the causes of root rot in guava decline in Brazil included colonisation by nematodes (*M. enterolobii* syn. *M. mayaguensis*) and a *Fusarium* sp., identified upon isolation as *F. solani*. Infestation of roots by the nematode appeared to predispose roots to infection by *F. solani*, as the latter species was only isolated from nematode-infected roots. The authors found that *F. solani* isolates inoculated into the roots of guava plants initiated infections in trees that had been pre-inoculated with the nematode, but not those that had been physically damaged using a knife. Therefore, it appears that physical damage alone by the *M. enterolobii* did not account for the predisposition of guava colonised by the nematodes to *F. solani* infection. Khan et al. (1995) had earlier shown that inoculation of papaya with both the nematode *M. incognita* and *F. solani* caused a greater decrease in plant growth than inoculation with either species alone. They also showed that the level of root rot (caused by *F. solani*) could be

Canker infections are initiated on species such as cocoa and durian by the same causal pathogen of fruit rots and leaf blights, *Phytophthora palmivora*. Other *Phytophthora* species may be associated with cankers, but *P. palmivora* is the most prevalent species of the genus on tropical tree crops. On cocoa, cankers are moist, wine-red lesions under the bark that expand in diameter during the wet season (Guest, 2007; McMahon et al., 2010). If cankers girdle the whole stem, sudden death results with the leaves still attached to the tree. Particularly wet conditions encourage Phytophthora stem canker, which can be a serious problem in areas prone to water logging or flooding. The presence of susceptible hosts, lack of pruning or management to enhance air circulation and lack of sanitation of sources of inoculum, such as infected pods can all lead to increased incidence of the disease. Since old and apparently weaker trees are more susceptible (author's observation) it is possible that poor nutrition might encourage cankers to develop. Low zinc (Zn) nutrition has been

suggested to predispose plants to Phytophthora infection (Nelson et al., 2011).

control plants which were not supplied with Si.

decreased by the application of NPK fertiliser.

Phytophthora *stem canker*

and other tree crops. It is a serious pathogen of rubber trees in Malaysia. Red root disease caused by *Ganoderma philippii* (syn. *G. pseudoferreum*) another basidiomycete, is also an important rubber pathogen in Malaysia and India. The use of arsenic-containing sprays as a control method for root rots creates environmental concerns. As for other root pathogens, sanitation of areas prior to planting is a necessary control measure.

*Ganoderma orbiforme* (syn. *G. boninense*) is the most severe pathogen on oil palm in southeast Asia (Susanto et al., 2005; Flood et al., 2005; www.dfid.gov.uk). Sanitation is particularly important and diseased trees, including the roots, are dug out mechanically, and shredded for composting. In a recently established trial in Sumatra, Indonesia, shredded plant material and empty fruit bunches are being used to prepare compost using microbial promoters, particularly *Trichoderma* spp., which the trial aims to test by soil application in order to assess their effect on root rot disease (Agus Purwantara, pers. comm.). Srinivasulu (2003) reported a higher incidence of *Ganoderma* spp. infection of coconut growing on sandy and red soils (which had a low organic matter content) than on black soils (with a higher organic matter content). Amendment of soil with calcium nitrate has been used to reduce Ganoderma basal stem rot in coconut palms (Kandan et al., 2010). In a plot of coconut trees affected by this disease, Kharthikaya et al. (2006) demonstrated that a combined treatment of frequent irrigation, soil applications of neem cake, *Trichoderma viride*, *Pseudomonas fluorescens* and a fungicide prevented the spread of the pathogen, *G. lucidum*, and led to the recovery of 42% of diseased palm trees.

*Phytophthora* spp. cause root rots in crops such as avocado and citrus. Root rot in avocado caused by *P. cinnamomi* becomes particularly severe under conditions of flooding (Ploetz, 2007). Phytophthora root rot in citrus is associated with citrus leaf miner damage and Diaprepes root weevil (Ploetz, 2007). The form of nitrogen available to citrus trees appears to affect the severity of this disease. Root rot of citrus was shown to increase in the presence of ammonium-N but decreased by supplying nitrate-N (Menge and Nemec, 1997). Root rots are also caused by *Rosellinia* spp. on crops such as avocado, citrus and banana – they are favoured by acidic soil conditions (Ploetz, 2007).

#### *Nematodes*

Nematodes are generally favoured by coarse-textured soils that are low in organic matter and biological activity (Desaeger et al., 2004). For example, bananas became more susceptible to nematodes when grown in degraded soil that had lost much of its original organic matter (Page and Bridges, 1993) and nematode attack on maize was more damaging in unfertilised, than in fertilised plots (Desaeger et al., 2004). The intensity of crop production can also influence nematode populations. In Costa Rica, Avelino et al. (2009) examined the conditions that influence populations of two nematode species, *Meloidogyne exigua* (root-knot nematode) and *Pratylenchus coffeae* colonising roots of coffee. The two species had specific preferences of altitude and soils, with low *M. exigua* populations being associated with non-sandy soils with a high K and Zn content, but high populations of both species occurred on farms which had inter-row planting distances of less than 0.9 m, irrespective of environmental conditions. This, the authors suggest, indicates that intensification of coffee production provides conditions favourable for nematode reproduction and transmission.

Nutrient supply and organic amendments can have direct impacts on nematode populations and infection. In a guava growing area of Brazil, the numbers of juveniles of the root-knot nematode of guava, *M. enterolobii* (syn. *M. mayaguensis*) in naturally infested areas, were decreased by manure application to the soil (Souza et al., 2006). As mentioned above (Section 4.2), silicon (Si) supplied to coffee plants was shown to decrease the number of rust lesions on leaves; Si also has an effect on resistance to nematodes. Silva et al. (2010) found that a lower number of *M. exigua* galls and eggs occurred in the roots of coffee (*C. arabica*) plants that had been inoculated with the nematode and provided with Si, compared to control plants which were not supplied with Si.

Nematode infections may predispose tree crops to infections by other pathogens creating disease complexes (Desaeger et al., 2004). Generally, nematodes may have a number of roles in facilitating disease development acting as vectors and wounding agents. They may affect the susceptibility of the host to other pathogens, or influence rhizosphere ecology (Desaeger et al., 2004). Melendez and Powell (1969) reported that nematode infection of roots of tobacco plants caused a soil-inhabiting *Trichoderma* sp. to become pathogenic. Vascular wilt pathogens are particularly encouraged by endoparasitic nematodes, while cortical root pathogens are encouraged by ectoparasitic nematodes (Hillocks and Waller, 1997). The nematode species, *Radopholus similis* causes root rot in, among other crops, banana, avocado, coconut, coffee and sugar cane. In banana, *Fusarium oxysporum* infections of the root were associated with roots infested with *R. similis* (Blake, 1966). Gomes et al. (2011) reported that the causes of root rot in guava decline in Brazil included colonisation by nematodes (*M. enterolobii* syn. *M. mayaguensis*) and a *Fusarium* sp., identified upon isolation as *F. solani*. Infestation of roots by the nematode appeared to predispose roots to infection by *F. solani*, as the latter species was only isolated from nematode-infected roots. The authors found that *F. solani* isolates inoculated into the roots of guava plants initiated infections in trees that had been pre-inoculated with the nematode, but not those that had been physically damaged using a knife. Therefore, it appears that physical damage alone by the *M. enterolobii* did not account for the predisposition of guava colonised by the nematodes to *F. solani* infection. Khan et al. (1995) had earlier shown that inoculation of papaya with both the nematode *M. incognita* and *F. solani* caused a greater decrease in plant growth than inoculation with either species alone. They also showed that the level of root rot (caused by *F. solani*) could be decreased by the application of NPK fertiliser.

#### Phytophthora *stem canker*

258 Plant Pathology

and other tree crops. It is a serious pathogen of rubber trees in Malaysia. Red root disease caused by *Ganoderma philippii* (syn. *G. pseudoferreum*) another basidiomycete, is also an important rubber pathogen in Malaysia and India. The use of arsenic-containing sprays as a control method for root rots creates environmental concerns. As for other root pathogens,

*Ganoderma orbiforme* (syn. *G. boninense*) is the most severe pathogen on oil palm in southeast Asia (Susanto et al., 2005; Flood et al., 2005; www.dfid.gov.uk). Sanitation is particularly important and diseased trees, including the roots, are dug out mechanically, and shredded for composting. In a recently established trial in Sumatra, Indonesia, shredded plant material and empty fruit bunches are being used to prepare compost using microbial promoters, particularly *Trichoderma* spp., which the trial aims to test by soil application in order to assess their effect on root rot disease (Agus Purwantara, pers. comm.). Srinivasulu (2003) reported a higher incidence of *Ganoderma* spp. infection of coconut growing on sandy and red soils (which had a low organic matter content) than on black soils (with a higher organic matter content). Amendment of soil with calcium nitrate has been used to reduce Ganoderma basal stem rot in coconut palms (Kandan et al., 2010). In a plot of coconut trees affected by this disease, Kharthikaya et al. (2006) demonstrated that a combined treatment of frequent irrigation, soil applications of neem cake, *Trichoderma viride*, *Pseudomonas fluorescens* and a fungicide prevented the spread of the pathogen, *G. lucidum*, and led to the recovery of

*Phytophthora* spp. cause root rots in crops such as avocado and citrus. Root rot in avocado caused by *P. cinnamomi* becomes particularly severe under conditions of flooding (Ploetz, 2007). Phytophthora root rot in citrus is associated with citrus leaf miner damage and Diaprepes root weevil (Ploetz, 2007). The form of nitrogen available to citrus trees appears to affect the severity of this disease. Root rot of citrus was shown to increase in the presence of ammonium-N but decreased by supplying nitrate-N (Menge and Nemec, 1997). Root rots are also caused by *Rosellinia* spp. on crops such as avocado, citrus and banana – they are

Nematodes are generally favoured by coarse-textured soils that are low in organic matter and biological activity (Desaeger et al., 2004). For example, bananas became more susceptible to nematodes when grown in degraded soil that had lost much of its original organic matter (Page and Bridges, 1993) and nematode attack on maize was more damaging in unfertilised, than in fertilised plots (Desaeger et al., 2004). The intensity of crop production can also influence nematode populations. In Costa Rica, Avelino et al. (2009) examined the conditions that influence populations of two nematode species, *Meloidogyne exigua* (root-knot nematode) and *Pratylenchus coffeae* colonising roots of coffee. The two species had specific preferences of altitude and soils, with low *M. exigua* populations being associated with non-sandy soils with a high K and Zn content, but high populations of both species occurred on farms which had inter-row planting distances of less than 0.9 m, irrespective of environmental conditions. This, the authors suggest, indicates that intensification of coffee production provides conditions favourable for nematode

Nutrient supply and organic amendments can have direct impacts on nematode populations and infection. In a guava growing area of Brazil, the numbers of juveniles of the root-knot

sanitation of areas prior to planting is a necessary control measure.

42% of diseased palm trees.

reproduction and transmission.

*Nematodes* 

favoured by acidic soil conditions (Ploetz, 2007).

Canker infections are initiated on species such as cocoa and durian by the same causal pathogen of fruit rots and leaf blights, *Phytophthora palmivora*. Other *Phytophthora* species may be associated with cankers, but *P. palmivora* is the most prevalent species of the genus on tropical tree crops. On cocoa, cankers are moist, wine-red lesions under the bark that expand in diameter during the wet season (Guest, 2007; McMahon et al., 2010). If cankers girdle the whole stem, sudden death results with the leaves still attached to the tree. Particularly wet conditions encourage Phytophthora stem canker, which can be a serious problem in areas prone to water logging or flooding. The presence of susceptible hosts, lack of pruning or management to enhance air circulation and lack of sanitation of sources of inoculum, such as infected pods can all lead to increased incidence of the disease. Since old and apparently weaker trees are more susceptible (author's observation) it is possible that poor nutrition might encourage cankers to develop. Low zinc (Zn) nutrition has been suggested to predispose plants to Phytophthora infection (Nelson et al., 2011).

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 261

A number of studies have been conducted reporting the isolation of potential microbial biocontrol agents from the soils in which tropical perennials are grown. Examples are the identification of five *Trichoderma* species, selected from 25 isolates from mango orchards in Mexico, which had an inhibitory effect on the growth of *Fusarium* spp. *in vitro* (Michel-Acev et al., 2001), the isolation of *Trichoderma* spp. demonstrated to have *in vitro* inhibitory effects on the cocoa pathogen, *Moniliophthora perniciosa* (Rivas-Cordero, 2010), on *Ganoderma orbiforme* (syn. *G. boninense*) (Siddiquee et al., 2009) and on *Lasiodiplodia theobromae* and *Colletotrichum musae,* isolated from infected banana (Samuels, 2006; Sangeetha 2009 ). Lower

levels of disease caused by *G. orbiforme* in infected oil palm seedlings were recorded

**Impact on disease**

No. of root lesions reduced

Reduce number of lesions

Reduce incidence by 52% up to 100%

Root rot severity index 1.2 compared to 5 (control)

Reduced infection

Reduce incidence from 95% to

Reduce leaf spot

diameter by over 70%

5%

Disease severity index 5% cf. 95% (control)

**Notes Source** 

Izzati et al. , 2008

Paparu, 2008

Vaast and Craswell, 1997

Otieno et al., 2003

Vawdrey et al., 2002

Bhai and Sarma, 2009

Widmer et al., 1998

Okigbo and Osuinde, 2003

Oil palm seedlings treated

Defencerelated genes upregulated

Four *T. harzanium* isolates effective

Treatment also lowered soil moisture content

*T. harzianum* applied with manure also effective

But some wastes toxic to

plants

*In vitro* inhibition by *B. subtilis* also verified

High P tissue level in mycorrhizal plants

**Type of treatment**

*T. harzianum* conidia spray

Non-pathogenic *F. oxysporum* isolate

Pre-inoculation of coffee with AM fungi

*T. harzianum* applied to detached tea stems

Sawdust/urea

*T. harzianum*

Composted municipal waste

Apply *B. subtilis* isolate *in vivo*

Table 2. Examples of impacts on diseases by soil amendments and other treatments applied

mix

**Crop Disease (causal pathogen)**

**Oil palm** Basal stem rot

(*G. orbiforme*)

**Banana** Nematode infection (*R. similis*)

**Coffee** Nematode infection (*P. coffea*)

(*Armillaria* sp.)

(*P. palmivora*)

(*P. nicotianae*)

 (*P. mangiferae, L. theobromae, M. mangiferae*)

**Cardamon** Root rot (*P. meadii*) Neem cake plus

**Tea** Rot

**Papaya** Root rot

**Citrus** Root rot

**Mango** Leaf spot

to tropical trees crops.

#### **5. Soil amendments with microbial species to control disease**

Application of composts to soils has been shown to have a suppressive effect on soil-borne diseases such as damping off, root rots and wilts, both in controlled glasshouse experiments and in the field (Noble and Coventry, 2005). Loss of disease suppression occurs if the composted materials are sterilised indicating that their suppressive effect is mainly biological (Bonanomi et al., 2010; Noble and Coventry, 2005). In Papua New Guinea, the time in which *P. palmivora* inoculum remained viable was found to be shorter in soils under cocoa leaf litter mulch than under grass litter: possibly conditions in the soils under leaf litter were more favourable to microorganisms antagonistic to the pathogen (Konam and Guest, 2002). McDonald et al. (2007) showed that the factor(s) conferring disease suppression of *P. cinnamomi* in avocado orchards can be transferred from suppressive to conducive soils. Suppressive soils may be effective in reducing disease due to either a high total microbial activity or to the presence of particular antagonistic species (Weller et al., 2002). Reeleder et al. (2003) identified biological parameters as being the best predictors of the capacity of soils for disease suppression. These include microbial biomass, substrate respiration, fluorescein diacetate (FDA) activity and populations of bacteria, including fluorescent pseudomonads, and of antagonist fungi, particularly *Trichoderma* spp. But the mechanisms leading to pathogen suppression and the antagonistic organisms involved remain little understood. The employment of molecular techniques to track changes in microbial communities in amended soils and to select potential biocontrol agents has been proposed by some researchers (Noble and Coventry, 2005; Reeleder et al, 2003).

Various studies have demonstrated disease suppression following soil amendments with organic materials or by treatments with antagonist microorganisms isolated from soils (Table 2). Root rot caused by *P. nicotianae* in Florida citrus orchards was reduced by treatments with composted municipal waste (Widmer et al., 1998). However, the suppressive activity of the compost was lost after storage for three months or more. Also, some sources of waste contained toxins that impaired plant growth. Vawdrey et al (2002) tested different soil amendments for their effect on root rot of papaya, caused by *Phytophthora palmivora*. They found in both pot and field experiments that a sawdust/urea preparation was more effective in reducing disease and *P. palmivora* populations, than the other organic materials tested, such as molasses. A lower soil moisture level in sawdust/urea treated soils might partly explain the suppressive effect of this treatment (Vawdrey et al., 2002). Organic amendments based on preparations from the neem tree (*Azadirachta indica*) have been shown to be effective in disease suppression, and to have nemiticidal properties (Agbenin, 2004). Applications of neem cake and other organic materials in combination with *Trichoderma harzianum*, reduced both the populations of *P. meadii* and their infection of cardamon plants (Bhai and Sarma, 2009). Peng et al. (1999) compared the effect of conducive and suppressive soils on disease severity of Fusarium wilt in Cavendish banana plantlets. The conducive soil had higher populations of filamentous fungi than the suppressive soil, which had greater numbers of bacteria and actinomyctes. They reported that, compared to the conducive soil, the suppressive soil reduced *Fusarium oxysporum* chlamydospore germination by 41% and decreased disease severity by over 50%. However, supplying Ca compounds or Fe in a chelated form to both suppressive and conducive soils decreased Fusarium wilt in the plantlets by 33-50%.

Application of composts to soils has been shown to have a suppressive effect on soil-borne diseases such as damping off, root rots and wilts, both in controlled glasshouse experiments and in the field (Noble and Coventry, 2005). Loss of disease suppression occurs if the composted materials are sterilised indicating that their suppressive effect is mainly biological (Bonanomi et al., 2010; Noble and Coventry, 2005). In Papua New Guinea, the time in which *P. palmivora* inoculum remained viable was found to be shorter in soils under cocoa leaf litter mulch than under grass litter: possibly conditions in the soils under leaf litter were more favourable to microorganisms antagonistic to the pathogen (Konam and Guest, 2002). McDonald et al. (2007) showed that the factor(s) conferring disease suppression of *P. cinnamomi* in avocado orchards can be transferred from suppressive to conducive soils. Suppressive soils may be effective in reducing disease due to either a high total microbial activity or to the presence of particular antagonistic species (Weller et al., 2002). Reeleder et al. (2003) identified biological parameters as being the best predictors of the capacity of soils for disease suppression. These include microbial biomass, substrate respiration, fluorescein diacetate (FDA) activity and populations of bacteria, including fluorescent pseudomonads, and of antagonist fungi, particularly *Trichoderma* spp. But the mechanisms leading to pathogen suppression and the antagonistic organisms involved remain little understood. The employment of molecular techniques to track changes in microbial communities in amended soils and to select potential biocontrol agents has been

**5. Soil amendments with microbial species to control disease** 

proposed by some researchers (Noble and Coventry, 2005; Reeleder et al, 2003).

conducive soils decreased Fusarium wilt in the plantlets by 33-50%.

Various studies have demonstrated disease suppression following soil amendments with organic materials or by treatments with antagonist microorganisms isolated from soils (Table 2). Root rot caused by *P. nicotianae* in Florida citrus orchards was reduced by treatments with composted municipal waste (Widmer et al., 1998). However, the suppressive activity of the compost was lost after storage for three months or more. Also, some sources of waste contained toxins that impaired plant growth. Vawdrey et al (2002) tested different soil amendments for their effect on root rot of papaya, caused by *Phytophthora palmivora*. They found in both pot and field experiments that a sawdust/urea preparation was more effective in reducing disease and *P. palmivora* populations, than the other organic materials tested, such as molasses. A lower soil moisture level in sawdust/urea treated soils might partly explain the suppressive effect of this treatment (Vawdrey et al., 2002). Organic amendments based on preparations from the neem tree (*Azadirachta indica*) have been shown to be effective in disease suppression, and to have nemiticidal properties (Agbenin, 2004). Applications of neem cake and other organic materials in combination with *Trichoderma harzianum*, reduced both the populations of *P. meadii* and their infection of cardamon plants (Bhai and Sarma, 2009). Peng et al. (1999) compared the effect of conducive and suppressive soils on disease severity of Fusarium wilt in Cavendish banana plantlets. The conducive soil had higher populations of filamentous fungi than the suppressive soil, which had greater numbers of bacteria and actinomyctes. They reported that, compared to the conducive soil, the suppressive soil reduced *Fusarium oxysporum* chlamydospore germination by 41% and decreased disease severity by over 50%. However, supplying Ca compounds or Fe in a chelated form to both suppressive and A number of studies have been conducted reporting the isolation of potential microbial biocontrol agents from the soils in which tropical perennials are grown. Examples are the identification of five *Trichoderma* species, selected from 25 isolates from mango orchards in Mexico, which had an inhibitory effect on the growth of *Fusarium* spp. *in vitro* (Michel-Acev et al., 2001), the isolation of *Trichoderma* spp. demonstrated to have *in vitro* inhibitory effects on the cocoa pathogen, *Moniliophthora perniciosa* (Rivas-Cordero, 2010), on *Ganoderma orbiforme* (syn. *G. boninense*) (Siddiquee et al., 2009) and on *Lasiodiplodia theobromae* and *Colletotrichum musae,* isolated from infected banana (Samuels, 2006; Sangeetha 2009 ). Lower levels of disease caused by *G. orbiforme* in infected oil palm seedlings were recorded


Table 2. Examples of impacts on diseases by soil amendments and other treatments applied to tropical trees crops.

Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 263

had been inoculated with mycorrhizal fungi (Elsen et al., 2003). In a study in citrus orchards in Thailand, increased growth, P uptake and resistance to root rot caused by *Phytophthora nicotianae* was shown to result from the inoculation of citrus trees with a species of AM fungus, *Glomus etunicatum*, isolated from local citrus orchard (Watanarojanaporn et al., 2011). A second AM fungus isolate, identified as *Acaulospora tuberculata,* also conferred resistance to *P. nicotianae* disease. Mycorrhizal fungi may compete for infection sites with the pathogen and/or they may impede access to nutrients by the pathogen (Azco'n-Aquila and Barea, 1996).

Soil function, plant nutritional status and cultural management practices have a strong influence on the incidence and severity of many diseases of tropical perennials. Diseases are influenced, not only by the general nutritional status of the plant (i.e. by an adequate supply of macronutrients), but also by individual nutrients. Soil pH is particularly important for a number of reasons, including its effect on availability of cations to the plant, pathogen or antagonists. It also influences microbial ecology, with a number of potential antagonists among bacterial and actinomycete species being favoured by a higher soil pH. Since tropical perennial crops and the use of inorganic fertiliser can lower soil pH, liming and/or compost treatments are strategies that can be adopted for disease mitigation in these crops. Composting farm waste, not only returns nutrients to the farm, but also improves farm sanitation as it kills larvae of pests and pathogen spores and other forms of inoculum. Nutrient elements supplied in mineral fertilisers can interact and care needs to be taken in their application. As mentioned earlier, some fertilisers can increase concentrations of Cl in plant tissues and decrease the K/Cl ratio. Also mentioned previously (Section 2), applications of Mg or K in excess can reduce the uptake of other basic cations, particularly Ca. To complement the use of soil amendments as a way to increase production and decrease pest and pathogen damage, it is important that tropical perennial crops are managed properly with cultural methods such as pruning, shade regulation, soil drainage

Preparation of this review paper was supported by funding provided by the Australian

Abd-Elgawad, M. M., N. S. El-Mougy, N. G. El-Gamal, M. M. Abdel-Kader, and M. M.

and Fruit Quality." *Revista Mexicana de Fitopatologia* 21, no. 1 (2003): 46-55. Adebitan, S. A. "Effects of Phosphorus and Weed Interference on Anthracnose of Cowpea in

Agbenin, O. N. "Potentials of Organic Amendments in the Control of Plant Parasitic

Orchards." *Journal of Plant Protection Research* 50, no. 4 (2010): 477-84. Acosta-Ramos, M., D. H. Noriega-Cantu, D. Nieto-Angel, and D. Teliz-Ortiz. "Effect of

Nigeria." *Fitopatologia Brasileira* 21, no. 2 (1996): 173-79.

Nematodes." *Plant Protection Science* 39, no. 1 (2004): 21-25.

Mohamed. "Protective Treatments against Soilborne Pathogens in Citrus

Integrated Mango (*Mangifera Indica* L.) Management on the Incidence of Diseases

**6. Conclusion** 

and sanitation.

**8. References** 

**7. Acknowledgement** 

Centre for International Agricultural Research (ACIAR).

following treatment of the seedlings with *T. harzianum* conidia (Izzati et al. 2008). Tea stems inoculated with *T. harzianum* demonstrated resistance to infection by *Armillaria* sp. (Otieno et al., 2003). Muleta et al. (2007) found that among isolates of rhizobacteria isolated from soils under coffee, some *Pseudomonas* and *Bacillus* species strongly inhibited the *in vitro* growth of *Fusarium* spp., including *F. stilboides* and *F. oxysporum*. Such rhizobacteria have potential as biocontrol agents of coffee wilt diseases. Using an integrated approach to management of *Fusarium* spp. and the citrus nematode, *Tylenchulus semipenetrans*, in Egypt, Abd-Elgawad et al. (2010) showed that the application of bacterial isolates contributed to a reduction of populations of these pathogens.

In a study of leaf spot disease of mango in Nigeria, Okigbo and Osuinde (2003) demonstrated the pathogenicity of three fungi species isolated from the leaf lesions (*Pestalotiopsis mangiferae*, *Lasiodiplodia theobromae* and *Macrophoma mangiferae*) by inoculating them individually onto healthy mango leaves. In addition, the authors isolated a bacterium identified as *Bacillus subtilis* from soil under mango trees and showed that it inhibited growth *in vitro* of the three causal pathogens and also reduced disease severity *in vivo* when applied to soil in the field. In tea plants infected with *L. theobromae*, *in vivo* control of the disease was demonstrated by pre-treatment of the plants with bacteria that had been isolated from the tea rhizosphere and shown to have *in vitro* antagonistic activity (Purkayastha et al., 2010). Stirling et al. (1992) isolated fluorescent pseudomonads from avocado soils suppressive to *P. cinnamomi* (causing root rot) and demonstrated their *in vitro* antagonism to the pathogen. *In vivo* control of the root-knot nematode on coffee roots, *Meloidogyne incognita*, by the application of an obligate bacterial parasite of the nematode, a strain of *Pasteuria penetrans*, was demonstrated by Carneiro et al. (2007). The colonisation of banana roots by the nematode *Radopholus similis* could be decreased by inoculation of the banana plants with a non-pathogenic isolate of *F. oxysporum* (Paparu et al., 2008). The authors demonstrated that inoculation of the fungus caused the up-regulation of a number of defence-related genes in the host plant (the expression of some of these genes was also increased by inoculation with the nematode). However, the means by which biocontrol agents exert antagonistic effects towards pathogens may include a variety of other mechanisms, including direct antibiosis and competition.

#### *Role of mycorrhizae*

Most plants form symbiotic associations with fungi, forming mycorrhizae. In tropical perennial crop species such associations mainly occur with arbuscular mycorrhizal (AM) fungi, but associations with other taxonomic groups of fungi, forming ectomycorrhizae, are also found. In fact, it could be said that, under natural conditions, plants have mycorrhizae rather than roots (Azco'n-Aguilar and Barea, 1996). Mycorrhizal fungi have an irreplaceable role in supplying nutrients to the plants, particularly of phosphorus (P), which is often unavailable for direct uptake by plant roots. They can also reduce disease severity, particularly of diseases caused by soil-borne pathogens, such as nematodes, in addition to conferring tolerance to drought and salinity (Andrade et al., 2009). Increased tolerance to nematodes has been reported in perennial crop species inoculated with mycorrhizal fungi. Vaast et al. (1997) reported enhanced resistance to *Pratylenchus coffeae* in coffee plants inoculated with AM fungi, with fewer lesions occurring in the AM fungi-inoculated roots (see Section 2). Similarly, an increase in resistance to nematodes was reported in banana plants that had been inoculated with mycorrhizal fungi (Elsen et al., 2003). In a study in citrus orchards in Thailand, increased growth, P uptake and resistance to root rot caused by *Phytophthora nicotianae* was shown to result from the inoculation of citrus trees with a species of AM fungus, *Glomus etunicatum*, isolated from local citrus orchard (Watanarojanaporn et al., 2011). A second AM fungus isolate, identified as *Acaulospora tuberculata,* also conferred resistance to *P. nicotianae* disease. Mycorrhizal fungi may compete for infection sites with the pathogen and/or they may impede access to nutrients by the pathogen (Azco'n-Aquila and Barea, 1996).

#### **6. Conclusion**

262 Plant Pathology

following treatment of the seedlings with *T. harzianum* conidia (Izzati et al. 2008). Tea stems inoculated with *T. harzianum* demonstrated resistance to infection by *Armillaria* sp. (Otieno et al., 2003). Muleta et al. (2007) found that among isolates of rhizobacteria isolated from soils under coffee, some *Pseudomonas* and *Bacillus* species strongly inhibited the *in vitro* growth of *Fusarium* spp., including *F. stilboides* and *F. oxysporum*. Such rhizobacteria have potential as biocontrol agents of coffee wilt diseases. Using an integrated approach to management of *Fusarium* spp. and the citrus nematode, *Tylenchulus semipenetrans*, in Egypt, Abd-Elgawad et al. (2010) showed that the application of bacterial isolates contributed to a

In a study of leaf spot disease of mango in Nigeria, Okigbo and Osuinde (2003) demonstrated the pathogenicity of three fungi species isolated from the leaf lesions (*Pestalotiopsis mangiferae*, *Lasiodiplodia theobromae* and *Macrophoma mangiferae*) by inoculating them individually onto healthy mango leaves. In addition, the authors isolated a bacterium identified as *Bacillus subtilis* from soil under mango trees and showed that it inhibited growth *in vitro* of the three causal pathogens and also reduced disease severity *in vivo* when applied to soil in the field. In tea plants infected with *L. theobromae*, *in vivo* control of the disease was demonstrated by pre-treatment of the plants with bacteria that had been isolated from the tea rhizosphere and shown to have *in vitro* antagonistic activity (Purkayastha et al., 2010). Stirling et al. (1992) isolated fluorescent pseudomonads from avocado soils suppressive to *P. cinnamomi* (causing root rot) and demonstrated their *in vitro* antagonism to the pathogen. *In vivo* control of the root-knot nematode on coffee roots, *Meloidogyne incognita*, by the application of an obligate bacterial parasite of the nematode, a strain of *Pasteuria penetrans*, was demonstrated by Carneiro et al. (2007). The colonisation of banana roots by the nematode *Radopholus similis* could be decreased by inoculation of the banana plants with a non-pathogenic isolate of *F. oxysporum* (Paparu et al., 2008). The authors demonstrated that inoculation of the fungus caused the up-regulation of a number of defence-related genes in the host plant (the expression of some of these genes was also increased by inoculation with the nematode). However, the means by which biocontrol agents exert antagonistic effects towards pathogens may include a variety of other

Most plants form symbiotic associations with fungi, forming mycorrhizae. In tropical perennial crop species such associations mainly occur with arbuscular mycorrhizal (AM) fungi, but associations with other taxonomic groups of fungi, forming ectomycorrhizae, are also found. In fact, it could be said that, under natural conditions, plants have mycorrhizae rather than roots (Azco'n-Aguilar and Barea, 1996). Mycorrhizal fungi have an irreplaceable role in supplying nutrients to the plants, particularly of phosphorus (P), which is often unavailable for direct uptake by plant roots. They can also reduce disease severity, particularly of diseases caused by soil-borne pathogens, such as nematodes, in addition to conferring tolerance to drought and salinity (Andrade et al., 2009). Increased tolerance to nematodes has been reported in perennial crop species inoculated with mycorrhizal fungi. Vaast et al. (1997) reported enhanced resistance to *Pratylenchus coffeae* in coffee plants inoculated with AM fungi, with fewer lesions occurring in the AM fungi-inoculated roots (see Section 2). Similarly, an increase in resistance to nematodes was reported in banana plants that

reduction of populations of these pathogens.

mechanisms, including direct antibiosis and competition.

*Role of mycorrhizae* 

Soil function, plant nutritional status and cultural management practices have a strong influence on the incidence and severity of many diseases of tropical perennials. Diseases are influenced, not only by the general nutritional status of the plant (i.e. by an adequate supply of macronutrients), but also by individual nutrients. Soil pH is particularly important for a number of reasons, including its effect on availability of cations to the plant, pathogen or antagonists. It also influences microbial ecology, with a number of potential antagonists among bacterial and actinomycete species being favoured by a higher soil pH. Since tropical perennial crops and the use of inorganic fertiliser can lower soil pH, liming and/or compost treatments are strategies that can be adopted for disease mitigation in these crops. Composting farm waste, not only returns nutrients to the farm, but also improves farm sanitation as it kills larvae of pests and pathogen spores and other forms of inoculum. Nutrient elements supplied in mineral fertilisers can interact and care needs to be taken in their application. As mentioned earlier, some fertilisers can increase concentrations of Cl in plant tissues and decrease the K/Cl ratio. Also mentioned previously (Section 2), applications of Mg or K in excess can reduce the uptake of other basic cations, particularly Ca. To complement the use of soil amendments as a way to increase production and decrease pest and pathogen damage, it is important that tropical perennial crops are managed properly with cultural methods such as pruning, shade regulation, soil drainage and sanitation.

#### **7. Acknowledgement**

Preparation of this review paper was supported by funding provided by the Australian Centre for International Agricultural Research (ACIAR).

#### **8. References**


Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 265

Chuang, T Y. "Suppressive Soil of Banana Fusarium Wilt in Taiwan." *Plant Protection Bulletin* 

Cooper, Richard M, and Jane S Williams. "Elemental Sulphur as an Induced Antifungal

Corden, M. E. "Influence of Calcium Nutrition on Fusarium Wilt of Tomato and

Crane, J. H., and C. W. Campbell. "The Mango." *Fact Sheets, Horticultural Sciences Department,* 

da Silva Moraes, Wilson, Hilario Antonio de Castro, Juliana Domingues Lima, Eloisa

Datnoff, L. E., F. A. Rodrigues, and K.W. Seebold. "Silicon and Plant Disease." In *Mineral* 

Huber, 233-46. St. Paul, Minn: American Phytopathological Society, 2007. Desaeger, J. , Meka R. Rao, and J. Bridge. "Nematodes and Other Soilborne Pathogens in

Dominguez, J., M. A. Negrin, and C. M. Rodriguez. "Aggregate Water-Stability, Particle-Size

Duffy, B. "Zinc and Plant Disease." In *Mineral Nutrition and Plant Disease*, edited by L. E.

Edington, L.V. , and J.C. Walker. "Influence of Calcium and Boron Nutrition on Development of Fusarium Wilt of Tomato." *Phytopathology* 48 (1958): 324-26. Elsen, A., H. Baimey, R. Swennen, and D. De Waele. "Relative Mycorrhizal Dependency and

Emechebe, A.M. "The Effect of Soil Moisture and of N, P and K on Incidence of Infection

Engelhard, Arthur W. *Soilborne Plant Pathogens: Management of Diseases with Macro- and* 

Evans, I., E. Solberg, and D. M. Huber. "Copper and Plant Disease." In *Mineral Nutrition and* 

Flood, J., L. Keenan, S. Wayne, and Y. Hasan. "Studies on Oil Palm Trunks as Sources of

Gaur, R. B., and P. K. Vaidya. "Reduction of Root Rot of Chickpea by Soil Application of Phosphorus and Zinc." *International Chickpea Newsletter* 9 (1983): 17-18.

Nematode Susceptibility." *Plant and Soil* 256, no. 2 (2003): 303-13.

*Microelements*. St. Paul, Minn: APS press, 1989.

Paul, Minn: American Phytopathological Society, 2007. Flood, J. *Coffee Wilt Disease*. Wallingford: CAB International, 2010.

Infection in the Field." *Mycopathologia* 159, no. 1 (2005): 101-07.

Polygalacturonase Activity." *Phytopathology* 55 (1965): 222-24.

*Florida Cooperative Extension Service* (1994).

Substance in Plant Defence." *Journal of experimental botany* 55, no. 404 (2004): 1947-

Aparecida das Gracas Leite, and Mauricio de Souza. "Susceptibility of Three Citrus Species to *Dothiorella Gregaria* Sacc. In Function of the Nutriconal State." *Ciencia* 

*Nutrition and Plant Disease*, edited by L. E. Datnoff, Wade H. Elmer and D. M.

Agroforestry." In *Below-Ground Interactions in Tropical Agroecosystems: Concepts and Models with Multiple Plant Components*, edited by Meine van Noordwijk, Georg

and Soil Solution Properties in Conducive and Suppressive Soils to Fusarium Wilt of Banana from Canary Islands (Spain)." *Soil Biology & Biochemistry* 33, no. 4-5

Datnoff, Wade H. Elmer and D. M. Huber, 155-78. St. Paul, Minn: American

Mycorrhiza-Nematode Interaction in Banana Cultivars ( *Musa* Spp.) Differing in

with Cacao Seedlings Inoculated with *Verticillium Dahliae*." *Plant & Soil* 54, no. 1

*Plant Disease*, edited by L. E. Datnoff, Wade H. Elmer and D. M. Huber, 177-88. St.

33, no. 1 (1991): 133-41.

*Rural* 37, no. 1 (2007): 7-12.

(2001): 449-55.

(2006): 143-47.

Cadisch and C.K. Ong: CABI, 2004.

Phytopathological Society, 2007.

53.

Agrios, G. N. *Plant Pathology*. Amsterdam: Elsevier Academic Press, 2005.


Andrade, S. A. L., P. Mazzafera, M. A. Schiavinato, and A. P. D. Silveira. "Arbuscular

Anonymous. "On Organic Coffee Farm, Complex Interactions Keep Pests under Control."

Avelino, J. , M. Bouvret, L. Salazar, and C. Cilas. "Relationships between Agro-Ecological

Avelino, J. , H. Zelaya, A. Merlo, A. Pineda, M. Ordonez, and S. Savary. "The Intensity of a

Azco'n-Aguilar, C., and J .M. Barea. "Arbuscular Mycorrhizas and Biological Control of Soil-

Beer, J. "Litter Production and Nutrient Cycling in Coffee(*Coffea Arabica*) and Cacao

Beer, J., R. Muschler, D. Kass, and E. Somarriba. "Shade Management in Coffee and Cacao

Bell, A. A. "The Role of Nutrition in Diseases of Cotton." In *Soilborne Plant Pathogens:* 

Bhai, R. S., and Y. R. Sarma. "Effect of Organic Amendments on the Proliferation Stability of

Bonanomi, G., V. Antignani, M. Capodilupo, and F. Scala. "Identifying the Characteristics of

Buresh, R. J., P.C. Smithson, and D.T. Hellums. "Building Soil P Capital in Africa." In

Calhoun, 111-49. Madison, Wisconsin: Soil Science Society of America, 1997. Cakmak, I. , C. Hengeler, and H. Marschner. "Changes in Phloem Export of Sucrose in

Carneiro, R. M. D. G., L. F. G. de Mesquita, P.A. S. Cirotto, Fabiane C Mota, Maria Ritta A

Chuang, T-Y. "Studies on the Soils Suppressive to Banana Fusarium Wilt Ii. Nature of

*Helicotylenchus Multicinctus*." *Nematologica* 12, no. 1 (1966): 129-37.

Plants." *Journal of Experimental Botany* 45 (1994): 1251-57.

on Coffee." *Nematology* 9, no. Part 6 (2007): 845-51.

*Plant Protection Bulletin* 30, no. 2 (1988): 125-34.

Plantations." *Agroforestry Systems* 38, no. 1 (1998): 139-64.

Engelhard, 167-204. St. Paul, Minn: APS press, 1989.

*Biochemistry* 42, no. 2 (2010): 136-44.

Mycorrhizal Association in Coffee." *The Journal of Agricultural Science* 147, no. 2

Factors and Population Densities of *Meloidogyne Exigua* and *Pratylenchus Coffeae* Sensu Lato in Coffee Roots, in Costa Rica." *Applied Soil Ecology* 43, no. 1 (2009): 95-

Coffee Rust Epidemic Is Dependent on Production Situations." *Ecological Modelling* 

Borne Plant Pathogens - an Overview of the Mechanisms Involved." *Mycorrhiza* 6

(*Theobroma Cacao*) Plantations with Shade Trees." *Agroforestry Systems* 7 (1988): 103-

*Management of Diseases with Macro- and Microelements* edited by Arthur W.

*Trichoderma Harzianum* and Suppression of *Phytophthora Meadii* in Cardamom Soils in Relation to Soil Microflora." *Journal of Biological Control* 23, no. 2 (2009): 163-67. Blake, C.D. "The Histological Changes in Banana Roots Caused by *Radopholus Similis* and

Organic Soil Amendments That Suppress Soilborne Plant Diseases." *Soil Biology &* 

*Replenishing Soil Fertility in Africa*, edited by R.J. Buresh, P.A. Sanchez and F.G.

Leaves in Response to Phosphorus, Potassium and Magnesium Deficiency in Bean

Almeida, and Maria Celia Cordeiro. "The Effect of Sandy Soil, Bacterium Dose and Time on the Efficacy of *Pasteuria Penetrans* to Control *Meloidogyne Incognita* Race 1

Suppression to Race 4 of Fusarium-Oxysporum-F-Sp-Cubense in Taiwan Soils."

Agrios, G. N. *Plant Pathology*. Amsterdam: Elsevier Academic Press, 2005.

*U.S. News & World Report*, no. Journal Article (2010): 1.

(2009): 105-15.

(1996): 457-64.

197, no. 3-4 (2006): 431-47.

105.

14.


Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 267

Keane, P. J., and C. A. J. Putter. *Cocoa Pest and Disease Management in Southeast Asia and* 

Keane, P.J. "Epidemiology of Vascular-Streak Dieback of Cocoa." *Annals of Applied Biology* 98

Keane, P.J., M.T. Flentje, and K.P. Lamb. "Investigation of Vascular-Streak Dieback of Cocoa in Papua New Guinea." *Australian Journal of Biological Sciences* 25 (1972): 553-64. Keane, Philip, and David Guest. "Vascular-Streak Dieback: A New Encounter Disease of

Khan, Tabreiz A., and Shabana T. Khan. "Effect of Npk on Disease Complex of Papaya

Kohler, F. , F. Pellegrin, G. Jackson, and E. McKenzie. *Diseases of Cultivated Crops in Pacific* 

Konam, J.K., and D. I. Guest. "Role of Flying Beetles (Coleoptera: Scolytidae and Nitidulae)

Konam, John K., and David I. Guest. "Leaf Litter Mulch Reduces the Survival of *Phytophthora* 

Krauss, A. "Importance of Balanced Fertilization to Meet the Nutrient Demand of Industrial

Maclean, R. H., J. A. Litsinger, K. Moody, and A. K. Watson. "The Impact of Alley Cropping

Mbenoun, M., E. H. Momo Zeutsa, G. Samuels, F. Nsouga Amougou, and S. Nyasse.

McDonald, V., E. Pond, M. Crowley, B. McKee, and J. Menge. "Selection for and Evaluation

McMahon, P. J., A. Purwantara, A. Wahab, M. Imron, S. Lambert, P. J. Keane, and D. I.

Melendez, K., and N.T. Powell. "The Influence of *Meloidogyne* on Root Decay in Tobacco

Caused by *Pythium* and *Trichoderma*." *Biological Abstracts* 59 (1969).

Marschner, Horst. *The Mineral Nutrition of Higher Plants*. London: Academic Press, 1995. Martinati, J. C., R. Harakava, S. D. Guzzo, and S. M. Tsai. "The Potential Use of a Silicon

Production in Cameroon." *Plant Pathology* 57, no. 2 (2008): 381-81.

2003, : International Potash Institute, Basel, Switzerland, 2003.

*Island Countries*: South Pacific Commission, 1997.

*Australasian Plant Pathology* 33 (2004): 55-59.

*Agroforestry Systems* 20, no. 3 (1992): 213-28.

*Phytopathology* 156, no. 7-8 (2008): 458-63.

*Plant & Soil* 299, no. 1-2 (2007): 17-28.

*Australasian Plant Pathology* 39, no. 2 (2010): 170-75.

Nations, 1992.

(1981): 227-41.

13, no. 1 (1995): 29-34.

31, no. 4 (2002): 381-83.

57.

*Australasia*. Vol. 112. Rome: Food and Agriculture Organization of the United

Cacao in Papua New Guinea and Southeast Asia Caused by the Obligate Basidiomyceteoncobasidium Theobromae." *Phytopathology* 97, no. 12 (2007): 1654-

Caused by *Meloidogyne Incognita* and *Fusarium Solani*." *Pakistan Journal of Nematology* 

in the Spread of Phytophthora Pod Rot of Cocoa in Papua New Guinea."

*Palmivora* under Cocoa Trees in Papua New Guinea." *Australasian Plant Pathology* 

and Plantation Crops." In *International Workshop Importance of potash fertilizers for sustainable production of plantation and food crops* Colombo, Sri Lanka,1-2 December

*Gliricidia Sepium* and *Cassia Spectabilis* on Upland Rice and Maize Production."

Source as a Component of an Ecological Management of Coffee Plants." *Journal of* 

"Dieback Due to Lasiodiplodia Theobromae, a New Constraint to Cocoa

of an Avocado Orchard Soil Microbially Suppressive to *Phytophthora Cinnamomi*."

Guest. "Phosphonate Applied by Trunk Injection Controls Stem Canker and Decreases Phytophthora Pod Rot (Black Pod) Incidence in Cocoa in Sulawesi."


Gomes, Vicente Martins, Ricardo Moreira Souza, Vicente Mussi-Dias, Silvaldo Felipe da

Guest, D. I., and P.J. Keane. "Vascular-Streak Dieback: A New Encounter Disease of Cacao in

Guest, David. "Black Pod: Diverse Pathogens with a Global Impact on Cocoa Yield."

Haneklaus, S., E. Bloem, and E. Schnug. "Sulfur and Plant Disease." In *Mineral Nutrition and* 

Harrington, T. "Ceratocystis Wilt: Taxonomy and Nomenclature." CABI Publishing,

Hartemink, A.E. "Nutrient Stocks, Nutrient Cycling and Soil Changes in Cocoa Ecosystems:

Hillocks, R. J. , and J.M. Walller, eds. *Soilborne Diseases of Tropical Crops*: CABI Publishing,

Holliday, Paul. *Fungus Diseases of Tropical Crops*. New York: Cambridge University Press,

Huber, D. M., and I. A. Thompson. "Nitrogen and Plant Disease." In *Mineral Nutrition and* 

Izzati, Mohd Zainudin Nur Ain, and Faridah Abdullah. "Disease Suppression in

Jones, P. J., A. W. Engelhard, and S. S. Woltz. "Management of Fusarium Wilt of Vegetables

Jones, P. J., and S. S. Woltz. "Effect of Soil Ph and Micronutrient Amendments on *Verticillium* and *Fusarium* Wilt of Tomato." *Plant Disease Report* 56 (1972): 151-53. Kader, Adel A., ed. *Postharvest Technology of Horticultural Crops*. 3rd ed: University of

Kalim, S., Y. P. Luthra, and S. K. Gandhi. "Role of Zinc and Manganese in Resistance of Cowpea Rot." *Journal of Plant Diseases & Protection* 110 (2003): 235-43. Kandan, A., R. Bhaskaran, and R. Samiyappan. "Ganoderma - a Basal Stem Rot Disease of

Kannan, C., M. Karthik, and K. Priya. "*Lasiodiplodia Theobromae* Causes a Damaging Dieback

Karthikeyan, G., T. Raguchander, and R. Rabindran. "Integrated Management of Basal Stem

*Plant Disease*, edited by L. E. Datnoff, Wade H. Elmer and D. M. Huber, 31-44. St.

Ganoderma-Infected Oil Palm Seedlings Treated with Trichoderma Harzianum."

and Ornamentals by Macro- and Microelement Nutrition." In *Soilborne Plant Pathogens: Management of Diseases with Macro- and Microelements*, edited by Arthur

Coconut Palm in South Asia and Asia Pacific Regions." *Archives of Phytopathology &* 

Rot/Ganoderma Disease of Coconut in India." *Crop Research* 32, no. 1 (2006): 121-23.

http://www.public.iastate.edu/~tcharrin/CABIinfo.html (2004).

1 (2011): 45-50.

*Phytopathology* 97, no. 12 (2007): 1650-53.

Paul, Minn: American Phytopathological Society, 2007.

A Review." *Advances in Agronomy* 86 (2005): 227-52.

Paul, Minn: American Phytopathological Society, 2007.

W. Engelhard, 18-32. St. Paul, Minn: APS press, 1989.

of Cocoa in India." *Plant Pathology* 59, no. 2 (2010): 410-10.

*Plant Protection Science* 44, no. 3 (2008): 101-07.

*Plant Protection* 43, no. 15 (2010): 1445-49.

57.

1997.

1980.

California, 2002.

Silveira, and Claudia Dolinski. "Guava Decline: A Complex Disease Involving Meloidogyne Mayaguensis and Fusarium Solani." *Journal of Phytopathology* 159, no.

Papua New Guinea and Southeast Asia Caused by the Obligate Basidiomyceteoncobasidium Theobromae." *Phytopathology* 97, no. 12 (2007): 1654-

*Plant Disease*, edited by L. E. Datnoff, Wade H. Elmer and D. M. Huber, 101-18. St.


Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 269

Otieno, Washington, Michael Jeger, and Aad Termorshuizen. "Effect of Infesting Soil with

Page, S.L.J., and J. Bridge. "Plant Nematodes and Sustainability in Tropical Agriculture."

Palm, C.A. "Contribution of Agroforestry Trees to Nutrient Requirements of Intercropped

Palti, J. *Cultural Practices and Infectious Crop Diseases*. Vol. 9. New York: Springer-Verlag,

Paparu, Pamela, Thomas Dubois, Danny Coyne, and Altus Viljoen. "Defense-Related Gene

Peng, H X, K Sivasithamparam, and D W Turner. "Chlamydospore Germination and

Pennypacker, B. W. "The Role of Mineral Nutrition in the Control of Verticillium Wilt." In

Ploetz, R. C., ed. *Diseases of Tropical Fruit Crops*. Wallingford, Oxon, UK: CAB International

Ploetz, R. C. "Diseases of Tropical Perennial Crops: Challenging Problems in Diverse

Ploetz, Randy C. "Fusarium-Induced Diseases of Tropical, Perennial Crops." *Phytopathology* 

Pozza, Adelia A A, Paulo T G Guimaraes, Edson A Pozza, Marcelo M Romaniello, and

Prabhu, A. S., N. K. Fageria, D. M. Huber, and F. A. Rodrigues. "Potassium and Plant

Prakash, O M. "Integrated Management of Mango Seedling (Planting Material) Diseases:

Prior, C. "Cocoa Quarantine: Measures to Prevent the Spread of Vascular-Streak Dieback in

Purkayastha, G D, A Saha, and D Saha. "Characterization of Antagonistic Bacteria Isolated

Agents in Tea." *Journal of Mycology & Plant Pathology* 40, no. 1 (2010): 27-37.

Janice G de Carvalho. "Effect of Substrata and Fertilizations of Coffee Seedlings in Tubes in Production and Brown Eye Spot Intensity." *Summa Phytopathologica* 27, no.

Disease." In *Mineral Nutrition and Plant Disease*, edited by L. E. Datnoff, Wade H. Elmer and D. M. Huber, 57-78. St. Paul, Minn: American Phytopathological Society,

from Tea Rhizosphere in Sub-Himalayan West Bengal as Potential Biocontrol

edited by Arthur W. Engelhard, 33-45. St. Paul, Minn: APS press, 1989.

Perrenoud, S. "Potassium and Plant Health." *Soil Science* 127, no. 1 (1977): 63. Perrenoud, S. "Potassium and Plant Health." *IPI - Research Topics* (1990).

Environments." *PLANT DISEASE* 91, no. 6 (2007): 644-63.

Present Status." *Biological Memoirs* 26, no. 1 (2000): 37-43.

Planting Material." *Plant Pathology* 34 (1985): 603-08.

*Armillaria*." *BIOLOGICAL CONTROL* 26, no. 3 (2003): 293-301.

*Experimental Agriculture* 29, no. 2 (1993): 139-54.

pp105-24, 1995.

(1999): 1363-74.

2003.

2007.

71, no. 4-6 (2008): 149-57.

96, no. 6 (2006): 648-52.

4 (2001): 370-74.

1981.

*Trichoderma Harzianum* and Amendment with Coffee Pulp on Survival of

Plants." In *Agroforestry: Science, Policy and Practice*, edited by Fergus L. Sinclair,

Expression in Susceptible and Tolerant Bananas (*Musa* Spp.) Following Inoculation with Non-Pathogenic *Fusarium Oxysporum* Endophytes and Challenge with *Radopholus Similis*." *PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY* 

Fusarium Wilt of Banana Plantlets in Suppressive and Conducive Soils Are Affected by Physical and Chemical Factors." *Soil Biology & Biochemistry* 31, no. 10

*Soilborne Plant Pathogens: Management of Diseases with Macro- and Microelements*,


Menge, J. A., and S. Nemec. "Citrus." In *Soilborne Diseases of Tropical Crops*. Wallingford,

Mengel, Konrad, and Ernest A. Kirkby. *Principles of Plant Nutrition*. Bern: International

Michel-Aceves, A. C., O. Rebolledo-Dominguez, R. Lezama-Gutierrez, M. E. Ochoa-Moreno,

Mobambo, K. N., K. Zuofa, F. Gauhl, M. O. Adeniji, and C. Pasberggauhl. "Effect of Soil

Mossu, G. *Le Cacaoyer*, Le Technicien D'agriculture Tropicale Paris Maisonneuve et Larose

Muleta, D, F Assefa, and U Granhall. "In Vitro Antagonism of Rhizobacteria Isolated from

Muraleedharam, N., and Z.M. Chen. "Pests and Diseases of Tea and Their Management."

Murray, D.B. "Shade and Nutrition." In *Cocoa*, edited by G.A.R. Wood and R.A. Lass, 105-24.

Nasir, N., P. A. Pittaway, and K. G. Pegg. "Effect of Organic Amendments and Solarisation

Nelson, P.N. , M.J. Webb, S. Berthelsen, G. Curry, Yinil, D., and Fidelis C. *Nutritional Status* 

Nelson, S. "Cercospora Leaf Spot and Berry Blotch of Coffee." *Plant Disease* (2008a),

Nelson, S. , D. Scmitt, and V.E. Smith. "Managing Coffee Nematode Decline." *Plant Disease*

Noble, R., and E. Coventry. "Suppression of Soil-Borne Plant Diseases with Composts: A

Okigbo, Ralph N., and Maria I. Osuinde. "Fungal Leaf Spot Diseases of Mango (*Mangifera* 

*Indica* L.) in Southeastern Nigeria and Biological Control with *Bacillus Subtilis*."

Nelson, S.C. "Cephaleuros Species, the Plant-Parasitic Green Algae." *Plant Disease* (2008b). Nelson, S.C., R. C. Ploetz, and A. K. Kepler. "Musa Species (Banana and Plantain)." *Species* 

J. C. Mesina-Escamilla, and G. J. Samuels. "*Trichoderma* Species in Soils Cultivated with Mango and Affected by Mango Malformation, and Its Inhibitory Potential on *Fusarium Oxysporum* and *F. Subglutinans*." *Revista Mexicana de Fitopatologia* 19, no. 2

Fertility on Host Response to Black Leaf Streak of Plantain (Musa Spp, Aab Group) under Traditional Farming Systems in Southeastern Nigeria." *International Journal of* 

*Coffea Arabica* L. Against Emerging Fungal Coffee Pathogens." *Engineering in Life* 

on Fusarium Wilt in Susceptible Banana Plantlets, Transplanted into Naturally Infested Soil." *Australian Journal of Agricultural Research* 54, no. 3 (2003): 251-57. Ndo, Eunice Golda Daniele, Faustin Bella-Manga, Sali Atanga Ndindeng, Michel Ndoumbe-

Nkeng, Ajong Dominic Fontem, and Christian Cilas. "Altitude, Tree Species and Soil Type Are the Main Factors Influencing the Severity of *Phaeoramularia* Leaf and Fruit Spot Disease of Citrus in the Humid Zones of Cameroon." *European Journal of* 

*of Cocoa in Papua New Guinea*. Edited by Australian Centre for International Agricultural Research. Vol. 76, Aciar Technical Reports Canberra: Australian

Oxon, UK: CAB International, 1997.

*Pest Management* 40 no. 1 (1994): 75-80.

*Journal of Plantation Crops* 25, no. 1 (1997): 15-43.

*Plant Pathology* 128, no. 3 (2010): 385-97.

*Profiles for Pacific Island Agroforestry* (2006).

*Plant Protection Science* 39, no. 2 (2003): 70-77.

Centre for International Agricultural Research (2011)

www.ctahr.hawaii.edu/oc/freepubs/pdf/PD41.pdf.

(2002), www.ctahr.hawaii.edu/oc/freepubs/pdf/PD.23.pdf.

Review." *Biocontrol Science & Technology* 15, no. 1 (2005): 3-20.

*Sciences* 7, no. 6 (2007): 577-86.

London: Longman, 1975

Potash Institute, 1982.

(2001): 154-60.

1990.


Effect of Nutrition and Soil Function on Pathogens of Tropical Tree Crops 271

Shahbaz, M., Z. Iqbal, A. Saleem, and M.A. Anjum. "Association of *Lasiodiplodia Theobromae*

Shen, G H, Q H Xue, M Tang, Q Chen, L N Wang, C M Duan, L Xue, and J Zhao. "Inhibitory

Siddiqui, I A, S S Shaukat, and M Hamid. "Role of Zinc in Rhizobacteria-Mediated

Silva, R V, R D L Oliveira, K J T Nascimento, and F A Rodrigues. "Biochemical Responses of

Smyth, T.J., and D.K. Cassell. "Synthesis of Long-Term Soil Management Research on

Somarriba, Eduardo, and John Beer. "Productivity of *Theobroma Cacao* Agroforestry Systems

Souza, Ricardo M, Maciel S Nogueira, Inorbert M Lima, Marcelo Melarato, and Claudia M

Staver, C., F. Guharay, D. Monterroso, and R. G. Muschler. "Designing Pest-Suppressive

Stirling, A. M., A. C. Hayward, and K. G. Pegg. "Evaluation of the Biological Control

Stover, R. H. . *Banana, Plantain and Abaca Diseases*. Kew, Surrey, UK: Commonwealth

Streeter, T. C., Rengel. Z., S. M. Neate, and R. D. Graham. "Zinc Fertilisation Increases

Susanto, A., P. S. Sudharto, and R. Y. Purba. "Enhancing Biological Control of Basal Stem

Thompson, I. A., and D. M. Huber. "Manganese and Plant Disease." In *Mineral Nutrition and* 

Turner, P. D. "Some Factors in the Control of Root Diseases of Oil Palm." In *Root Diseases and* 

*AUSTRALASIAN PLANT PATHOLOGY* 21, no. 4 (1992): 133-42.

Paul, Minn: American Phytopathological Society, 2007.

Berkely, LA: Univ of California, 1970.

*Journal of Plant Diseases & Protection* 117, no. 4 (2010): 180-84.

*Botany* 41, no. 1 (2009): 359-68.

*Phytopathology* 150, no. 10 (2002): 569-75.

*Pathology* 59, no. 3 (2010): 586-93.

Raton: Lewis Publishers, 1995.

*Protection* 31, no. 1 (2003): 48-50.

Mycological Institute 1972

(2001): 233-42.

1 (2005): 153-57.

*Agroforestry Systems* 53, no. 2 (2001): 151-70.

2 (2011): 109-21.

with Different Decline Disorders in Mango (*Mangifera Indica* L.)." *Pakistan Journal of* 

Effects of Potassium Silicate on Five Soil-Borne Phytopathogenic Fungi in Vitro."

Suppression of Root-Infecting Fungi and Root-Knot Nematode." *Journal of* 

Coffee Resistance against Meloidogyne Exigua Mediated by Silicon." *Plant* 

Ultisols and Oxisols in the Amazon." In *Soil Management: Experimental Basis for Sustainability and Environmental Quality*, edited by R. Lal and B. A. Stewart. Boca

with Timber or Legume Service Shade Trees." *AGROFORESTRY SYSTEMS* 81, no.

Dolinski. "Management of the Guava Root-Knot Nematode in Sao Joao Da Barra, Brazil, and Report of New Hosts." *Nematologia Brasileira* 30, no. 2 (2006): 165-69. Srinivasulu, B, K Aruna, D V R Rao, and H Hameedkhan. "Epidemiology of Basal Stem Rot

(Ganoderma Wilt) Disease of Coconut in Andhra Pradesh." *Indian Journal of Plant* 

Multistrata Perennial Crop Systems: Shade-Grown Coffee in Central America."

Potential of Bacteria Isolated from a Soil Suppressive to *Phytophthora Cinnamomi*."

Tolerance to *Rhizoctonia Solani* (Ag 8) in *Medicago Trunculata*." *Plant & Soil* 228

Rot Disease (*Ganoderma Boninense*) in Oil Palm Plantations." *Mycopathologia* 159, no.

*Plant Disease*, edited by L. E. Datnoff, Wade H. Elmer and D. M. Huber, 139-54. St.

*Soil-Borne Pathogens*, edited by T. A. Tousson, R. V. Bega and P. E. Nelson, 194-200.


Reeleder, R D. "Fungal Plant Pathogens and Soil Biodiversity." *Canadian Journal of Soil Science* 

Resende, M L V, J Flood, J D Ramsden, M G Rowan, M H Beale, and R M Cooper. "Novel

Reuveni, R, and M Reuveni. "Foliar-Fertilizer Therapy: A Concept in Integrated Pest

Rice, R. W. "The Physiological Role of Minerals in Plants." In *Mineral Nutrition and Plant* 

Rivas Cordero, M., and D. Pavone Maniscalco. "Diversity of *Trichoderma* Spp. On *Theobroma* 

*Crinipellis Perniciosa* (Stahel) Singer." *Interciencia* 35, no. 10 (2010): 777-83. Rubini, M.R. , R.T. Silva-Ribeiro, A.W.V. Pomella, C.S. Maki, W.L. Araujo, D.R. dos

Rutherford, Mike A. "Current Knowledge of Coffee Wilt Disease, a Major Constraint to Coffee Production in Africa." *Phytopathology* 96, no. 6 (2006): 663-66. Samuels, Gary J., Adnan Ismaiel, Ade Rosmana, Muhammad Junaid, David Guest, Peter

Samuels, Gary J., Carmen Suarez, Karina Solis, Keith A. Holmes, Sarah E. Thomas, Adnan

Sangeetha, Ganesan, Swaminathan Usharani, and Arjunan Muthukumar. "Biocontrol with

Santos, Florisvalda da Silva, Paulo Estevao de Souza, Edson Ampelio Pozza, Julio Cesar

Schroth, G., U. Krauss, L. Gasparotto, J. A. Duarte Aguilar, and K. Vohland. "Pests and

Schulz, Barbara, and Christine Boyle. "The Endophytic Continuum." *Mycological Research* 

Conventional Systems." *Summa Phytopathologica* 34, no. 1 (2008): 48-54. Schroth, G. , J. Lehmann, M. R. L. Rodrigues, E. Barros, and J. L. V. Macêdo. "Plant-Soil

*Phytopathologia Mediterranea* 48, no. 2 (2009): 214-25.

Phytoalexins Including Elemental Sulphur in the Resistance of Cocoa (Theobroma Cacao L.) to Verticillium Wilt (Verticillium Dahliae Kleb.)." *Physiological &* 

*Disease*, edited by L. E. Datnoff, Wade H. Elmer and D. M. Huber, 9-30. St. Paul,

*Cacao* L. Fields in Caraboro State, Venezuela, and Its Biocontrol Capacity on

Santos, and J.L. Azevedo. "Diversity of Endphytic Fungal Community of Cacao (*Theobroma Cacao* L.) and Biological Control of *Crinipellis Perniciosa*, Causal Agent of Witches' Broom Disease." *International Journal of Biological Science* 1 (2005): 24-33.

McMahon, Philip Keane, Agus Purwantara, Smilja Lambert, Marianela Rodriguez-Carres, and Marc A. Cubeta. "Vascular Streak Dieback of Cacao in Southeast Asia and Melanesia: In Planta Detection of the Pathogen and a New Taxonomy." *Fungal* 

Ismaiel, and Harry C. Evans. "Trichoderma Theobromicola and T. Paucisporum: Two New Species Isolated from Cacao in South America." *Mycological Research* 110,

*Trichoderma* Species for the Management of Postharvest Crown Rot of Banana."

Miranda, Sarah Silva Barreto, and Vanessa Cristina Theodoro. "Progress of Brown Eye Spot (*Cercospora Coffeicola* Berkeley & Cooke) in Coffee Trees in Organic and

Interactions in Multistrata Agroforestry in the Humid Tropics." *Agroforestry Systems* 

Diseases in Agroforestry Systems of the Humid Tropics." *Agroforestry Systems* 50,

83, no. 3 (2003): 331-36.

*Molecular Plant Pathology* 48, no. 5 (1996): 347-59.

Minn: American Phytopathological Society, 2007.

*biology*, no. Journal Article (2011).

no. Pt 4 (2006): 381-92.

53 (2001): 85-102.

no. 3 (2000): 199-241.

109, no. 6 (2005): 661-86.

Management." *Crop Protection* 17, no. 2 (1998): 111-18.


**11** 

*India* 

**Current Advances in the Fusarium Wilt Disease** 

Banana (*Musa* spp.) is the fourth most important global food commodity after rice, wheat and maize in terms of gross value production. At present, it is grown in more than 120 countries throughout tropical and subtropical regions and it is the staple food for more than 400 million people (Molina and Valmayor, 1999). Among the production constraints, Fusarium wilt caused by the fungus *Fusarium oxysporum* f.sp *cubense* (Foc) is the most devastating disease affecting commercial and subsistence of banana production through out the banana producing areas of the world (Ploetz, 2005). The disease is ranked as one of the top 6 important plant diseases in the world (Ploetz & Pegg, 1997). In terms of crop destruction, it ranks with the few most devastating diseases such as wheat rust and potato blight (Carefoot and sprott, 1969). The disease almost destroyed the banana export industry, built on the Gros Michel variety, in Central America during the 1950's (Stover, 1962). In addition, the widely grown clones in the ABB 'Bluggoe' and AAA 'Gros Michel and Cavendish' sub groups are also highly susceptible to this disease worldwide. Presently, Fusarium wilt has been reported in all banana growing regions of the world (Asia, Africa, Australia and the tropical Americas) except some islands in the South Pacific, the Mediterranean, Melanesia, and Somalia (Stover, 1962; Anonymous, 1977;

The fungus *Foc* is the soilborne hyphomycete and is one of more than 100 formae speciales of *F. oxysporum* that causes vascular wilts of flowering plants (Domsch et al. 1980; Nelson et al. 1983). Although Fusarium wilt probably originated in Southeast Asia, (Ploetz and Pegg, 1997), the disease was first discovered at Eagle Farm, Brisbane, Queensland, Australia in 1876 in banana plants var. Sugar (Silk AAB) (Bancroft, 1876). The fungus infects the roots of banana plants, colonizing the vascular system of the rhizome and pseudostem, and inducing characteristic wilting symptoms mostly after 5-6 months of planting and the symptoms are expressed both externally and internally (Wardlaw, 1961; Stover, 1962). Generally, infected plants produce no bunches and if produced, the fruits are very small and only few fingers develop. Fruits ripen irregularly and the flesh is pithy and acidic. The fungus survives in soil for up to 30 years as chlamydospores in infested plant material or in the roots of alternative

Since the discovery of Fusarium wilt of banana, though various control strategies like soil fumigation (Herbert and Marx, 1990); fungicides (Lakshmanan et al., 1987); crop rotation

**1. Introduction** 

Ploetz and Pegg, 2000).

hosts (Ploetz, 2000).

**Management in Banana with Emphasis** 

**on Biological Control** 

R. Thangavelu and M.M. Mustaffa

*National Research Centre for Banana, Trichirapalli* 


### **Current Advances in the Fusarium Wilt Disease Management in Banana with Emphasis on Biological Control**

R. Thangavelu and M.M. Mustaffa *National Research Centre for Banana, Trichirapalli India* 

#### **1. Introduction**

272 Plant Pathology

Vaast, P., E. P. Caswell-Chen, and R. J. Zasoski. "Influences of a Root-Lesion Nematode,

Vandermeer, John, Ivette Perfecto, and Stacy Philpott. "Ecological Complexity and Pest

Vawdrey, L. L., T. M. Martin, and J. De Faveri. "The Potential of Organic and Inorganic Soil

Waggoner, P.E., and A.E. Dimond. "Production and Role of Extracellular Pectic Enzymes of *Fusarium Oxysporum* F. Lycopersici." *Phytopathology* 45 (1955): 79-87.

Waller, J. M., M. Bigger, and R. J. Hillocks. *Coffee Pests, Diseases and Their Management*.

Watanarojanaporn, Nantida, Nantakorn Boonkerd, Sopone Wongkaew, Phrarop

Weller, David M., Jos M. Raaijmakers, Brian B. McSpadden Gardener, and Linda S.

Wellman, F.L. "More Diseases on Crops in the Tropics Than in Temperate Zones." *Ceiba* 14

Widmer, T L, J H Graham, and D J Mitchell. "Composted Municipal Waste Reduces

Williams, Jane S, Sharon A Hall, Malcolm J Hawkesford, Michael H Beale, and Richard M

Yi, Qi-fe, Fu-wu Xin, and Xiu-lin Ye. "Effects of Increasing Phosphate and Potassium

Zheng, Q., and R. Ploetz. "Genetic Diversity in the Mango Malformation Pathogen and Development of a Pcr Assay." *Plant Pathology* 51, no. 2 (2002): 208-16.

a Fungal Vascular Pathogen." *Plant Physiology* 128, no. 1 (2002): 150-59. Woltz, S. S., and J. P. Jones. "Nutritional Requirements of *Fusarium Oxysporum*: Basis for a

Infection of Citrus Seedlings by *Phytophthora Nicotianae*." *PLANT DISEASE* 82, no. 6

Cooper. "Elemental Sulfur and Thiol Accumulation in Tomato and Defense against

Disease Control System." In *Diseases, Biology and Taxonomy*, edited by P.E. Nelson, T.A. Tousson and R.J. Cook, 340-49. University Park, PA: Penn State Univ Press,

Fertilizers on the Control of Cymbidium Anthracnose." *Journal of Tropical &* 

Wellman, F.L. *Tropical American Plant Disease*. Metuchen, NJ: The Scarecrow Press, 1972. Wessel, M. "Shade and Nutrition of Cocoa." In *Cocoa*, edited by G.A.R. and Lass Wood, R.A.

Prommanop, and Neung Teaumroong. "Selection of Arbuscular Mycorrhizal Fungi for Citrus Growth Promotion and *Phytophthora* Suppression." *Scientia Horticulturae* 

Thomashow. "Microbial Populations Responsible for Specific Soil Suppressiveness to Plant Pathogens." *Annual review of phytopathology* 40, no. Journal Article (2002):

2 (1997): 130-35.

Service." *BioScience* 60, no. 7 (2010): 527-37.

Walker, J. C. *Plant Pathology*. New York: McGraw-Hill, 1972.

Essex: Longman Scientific and Technical, 1985.

Wrigley, G. *Coffee*: Harlow: Longman Scientific and Technical., 1988.

*Subtropical Botany* 11, no. 2 (2003): 157-60.

Cambridge, MA: CABI Pub, 2007.

128, no. 4 (2011): 423-33.

309-09.

(1968): 17-28.

(1998): 683-88.

1981.

*Australasian Plant Pathology* 31, no. 4 (2002): 391-99.

*Pratylenchus Coffeae*, and Two Arbuscular Mycorrhizal Fungi, *Acaulospora Mellea* and *Glomus Clarum* on Coffee (*Coffea Arabica* L.)." *Biology and Fertility of Soils* 26, no.

Control in Organic Coffee Production: Uncovering an Autonomous Ecosystem

Amendments, and a Biological Control Agent (*Trichoderma* Sp.) for the Management of Phytophthora Root Rot of Papaw in Far Northern Queensland."

> Banana (*Musa* spp.) is the fourth most important global food commodity after rice, wheat and maize in terms of gross value production. At present, it is grown in more than 120 countries throughout tropical and subtropical regions and it is the staple food for more than 400 million people (Molina and Valmayor, 1999). Among the production constraints, Fusarium wilt caused by the fungus *Fusarium oxysporum* f.sp *cubense* (Foc) is the most devastating disease affecting commercial and subsistence of banana production through out the banana producing areas of the world (Ploetz, 2005). The disease is ranked as one of the top 6 important plant diseases in the world (Ploetz & Pegg, 1997). In terms of crop destruction, it ranks with the few most devastating diseases such as wheat rust and potato blight (Carefoot and sprott, 1969). The disease almost destroyed the banana export industry, built on the Gros Michel variety, in Central America during the 1950's (Stover, 1962). In addition, the widely grown clones in the ABB 'Bluggoe' and AAA 'Gros Michel and Cavendish' sub groups are also highly susceptible to this disease worldwide. Presently, Fusarium wilt has been reported in all banana growing regions of the world (Asia, Africa, Australia and the tropical Americas) except some islands in the South Pacific, the Mediterranean, Melanesia, and Somalia (Stover, 1962; Anonymous, 1977; Ploetz and Pegg, 2000).

> The fungus *Foc* is the soilborne hyphomycete and is one of more than 100 formae speciales of *F. oxysporum* that causes vascular wilts of flowering plants (Domsch et al. 1980; Nelson et al. 1983). Although Fusarium wilt probably originated in Southeast Asia, (Ploetz and Pegg, 1997), the disease was first discovered at Eagle Farm, Brisbane, Queensland, Australia in 1876 in banana plants var. Sugar (Silk AAB) (Bancroft, 1876). The fungus infects the roots of banana plants, colonizing the vascular system of the rhizome and pseudostem, and inducing characteristic wilting symptoms mostly after 5-6 months of planting and the symptoms are expressed both externally and internally (Wardlaw, 1961; Stover, 1962). Generally, infected plants produce no bunches and if produced, the fruits are very small and only few fingers develop. Fruits ripen irregularly and the flesh is pithy and acidic. The fungus survives in soil for up to 30 years as chlamydospores in infested plant material or in the roots of alternative hosts (Ploetz, 2000).

> Since the discovery of Fusarium wilt of banana, though various control strategies like soil fumigation (Herbert and Marx, 1990); fungicides (Lakshmanan et al., 1987); crop rotation

Current Advances in the Fusarium Wilt Disease

and Baliad, 2005).

**3.** *Pseudomonas* **spp.** 

Management in Banana with Emphasis on Biological Control 275

reported that *T. viride* and *P. fluorescens* were equally effective in reducing the wilt incidence. Inoculation of potted abaca plants with *Trichoderma viride* and yeast showed 81.76% and 82.52% reduction of wilt disease severity respectively in the antagonist treated plants. (Bastasa

Similarly, soil application of *T. viride* NRCB1 as chaffy grain formulation significantly reduced the external (up to 78%) and internal symptoms (up to 80 %) of Fusarium wilt disease in tissue cultured as well as sucker derived plants of banana cv. Rasthali (Silk-AAB) and increased the plant growth parameters significantly as compared to the talc powder

The possible mechanisms involved in the reduction of Fusarium wilt severity due to *Trichoderma* spp. treatment might be the mycoparasitism, spatial and nutrient competition, antibiosis by enzymes and secondary metabolites, and induction of plant defence system. The mycoparasitism involves in coiling, disorganization of host cell contents and penetration of the host (Papavisas, 1985; University of Sydney, 2003). During the mycoparasitism, *Trichoderma* spp. parasitizes the hyphae of the pathogen and produce extracellular enzymes such as proteolytic enzymes, β-1, 3- glucanolytic enzymes and chitinase etc., which cause lysis of the pathogen. The toxic metabolites such as extracellular enzymes, volatiles and antibiotics like gliotoxin and viridin which are highly fungistatic substances (Weindling, 1941) are considered as elements involved in antibiosis. In addition, *Trichoderma* spp. could compete and sequester ions of iron (the ions are essential for the plant pathogen,) by releasing compounds known as siderophores (Srinivasan et al. 1992). There are several reports demonstrating control of a wide range of plant pathogens including *Fusarium* spp. by *Trichoderma* spp. by elicitation of induced systemic or localized resistance which occur due to the interaction of bioactive molecules such as proteins avr-like proteins and cell wall fragments released by the action of extracellular enzymes during mycoparasitic reaction. Thangavelu and Musataffa, (2010) reported that the application of *T. viride* NRCB1 as rice chaffy grain formulation and challenge inoculation with *Foc* in cv. Rasthali resulted in the induction of defense related enzymes such as Peroxidase and Penylalanine Ammonia lyase (PAL) and also the total phenolic content significantly higher (>50%) as compared to control and *Foc* alone inoculated banana plants and the induction was maximum at 4-6th day after treatment. They suggested that this increased activities of these lytic enzymes and thus increased content of phenols in the *T. viride* applied plants might have induced resistance against *Foc* by either making physical barrier stronger or chemically impervious to the hydrolytic enzymes produced by the pathogen (Thangavelu and Mustaffa, 2010). Morpurgo et al. (1994) reported that the activity of peroxidase was at least five times higher in the roots and corm tissues of *Foc* resistant banana variety than in the susceptible variety. Inoculation of resistant plants with *Foc* resulted in 10-fold increase in PO activity after seven days of inoculation, whereas the susceptible variety exhibited only a slight increase in PO activity.

*Pseudomonas* spp. are particularly suitable for application as agricultural biocontrol agents since they can use many exudates compounds as a nutrient source (Lugtenberg et al.1999a); abundantly present in natural soils, particularly on plant root systems, (Sands & Rovira, 1971); high growth rate, possess diverse mechanisms of actions towards phytopathogens

formulation under pot culture and field conditions (Thangavelu and Mustaffa, 2010).

(Hwang, 1985; Su et al., 1986), flood –fallowing (Wardlaw, 1961; Stover, 1962) and organic amendments (Stover, 1962) have been evolved and attempted, yet, the disease could not be controlled effectively except by planting of resistant cultivars (Moore et al., 1999). Planting of resistant varieties also cannot be implemented because of consumer preference (Viljoen, 2002). Under these circumstances, use of antagonistic microbes which protect and promote plant growth by colonizing and multiplying in both rhizosphere and plant system could be a potential alternative approach for the management of Fusarium wilt of banana.

Besides, biological control of Fusarium wilt disease has become an increasingly popular disease management consideration because of its environmental friendly nature which offers a potential alternative to the use of resistant banana varieties and the discovery of novel mechanisms of plant protection associated with certain microorganisms (Weller et al., 2002; Fravel et al., 2003). Biological control of soil borne diseases caused especially by *Fusarium oxysporum* is well documented (Marois et al., 1981; Sivan and Chet, 1986; Larkin and Fravel, 1998; Thangavelu et al., 2004). Several reports have previously demonstrated the successful use different species of *Trichoderma, Pseudomonas, Streptomyces*, non pathogenic *Fusarium* (np*Fo*) of both rhizospheric and endophytic in nature against Fusarium wilt disease under both glass house and field conditions (Lemanceau & Alabouvette, 1991; Alabouvette et al.1993; Larkin & Fravel, 1998; Weller et al. 2002; Sivamani and Gnanamanickam, 1988; Thangavelu et al. 2001; Rajappan et al. 2002; Getha et al. 2005). The details on the effect of these biocontrol agents in controlling Fusarium wilt disease of banana are discussed in detail hereunder.

#### **2.** *Trichoderma* **spp.**

*Trichoderma* spp., are free-living fungi that are common in soil and root ecosystems. They are highly interactive in root, soil and foliar environments. They produce or release a variety of compounds that induce localized or systemic resistance responses in plants. This fungal biocontrol agent has long been recognized as biological agents, for the control of plant disease and for their ability to increase root growth and development, crop productivity, resistance to abiotic stresses, and uptake and use of nutrients. It can be efficiently used as spores (especially, conidia), which are more tolerant to adverse environmental conditions during product formulation and field use, in contrast to their mycelial and chlamydospore forms as microbial propagules (Amsellem et al. 1999). However, the presence of a mycelial mass is also a key component for the production of antagonistic metabolites (Benhamou and Chet 1993; Yedidia et al. 2000). Several reports indicate that *Trichoderma* species can effectively suppress Fusarium wilt pathogens (Sivan and Chet, 1986; Thangavelu et al. 2004). Thangavelu (2002) reported that application of *T. harzianum* Th-10, as dried banana leaf formulation @ 10 g/plant containing 4X1031 cfu/g in basal + top dressing on 2, 4 and 6 months after planting in cv. Rasthali recorded the highest reduction of disease incidence (51.16%) followed by *Bacillus subtilis* or *Pseudomonas fluorescens* (41.17%) applications as talc based formulation under both glass house and field conditions. The talc based formulation of *T*. *harzianum* Th-10 and fungicide treatment recorded only 40.1% and 18.1% reduction of the disease respectively compared to control. In the Fusarium wilt-nematode interaction system also, soil application of biocontrol agents reduced significantly the wilt incidence and also the root lesion and root knot index. In addition to this, 50 to 82% of reduction in nematode population *viz., Pratylenchus coffeae* and *Meloidogyne incognita* was also noted due to application of bioagents and the maximum reduction was due to *T. harzianum* treatment (Thangavelu, 2002). Raghuchander et al. (1997)

(Hwang, 1985; Su et al., 1986), flood –fallowing (Wardlaw, 1961; Stover, 1962) and organic amendments (Stover, 1962) have been evolved and attempted, yet, the disease could not be controlled effectively except by planting of resistant cultivars (Moore et al., 1999). Planting of resistant varieties also cannot be implemented because of consumer preference (Viljoen, 2002). Under these circumstances, use of antagonistic microbes which protect and promote plant growth by colonizing and multiplying in both rhizosphere and plant system could be a potential alternative approach for the management of Fusarium wilt of banana. Besides, biological control of Fusarium wilt disease has become an increasingly popular disease management consideration because of its environmental friendly nature which offers a potential alternative to the use of resistant banana varieties and the discovery of novel mechanisms of plant protection associated with certain microorganisms (Weller et al., 2002; Fravel et al., 2003). Biological control of soil borne diseases caused especially by *Fusarium oxysporum* is well documented (Marois et al., 1981; Sivan and Chet, 1986; Larkin and Fravel, 1998; Thangavelu et al., 2004). Several reports have previously demonstrated the successful use different species of *Trichoderma, Pseudomonas, Streptomyces*, non pathogenic *Fusarium* (np*Fo*) of both rhizospheric and endophytic in nature against Fusarium wilt disease under both glass house and field conditions (Lemanceau & Alabouvette, 1991; Alabouvette et al.1993; Larkin & Fravel, 1998; Weller et al. 2002; Sivamani and Gnanamanickam, 1988; Thangavelu et al. 2001; Rajappan et al. 2002; Getha et al. 2005). The details on the effect of these biocontrol agents in

controlling Fusarium wilt disease of banana are discussed in detail hereunder.

*Trichoderma* spp., are free-living fungi that are common in soil and root ecosystems. They are highly interactive in root, soil and foliar environments. They produce or release a variety of compounds that induce localized or systemic resistance responses in plants. This fungal biocontrol agent has long been recognized as biological agents, for the control of plant disease and for their ability to increase root growth and development, crop productivity, resistance to abiotic stresses, and uptake and use of nutrients. It can be efficiently used as spores (especially, conidia), which are more tolerant to adverse environmental conditions during product formulation and field use, in contrast to their mycelial and chlamydospore forms as microbial propagules (Amsellem et al. 1999). However, the presence of a mycelial mass is also a key component for the production of antagonistic metabolites (Benhamou and Chet 1993; Yedidia et al. 2000). Several reports indicate that *Trichoderma* species can effectively suppress Fusarium wilt pathogens (Sivan and Chet, 1986; Thangavelu et al. 2004). Thangavelu (2002) reported that application of *T. harzianum* Th-10, as dried banana leaf formulation @ 10 g/plant containing 4X1031 cfu/g in basal + top dressing on 2, 4 and 6 months after planting in cv. Rasthali recorded the highest reduction of disease incidence (51.16%) followed by *Bacillus subtilis* or *Pseudomonas fluorescens* (41.17%) applications as talc based formulation under both glass house and field conditions. The talc based formulation of *T*. *harzianum* Th-10 and fungicide treatment recorded only 40.1% and 18.1% reduction of the disease respectively compared to control. In the Fusarium wilt-nematode interaction system also, soil application of biocontrol agents reduced significantly the wilt incidence and also the root lesion and root knot index. In addition to this, 50 to 82% of reduction in nematode population *viz., Pratylenchus coffeae* and *Meloidogyne incognita* was also noted due to application of bioagents and the maximum reduction was due to *T. harzianum* treatment (Thangavelu, 2002). Raghuchander et al. (1997)

**2.** *Trichoderma* **spp.** 

reported that *T. viride* and *P. fluorescens* were equally effective in reducing the wilt incidence. Inoculation of potted abaca plants with *Trichoderma viride* and yeast showed 81.76% and 82.52% reduction of wilt disease severity respectively in the antagonist treated plants. (Bastasa and Baliad, 2005).

Similarly, soil application of *T. viride* NRCB1 as chaffy grain formulation significantly reduced the external (up to 78%) and internal symptoms (up to 80 %) of Fusarium wilt disease in tissue cultured as well as sucker derived plants of banana cv. Rasthali (Silk-AAB) and increased the plant growth parameters significantly as compared to the talc powder formulation under pot culture and field conditions (Thangavelu and Mustaffa, 2010).

The possible mechanisms involved in the reduction of Fusarium wilt severity due to *Trichoderma* spp. treatment might be the mycoparasitism, spatial and nutrient competition, antibiosis by enzymes and secondary metabolites, and induction of plant defence system. The mycoparasitism involves in coiling, disorganization of host cell contents and penetration of the host (Papavisas, 1985; University of Sydney, 2003). During the mycoparasitism, *Trichoderma* spp. parasitizes the hyphae of the pathogen and produce extracellular enzymes such as proteolytic enzymes, β-1, 3- glucanolytic enzymes and chitinase etc., which cause lysis of the pathogen. The toxic metabolites such as extracellular enzymes, volatiles and antibiotics like gliotoxin and viridin which are highly fungistatic substances (Weindling, 1941) are considered as elements involved in antibiosis. In addition, *Trichoderma* spp. could compete and sequester ions of iron (the ions are essential for the plant pathogen,) by releasing compounds known as siderophores (Srinivasan et al. 1992). There are several reports demonstrating control of a wide range of plant pathogens including *Fusarium* spp. by *Trichoderma* spp. by elicitation of induced systemic or localized resistance which occur due to the interaction of bioactive molecules such as proteins avr-like proteins and cell wall fragments released by the action of extracellular enzymes during mycoparasitic reaction. Thangavelu and Musataffa, (2010) reported that the application of *T. viride* NRCB1 as rice chaffy grain formulation and challenge inoculation with *Foc* in cv. Rasthali resulted in the induction of defense related enzymes such as Peroxidase and Penylalanine Ammonia lyase (PAL) and also the total phenolic content significantly higher (>50%) as compared to control and *Foc* alone inoculated banana plants and the induction was maximum at 4-6th day after treatment. They suggested that this increased activities of these lytic enzymes and thus increased content of phenols in the *T. viride* applied plants might have induced resistance against *Foc* by either making physical barrier stronger or chemically impervious to the hydrolytic enzymes produced by the pathogen (Thangavelu and Mustaffa, 2010). Morpurgo et al. (1994) reported that the activity of peroxidase was at least five times higher in the roots and corm tissues of *Foc* resistant banana variety than in the susceptible variety. Inoculation of resistant plants with *Foc* resulted in 10-fold increase in PO activity after seven days of inoculation, whereas the susceptible variety exhibited only a slight increase in PO activity.

#### **3.** *Pseudomonas* **spp.**

*Pseudomonas* spp. are particularly suitable for application as agricultural biocontrol agents since they can use many exudates compounds as a nutrient source (Lugtenberg et al.1999a); abundantly present in natural soils, particularly on plant root systems, (Sands & Rovira, 1971); high growth rate, possess diverse mechanisms of actions towards phytopathogens

Current Advances in the Fusarium Wilt Disease

98.76% vascular discolouration (Ayyadurai et al. 2006).

*oxysporum* f. sp. *cubense* as compared to *P. fluorescens* alone inoculated soil.

bio-fertilizer and bio-control agents for banana (Weber et al. 2007).

**4.** *Bacillus* **spp.** 

Management in Banana with Emphasis on Biological Control 277

against *Foc*. Of the 56 fluorescent pseudomonad isolates obtained from banana rhizosphere, *Pseudomonas aeruginosa* strain FP10 displayed the most potent antibiosis towards the *Foc*. This strain was found to produce IAA, siderophores and phosphate-solubilizing enzyme which indicated that this strain is having potential of plant-growth-promoting ability. The presence of DAPG gene (ph1D) in the strain FP10 was confirmed by PCR and the production of DAPG was confirmed by TLC, HPLC and FT-IR analyses. The *in-vivo* bioassay carried out showed that the banana plants received with pathogen and the strain FP10 exhibited increased height (30.69cm) and reduced vascular discolouration (24.49%), whereas, the pathogen *Foc* alone-inoculated plants had an average height of 21.81 cm and

Saravanan and Muthusamy (2006) reported that soil application of talc-based formulation of *P. fluorescens* at 15 g/plant in banana, suppressed Fusarium wilt disease significantly (30.20 VDI) as compared to pathogen *Foc* alone-inoculated plants (88.89 VDI). It was found that the ability of *P. fluorescens* to suppress Fusarium wilt pathogens depends on their ability to produce antibiotic metabolites particularly 2, 4- Diacetylphloroglucinol (DAPG). The metabolite DAPG extracted from the rhizosphere of *P. fluorescens* applied to soil showed significant inhibition of growth and spore germination of *Foc*. They also showed that the quantity of DPAG production was less in the extracts of soil, inoculated with *P. fluorescens* and challenge inoculated with *F.* 

In plants pretreated with *P. fluorescens* and challenged with pathogen *Foc,* there was reduction in the number of *Foc* colonies (14 numbers) as compared to the plants treated with *Foc* alone (41 number). A 72% reduction in the pathogen infection was noticed as a result of *P.fluorescens* treatment. Colonies of *P.fluorescens* in plants challenged with *F. oxysporum* were reduced to 33 in number, perhaps due to competition for infection loci (Sukhada et al. 2004). Electron microscopic studies revealed that in the root samples of bacteria treated and pathogen challenge inoculated plants, there was extensive fungal proliferation in the cortex and had wall appositions made of electron-dense materials lining the host cortical cell wall. The wall appositions formed were highly significant in restricting the further growth of the fungus. They opined that electron-dense materials might have been produced either by the bacteria or the host tissue in response to the attacking pathogen. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue (Sukhada et al. 2004). Pre-inoculated *P. fluorescens* helped the banana plant to resist pathogen attack to some extent due to the structural modification of the root system and due to the accumulation of newly formed electron-dense molecules, which may be providing the defense mechanism to the host plant. Treatment of 'Maçã' banana (*Musa spp.;* group ABB) with endophytic diazotrophic bacteria *Herbaspirillum* (BA234) and *Burkholderia*  (AB202) also resulted in significant reduction of *Foc* unit propagules as well as increase in biomass of the plant in four and two months after plant inoculation with AB202 and BA234 respectively suggesting that these endophytic diazotrophic bacteria may be used as potential

*Bacillus subtilis* has been identified as a potential biological control agent. These strains could produce a wide range of antifungal compounds, such as subtilin, TasA, subtilosin, bacilysin,

including the production of a wide range of antagonistic metabolites (Lugtenberg et al. 1991; Dowling & O'Gara, 1994; Dunlap et al.1996; Lugtenberg et al., 1999b), easy to grow *in vitro*  and subsequently can be reintroduced into the rhizosphere (Lugtenberg et al. 1994; Rhodes & Powell, 1994) and capable of inducing a systemic resistance to pathogens (van Loon et al . 1998; Pieterse et al. 2001).

Several studies have investigated the ability of *P. fluorescens* to suppress Fusarium wilt disease of banana. Fluorescent pseudomonad species such as *Pseudomonas fluorescens* (Sakthivel and Gnanamanickam 1987), *Pseudomonas putida* (de Freitas and Germida 1991), *Pseudomonas chlororaphis* (Chin-A-Woeng et al. 1998) and *Pseudomonas aeruginosa* (Anjaiah et al. 2003) have been used to suppress pathogens as well as to promote growth and yield in many crop plants. Sivamani and Gnanamanickam (1988) reported that the seedlings of *Musa balbisiana* treated with *P.fluorescens* showed less severe wilting and internal discoloration due to *Foc* infection in green house experiments. The bacterized seedlings also showed better root growth and enhanced plant height.

Thangavelu et al. (2001) demonstrated that *P. fluorescens* strain pf10, which was isolated from the rhizosphere of banana roots, was able to detoxify the fusaric acid produced by *Foc* race-1 and reduced wilt incidence by 50%. Dipping of suckers in the suspension of *P. fluorescens* along with the application of 500 g of wheat bran and saw dust inoculation (1: 3) of the respective bio-control agent effectively reduced Fusarium wilt incidence in banana (Raghuchander et al.1997). Rajappan et al. (2002) reported that the talc based powder formulation of *P. fluorescens* strain pf1 was effective against *Foc* in the field. *Pseudomonas fluorescens* strain WCS 417, known for its ability to suppress other Fusarium wilt diseases, reduced the disease incidence by 87 4% in Cavendish bananas in glasshouse trials (Nel et al. 2006). Saravanan et al. (2003) demonstrated that either basal application of neem cake at 0.5 kg/plant + sucker dipping in spore suspension of *P. fluorescens* for 15 min+ soil application of *P. fluorescens* at 10 g/plant at 3,5 and 7 months after planting or the basal application of neem cake at 0.5 kg/plant + soil application of *P. fluorescens* at 10 g/plant at 3, 5 and 7 months after planting showed the greatest suppression of wilt disease in two field trials conducted in Tamil Nadu, India.

Fishal et al. (2010) assessed the ability of two endophytic bacteria originally isolated from healthy oil palm roots, *Pseudomonas* sp. (UPMP3) and *Burkholderia* sp. (UPMB3) to induce resistance in susceptible Berangan banana against *Fusarium oxysporum* f. sp. *cubense* race 4 (FocR4) under glasshouse conditions. The study showed that pre-inoculation of banana plants with *Pseudomonas* sp UPMP3 recorded 51% reduction of Fusarium wilt disease severity, whereas, the combined application of UPMP3+UPMB3 and single application of UPMB3 alone recorded only 39 and 38% reduction of Fusarium wilt disease severity respectively. Ting et al. (2011) reported that among six endobacteria isolates, only two isolates (*Herbaspirillum* spp and *Pseudomonas* spp.) produced volatile compounds which were capable of inhibiting the growth of *Foc* race 4. The compounds were identified as 2 pentane 3-methyl, methanethiol and 3-undecene. They found that the isolate *Herbaspirillum* spp. recorded 20.3% inhibition of growth of *Foc* race 4 as its volatile compounds contained all the three compounds whereas *Pseudomonas* isolate AVA02 recorded only 1.4% of growth inhibition of race 4 *Foc* as its volatile compounds contained only methanethiol and 3 undecene. They concluded that the presence of all these three compounds especially 2 pentane 3-methyl and also in high quantity is very important for the antifungal activity

including the production of a wide range of antagonistic metabolites (Lugtenberg et al. 1991; Dowling & O'Gara, 1994; Dunlap et al.1996; Lugtenberg et al., 1999b), easy to grow *in vitro*  and subsequently can be reintroduced into the rhizosphere (Lugtenberg et al. 1994; Rhodes & Powell, 1994) and capable of inducing a systemic resistance to pathogens (van Loon et al .

Several studies have investigated the ability of *P. fluorescens* to suppress Fusarium wilt disease of banana. Fluorescent pseudomonad species such as *Pseudomonas fluorescens* (Sakthivel and Gnanamanickam 1987), *Pseudomonas putida* (de Freitas and Germida 1991), *Pseudomonas chlororaphis* (Chin-A-Woeng et al. 1998) and *Pseudomonas aeruginosa* (Anjaiah et al. 2003) have been used to suppress pathogens as well as to promote growth and yield in many crop plants. Sivamani and Gnanamanickam (1988) reported that the seedlings of *Musa balbisiana* treated with *P.fluorescens* showed less severe wilting and internal discoloration due to *Foc* infection in green house experiments. The bacterized seedlings also showed

Thangavelu et al. (2001) demonstrated that *P. fluorescens* strain pf10, which was isolated from the rhizosphere of banana roots, was able to detoxify the fusaric acid produced by *Foc* race-1 and reduced wilt incidence by 50%. Dipping of suckers in the suspension of *P. fluorescens* along with the application of 500 g of wheat bran and saw dust inoculation (1: 3) of the respective bio-control agent effectively reduced Fusarium wilt incidence in banana (Raghuchander et al.1997). Rajappan et al. (2002) reported that the talc based powder formulation of *P. fluorescens* strain pf1 was effective against *Foc* in the field. *Pseudomonas fluorescens* strain WCS 417, known for its ability to suppress other Fusarium wilt diseases, reduced the disease incidence by 87 4% in Cavendish bananas in glasshouse trials (Nel et al. 2006). Saravanan et al. (2003) demonstrated that either basal application of neem cake at 0.5 kg/plant + sucker dipping in spore suspension of *P. fluorescens* for 15 min+ soil application of *P. fluorescens* at 10 g/plant at 3,5 and 7 months after planting or the basal application of neem cake at 0.5 kg/plant + soil application of *P. fluorescens* at 10 g/plant at 3, 5 and 7 months after planting showed the greatest suppression of wilt disease in two field trials

Fishal et al. (2010) assessed the ability of two endophytic bacteria originally isolated from healthy oil palm roots, *Pseudomonas* sp. (UPMP3) and *Burkholderia* sp. (UPMB3) to induce resistance in susceptible Berangan banana against *Fusarium oxysporum* f. sp. *cubense* race 4 (FocR4) under glasshouse conditions. The study showed that pre-inoculation of banana plants with *Pseudomonas* sp UPMP3 recorded 51% reduction of Fusarium wilt disease severity, whereas, the combined application of UPMP3+UPMB3 and single application of UPMB3 alone recorded only 39 and 38% reduction of Fusarium wilt disease severity respectively. Ting et al. (2011) reported that among six endobacteria isolates, only two isolates (*Herbaspirillum* spp and *Pseudomonas* spp.) produced volatile compounds which were capable of inhibiting the growth of *Foc* race 4. The compounds were identified as 2 pentane 3-methyl, methanethiol and 3-undecene. They found that the isolate *Herbaspirillum* spp. recorded 20.3% inhibition of growth of *Foc* race 4 as its volatile compounds contained all the three compounds whereas *Pseudomonas* isolate AVA02 recorded only 1.4% of growth inhibition of race 4 *Foc* as its volatile compounds contained only methanethiol and 3 undecene. They concluded that the presence of all these three compounds especially 2 pentane 3-methyl and also in high quantity is very important for the antifungal activity

1998; Pieterse et al. 2001).

better root growth and enhanced plant height.

conducted in Tamil Nadu, India.

against *Foc*. Of the 56 fluorescent pseudomonad isolates obtained from banana rhizosphere, *Pseudomonas aeruginosa* strain FP10 displayed the most potent antibiosis towards the *Foc*. This strain was found to produce IAA, siderophores and phosphate-solubilizing enzyme which indicated that this strain is having potential of plant-growth-promoting ability. The presence of DAPG gene (ph1D) in the strain FP10 was confirmed by PCR and the production of DAPG was confirmed by TLC, HPLC and FT-IR analyses. The *in-vivo* bioassay carried out showed that the banana plants received with pathogen and the strain FP10 exhibited increased height (30.69cm) and reduced vascular discolouration (24.49%), whereas, the pathogen *Foc* alone-inoculated plants had an average height of 21.81 cm and 98.76% vascular discolouration (Ayyadurai et al. 2006).

Saravanan and Muthusamy (2006) reported that soil application of talc-based formulation of *P. fluorescens* at 15 g/plant in banana, suppressed Fusarium wilt disease significantly (30.20 VDI) as compared to pathogen *Foc* alone-inoculated plants (88.89 VDI). It was found that the ability of *P. fluorescens* to suppress Fusarium wilt pathogens depends on their ability to produce antibiotic metabolites particularly 2, 4- Diacetylphloroglucinol (DAPG). The metabolite DAPG extracted from the rhizosphere of *P. fluorescens* applied to soil showed significant inhibition of growth and spore germination of *Foc*. They also showed that the quantity of DPAG production was less in the extracts of soil, inoculated with *P. fluorescens* and challenge inoculated with *F. oxysporum* f. sp. *cubense* as compared to *P. fluorescens* alone inoculated soil.

In plants pretreated with *P. fluorescens* and challenged with pathogen *Foc,* there was reduction in the number of *Foc* colonies (14 numbers) as compared to the plants treated with *Foc* alone (41 number). A 72% reduction in the pathogen infection was noticed as a result of *P.fluorescens* treatment. Colonies of *P.fluorescens* in plants challenged with *F. oxysporum* were reduced to 33 in number, perhaps due to competition for infection loci (Sukhada et al. 2004). Electron microscopic studies revealed that in the root samples of bacteria treated and pathogen challenge inoculated plants, there was extensive fungal proliferation in the cortex and had wall appositions made of electron-dense materials lining the host cortical cell wall. The wall appositions formed were highly significant in restricting the further growth of the fungus. They opined that electron-dense materials might have been produced either by the bacteria or the host tissue in response to the attacking pathogen. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue (Sukhada et al. 2004). Pre-inoculated *P. fluorescens* helped the banana plant to resist pathogen attack to some extent due to the structural modification of the root system and due to the accumulation of newly formed electron-dense molecules, which may be providing the defense mechanism to the host plant. Treatment of 'Maçã' banana (*Musa spp.;* group ABB) with endophytic diazotrophic bacteria *Herbaspirillum* (BA234) and *Burkholderia*  (AB202) also resulted in significant reduction of *Foc* unit propagules as well as increase in biomass of the plant in four and two months after plant inoculation with AB202 and BA234 respectively suggesting that these endophytic diazotrophic bacteria may be used as potential bio-fertilizer and bio-control agents for banana (Weber et al. 2007).

#### **4.** *Bacillus* **spp.**

*Bacillus subtilis* has been identified as a potential biological control agent. These strains could produce a wide range of antifungal compounds, such as subtilin, TasA, subtilosin, bacilysin,

Current Advances in the Fusarium Wilt Disease

with *Foc* alone (41 number).

absence of the biocontrol strain S96 (Cao et al. 2005).

**6. General mode of action of antagonistic bacteria** 

Management in Banana with Emphasis on Biological Control 279

2004). Similarly in 2005, out of 131 endophytic actinomycete strains isolated from banana roots, the most frequently isolated and siderophore producing endophytic *Streptomyces* sp. strain S96 was found to be highly antagonistic to *Foc*. The subsequent *in vivo* biocontrol assays carried out showed that the disease severity index of Fusarium wilt was significantly reduced and mean fresh weight of plantlets increased compared to those grown in the

Generally biocontrol agents can antagonize soil-borne pathogens through the following strategies: (1) Competition for niches and nutrients (niche exclusion), (2) Production of secondary metabolites which are used in direct antagonism (3) Growth promotion by

Antagonistic bacteria are more effective against root pathogens only if they have a strong ability to colonize the root system (Weller, 1988) and also the fungal hyphae. This is widely believed to be essential for biocontrol (Weller et al. 1983; deWeger et al. 1987; Parke, 1990). The scanning and transmission electron microscopy study revealed that colonization on banana roots, on the hyphal surface and macrospores of *Foc* fungus race 4, by the endophyte *Burkholderia cepacia*. The study also showed that *B. cepacia* exists mainly in the intercellular space of the banana root tissues. Benhamou et al. (1996) provided evidence that root colonization by the endophytic bacterium *Pseudomonas fluorescens,* involved in a sequence of events that included bacterial attachment to the plant roots, proliferation along the elongation root, and local penetration of the epidermis. M'Piga et al. (1997) also confirmed the entry of *P. fluorescens* into the root system and their colonization inside. Once inside the host tissue, these bacteria produce an array of antifungal metabolites like siderophores and different antibiotics like phenazine-1 carboxylic acid, and 2, 4-diacetylphloroglucinol preventing the further advancement of the fungus (Beckman et al. 1982; Mueller & Beckmann,1988) by inducing severe cell disturbances in pathogenic fungi (Dowling & O'Gara, 1994). Sukhada et al. (2004) also located the colonies of *P. fluorescens* and *Foc* in banana using respective FITC-conjugated antibodies. They found that the bacterial population was relatively greater towards the cortex region of the root as compared to the stele region. In plants pretreated with *P. fluorescens* and challenged with *Foc*, there was reduction in the number of *Foc* colonies (14 numbers) as compared to the plants treated

Competition for nutrients such as carbon, nitrogen or iron is one of the mechanisms through which biocontrol strains can reduce the ability of fungal pathogens to propagate in the soil (Alabouvette, 1986; Buyer & Leong, 1986; Leong, 1986; Loper & Buyer, 1991; Fernando et al., 1996; Handelsman & Stabb, 1996). Already established (pre-emptive competitive exclusion) or aggressively colonizing biocontrol bacteria can therefore prevent the establishment and subsequent deleterious effects of a pathogen. Most organisms, including fluorescent *Pseudomonas* species, take up ferric ions through high-affinity iron chelators, designated as siderophores that are released from bacterial cells under Fe3+ limiting conditions. The role of siderophores produced by pseudomonads has been well correlated with the biocontrol of disease suppressive soils and on the plant growth by supplying the plant with sequestered iron. Kloepper et al. (1980) reported that inhibition of the wilt pathogen was attributable to iron deprivation caused by pseudomonad siderophores compounds produced in low-iron

changing the physiology of the plant and (4) Induction of resistance to disease

mycobacillin and some enzymes, which can degrade fungal cell wall (Berg et al. 2001). It was suggested that these antibiotic production plays a major role in plant disease suppression (Knox et al. 2000; Leelasuphakul et al. 2006). In addition, some antagonistic mechanisms of these *Bacillus* species involves in the competition for nutrients and space, the induction of plant resistance, etc. (Guerra-Cantera et al., 2005; Van loon et al., 1998).

Sun et al. (2011) isolated an antagonistic *Bacillus* strain, KY-21 from the soil of banana's rhizosphere and tested against *Foc* both under *in-vitro* and *in-vivo* conditions. Under lab condition, mycelium growth of the pathogen was seriously inhibited after treatment with the fermentation filtrate of KY-21. The microscopic examination of mycelium revealed that the tips of the hypha were deformed into spherical structures that were remarkably constricted by dual culture. Besides, the inoculation of banana plants with *Bacillus* strain, KY-21 also increased the activities of polyphenol oxidase (PPO) and peroxidase (POD) significantly compared to control. The *in-vivo* biocontrol assays showed that at 60 days after *Foc* inoculation, the plantlets treated with KY-21 exhibited 35% severe wilt symptom and 18.3% severe vascular discoloration as against 68.4% and 48.3% of severe wilt symptom and severe vascular discoloration respectively in control plantlets. Besides, plantlets inoculated with KY-21 showed significantly reduced development of disease as compared to the control.

#### **5.** *Actinomycetes*

Actinomycetes particularly *Streptomyces* spp. are important soil dwelling microorganisms, generally saprophytic, spend majority of their life cycle as spores and are best known for their ability to produce antibiotics. They may influence plant growth and protect plant roots against invasion by root pathogenic fungi (Crawford et al. 1993). Streptomyces species have been used extensively in the biological control of several formae speciales of *F. oxysporum*, which caused wilt disease in many plant species (Reddi and Rao 1971; Lahdenpera and Oy, 1987; Smith et al. 1990). *Streptomyces violaceusniger* strain G10 isolated from a coastal mangrove (*Rhizophora apiculata* (Blume)] stand, was shown to exhibit strong *in-vitro* antagonism toward several plant pathogenic fungi including *Foc* race 4. Under *in-vivo* bioassay, treating the planting hole and roots of tissue-culture-derived 'Novaria' banana plantlets with *Streptomyces* sp. strain g10 suspension (108 cfu/ml), resulted in 47% reduction of leaf symptom index (LSI) and 53% of rhizome discoloration index (RDI) with reduced wilt severity when the plantlets were inoculated with 104 spores/ml *Foc* race 4 compared to untreated plantlets. However, the reduction in disease severity was not significant when plantlets were inoculated with a higher concentration (106 spores/ml) of Foc race 4 (Getha et al. 2005). Getha and Vikineswary (2002) studied the interaction between *Streptomyces violaceusniger* strain g10 and *F. oxysporum* f.sp. *cubense* and demonstrated the production of antifungal metabolites especially antibiotics by the antagonists which caused swelling, distortion, excessive branching and lysis of hyphae and inhibition of spore germination of *Foc* pathogen by the antagonist.

Among 242 actinomycete strains, isolated from the interior of leaves and roots of healthy and wilting banana plants, *Streptomyces griseorubiginosus*-like strains were the most frequently encountered strains. The screening of these strains for antagonistic activity against *Fusarium oxysporum* f. sp. *cubense* revealed that 50% of the *Streptomyces* strains isolated from healthy trees especially from the roots had antagonistic activities against *Foc* and only 27% of strains isolated from wilting trees showed the same activity (Cao et al.

mycobacillin and some enzymes, which can degrade fungal cell wall (Berg et al. 2001). It was suggested that these antibiotic production plays a major role in plant disease suppression (Knox et al. 2000; Leelasuphakul et al. 2006). In addition, some antagonistic mechanisms of these *Bacillus* species involves in the competition for nutrients and space, the

Sun et al. (2011) isolated an antagonistic *Bacillus* strain, KY-21 from the soil of banana's rhizosphere and tested against *Foc* both under *in-vitro* and *in-vivo* conditions. Under lab condition, mycelium growth of the pathogen was seriously inhibited after treatment with the fermentation filtrate of KY-21. The microscopic examination of mycelium revealed that the tips of the hypha were deformed into spherical structures that were remarkably constricted by dual culture. Besides, the inoculation of banana plants with *Bacillus* strain, KY-21 also increased the activities of polyphenol oxidase (PPO) and peroxidase (POD) significantly compared to control. The *in-vivo* biocontrol assays showed that at 60 days after *Foc* inoculation, the plantlets treated with KY-21 exhibited 35% severe wilt symptom and 18.3% severe vascular discoloration as against 68.4% and 48.3% of severe wilt symptom and severe vascular discoloration respectively in control plantlets. Besides, plantlets inoculated with KY-21 showed

Actinomycetes particularly *Streptomyces* spp. are important soil dwelling microorganisms, generally saprophytic, spend majority of their life cycle as spores and are best known for their ability to produce antibiotics. They may influence plant growth and protect plant roots against invasion by root pathogenic fungi (Crawford et al. 1993). Streptomyces species have been used extensively in the biological control of several formae speciales of *F. oxysporum*, which caused wilt disease in many plant species (Reddi and Rao 1971; Lahdenpera and Oy, 1987; Smith et al. 1990). *Streptomyces violaceusniger* strain G10 isolated from a coastal mangrove (*Rhizophora apiculata* (Blume)] stand, was shown to exhibit strong *in-vitro* antagonism toward several plant pathogenic fungi including *Foc* race 4. Under *in-vivo* bioassay, treating the planting hole and roots of tissue-culture-derived 'Novaria' banana plantlets with *Streptomyces* sp. strain g10 suspension (108 cfu/ml), resulted in 47% reduction of leaf symptom index (LSI) and 53% of rhizome discoloration index (RDI) with reduced wilt severity when the plantlets were inoculated with 104 spores/ml *Foc* race 4 compared to untreated plantlets. However, the reduction in disease severity was not significant when plantlets were inoculated with a higher concentration (106 spores/ml) of Foc race 4 (Getha et al. 2005). Getha and Vikineswary (2002) studied the interaction between *Streptomyces violaceusniger* strain g10 and *F. oxysporum* f.sp. *cubense* and demonstrated the production of antifungal metabolites especially antibiotics by the antagonists which caused swelling, distortion, excessive branching and lysis of hyphae and inhibition of spore germination of

Among 242 actinomycete strains, isolated from the interior of leaves and roots of healthy and wilting banana plants, *Streptomyces griseorubiginosus*-like strains were the most frequently encountered strains. The screening of these strains for antagonistic activity against *Fusarium oxysporum* f. sp. *cubense* revealed that 50% of the *Streptomyces* strains isolated from healthy trees especially from the roots had antagonistic activities against *Foc* and only 27% of strains isolated from wilting trees showed the same activity (Cao et al.

induction of plant resistance, etc. (Guerra-Cantera et al., 2005; Van loon et al., 1998).

significantly reduced development of disease as compared to the control.

**5.** *Actinomycetes* 

*Foc* pathogen by the antagonist.

2004). Similarly in 2005, out of 131 endophytic actinomycete strains isolated from banana roots, the most frequently isolated and siderophore producing endophytic *Streptomyces* sp. strain S96 was found to be highly antagonistic to *Foc*. The subsequent *in vivo* biocontrol assays carried out showed that the disease severity index of Fusarium wilt was significantly reduced and mean fresh weight of plantlets increased compared to those grown in the absence of the biocontrol strain S96 (Cao et al. 2005).

#### **6. General mode of action of antagonistic bacteria**

Generally biocontrol agents can antagonize soil-borne pathogens through the following strategies: (1) Competition for niches and nutrients (niche exclusion), (2) Production of secondary metabolites which are used in direct antagonism (3) Growth promotion by changing the physiology of the plant and (4) Induction of resistance to disease

Antagonistic bacteria are more effective against root pathogens only if they have a strong ability to colonize the root system (Weller, 1988) and also the fungal hyphae. This is widely believed to be essential for biocontrol (Weller et al. 1983; deWeger et al. 1987; Parke, 1990). The scanning and transmission electron microscopy study revealed that colonization on banana roots, on the hyphal surface and macrospores of *Foc* fungus race 4, by the endophyte *Burkholderia cepacia*. The study also showed that *B. cepacia* exists mainly in the intercellular space of the banana root tissues. Benhamou et al. (1996) provided evidence that root colonization by the endophytic bacterium *Pseudomonas fluorescens,* involved in a sequence of events that included bacterial attachment to the plant roots, proliferation along the elongation root, and local penetration of the epidermis. M'Piga et al. (1997) also confirmed the entry of *P. fluorescens* into the root system and their colonization inside. Once inside the host tissue, these bacteria produce an array of antifungal metabolites like siderophores and different antibiotics like phenazine-1 carboxylic acid, and 2, 4-diacetylphloroglucinol preventing the further advancement of the fungus (Beckman et al. 1982; Mueller & Beckmann,1988) by inducing severe cell disturbances in pathogenic fungi (Dowling & O'Gara, 1994). Sukhada et al. (2004) also located the colonies of *P. fluorescens* and *Foc* in banana using respective FITC-conjugated antibodies. They found that the bacterial population was relatively greater towards the cortex region of the root as compared to the stele region. In plants pretreated with *P. fluorescens* and challenged with *Foc*, there was reduction in the number of *Foc* colonies (14 numbers) as compared to the plants treated with *Foc* alone (41 number).

Competition for nutrients such as carbon, nitrogen or iron is one of the mechanisms through which biocontrol strains can reduce the ability of fungal pathogens to propagate in the soil (Alabouvette, 1986; Buyer & Leong, 1986; Leong, 1986; Loper & Buyer, 1991; Fernando et al., 1996; Handelsman & Stabb, 1996). Already established (pre-emptive competitive exclusion) or aggressively colonizing biocontrol bacteria can therefore prevent the establishment and subsequent deleterious effects of a pathogen. Most organisms, including fluorescent *Pseudomonas* species, take up ferric ions through high-affinity iron chelators, designated as siderophores that are released from bacterial cells under Fe3+ limiting conditions. The role of siderophores produced by pseudomonads has been well correlated with the biocontrol of disease suppressive soils and on the plant growth by supplying the plant with sequestered iron. Kloepper et al. (1980) reported that inhibition of the wilt pathogen was attributable to iron deprivation caused by pseudomonad siderophores compounds produced in low-iron

Current Advances in the Fusarium Wilt Disease

**7. Non-pathogenic Fusarium (np***Fo***)** 

banana plants.

Management in Banana with Emphasis on Biological Control 281

Several endophytic isolates of non-pathogenic *F. oxysporum* (np*Fo*) derived from symptomless banana roots provided some degree of protection against *Foc* race-4 for the Cavendish cultivar Williams in the green house (Gerlach et al.1999). Similarly, pretreatment of banana plants with endophytic bacterial strain UPM39B3 (*Serratia*) and fungal strain UPM31P1 (*Fusarium oxysporum*), isolated from the roots of wild bananas either singly or in combination resulted in significant increase in plant growth parameters in the FocR4 inoculated plants than the diseased plantlets that were not infected with endophytes (Ting et al. 2009). It was also observed that the diseased plantlets benefited from the improved plant growth were able to survive longer than diseased plantlets without endophytes. Nel et al. (2006) evaluated several np*Fo* and *Trichoderma* isolates obtained from suppressive soils in South Africa for the suppression of Fusarium wilt disease under glass house conditions. The results of the study indicated that two of the nonpathogenic *F. oxysporum* isolates, CAV 255 and CAV 241 recorded 87.4 and 75.0% reduction of Fusarium wilt incidence respectively. Forsyth et al. (2006) isolated three nonpathogenic *F. oxysporum* isolates from the roots of banana grown in Fusarium wilt suppressive soils and evaluated for their capability for suppressing Fusarium wilt of banana in glasshouse trials. The results showed that among the three np*Fo* isolates examined, one isolate BRIP 29089, was associated with a significant reduction in internal disease symptom development, with 25 % of plants showing mild vascular discoloration caused by *Foc* race 1 and race 4 in Lady Finger and Cavendish (cv. Williams) group of banana respectively. Interestingly, Cavendish plants treated with isolate BRIP 45952, and inoculated with *Fo*c, displayed a significant increase in internal symptom development, with 50 % of the plants showing severe vascular discolouration. Hence, it is important to understand that np*Fo* can either reduce or increase the disease severity based on the nature of the strains used and hence one should be cautious while selecting strain for disease control (Forsyth et al. 2006). Ting et al. (2008) demonstrated the potential of endophytic microorganisms in promoting the growth parameters (plant height, pseudostem diameter, root mass and total number of leaves) of their host plant by artificially introducing five isolates of bacterial and fungal strains isolated from the roots of wild bananas into both healthy and diseased banana plantlets (Berangan cv. Intan). The results indicated that among the five isolates tested the bacterial isolate UPM39B3 (*Serratia*) and fungal isolate UPM31P1 (*Fusarium oxysporum*) showed tolerance towards Fusarium wilt via improving vegetative growth of the plant. This ''tolerance'' to disease may also be attributed to direct inhibition of the pathogen through the production of antifungal compounds (White and Cole 1985; Koshino et al. 1989). Thangavelu and Jayanthi (2009) selected two np*Fo* isolates (Ro-3 and Ra-1) out of 33 obtained from banana rhizosphere soil based on mycelial growth and spore germination under *in-vitro* condition. These two np*Fo* isolates were evaluated under both pot culture and field conditions by application: (i) at planting; (ii) at planting + 2 months after planting; and (iii) at planting + 2 months after planting + 4 months after planting; in tissue-cultured as well as in sucker derived plants of cv. Rasthali (Silk-AAB). The result showed that soil application of Ro3 np*Fo* isolate three times in both tissue-cultured and sucker derived plants of banana registered 89% reduction of Fusarium wilt severity and significant increase in plant growth parameters when compared with Foc alone inoculated

environments that function in iron transport. It is suggested that the management of Fe availability in the infection court, through Fe competition, can induce suppressiveness to a Fusarium wilt pathogen.

Dowling and O'Gara (1994) reported that bacterial endophytes like *P. fluorescens* produced an array of antifungal metabolites like siderophores and different antibiotics like phenazine-1 carboxylic acid, and 2, 4-diacetylphloroglucinol that could induce severe cell disturbances in a number of pathogenic fungi. These compounds have direct effect on the growth of the pathogens. Biocontrol bacteria producing chitinase (Shapira et al., 1989; Dunne et al., 1996; Ross et al., 2000), protease (Dunlap et al., 1997; Dunne et al., 1998), cellulase (Chatterjee et al., 1995) or β glucanases (RuizDuenas & Martinez, 1996; Jijakli & Lepoivre, 1998) were shown to suppress plant diseases as these enzymes are involved in the breakdown of fungal cell walls by degrading cell wall constituents such as glucans and chitins, resulting in the destruction of pathogen structures or propagules. The bacteria also play a major role in growth promotion by producing phytohormones such as auxins, gibberellins, cytokinins and ethylene (García de Salamone et al., 2001; Remans et al., 2008). Besides promoting growth, they induce resistance in plants against pest and disease. There are two types of induced resistance exist called Systemic acquired resistance (SAR) and Induced systemic resistance (ISR). SAR is dependent on the salicylic acid pathway and is mainly associated with pathogen attack or in response to the exogenous application of chemicals such as salicylic acid and produces pathogenesis-related (PR) proteins such as β -1,3-glucanases, endo-chitinases and thaumatin-like proteins (Ward et al. 1991; Uknes et al. 1992; Rahimi et al. 1996; Van Pelt-Heerschap et al. 1999).Bacteria induced defenses in plants are expressed through structural and biochemical mechanisms. Structural mechanisms include the reinforcement of plant cell walls by deposition of newly formed molecules of callose, lignin and phenolic , occlusion of colonized vessels by gels, gums and tyloses (He et al. 2002; Jeun et al. 2004 Gordon and Martyn 1997; Olivain and Alabouvette, 1999). Whereas, the biochemical mechanism of resistance includes accumulation of secondary metabolites such as phytoalexins and production of PR proteins such as β -1,3-glucanases and chitinases. In the case of induced systemic resistance (ISR), the resistance induced only after the colonization of plant roots by bacteria. After colonization, they produce secondary metabolites and volatiles and defense related enzymes (Stougard, 2000; Han et al., 2006), which give resistance to plants. The level of defense related enzymes are known to play a crucial role in the degree of host resistance. Peroxidase (PO) and Polyphenol oxidase (PPO) are believed to be one of the most important factors of the plant's biochemical defense against pathogens, and are actively involved in the self-regulation of plant metabolism after infection (Kavitha and Umesha, 2008; Dutta et al., 2008). Peroxidase is involved in substrate oxidation and cell wall lignifications; the PPO can oxidize phenolic compounds to quinines. Both of these defense mechanisms are associated with disease resistance. ISR elicited by PGPR has shown promise in managing a wide spectrum of plant pathogens in several plant species under greenhouse and field environments (Radjacommare et al., 2004; Thangavelu et al. 2004; Murphy et al., 2003). Fishal et al. (2010) observed increased accumulation of resistance-related enzymes such as peroxidase (PO), phenylalanine ammonia lyase (PAL), lignithioglycolic acid (LTGA), and pathogenesis-related (PR) proteins (chitinase and β-1, 3 glucanase) in banana plantlets treated with endophytic bacteria UPMP3 and UPMB3 singly or as mixture under glasshouse conditions.

#### **7. Non-pathogenic Fusarium (np***Fo***)**

280 Plant Pathology

environments that function in iron transport. It is suggested that the management of Fe availability in the infection court, through Fe competition, can induce suppressiveness to a

Dowling and O'Gara (1994) reported that bacterial endophytes like *P. fluorescens* produced an array of antifungal metabolites like siderophores and different antibiotics like phenazine-1 carboxylic acid, and 2, 4-diacetylphloroglucinol that could induce severe cell disturbances in a number of pathogenic fungi. These compounds have direct effect on the growth of the pathogens. Biocontrol bacteria producing chitinase (Shapira et al., 1989; Dunne et al., 1996; Ross et al., 2000), protease (Dunlap et al., 1997; Dunne et al., 1998), cellulase (Chatterjee et al., 1995) or β glucanases (RuizDuenas & Martinez, 1996; Jijakli & Lepoivre, 1998) were shown to suppress plant diseases as these enzymes are involved in the breakdown of fungal cell walls by degrading cell wall constituents such as glucans and chitins, resulting in the destruction of pathogen structures or propagules. The bacteria also play a major role in growth promotion by producing phytohormones such as auxins, gibberellins, cytokinins and ethylene (García de Salamone et al., 2001; Remans et al., 2008). Besides promoting growth, they induce resistance in plants against pest and disease. There are two types of induced resistance exist called Systemic acquired resistance (SAR) and Induced systemic resistance (ISR). SAR is dependent on the salicylic acid pathway and is mainly associated with pathogen attack or in response to the exogenous application of chemicals such as salicylic acid and produces pathogenesis-related (PR) proteins such as β -1,3-glucanases, endo-chitinases and thaumatin-like proteins (Ward et al. 1991; Uknes et al. 1992; Rahimi et al. 1996; Van Pelt-Heerschap et al. 1999).Bacteria induced defenses in plants are expressed through structural and biochemical mechanisms. Structural mechanisms include the reinforcement of plant cell walls by deposition of newly formed molecules of callose, lignin and phenolic , occlusion of colonized vessels by gels, gums and tyloses (He et al. 2002; Jeun et al. 2004 Gordon and Martyn 1997; Olivain and Alabouvette, 1999). Whereas, the biochemical mechanism of resistance includes accumulation of secondary metabolites such as phytoalexins and production of PR proteins such as β -1,3-glucanases and chitinases. In the case of induced systemic resistance (ISR), the resistance induced only after the colonization of plant roots by bacteria. After colonization, they produce secondary metabolites and volatiles and defense related enzymes (Stougard, 2000; Han et al., 2006), which give resistance to plants. The level of defense related enzymes are known to play a crucial role in the degree of host resistance. Peroxidase (PO) and Polyphenol oxidase (PPO) are believed to be one of the most important factors of the plant's biochemical defense against pathogens, and are actively involved in the self-regulation of plant metabolism after infection (Kavitha and Umesha, 2008; Dutta et al., 2008). Peroxidase is involved in substrate oxidation and cell wall lignifications; the PPO can oxidize phenolic compounds to quinines. Both of these defense mechanisms are associated with disease resistance. ISR elicited by PGPR has shown promise in managing a wide spectrum of plant pathogens in several plant species under greenhouse and field environments (Radjacommare et al., 2004; Thangavelu et al. 2004; Murphy et al., 2003). Fishal et al. (2010) observed increased accumulation of resistance-related enzymes such as peroxidase (PO), phenylalanine ammonia lyase (PAL), lignithioglycolic acid (LTGA), and pathogenesis-related (PR) proteins (chitinase and β-1, 3 glucanase) in banana plantlets treated with endophytic bacteria UPMP3 and UPMB3 singly

Fusarium wilt pathogen.

or as mixture under glasshouse conditions.

Several endophytic isolates of non-pathogenic *F. oxysporum* (np*Fo*) derived from symptomless banana roots provided some degree of protection against *Foc* race-4 for the Cavendish cultivar Williams in the green house (Gerlach et al.1999). Similarly, pretreatment of banana plants with endophytic bacterial strain UPM39B3 (*Serratia*) and fungal strain UPM31P1 (*Fusarium oxysporum*), isolated from the roots of wild bananas either singly or in combination resulted in significant increase in plant growth parameters in the FocR4 inoculated plants than the diseased plantlets that were not infected with endophytes (Ting et al. 2009). It was also observed that the diseased plantlets benefited from the improved plant growth were able to survive longer than diseased plantlets without endophytes. Nel et al. (2006) evaluated several np*Fo* and *Trichoderma* isolates obtained from suppressive soils in South Africa for the suppression of Fusarium wilt disease under glass house conditions. The results of the study indicated that two of the nonpathogenic *F. oxysporum* isolates, CAV 255 and CAV 241 recorded 87.4 and 75.0% reduction of Fusarium wilt incidence respectively. Forsyth et al. (2006) isolated three nonpathogenic *F. oxysporum* isolates from the roots of banana grown in Fusarium wilt suppressive soils and evaluated for their capability for suppressing Fusarium wilt of banana in glasshouse trials. The results showed that among the three np*Fo* isolates examined, one isolate BRIP 29089, was associated with a significant reduction in internal disease symptom development, with 25 % of plants showing mild vascular discoloration caused by *Foc* race 1 and race 4 in Lady Finger and Cavendish (cv. Williams) group of banana respectively. Interestingly, Cavendish plants treated with isolate BRIP 45952, and inoculated with *Fo*c, displayed a significant increase in internal symptom development, with 50 % of the plants showing severe vascular discolouration. Hence, it is important to understand that np*Fo* can either reduce or increase the disease severity based on the nature of the strains used and hence one should be cautious while selecting strain for disease control (Forsyth et al. 2006). Ting et al. (2008) demonstrated the potential of endophytic microorganisms in promoting the growth parameters (plant height, pseudostem diameter, root mass and total number of leaves) of their host plant by artificially introducing five isolates of bacterial and fungal strains isolated from the roots of wild bananas into both healthy and diseased banana plantlets (Berangan cv. Intan). The results indicated that among the five isolates tested the bacterial isolate UPM39B3 (*Serratia*) and fungal isolate UPM31P1 (*Fusarium oxysporum*) showed tolerance towards Fusarium wilt via improving vegetative growth of the plant. This ''tolerance'' to disease may also be attributed to direct inhibition of the pathogen through the production of antifungal compounds (White and Cole 1985; Koshino et al. 1989). Thangavelu and Jayanthi (2009) selected two np*Fo* isolates (Ro-3 and Ra-1) out of 33 obtained from banana rhizosphere soil based on mycelial growth and spore germination under *in-vitro* condition. These two np*Fo* isolates were evaluated under both pot culture and field conditions by application: (i) at planting; (ii) at planting + 2 months after planting; and (iii) at planting + 2 months after planting + 4 months after planting; in tissue-cultured as well as in sucker derived plants of cv. Rasthali (Silk-AAB). The result showed that soil application of Ro3 np*Fo* isolate three times in both tissue-cultured and sucker derived plants of banana registered 89% reduction of Fusarium wilt severity and significant increase in plant growth parameters when compared with Foc alone inoculated banana plants.

Current Advances in the Fusarium Wilt Disease

was associated with host mineral nutrition (Ploetz, 2000).

**10. Integrated approach of Fusarium wilt management** 

In general, most of the available approaches for biocontrol of plant diseases are involved in the use of a single biocontrol agent to a single pathogen (Raupach and Kloepper, 1998). This has led to inconsistent performance of biocontrol agents and poor activity in all soil environments in which they are applied or against all pathogens that attack the host plant. To overcome these problems, applications of mixtures of biocontrol agents having multiple mode of actions are advocated particularly under field conditions, where they are highly influenced by abiotic and biotic conditions (Duffy et al., 1996; Raupach and Kloepper, 1998; Guetsky et al., 2001). Integration of biocontrol with agronomic practices may also improve the efficacy of the biocontrol organisms and the health of the host plants, which may be sensitive to environmental changes. Under this situation, compatible interactions are an important pre-requisite for the successful development of an integrated approach for the control of plant diseases. In the case of banana, integration of multiple control methods was more effective than single method for controlling Fusarium wilt disease in banana. Saravanan et al. (2003) carried out both *in-vitro* and *in-vivo* studies with biocontrol agents along with organic manures to develop integrated disease management practices to control Fusarium wilt disease. They found that basal application of neem cake at 0.5 kg/plant + sucker dipping in spore suspension of *Peudomonas fluorescens* for 15 min+soil application of *P. fluorescens* at 10 g/plant at 3,5 and 7 months after planting showed the greatest suppression of wilt disease and this was on par with basal application of neem cake at 0.5

Management in Banana with Emphasis on Biological Control 283

Fusarium wilt has been reported in many regions of the world. Although the suppression has generally been shown to be due to soil physical structure (type of soil, drainage condition, presence of montmorillonoid soils and pH) nutritional status and microbial composition (Fungi, bacteria and Actinomycetes) and biological factors also said to play a major role (Scher and Baker, 1982; Alabouvette et al. 1993). Biological control of Fusarium wilts of numerous crops by application of antagonistic fungi and bacteria isolated from suppressive soils has been accomplished during the last two decades all over the world (Leeman et al., 1996; Lemanceau et al., 1992; Park et al., 1988; Raaijmakers et al., 1995). Most of the studies have found that non-pathogenic strains of *F. oxysporum* are associated with the natural suppressiveness of soil to Fusarium wilt diseases (Smith and Snyder, 1971; Alabouvette, 1990; Postma & Rattink, 1992). These np*Fo* colonize the plant rhizosphere and roots without inducing any symptoms in the plants (Olivain and Alabouvette, 1997). Nel et al. (2006) evaluated the ability of non-pathogenic *F. oxysporum* and *Trichoderma* isolates from suppressive soils in South Africa to suppress Fusarium wilt of banana in the glasshouse. The results revealed that only npFo isolates CAV 255 and CAV 241, reduced Fusarium wilt incidence by 874 and 750%, respectively. Smith et al. (1999) proposed that application of biocontrol agents isolated from banana roots grown in Fusarium wilt suppressive soil of tissue culture plantlets in the nursery. By application of these biocontrol agents, the banana roots had a better chance of protection against *Foc*. Generally, the microbial activity in suppressive soil is influenced by type of clay minerals present in the soil. In tropical America, a close relationship was found between suppression of Fusarium wilt and presence of clay (montmorillonoid type) soils, where as in the Canary Islands, suppression

The modes of actions of non- pathogenic Fusarium isolates suggested commonly are: competition for nutrients (Couteaudier and Alabouvette, 1990), competition for infection sites at the root surface or inside the roots (Fravel et al. 2003) production of secondary metabolites, which cause antibiosis and antixenosis and induced resistance (Clay, 1991; Dubois et al. 2006). Some endophytes with growth promoting properties are also useful in enhancing tolerance to diseases by growth promotion (Ting et al. 2009).

Although the non-pathogenic Fusarium isolates are useful in controlling the Fusarium wilt disease, the main concern are: i) whether the biocontrol agent is truly nonpathogenic, ii) whether it may be pathogenic on a species of plant on which it has not yet been tested and iii) whether the biocontrol agent could become pathogenic in the future.

#### **8. Biocontrol agents for tissue cultured plants**

In the case of micro-propagated banana plants, its usage as planting material leads to a reduction in the spread of *Foc*, but at the same time, resulted in enhanced susceptibility to *Foc* under field conditions (Smith et al. 1998) due to the loss of native endophytes during tissue culture, including beneficial plant growth promoting rhizobacteria and fungi (Nowak, 1998; Smith et al.1998). Therefore, biotization of tissue culture plantlets with native effective non-pathogenic endophytic microbes including mycorrhizal fungi during first or second stage hardening but before planting, enhance plant resistance to tissue cultured plants against Fusarium wilt (Nowak, 1998). Lian et al. (2009) reported that reintroduction of naturally occurring endophytes to tissue culture banana plantlets resulted in a substantial reduction in the infection and severity of Fusarium wilt disease (67%) as well as increased plant growth parameters (height, girth, leaf area). Arbuscular mycorrhiza (AM) fungi are the most beneficial symbiotic fungi, increases nutrient uptake ability of the plant roots, by enhancing the water transport in the plant thus increasing the growth and yield. Besides, these fungi have also been shown to provide physical barrier against invading pathogens and thus reduce disease severity in short-term green house studies. The application of *Glomus* spp to micropropagated banana plantlets (Grand Naine) reduced the internal and external symptoms of *Foc* race 4 and enhanced plant development and nutrient uptake of the plants (Jaizme-vega et al. 1998). Jie et al. (2009) re-introduced mixture of naturally-occurring uncultivated endophytes (dominated by γ-Proteobacteria) isolated from native healthy banana plant into tissue culture banana plantlets led to 67% suppression rate of wilt disease at the fifth month after pathogen infection on plantlets in the greenhouse. In addition to disease suppression, growth of host plantlets was also promoted with the inoculation of these endophytes both in pathogen- infected and healthy control plants. They proposed that the suppression of wilt disease was due to increased activities of PPO, POD and SOD enzymes in the plantlets inoculated with endophytic communities.

#### **9. Suppressive soil for the biological control of Fusarium wilt**

Suppressive soils are sites where, despite the presence of a virulent pathogen and susceptible host, disease either does not develop, or the severity and spread of disease through the site is restricted (Alabouvette et al. 1993). This type of suppressive soils for

The modes of actions of non- pathogenic Fusarium isolates suggested commonly are: competition for nutrients (Couteaudier and Alabouvette, 1990), competition for infection sites at the root surface or inside the roots (Fravel et al. 2003) production of secondary metabolites, which cause antibiosis and antixenosis and induced resistance (Clay, 1991; Dubois et al. 2006). Some endophytes with growth promoting properties are also useful in

Although the non-pathogenic Fusarium isolates are useful in controlling the Fusarium wilt disease, the main concern are: i) whether the biocontrol agent is truly nonpathogenic, ii) whether it may be pathogenic on a species of plant on which it has not yet been tested and

In the case of micro-propagated banana plants, its usage as planting material leads to a reduction in the spread of *Foc*, but at the same time, resulted in enhanced susceptibility to *Foc* under field conditions (Smith et al. 1998) due to the loss of native endophytes during tissue culture, including beneficial plant growth promoting rhizobacteria and fungi (Nowak, 1998; Smith et al.1998). Therefore, biotization of tissue culture plantlets with native effective non-pathogenic endophytic microbes including mycorrhizal fungi during first or second stage hardening but before planting, enhance plant resistance to tissue cultured plants against Fusarium wilt (Nowak, 1998). Lian et al. (2009) reported that reintroduction of naturally occurring endophytes to tissue culture banana plantlets resulted in a substantial reduction in the infection and severity of Fusarium wilt disease (67%) as well as increased plant growth parameters (height, girth, leaf area). Arbuscular mycorrhiza (AM) fungi are the most beneficial symbiotic fungi, increases nutrient uptake ability of the plant roots, by enhancing the water transport in the plant thus increasing the growth and yield. Besides, these fungi have also been shown to provide physical barrier against invading pathogens and thus reduce disease severity in short-term green house studies. The application of *Glomus* spp to micropropagated banana plantlets (Grand Naine) reduced the internal and external symptoms of *Foc* race 4 and enhanced plant development and nutrient uptake of the plants (Jaizme-vega et al. 1998). Jie et al. (2009) re-introduced mixture of naturally-occurring uncultivated endophytes (dominated by γ-Proteobacteria) isolated from native healthy banana plant into tissue culture banana plantlets led to 67% suppression rate of wilt disease at the fifth month after pathogen infection on plantlets in the greenhouse. In addition to disease suppression, growth of host plantlets was also promoted with the inoculation of these endophytes both in pathogen- infected and healthy control plants. They proposed that the suppression of wilt disease was due to increased activities of PPO, POD and SOD enzymes in the plantlets

enhancing tolerance to diseases by growth promotion (Ting et al. 2009).

iii) whether the biocontrol agent could become pathogenic in the future.

**8. Biocontrol agents for tissue cultured plants** 

inoculated with endophytic communities.

**9. Suppressive soil for the biological control of Fusarium wilt** 

Suppressive soils are sites where, despite the presence of a virulent pathogen and susceptible host, disease either does not develop, or the severity and spread of disease through the site is restricted (Alabouvette et al. 1993). This type of suppressive soils for Fusarium wilt has been reported in many regions of the world. Although the suppression has generally been shown to be due to soil physical structure (type of soil, drainage condition, presence of montmorillonoid soils and pH) nutritional status and microbial composition (Fungi, bacteria and Actinomycetes) and biological factors also said to play a major role (Scher and Baker, 1982; Alabouvette et al. 1993). Biological control of Fusarium wilts of numerous crops by application of antagonistic fungi and bacteria isolated from suppressive soils has been accomplished during the last two decades all over the world (Leeman et al., 1996; Lemanceau et al., 1992; Park et al., 1988; Raaijmakers et al., 1995). Most of the studies have found that non-pathogenic strains of *F. oxysporum* are associated with the natural suppressiveness of soil to Fusarium wilt diseases (Smith and Snyder, 1971; Alabouvette, 1990; Postma & Rattink, 1992). These np*Fo* colonize the plant rhizosphere and roots without inducing any symptoms in the plants (Olivain and Alabouvette, 1997). Nel et al. (2006) evaluated the ability of non-pathogenic *F. oxysporum* and *Trichoderma* isolates from suppressive soils in South Africa to suppress Fusarium wilt of banana in the glasshouse. The results revealed that only npFo isolates CAV 255 and CAV 241, reduced Fusarium wilt incidence by 874 and 750%, respectively. Smith et al. (1999) proposed that application of biocontrol agents isolated from banana roots grown in Fusarium wilt suppressive soil of tissue culture plantlets in the nursery. By application of these biocontrol agents, the banana roots had a better chance of protection against *Foc*. Generally, the microbial activity in suppressive soil is influenced by type of clay minerals present in the soil. In tropical America, a close relationship was found between suppression of Fusarium wilt and presence of clay (montmorillonoid type) soils, where as in the Canary Islands, suppression was associated with host mineral nutrition (Ploetz, 2000).

#### **10. Integrated approach of Fusarium wilt management**

In general, most of the available approaches for biocontrol of plant diseases are involved in the use of a single biocontrol agent to a single pathogen (Raupach and Kloepper, 1998). This has led to inconsistent performance of biocontrol agents and poor activity in all soil environments in which they are applied or against all pathogens that attack the host plant. To overcome these problems, applications of mixtures of biocontrol agents having multiple mode of actions are advocated particularly under field conditions, where they are highly influenced by abiotic and biotic conditions (Duffy et al., 1996; Raupach and Kloepper, 1998; Guetsky et al., 2001). Integration of biocontrol with agronomic practices may also improve the efficacy of the biocontrol organisms and the health of the host plants, which may be sensitive to environmental changes. Under this situation, compatible interactions are an important pre-requisite for the successful development of an integrated approach for the control of plant diseases. In the case of banana, integration of multiple control methods was more effective than single method for controlling Fusarium wilt disease in banana. Saravanan et al. (2003) carried out both *in-vitro* and *in-vivo* studies with biocontrol agents along with organic manures to develop integrated disease management practices to control Fusarium wilt disease. They found that basal application of neem cake at 0.5 kg/plant + sucker dipping in spore suspension of *Peudomonas fluorescens* for 15 min+soil application of *P. fluorescens* at 10 g/plant at 3,5 and 7 months after planting showed the greatest suppression of wilt disease and this was on par with basal application of neem cake at 0.5

Current Advances in the Fusarium Wilt Disease

1. *Trichoderma viride* 

2. *Pseudomonas* spp.

4 *P. fluorescens* 

5. *Bacillus* spp.

11 γ-Proteobacteria

12. *P. fluorescens* 

13. *Bacillus subtilis* 

banana with their mode of action.

Management in Banana with Emphasis on Biological Control 285

Induction of defense related enzymes, production of

Production of volatiles (2- Pentane 3-methyl,

Diacetyl Phloroglucinol

Antibiotics, induction of defense related enzymes such as Peroxidase and Polyphenol

Increase in Polyphenol oxidase, Peroxidase, Superoxide dismutase,

Induction of defense related enzymes such as Peroxidase &Polyphenol oxidase

Induction of defense related enzymes such as Peroxidase &Polyphenol oxidase

Table 1. Summary of Bio-control agents used in the management of Fusarium wilt disease of

cortical cell wall

oxidase.

7. *Streptomyces violaceusniger* Production of Antibiotics Getha and

6. *Streptomyces violaceusniger* Production of Antibiotics Getha et al. 2005

8 Non-pathogenic Fusarium Plant growth promotion Ting et al. 2009 9 *Serratia* sp. Plant growth promotion Ting et al. 2008 10 *F. oxysporum* Plant growth promotion Ting et al. 2008

Competition for space, cell wall appositions lining the

methanethil and 3-undecene, antibiotics DAPG and Siderophore production.

Thangavelu and Mustaffa, 2010

Ting et al. 2011

Saravanan and Muthusamy, 2006

Sukhada et al. 2004

Sukhada et al. 2004

Vikineswary, 2002

Jie et al. 2009

Akila et al. 2011

Akila et al. 2011

**Sl. no Name of biocontrol agents Mode of action References** 

<sup>3</sup>*Pseudomonas aeruginosa* Production of antibiotics (2,4-

antibiotics

kg/plant + soil application of *P. fluorescens* at 10 g/plant at 3,5 and 7 months after planting. They also reported that *Trichoderma viride* applied as soil or sucker dipping or their combinations or along with the neem cake also had a significant reduction in disease index, but less than that of *P. fluorescens*. Raghuchander et al. (1997) reported that dipping of suckers in the suspension of *T.viride* along with application of 500 g of wheat bran and saw dust inoculation (1: 3) of the respective bio control agent effectively reduced Fusarium wilt incidence in banana. Kidane and Laing (2010) developed integrated method of controlling Fusarium wilt by integrating biological and agronomic control methods. Single and combined applications of non-pathogenic, endophytic *Fusarium oxysporum* N16 strain by dipping their roots in a spore suspension containing 107 cfu ml-1, *Trichoderma harzianum* Eco-T® (Plant Health Products (Pty) Ltd. KwaZulu-Natal, South Africa) @ 4L-pt at a concentration of 105 conidia ml-1 at the time of planting, monthly application of plants with 4 L of silicon solution per plant containing 900mg silicon L-1 and placing coarse macademia husks at the bottom of banana plants as mulching were tested against *F. oxysporum* f. sp. *cubense* on bananas under greenhouse and field conditions. The results showed that treatments involving combinations of nonpathogenic *F. oxysporum, T. harzianum* Eco-T®, silicon and mulch had significantly higher number of leaves, stem height and girth size than single applications of the treatments. They found that the mulching increased the growth of feeder roots and created a conducive microenvironment, thereby increased the microbial activity in the soil. The combined application of non-pathogenic Fusarium strain along with silicon also resulted in reduction of corm disease index by more than 50% and shoot yellowing and wilting by 80%. Therefore, integration of biocontrol with agronomic practices improved the efficacy of the biocontrol organisms and the health of the host plants. Recently Zhang et al. (2011) evaluated the effects of novel bio-fertilizers, which combined an amino acid fertilizer and mature pig manure compost with the antagonists *Paenibacillus polymyxa* SQR21, *Trichoderma harzianum* T37 and *Bacillus subtilis* N11 (isolated from the healthy banana roots) in a severely Fusarium wilt diseased field for the suppression of Fusarium wilt of banana as pot experiments. The results showed that the bio-organic fertilizers which contained the bio-agents significantly suppressed the incidence of wilt disease (by 64–82%), compared to the control. The best biocontrol effect was obtained in the treatment with the BIO2 that contains *Bacillus subtilis* N11. The reason for more effect might be due to the application of the antagonists in combination with suitable organic amendments.

Botanical fungicides are also gaining momentum as these are considered as an alternative source for chemicals in the management of soil borne pathogens. The active principles present in both bio-agents and botanicals may either act on the pathogen directly or induce systemic resistance in the host plants resulting in reduction of disease development (Paul and Sharma, 2002). Akila et al. (2011) tested two botanical fungicides from *Datura metel*-Wanis 20 EC and Damet 50 EC along with *Pseudomonas fluorescens*, Pf1 and *Bacillus subtilis*, TRC 54 individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). The reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase and polyphenol oxidase.

kg/plant + soil application of *P. fluorescens* at 10 g/plant at 3,5 and 7 months after planting. They also reported that *Trichoderma viride* applied as soil or sucker dipping or their combinations or along with the neem cake also had a significant reduction in disease index, but less than that of *P. fluorescens*. Raghuchander et al. (1997) reported that dipping of suckers in the suspension of *T.viride* along with application of 500 g of wheat bran and saw dust inoculation (1: 3) of the respective bio control agent effectively reduced Fusarium wilt incidence in banana. Kidane and Laing (2010) developed integrated method of controlling Fusarium wilt by integrating biological and agronomic control methods. Single and combined applications of non-pathogenic, endophytic *Fusarium oxysporum* N16 strain by dipping their roots in a spore suspension containing 107 cfu ml-1, *Trichoderma harzianum* Eco-T® (Plant Health Products (Pty) Ltd. KwaZulu-Natal, South Africa) @ 4L-pt at a concentration of 105 conidia ml-1 at the time of planting, monthly application of plants with 4 L of silicon solution per plant containing 900mg silicon L-1 and placing coarse macademia husks at the bottom of banana plants as mulching were tested against *F. oxysporum* f. sp. *cubense* on bananas under greenhouse and field conditions. The results showed that treatments involving combinations of nonpathogenic *F. oxysporum, T. harzianum* Eco-T®, silicon and mulch had significantly higher number of leaves, stem height and girth size than single applications of the treatments. They found that the mulching increased the growth of feeder roots and created a conducive microenvironment, thereby increased the microbial activity in the soil. The combined application of non-pathogenic Fusarium strain along with silicon also resulted in reduction of corm disease index by more than 50% and shoot yellowing and wilting by 80%. Therefore, integration of biocontrol with agronomic practices improved the efficacy of the biocontrol organisms and the health of the host plants. Recently Zhang et al. (2011) evaluated the effects of novel bio-fertilizers, which combined an amino acid fertilizer and mature pig manure compost with the antagonists *Paenibacillus polymyxa* SQR21, *Trichoderma harzianum* T37 and *Bacillus subtilis* N11 (isolated from the healthy banana roots) in a severely Fusarium wilt diseased field for the suppression of Fusarium wilt of banana as pot experiments. The results showed that the bio-organic fertilizers which contained the bio-agents significantly suppressed the incidence of wilt disease (by 64–82%), compared to the control. The best biocontrol effect was obtained in the treatment with the BIO2 that contains *Bacillus subtilis* N11. The reason for more effect might be due to the application of the antagonists in combination with

Botanical fungicides are also gaining momentum as these are considered as an alternative source for chemicals in the management of soil borne pathogens. The active principles present in both bio-agents and botanicals may either act on the pathogen directly or induce systemic resistance in the host plants resulting in reduction of disease development (Paul and Sharma, 2002). Akila et al. (2011) tested two botanical fungicides from *Datura metel*-Wanis 20 EC and Damet 50 EC along with *Pseudomonas fluorescens*, Pf1 and *Bacillus subtilis*, TRC 54 individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). The reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase and

suitable organic amendments.

polyphenol oxidase.


Table 1. Summary of Bio-control agents used in the management of Fusarium wilt disease of banana with their mode of action.

Current Advances in the Fusarium Wilt Disease

C

and micro conidia of *Foc*.

D

**11. Conclusion** 

Management in Banana with Emphasis on Biological Control 287

Fig. 1. A) External symptoms (yellowing and buckling of leaves) of Fusarium wilt infected banana plant. B) Brown vascular discoloration in the Pseudostem C) Brown vascular

discoloration in the corm of Fusarium wilt infected plant D) Microscopic view of both macro

Although several biocontrol agents including botanicals have been tried against Fusarium wilt disease, still this lethal disease could not be controlled completely. Besides most of the biocontrol experiments were conducted either under lab condition or green house conditions and only in few cases, field experiments were conducted. Therefore, most of the bioagents tested against Fusarium wilt of banana have not yet registered and reached the end users ie. banana growers. This is mainly because of lack of confidence on the efficacy and consistency of the bioagents in controlling the disease. Therefore, for evolving consistent and effective biological control methods for the management of Fusarium wilt disease are i) the *Foc* pathogen present in a particular area or country must be characterized thoroughly up to VCG level and the bio-agents isolated must be screened under both *in vitro*

B

A

C

286 Plant Pathology

A

B

Fig. 1. A) External symptoms (yellowing and buckling of leaves) of Fusarium wilt infected banana plant. B) Brown vascular discoloration in the Pseudostem C) Brown vascular discoloration in the corm of Fusarium wilt infected plant D) Microscopic view of both macro and micro conidia of *Foc*. D

#### **11. Conclusion**

Although several biocontrol agents including botanicals have been tried against Fusarium wilt disease, still this lethal disease could not be controlled completely. Besides most of the biocontrol experiments were conducted either under lab condition or green house conditions and only in few cases, field experiments were conducted. Therefore, most of the bioagents tested against Fusarium wilt of banana have not yet registered and reached the end users ie. banana growers. This is mainly because of lack of confidence on the efficacy and consistency of the bioagents in controlling the disease. Therefore, for evolving consistent and effective biological control methods for the management of Fusarium wilt disease are i) the *Foc* pathogen present in a particular area or country must be characterized thoroughly up to VCG level and the bio-agents isolated must be screened under both *in vitro*

Current Advances in the Fusarium Wilt Disease

process. Phytopathology, 83, 1062-1071

Applied Microbiology 91, 963-971.

& Biotechnology 20, 501–504.

Microbiology 61, 1959–1967.

Microbiology 36, 551–556.

11, 1069–1077.

30(2), 29-37

105-117.

261, 791–794.

Management in Banana with Emphasis on Biological Control 289

Bastasa, G.N., Baliad, A.A., 2005. Biological control of *Fusarium* wilt of abaca (*Fusarium* 

Beckman, C. H., Mueller, W.C., Tessier, B.J., Harrisson, N.A., 1982. Recognition and callose

Benhamou,N., Chet, I., 1993. Hyphal interactions between Trichoderma harzianum and

Benhamou, N.,. Belanger, R. R., Paulitz, T., 1996. Ultrastructural and cytochemical aspects

Berg. G., Fritze. A., Roskot. N., Smalla. K., 2001. Evaluation of potential biocontrol

Buyer, J, S., Leong, J., 1986. Iron transport-mediated antagonism between plant growth-

Cao, L., Qiu, Z., Dai, X., Tan, H., Lin, Y., Zhou, S., 2004. Isolation of endophytic

Cao, L., Qiu, Z., You, J., Tan, H., Zhou,S., 2005. Isolation and characterization of endophytic

Carefoot. G. L., Sprott, E. R., 1969. 'Famine on the Wind.' (Angus and Robertson: London) Chatterjee, A., Cui, Y., Liu, Y., Dumenyo, C. K., Chatterjee, A. K. 1995. Inactivation of *rsmA* 

Chin-A-Woeng, T.F.C., Bloemberg, G.V., Vander Bij, A.J., Vander Drift, K.M.G.M., Schripse-

Clay, K., 1991. Endophytes as antagonists of plant pests. In: Andrews J. H., Hirano, S. S.,

Couteaudier, Y., Alabouvette, C., 1990. Survival and inoculum potential of conidia and

Crawford, D.L., Lynch, J.M., Whipps, J.M., Ousley, M.A., 1993. Isolation and

Microbial ecology of leaves. Springer, New York, 331–357.

Environmental Microbiology 59, 3899–3905.

banana roots. FEMS Microbiology Letters 247, 147–152.

susceptible tomato plants. Physiological Plant Pathology 20, 1–10.

*oxysporum*) with *Trichoderma* and yeast. Philippine Journal of Crop Science (PJCS)

deposition in response to vascular infection in Fusarium wilt-resistant or

Rhizoctonia solani: Ultrastructure and gold cytochemistry of the mycoparasitic

of the interaction between *Pseudomonas fluorescens* and Ri T-DNA transformed pea roots: host response to colonization by *Phythium ultimum* Trow, Planta 199,

rhizobacteria from different host plants of *Verticillium dahliae* Kleb. Journal of

promoting and plant-deleterious *Pseudomonas* strains. *Journal of Biological Chemistry* 

actinomycetes from roots and leaves of banana (*Musa acuminata*) plants and their activities against *Fusarium oxysporum* f. sp. *Cubense*. World Journal of Microbiology

streptomycete antagonists of fusarium wilt pathogen from surface-sterilized

leads to overproduction of extracellular pectinases, cellulases, and proteases in *Erwinia carotovora* subsp. *carotovora* in the absence of the starvation/cell densitysensing signal, *N*-(3-oxohexanoyl) - L-homoserine lactone. Applied Environmental

ma, J., Kroon, B., Scheffer, R.J., Keel, C., 1998. Biocontrol by phenazine-1 carboxamide-producing Pseudomonas chlororaphis PCL1391 of tomato root rot caused by *Fusarium oxysporum* f. sp*. radicis lycopersici*. Mol Plant Microbe Interact

(eds. Huang, T.Y., 1991) Soil suppressive of banana *Fusarium* wilt in Taiwan. Plant

chlamydospores of *Fusarium oxysporum* f.sp. *lini* in soil. Canadian Journal of

characterization of actinomycete antagonists of a fungal root pathogen. Applied

and *in vivo* conditions ii) the bio-agents having multiple mode of actions and functions should be selected rather than selecting bioagents with one or two mode of actions. In addition, mixture of bioagents of different genera or mixture of fungal and bacterial bioagents along with or without fungicides or botanicals have to be tried to improve the level and extent of disease control under different environmental and soil conditions iii) the compatibility between bioagents or tolerance of bioagents to chemicals or botanicals must be tested, iv) suitable method of mass production and delivery system which support more number of propagules and long shelf life, easy to prepare and adopt must be selected, v) mass produced bioagents should be applied at right quantity (the initial inoculum level of bioagents should be more than the inoculum level of the pathogen) at the right place (at the soil around the rhizosphere) at the right time (before planting or at the time of planting and also at 2nd and 4th month after planting as booster application) and at the appropriate physiological state, vi) mass production and delivery system should be compatible with the production system of banana, vii) application of bioagents with other organic amendments which can support the survival and multiplication of bio-agents and vii) integration of biological control with other cultural or agronomic practices so that the Fusarium wilt disease can be controlled effectively.

#### **12. References**


and *in vivo* conditions ii) the bio-agents having multiple mode of actions and functions should be selected rather than selecting bioagents with one or two mode of actions. In addition, mixture of bioagents of different genera or mixture of fungal and bacterial bioagents along with or without fungicides or botanicals have to be tried to improve the level and extent of disease control under different environmental and soil conditions iii) the compatibility between bioagents or tolerance of bioagents to chemicals or botanicals must be tested, iv) suitable method of mass production and delivery system which support more number of propagules and long shelf life, easy to prepare and adopt must be selected, v) mass produced bioagents should be applied at right quantity (the initial inoculum level of bioagents should be more than the inoculum level of the pathogen) at the right place (at the soil around the rhizosphere) at the right time (before planting or at the time of planting and also at 2nd and 4th month after planting as booster application) and at the appropriate physiological state, vi) mass production and delivery system should be compatible with the production system of banana, vii) application of bioagents with other organic amendments which can support the survival and multiplication of bio-agents and vii) integration of biological control with other cultural or agronomic practices so that the Fusarium wilt

Akila, R., Rajendran, L., Harish, S,. Saveetha, K., Raguchander, T., Samiyappan. R., 2011.

Alabouvette, C., 1986. Fusarium wilt suppressive soils from the Chateaurenard region:

Alabouvette, C., 1990. Biological control of *Fusarium* wilt pathogens in suppressive soils. In:

Alabouvette, C., Lemanceau, P., Steinberg, C., 1993. Recent advances in the biological

Amsellem, Z., Zidack, N. K., Quimby, P. C., Jr., & Gressel, J. 1999. Long-term dry

Anjaiah, V., Cornelis, P., Koedam, N., 2003. Effect of genotype and root colonization in

Anonymous,1977. *Fusarium oxysporum* f. sp. *cubense*, Distribution maps of plant diseases. Map No. 31, 4th ed. Commonwealth Mycological Institute, Kew, England. Ayyadurai, N., Ravindra Naik., P Sreehari Rao. M., Sunish Kumar, R., Samrat, S.K.,

micropropagation of banana. Journal of Applied Microbiology 100, 926–937 Bancroft, J. 1876. Report of the board appointed to enquire into the cause of disease affecting

Combined application of botanical formulations and biocontrol agents for the management of *Fusarium oxysporum* f. sp. *cubense* (Foc) causing Fusarium wilt in

Horn by, D. (Ed.), Biological Control of Soil-borne Plant Pathogens. CAB

preservation of viable mycelia of two mycoherbicidal organisms. Crop Protection,

biological control of *Fusarium* wilts in pigeonpea and chickpea by *Pseudomonas* 

Manohar, M., Sakthivel, N., 2006. Isolation and characterization of a novel banana rhizosphere bacterium as fungal antagonist and microbial adjuvant in

livestock and plants. In: Votes and Proceedings 1877, Vol 3, Queensland, pp. 1011-

disease can be controlled effectively.

18, 643–649.

1038

banana. Biological Control 57, 175–183.

International, Wallingford, pp. 27–43.

reviews of a 10 year study. Agronomie 6, 273–284.

control of *Fusarium* wilts. Pesticides Science 37, 365–373.

*aeruginosa* PNA1. Canadian Journal of Microbiology 49, 85–91.

**12. References** 


Current Advances in the Fusarium Wilt Disease

Phytologist 157, 493–502.

Microbiology 47, 404–411.

Workshop. Dijon, France.

Biotechnology 32, 24-32.

Biotechnology. 28, 303 – 310.

Rev Phytopathol 35, 111–28.

1855–1869.

95.

Management in Banana with Emphasis on Biological Control 291

Fravel, D., Olivain, C., Alabouvette, C., 2003. *Fusarium oxysporum* and its biocontrol. New

García de Salamone, I.E., Hynes, R.K., Nelson, L. M., 2001. Cytokinin production by plant

Gerlach, K.S., Bentley, S., Moore, N.Y., Aitken, E.A.B., Pegg, K.G., 1999. Investigation of non-

Getha K., Vikineswary, S., Wong, W., Seki, T., Ward, A., Goodfellow, M., 2005. Evaluation of

Getha, K., Vikineswary, S., 2002. Antagonistic effects of *Streptomyces violaceusniger* strain G10

Gordon, T.R., Martyn, R. D. 1997. The evolutionary biology of *Fusarium oxysporum*. Annu

Guerra-Cantera MARV, Raymundo, A.K., (2005). Utilization of a polyphasic approach in the

Han, S.H., Lee, S.J., Moon, J.H., Yang, K.Y., Cho, B.H., Kim, K.Y., Kim, Y.W., Lee, M.C.,

Handelsman, J., Stabb, E.V., 1996. Biocontrol of soilborne plant pathogens. Plant Cell 8,

He, C.Y., Hsiang, T., Wolyn, D.J., 2002. Induction of systemic disease resistance and

Herbert, J.A., Marx, D., 1990. Short-term control of Panama disease in South Africa.

Hwang, S.C., 1985. Ecology and control of *Fusarium* wilt of banana. Plant Protection Bulletin

Jaizme-Vega M.C., Hernández, B.S., and Hernández, J.M., 1998. Interaction of arbuscular

Jeun, Y.C., Park, K.S., Kim, C., Fowler, W.D., Kloepper, J.W., 2004. Cytological observations

from the Philippines. World. J. Microb. Biot., 21: 635-644

to reduce the variability of biological control. Phytopathology 91, 621–627.

strains of *Fusarium oxysporum*. Plant Pathology 51, 225–30.

*tabaci* in tobacco. Interaction 19, 924–930.

Phytophylactica 22, 339–340.

(Taiwan) 27, 233-245.

Control 29, 34–42.

Guetsky, R., Shtienberg, D., Elad, Y., Dinoor, A., 2001. Combining biocontrol agents

growth promoting rhizobacteria and selected mutants. Canadian Journal of

pathogenic strains of *Fusarium oxysporum* for suppression of Fusarium wilt of banana in Australia. In: Alabouvette C, ed. Second International Fusarium

*Streptomyces* sp. strain G10 for suppression of Fusarium wilt and rhizosphere colonization in pot grown banana plantlets. Journal of Industrial Microbiology and

on *Fusarium oxysporum* f.sp. *cubense* race 4: Indirect evidence for the role of antibiosis in the antagonistic process. Journal of Industrial Microbiology &

taxonomic reassessment of antibiotic and enzyme-producing *Bacillus* spp. isolated

Anderson, A.J., Kim, Y.C., 2006. GacS-dependent production of 2R, 3R butanediol by Pseudomonas chlororaphis O6 is a major determinant for eliciting systemic resistance against Erwinia carotovora but not against *Pseudomonas syringae* pv.

pathogen defence responses in *Asparagus officinalis* inoculated with non-pathogenic

mycorrhizal fungi and the soil pathogen *Fusarium oxysporum* f.sp. *cubense* on the first stages of micropropagated Grande Naine banana. Acta Horticulturae 490, 285–

of cucumber plants during induced resistance elicited by rhizobacteria. Biol


de Freitas, J.R., Germida, J.J., 1991. *Pseudomonas cepacia* and *Pseudomonas putida* as winter

de Weger, L.A., van der Vlught, C.I.M., Wijfjes, A.H.M., Bakker, P.A.H.M., Schippers, B.,

Domsch, K. H., Gams, W., Anderson, T. H., 1980. Compendium of Soil Fungi, Vol. 1.

Dowling, D.N., O'Gara, F., 1994. Metabolites of Pseudomonas involved in the biocontrol of

Dubois, T., Gold, C. S., Paparu, P., Athman S., Kapindu, S., 2006. Tissue culture and the *in* 

Dunlap, C., Crowley, J. J, Moënne-Loccoz, Y., Dowling, D.N, de Bruijn FJ, O'Gara F. 1997.

Dunne, C., Delany, I., Fenton, A., O'Gara, F., 1996. Mechanisms involved in biocontrol by

Dunne, C., Moenne, L.Y., McCarthy, J., Higgins, P., Powell, J., Dowling, D., O'Gara, F., 1998.

Dutta, S., Mishra, A. K, Dileep Kumar, B.S., 2008. Induction of systemic resistance against

Fernando, W.G.D., Watson, A. K., Paulitz, T.C., 1996. The role of *Pseudomonas* spp. and

Fishal, E.M.M., Meon, S., Yun, W.M., 2010. Induction of Tolerance to Fusarium Wilt and

Forsyth, L. M., Smith, L.J., Aitken, E., A. B., 2006. Identification and Characterization of non-

rhizobacteria and rhizobia. Soil Biol. Biochem., 40: 452-461.

and topical issues, 3rd edn. Global Science Books, London, 397–409. Duffy, B.K., Simon, A., Weller, D.M., 1996. Combination of *Trichoderma koningii* with

Microbiology 37, 780–784.

Bacteriology 169, 2769–2773.

Academic Press, New York.

Plant–Microbe Interactions.

microbial inoculants. Agronomie 16, 721–729.

Agricultural Sciences in China 9, 1140-1149.

severity. Mycological Research 30, 1-7.

188–194.

299–307.

Pathology 102, 1–7.

plant disease. Trends in Biotechnology 3, 121–141.

wheat inoculants for biocontrol of *Rhizoctonia solani*. Canadian Journal of

Lugtenberg, B.J.J., 1987. Flagella of a plant growth-stimulating *Pseudomonas fluorescens* strain are required for colonization of potato roots. Journal of

*vitro* environment. Enhancing plants with endophytes: potential for ornamentals? In: Teixeira S. J., (ed) Floriculture, ornamental and plant biotechnology: advances

fluorescent pseudomonads for control of take-all on wheat. Phytopathology 86:

Biological control of *Pythium ultimum* by *Stenotrophomonas maltophilia* W81 is mediated by an extracellular proteolytic activity. Microbiology 143, 3921–3931. Dunlap, C., Delaney, I., Fenton, A., Lohrke. S., Moënne-Loccoz, Y., O'Gara, F., 1996. The

biotechnology and application of *Pseudomonas* inoculants for the biocontrol of phytopathogens, 441– 448. In: Stacey, G., Mullin, B., Gresshoff, P.M., eds. *Biology of plant microbe interactions.* St Paul, MN, USA: International Society for Molecular

Combining proteolytic and phloroglucinol-producingbacteria for improved biocontrol of *Pythium*-mediated damping-off of sugar beet. Plant Pathology 47,

fusarial wilt in pigeon pea through interaction of plant growth promoting

competition for carbon, nitrogen and iron in the enhancement of appressorium formation by *Colletotrichum coccodes* on velvetleaf. European Journal of Plant

Defense-Related Mechanisms in the Plantlets of Susceptible Berangan Banana Pre-Inoculated with *Pseudomonas* sp. (UPMP3) and *Burkholderia* sp. (UPMB3).

pathogenic *Fusarium oxysporum* capable of increasing and decreasing Fusarium wilt


Current Advances in the Fusarium Wilt Disease

Plant Pathology 50, 301–320.

Malaysia: INIBAP, 11–30.

Phytopathology 93, 1301–1307

acuminata Colla). Euphytica 75, 121-129.

fusarium wilt of banana. Plant Pathology 55, 217–223.

inoculants. *In vitro* cell development biology-Plant 34, 122-130

France, 423-436.

Management in Banana with Emphasis on Biological Control 293

Lugtenberg, B.J.J., de Weger, L.A., Bennett, J.W., 1991. Microbial stimulation of plant growth and protection from disease. Current Opinions in Biotechnology 2, 457–464. Lugtenberg, B.J.J., de Weger, L.A., Schippers, B., 1994. Bacterization to protect seed and

Woeng, T.F.C., van den Hondel, C., Kravchenko, L., Kuiper, I., Lagopodi, A.L., Mulders, I.,

Lugtenberg, B.J.J., Kravchenko, L.V., Simons, M. 1999a. Tomato seed and root exudate

M'Piga, P., Belanger, R.R., Paulitz, T.C., Benhamou, N., 1997. Increased resistance to

Marois, J.J., Mitchel, D.J., Somada, R.M. 1981. Biological control of Fusarium crown and root

Molina, A.B., Valmayor, R. V., 1999. Banana production systems in South East Asia. Bananas

Moore, N.Y., Pegg, K.G., Bentley, S., Smith, L.J., 1999. Fusarium wilt of banana: global

Morpurgo, R., Lopato, S.V., Afza, R., Novak, F.J., 1994. Selection parameters for resistance to

Mueller, W.C., Beckman C.H., 1988. Correlated light and EM studies of callose deposits in

f.sp. *lycopersici*. Physiological and Molecular Plant Pathology 33, 201–208. Murphy, J.F., Reddy, M.S., Ryu, C.M., Kloepper, J.W., Li, R., 2003. Rhizobacteria mediated

Nel, B., Steinberg, C., Labuschagne, N., Viljoen, A., 2006. The potential of nonpathogenic

Nelson, P. E., Toussoun, T. A., Marasas, W.F.O., 1983. *Fusarium* species: An illustrated Manual for identification. Pennsylvania State University Press, University Park. Nowak, J., 1998. Benefits of *in vitro* 'biotization' of plant tissue cultures with microbial

rhizosphere colonization. Environmental Microbiology 1, 439–446.

rot of tomato under field condition. Pytopathology 12, 1257-1260.

Phoelich, C., Ram, A., Tikhonovich, I., Tuinman, S., Wijffelman, C., Wijfjes A., 1999b. *Pseudomonas* genes and traits involved in tomato root colonization. In: De Wit PJGM, Bisseling, T., Stiekema, W.J., eds 1999. IC-MPMI Congress Proceedings: biology of plant–microbe interactions, Vol. 2. St Paul, MN, USA: International

sugars: composition, utilization by *Pseudomonas* bio-control strains and role in

*Fusarium oxysporum* f. sp. *radicis-lycopersici* in tomato plants treated with the endophytic bacterium *Pseudomonas fluorescens* strain 63–28. Physiology Molecular

and Food security, Pica C., Foure, E., Frison, E.A., (eds.), INIBAP, Montpellier,

problems and perspectives. In: Molina, A.B., Masdek, N.H.N. Liew, K.W, (eds). Banana Fusarium Wilt Management: Towards Sustainable Cultivation. Proceedings of the International Workshop on Banana Fusarium Wilt Disease. Kuala Lumpur,

*Fusarium oxysporum* f.sp.*cubense* race 1 and race 4 on diploid banana (Musa

vascular parenchyma cells of tomato plants inoculated with *Fusarium oxysporum*

growth promotion of tomato leads to protection against Cucumber mosaic virus.

*Fusarium oxysporum* and other biological control organisms for suppressing

rhizosphere against disease. BCPC Monograph 57, 293–302.

Society for Molecular Plant–Microbe Interactions, 324–330.

Lugtenberg, B.J.J., Dekkers, L.C., Bansraj, M., Bloemberg, G.V., Camacho, M., Chin-A-


Jie, L., Zifeng, W., Lixiang, C., Hongming, T., Patrik, I., Zide, J., Shining, Z., 2009. Artificial

originally derived from native banana plants. Biological control. 51, 427-434. Jijakli, M.H., Lepoivre, P., 1998. Characterization of an exo-beta-1, 3-glucanase produced by

Kidane, E.G., Laing, M.D., 2010. Integrated Control of Fusarium Wilt of Banana (*Musa* spp.)

Kavitha, R., Umesha, S., 2008. Regulation of defense-related enzymes associated with

Kloepper, J.L., Leong, J., Teintze, M., Schroth, M.N., 1980. *Pseudomonas* siderophores: a mechanism explaining disease-suppressive soils. Curr. Microbiol 4, 317–320. Knox O.G.G., Killham, K., Leifert, C., 2000. Effects of increased nitrate availability on the

Koshino, H., Terada, S., Yoshihara, T., Sakamura, S., Shimanuki, T., Sato, T., Tajimi, A., 1989.

Lahdenpera, M. L., Oy, K., 1987. The control of Fusarium wilt on carnation with a

Lakshmanan, P., Selvaraj, P., Mohan, S., 1987. Efficiency of different methods for the control

Larkin, R.. Fravel, D., 1998. Efficacy of various fungal and bacterial biocontrol organisms for

Leelasuphakul, W., Sivanunsakul, P., Phongpaichit, S., 2006. Purification, characterization

Leeman, M., Vanpelt, J.A., Den Ouden, F.M., Heinsbroek, M., Bakker, P.A.H.M., Schippers,

Lemanceau, P., Alabouvette, C., 1991. Biological control of *Fusarium* diseases by fluorescent *Pseudomonas* and non-pathogenic *Fusarium*. Crop Protection 10, 279-286. Lemanceau, P., Bakker, P.A.H.M., DeKogel, W.J., Alabouvette, C., Schippers, B., 1992. Effect

Leong, J., 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant

Lian, J., Wang, Z., Cao, L., Tan, H., Inderbitzin, P., Jiang, Z., Zhou, S., 2009. Articial

and synergistic activity of β-1,3- glucanase and antibiotic extract from an antagonistic Bacillus subtilis NSRS 89-24 against rice blast and sheath blight.

B., 1996. Iron availability affects induction of systematic resistance to fusarium wilt

of pseudobactin 358 production of *Pseudomonas putida* wcs 358 on suppression of fusarium wilt of carnations by non pathogenic Fusarium oxysporum FO47.

inoculation of banana tissue culture plantlets with indigenous endophytes originally derived from native banana plants. Biological Control 51, 427–434. Loper, J.E., Buyer, J.S., 1991. Siderophores in microbial interactions on plant surfaces.

the control of Fusarium wilt of tomato. Plant Disease 82, 1022-1028.

to radish by *Pseudomonas fluorescens*. Phytopathology 86, 149–155.

bacterial spot resistance in tomato. Phytoparasitica 36, 144-159.

Streptomyces preparation. Acta Horticult 216, 85– 92.

of Panama disease. Trop. Pest Manage 33, 373–376.

Applied Environmental Microbiology 58, 2978–2982.

Molecular Plant–Microbe Interaction 4, 5–13.

pathogens. Annual Review of Phytopathology 24, 187–209.

Enzym. Microb. Technol 38, 990-997.

335–343.

879, 315-321.

Ecol 15, 227-231.

771-772.

inoculation of banana tissue culture plantlets with indigenous endophytes

*Pichia anomala* strain K, antagonist of *Botrytis cinerea* on apples. Phytopathology 88,

In. Proc. IC on Banana & Plantain in Africa Eds: T. Dubois et al. Acta Horticulture,

control of plant pathogenic fungi by the soil bacterium Bacillus subtilis. Appl. Soil

A ring B aromatic sterol from stromata of *Ephichloe typhina*. Phytochemistry, 28,


Current Advances in the Fusarium Wilt Disease

BCPC Monograph 57, 303–310.

Current Microbiology 32, 151–155.

Applied Bacteriology 34, 261–275.

protection research 46, 241-254

80–87.

161.

1609–1616.

1567-1573.

1249.

1158–1164.

Management in Banana with Emphasis on Biological Control 295

Rajappan, K., Vidhyasekaran, P., Sethuraman, K., Baskaran, T. L., 2002. Development of

Raupach, G.S., Kloepper, J.W., 1998. Mixtures of plant growth-promoting rhizobacteria

Reddi, G. S., Rao. A. S., 1971. Antagonism of soil actinomycetes to some soil - borne plant

Remans, R., Beebe, S., Blair, M., Manrique, G., Tovar, E., Rao, I., Croonenborghs, A., Torres-

Rhodes, D.J., Powell, K.A., 1994. Biological seed treatments – the development process.

Ross, I. L., Alami, Y., Harvey, P.R., Achouak, W., Ryder, M.H., 2000. Genetic diversity and

RuizDuenas, F.J., Martinez, M.J., 1996. Enzymatic activities of *Trametes versicolar* and

Sakthivel, N., Gnanamanickam, S.S., 1987. Evaluation of *Pseudomonas fluorescens* for

Saravanan, T., Muthusamy, M., 2006. Influence of *Fusarium oxysporum* f. sp*. cubense* (e.f.

Saravanan, T., Muthusamy, M., Marimuthu, T., 2003. Development of integrated approach to manage the Fusarial wilt of banana. Crop Protection 22, 1117–1123. Scher, F. M., Baker, R., 1982. Effect of *Pseudomonas putida* and a synthetic iron chelator on

Shapira, R., Ordentlich, A., Chet, I., Oppenheim, A.B., 1989. Control of plant diseases by

Sivamani, E., Gnanamanickam, S. S., 1988. Biological control of *Fusarium oxysporum* f.sp. *cubense* in banana by inoculation with *Pseudomonas fluorescens*. Plant Soil 107, 3 9. Sivan, A., Chet, I., 1986. Biological control of *Fusarium* spp. in cotton, wheat and muskmelon

by *Trichoderma harzianum*. J.Phytopathol. 116, 39–47.

(Oryza sativa L.). Applied Environmental Microbiology 53, 2056–2059. Sands, D.C., Rovira, A.D., 1971. *Pseudomonas fluorescens* biotype G, the dominant fluorescent

pathogenic fungi. Indian Phytopathol 24, 649–657.

powder and capsule formulations of *Pseudomonas fluorescens* strain Pf-1 for the control of banana wilt. Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz 109,

enhance biological control of multiple cucumber pathogens. Phytopathology 88,

Gutierrez, R., El-Howeity, M., Michiels, J., Vanderleyden, J., 2008. Physiological and genetic analysis of root responsiveness to auxin-producing plant growthpromoting bacteria in common bean (*Phaseolus vulgaris* L.). Plant and Soil 302, 149–

biological control activity of novel species of closely related pseudomonads isolated from wheat field soils in South Australia. Applied Environmental Microbiology 66,

*Pleurotus eryngii* implicated in biocontrol of *Fusarium oxysporum* f. sp. *lycopersici*.

suppression of sheath rot disease and for the enhancement of grain yields in rice

pseudomonad in South Australian soils and wheat rhizospheres. Journal of

smith) Snyder and Hansen on 2, 4- diacetylphloroglucinol production by pseudomonas fluorescens migula in banana rhizosphere. Journal of plant

induction of soil suppressiveness to Fusarium wilt pathogen. Phytopathology 72,

chitinase expressed from cloned DNA in *Escherichia coli*. Phytopathology 79, 1246–


Olivain C, Alabouvette C., 1999. Process of tomato root colonization by a pathogenic strain

Olivain, C., Alabouvette, C., 1997. Colonization of tomato root by a non-pathogenic strain of

Papavizas, G.C., 1985. Trichoderma and Gliocladium: biology, ecology and potential for

Park, C.S., Paulitz, T.C., Baker, R., 1988. Biocontrol of Fusarium wilt of cucumber resulting

Parke, J. L., 1990. Population dynamics *oi Pseudomona.s cepacia* in the pea spermosphere in

Paul, P.K., Sharma, P.D., 2002. Azadirachta indica leaf extract induces resistance in barley against leaf stripe disease. Physiology and Molecular Plant Pathology 61, 3–13. Paul, P.K., Sharma, P.D., 2002. *Azadirachta indica* leaf extract induces resistance in barley against leaf stripe disease. Physiology and Molecular Plant Pathology 61, 3–13 Pieterse, C.M.J., van Pelt J.A., van Wees S.C.M., Ton, J., Leon-Kloosterziel K.M., Keurentjes

Ploetz, R. C., 2005. Panama disease, an old enemy rears its ugly head: Parts 1 and 2. In: Plant Health Progress, APSnet: Online doi:10.1094/PHP-2005-1221-01-RV. Ploetz, R. C., Pegg, K. G., 1997. *Fusarium* wilt of banana and Wallace's line: Was the disease

Ploetz, R. C., Pegg, K. G., 2000. Fusarium wilt. Pages 143-159 In: Diseases of Banana, Abacá

Ploetz, R.C., 2000. Panama disease: a classic and destructive disease of banana. Plant Health

Postma, J., Rattink, H., 1992. Biological control of Fusarium wilt of carnation with a non-

Raaijmakers, J.M., Leeman, M., van Oorschot, M.M.P., er Sluis, I.V., Schippers, b., bakker,

Radjacommare, R., Ramanathan, A., Kandan, A., Harish, S., Thambidurai, G., Sible, G.V.,

Raguchander, T., Jayashree, K., Samiyappan, R., 1997. Management of *Fusarium* wilt of

Rahimi, S., Perry, R. N., Wright, D.G. 1996. Identification of pathogenesis related proteins

relation to biocontroi of *Pythium.* Phytopathology 80, 1307-1311.

and expression. European Journal of Plant Pathology 107,51–61.

and Enset. D. R. Jones, ed. CABI Publishing, Wallingford, UK.

radish by *pseudomonas* spp. Phytopathology 85, 1075-1081.

susceptible fingermillet cultivars. Plant and Soil 266, 165–176.

species. Physiological Molecular Plant Pathology 49, 49–59.

New Phytologist 141, 497–510.

239-249.

205.

105.

Progress 10, 1–7.

*Fusarium oxysporum*. New Phytologist 137, 481-494.

biocontrol. Annu Rev Phytopathol 23, 23–54.

*Fusarium oxysporum*. Phytopathology 78, 190–4.

of *Fusarium oxysporum* f. sp. *lycopersici* in comparison with a non-pathogenic strain.

from interactions between *Pseudomonas putida* and non-pathogenic isolates of

J.J.B., Verhagen B.W.M., van Knoester, M,, dSI, Bakker, P.A.H.M., van Loon, L.C., 2001. Rhizobacteria-mediated induced systemic resistance: triggering, signalling

originally restricted to his Indo-Malayan region? Australasian Plant Pathology 26,

pathogenic isolate of *Fusarium oxysporum*. Canadian Journal of Botany 70, 1199–

P.A.h.m., 1995. Dose-response relationships in biological control of *Fusarium* wilt of

Ragupathy, N., Samiyappan, R., 2004. PGPR mediates induction of pathogenesis – related (PR) proteins against the infection of blast pathogen in resistant and

banana using antagonistic microorganisms. Journal of Biological Control 11, 101–

induced in leaves of potato plants infected with potato cyst nematodes, Globodera


Current Advances in the Fusarium Wilt Disease

13–21.

Management in Banana with Emphasis on Biological Control 297

Thangavelu,R., Jayanthi, A., 2009. RFLP analysis of rDNA-ITS regions of native non-

Ting, A.S.Y., Mah, S.W., Tee, C.S., 2011. Detection of potential volatile inhibitory compounds

Ting, A.S.Y., Meon,S., Kadir, J., Son Radu,S., Singh, G., 2008. Endophytic microorganisms as

Ting, A.S.Y., Sariah, M., Kadir, J., Gurmit, S., 2009. Field evaluation of Non- pathogenic

Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., 1992. Acquired

Van loon, L.C., Bakker, P.A., Pieterse, C.M., 1998. Systemic resistance induced by rhizospere

Van Pelt-Heerschap, H., Smit-Bakker, O., 1999. Analysis of defense-related proteins in stem

Ward, E. R, Uknes, S.J, Williams, S.C, Dincher, S.S, Wiederhold, D.L, Alexander, D.C, 1991.

Wardlaw, C.W., 1961. Banana diseases, including Plantains and Abaca. Longmans, Green

Weber, O.B., Celli R. Muniz, C.R., Aline O. Vitor, A.O., Freire, F.C.O., Valéria M. Oliveira,

Weindling, R. 1941. Experimental consideration of the mold toxin of *Gliocladium* and

Weller, D.M., 1983. Colonization of wheat roots by a fluorescent pseudomonad suppressive

Weller, D. M., 1988. Biological control of soilbome plant pathogens in the rhizosphere with

Weller, D.M., Raaijmakers, J.M., McSpadden Gardener,B.B., Thomashow, L.S., 2002.

White, J. F. Jr., Cole, G, T., 1985. Endophyte-host association in forage grasses. III. *In-vitro*  inhibition of fungi by *Acremonium coenophialum.* Mycologia, 77, 487-489. Viljoen, A., 2002. The status of Fusarium wilt (Panama disease) of banana in South Africa.

Microbial populations responsible for specific soil suppressiveness to plant

f. sp. *cubense* on plantlets of banana 'Maça' Plant Soil 298, 47–56

pathogens. Annual Review of Phytopathology 40, 309–48.

tissue of carnation inoculated with a virulent and avirulent race of Fusarium

Coordinate gene activity in response to agents that induce systemic acquired

V.M., 2007. Interaction of endophytic diazotrophic bacteria and *Fusarium oxysporum* 

f. sp*. cubense* race 4. World J Microbiol Biotechnol. 27, 229–235.

potential growth promoters of banana. BioControl 53, 541–553

den Berg. Acta Horticulture 828, 139-143.

http://bugs.bio.usyd.edu.au/plantpathology

resistance. Plant Cell 3, 1085–94.

*Trichoderma*. Phytopathology 31, 991-1003

to take-all. Phytopathology 73, 1548- 553.

bacteria. Ann. Rev, Phytopathol. 26, 379- 407.

South African Journal of Science 98, 341–344.

and Co. Ltd, London, 648.

resistance in Arabidopsis. Plant Cell 4, 645–56. University of Sydney. 2003. Disease management: Biological control.

bacteria. Annu. Rev. Phytopathol 36, 453-483.

oxysporum f. sp. dianthi. Eur J Plant Pathol 105,681–91.

pathogenic *Fusarium oxysporum* isolates and their field evaluation for the suppression of Fusarium wilt disease of banana. Australasian Plant Pathology 38,

produced by endobacteria with biocontrol properties towards *Fusarium oxysporum* 

*Fusarium oxysporum* isolates UPM31P1 and UPM39B3 for the control of *Fusarium* wilt in 'Pisang Berangan' (*Musa*, AAA). In. Proceedings on Banana crop protection for Sustainable Production and Improved Livelihoods (Eds. D. Jones and I. Van


Smith, J., Putnam, A., Nair, M., 1990. *In vitro* control of Fusarium diseases of *Asparagus* 

Smith, M. R., Hamil, S. D., Doogan, V. J., Daniells, J. W., 1999. Chracterization and early

Smith, M., Wiley, A., Searle, C., Langdon, P., Schaffer, B., Pegg, K., 1998. Micropropagated

Srinivasan, U., Staines, H. J., Bruce, A.,1992. Influence of media type on antagonistic modes of *Trichoderma* spp. against wood decay basidiomycetes, Mater. Org. 27, 301–321. Stougard, J., 2000. Regulators and regulation of legume root nodule development. Plant

Stover, R. H., 1962. Fusarial Wilt (Panama Disease) of Bananas and Other *Musa* Species.

Su, H. J., Hwang, S. C., Ko, W. H., 1986. Fusarial wilt of Cavendish bananas in Taiwan. Plant

Sukhada, M., Manamohan, M., Rawal, R.D., Chakraborty, S., Sreekantappa, H., Manjula, R.,

Sun, J.B., Peng, M., Wang, Y.G., Zhao P.J., Xia Q.Y., 2011. Isolation and characterization of

enzymes in banana. African Journal of Microbiology Research 5, 509-515. Suslow, T. V., 1982. Role of root-colonizing bacteria in plant growth. In: Mount, M. S., and

Thangavelu, R. 2002. Characterization of *Fsarium oxysporum* schlecht. f.sp. *cubense* (e.f. smith)

Thangavelu, R., and Mustaffa M.M., 2010. A Potential isolate of *Trichoderma viride*

Thangavelu, R., Palaniswami, A., Ramakrishnan, G., Sabitha, D., Muthukrishnan, S.,

Thangavelu, R., Palaniswami, A., Velazhahan, R., 2004. Mass production of *Trichoderma* 

Lakshmikantha, H.C., 2004. Interaction of *Fusarium oxysporum* f.sp. *cubense* with *Pseudomonas fluorescens* precolonized to banana roots. World Journal of

antagonistic bacteria against *Fusarium* wilt and induction of defense related

G.H. Lacy (eds), Phytopathogenic prokaryotes. Vol. I, pp. 187-223. Academic Press,

snyd. & hans. and Molecular Approaches for the Management of Fusarium Wilt of Banana. Ph.D. thesis. Tamil Nadu Agricultural University, Coimbatore,Tamil

NRCB1and its mass production for the effective management of *Fusarium* wilt disease in banana. Tree and Forestry Science and Biotechnology 4 (Special issue 2),

Velazhahan, R., 2001. Involvement of Fusaric acid detoxification by *Pseudomonas fluorescens* strain Pf10 in the biological control of Fusarium wilt of banana caused by *Fusarium oxysporum* f.sp*. cubense*. Journal of Plant Disease and Protection 108,

*harzianum* for managing *Fusarium* wilt of banana. Agriculture, Ecosytems and

of *Fusarium* wilt of sweet potato. Phytopathology 61, 1049-1051.

Commonwealth Mycological Institute, Kew, England.

Microbiology & Biotechnology 20, 651–655.

Journal of Experimental Agriculture 39,1017-023.

Chem 38, 1729–1733.

Physiology 124, 531–540.

Disease 70, 814–818.

Inc., New York,

76-84.

433-445.

Nadu, India,. 254 pp.

Environment 103, 259–263.

*officinalis* L. with a *Streptomyces* or its polyene antibiotic, faeriefungin. J Agric Food

detection of an off type from micropropagated Lady Finger bananas. Australian

bananas are more susceptible to *Fusarium* wilt than plants grown from conventional material. Australian Journal of Agricultural Research 49, 1133–1139. Smith, S. N., Snyder, W. C., 1971. Relationship of inoculum density and soil types to severity


**12** 

*Spain* 

**Epidemiology and Control of Plant Diseases** 

**The Case of Olive Knot Disease Caused by** 

*Pseudomonas savastanoi* pv. *savastanoi* (Gardan et al., 1992) (hereafter Psv, according to Vivian & Mansfield (1993)) is the causal agent of olive knot disease. It is considered one of the most serious diseases affecting olive trees (*Olea europaea* L.) in most olive growing regions worldwide and mainly in Mediterranean countries, where this crop has been growing for centuries. The disease can lead to severe damage in olive groves, causing serious losses in terms of production. This is probably the first disease clearly described in antiquity by Theophrastus (370-286 BC) (Iacobellis, 2001) and its bacterial etiology was known through the work of Savastano since 1887 (Smith & Rorer, 1904). However, there are currently many unknown facts about the epidemiology of this disease or its chemical control. Here we describe the most relevant studies performed on the epidemiology and chemical control of

Psv causes the formation of hyperplastic growth in olive trees, producing spherical knots on the trunk and branches, and less frequently on leaves and fruits (Sisto & Iacobellis, 1999; Smith, 1920). See details in figure 1. Psv infections in fresh wounds of olive trees start with a small cavity caused by the collapse of adjoining plant cells and are more frequent on trunks and branches, and rare on leaves and fruits. Subsequently, a proliferation of tissue follows the periphery of the cavity resulting in knot development (Smith, 1920; Surico, 1977). Tumor development is dependent on bacterial production of phytohormones indoleacetic acid and cytokinins (Comai & Kosuge, 1980; Iacobellis et al., 1994; Rodríguez-Moreno et al., 2008; Smidt & Kosuge, 1978; Surico et al., 1985). Besides, recent results have revealed that Psv strains contain two copies of all the genes involved in indoleacetic acid synthesis (Matas et al., 2009; Pérez-Martínez et al., 2008). It has been

**1. Introduction** 

olive knot.

**2. Biology of infection** 

*Pseudomonas savastanoi* **pv.** *savastanoi*

José M. Quesada1, Ramón Penyalver2 and María M. López2,\* *1Departamento de Protección Ambiental, Estación Experimental del Zaidín,* 

*Consejo Superior de Investigaciones Científicas (CSIC), Granada,* 

*Instituto Valenciano de Investigaciones Agrarias (IVIA), Valencia* 

*2Centro de Protección Vegetal y Biotecnología,* 

**Caused by Phytopathogenic Bacteria:** 


### **Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria: The Case of Olive Knot Disease Caused by**  *Pseudomonas savastanoi* **pv.** *savastanoi*

José M. Quesada1, Ramón Penyalver2 and María M. López2,\*

*1Departamento de Protección Ambiental, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas (CSIC), Granada, 2Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Valencia Spain* 

#### **1. Introduction**

298 Plant Pathology

Yedidia, I., Benhamou, N., Kapulnik, Y., Chet, I., 2000. Induction and accumulation of PR

*Trichoderma harzianum* strain T-203. Plant Physiol. Biochem. 38, 863-873. Zhang, N., Wu, K., He, X., Li, S., Zhang, Z., Shen, B., Yang, X., Zhang, R., Huang, Q., Shen,

colonization with *Bacillus subtilis* N11. Plant Soil. 344, 87–97

proteins activity during early stages of root colonization by the mycoparasite

Q., 2011. A new bioorganic fertilizer can effectively control banana wilt by strong

*Pseudomonas savastanoi* pv. *savastanoi* (Gardan et al., 1992) (hereafter Psv, according to Vivian & Mansfield (1993)) is the causal agent of olive knot disease. It is considered one of the most serious diseases affecting olive trees (*Olea europaea* L.) in most olive growing regions worldwide and mainly in Mediterranean countries, where this crop has been growing for centuries. The disease can lead to severe damage in olive groves, causing serious losses in terms of production. This is probably the first disease clearly described in antiquity by Theophrastus (370-286 BC) (Iacobellis, 2001) and its bacterial etiology was known through the work of Savastano since 1887 (Smith & Rorer, 1904). However, there are currently many unknown facts about the epidemiology of this disease or its chemical control. Here we describe the most relevant studies performed on the epidemiology and chemical control of olive knot.

#### **2. Biology of infection**

Psv causes the formation of hyperplastic growth in olive trees, producing spherical knots on the trunk and branches, and less frequently on leaves and fruits (Sisto & Iacobellis, 1999; Smith, 1920). See details in figure 1. Psv infections in fresh wounds of olive trees start with a small cavity caused by the collapse of adjoining plant cells and are more frequent on trunks and branches, and rare on leaves and fruits. Subsequently, a proliferation of tissue follows the periphery of the cavity resulting in knot development (Smith, 1920; Surico, 1977). Tumor development is dependent on bacterial production of phytohormones indoleacetic acid and cytokinins (Comai & Kosuge, 1980; Iacobellis et al., 1994; Rodríguez-Moreno et al., 2008; Smidt & Kosuge, 1978; Surico et al., 1985). Besides, recent results have revealed that Psv strains contain two copies of all the genes involved in indoleacetic acid synthesis (Matas et al., 2009; Pérez-Martínez et al., 2008). It has been

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

membrane vesicles from the pathogen surface (Rodríguez-Moreno et al., 2009).

the usually dry and hot summers of the olive-growing areas (Comai & Kosuge, 1980).

Subsequently, in old knots, plant cells collapse and form cavities containing large numbers of bacteria. Fisures reaching the knot surface develop inside these cavities, allowing bacteria to escape to the external surface of intact knots (Surico, 1977). However, it remains unclear how knot formation in the host benefits Psv. Knots may represent a favorable environment for bacteria to multiply and also protect them against extreme environmental conditions, such as

The causative agent of olive knot disease is not the only organism living in knots, because there are also white or yellow saprophytic bacteria characterized as *Pantoea agglomerans* (García de los Ríos, 1989), other species of enterobacteria and even putative human pathogenic bacteria (Ouzari et al., 2008). The etiologic agent of olive knot disease has only been isolated from 5 to 10% of olive knots, similarly to that observed in other Psv hosts species, such as oleander and ash (García de los Ríos, 1989). Four new bacterial species belonging to the genus *Pantoea* were proposed in a study of endophytic bacteria from olive knots associated to Psv (Rojas, 1999) and one of them known as *Erwinia toletana* has been accepted as new species (Rojas et al., 2004). These bacteria would be incorporated to the knot subsequently to the infection caused by the etiologic agent, according to García de los Ríos (1989). Indeed, a symbiotic relationship may exist between Psv and *Pantoea* (or may be other bacteria) because they are found together not only in knots but also as epiphytic bacteria in infected plants (Ercolani, 1978, 1991; Quesada et al., 2007). Furthermore, preliminary tests have shown that strains of an uncharacterized *Erwinia*, isolated from Psv-related olive

elements of xylem and phloem.

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 301

second phase, intact cells surrounding the pathogen suffer the effect of the hormones that Psv produces and increase in size (hypertrophy) followed by an abnormal cell division (hyperplasia). Finally, there is a differentiation of certain cells of the hyperplastic area,

During infection of young olive stems after inoculation, the bacteria multiply by a succession of phases which include a population increase, a stationary phase and a population decline. There is a clear parabolic trend whose maximum value depends on cultivar susceptibility (Varvaro & Surico, 1978). Pathogen multiplication inside tissues of micropropagated olive plants can reach densities of 107 to 108 cfu/ knot (Rodríguez-Moreno et al., 2008), values very similar to those previously described with 1-2 years old seedlings by Penyalver et al. (2006). The first reaction of tissue from the inoculated slit of a young stem is to renew or quickly increase cambium activity, although this depends on whether the inoculation takes place in winter, summer or spring (Surico, 1977). The increased activity of the cambium promotes the formation of two new tissue masses on both sides of the wound, which grow until their junction and form a knot. Differentiation of phloem and xylem elements, which are organized or not in vascular bundles, occurs within the new parenchyma tissue. Light microscopy shows the presence of vascular bundles of new formation in olive knots, connected with the stem vascular cylinder (Rodríguez-Moreno et al., 2009). Psv has been located in cavities formed after the collapse of intercellular plant cells, as well as in peripheral areas close to the epidermis or invading the newly formed xylem bundles (Rodríguez-Moreno et al., 2009). This could be related to the spread of the pathogen and its external output through plant exudates. Formation of bacterial aggregates, microcolonies and multilayer biofilms has been observed in knot sections by scanning electron microscopy. Besides, TEM analysis of knot sections shows the release of outer

reported that olive knots are also dependent on the *hrp/hrc* genes (Sisto et al., 2004), which encode the biosynthesis of a functional Type III Secretion System (TTSS). Recently, remarkable progress has been made in research into several aspects of the host-pathogen interaction of the causal agent of olive knot (Pérez-Martínez et al., 2008; Matas, 2010; Pérez-Martínez et al., 2010). Several putative virulence factors in Psv have been identified, including TTSS protein effectors and a variety of genes encoding known *P. syringae* virulence determinants (Pérez-Martínez et al., 2008). Analyses of TTSS protein effectors of Psv have recently shed light on the role of TTSS in pathogenicity and host range (Matas, 2010; Pérez-Martínez et al., 2010).

Fig. 1. Typical olive knot symptoms caused by *Pseudomonas savastanoi* pv. *savastanoi* on twigs (upper left), leaf (upper right), branches (lower left) and fruits (lower right).

Anatomical studies of knots have been performed in olive (Smith, 1920; Surico, 1977), oleander (Wilson & Magie, 1964; Wilson, 1965) and more recently, in buckthorn (Temsah et al., 2007a) and myrtle (Temsah et al., 2007b) by light microscopy. Rodríguez-Moreno et al. (2009) performed the first real-time monitoring of Psv disease development and the first illustrated description of the ultrastructure of Psv induced knots. They examined knot sections using a green fluorescent protein tagging a Psv strain, coupled with epifluorescence microscopy and scanning confocal electron microscopy. Additionally, scanning and TEM (transmission electron microscopy) were used for a detailed ultrastructural analysis within knot tissues (Rodríguez-Moreno et al., 2009).

Infection by Psv and subsequent knot formation in young twigs of oleander (*Nerium oleander*) requires vascular cambium activity (Wilson, 1965). The host invasion by the bacterium begins with the colonization of the infection site, followed by the disintegration and breakdown of adjacent plant cells that results in the formation of a large cavity around the area colonized by the bacteria. Curiously, this bacterium produces cell wall degrading enzymes *in vitro* such as cellulase, cellobiase, xylanase and peptinase (Magie, 1963). In a

reported that olive knots are also dependent on the *hrp/hrc* genes (Sisto et al., 2004), which encode the biosynthesis of a functional Type III Secretion System (TTSS). Recently, remarkable progress has been made in research into several aspects of the host-pathogen interaction of the causal agent of olive knot (Pérez-Martínez et al., 2008; Matas, 2010; Pérez-Martínez et al., 2010). Several putative virulence factors in Psv have been identified, including TTSS protein effectors and a variety of genes encoding known *P. syringae* virulence determinants (Pérez-Martínez et al., 2008). Analyses of TTSS protein effectors of Psv have recently shed light on the role of TTSS in pathogenicity and host range (Matas,

Fig. 1. Typical olive knot symptoms caused by *Pseudomonas savastanoi* pv. *savastanoi* on twigs

Anatomical studies of knots have been performed in olive (Smith, 1920; Surico, 1977), oleander (Wilson & Magie, 1964; Wilson, 1965) and more recently, in buckthorn (Temsah et al., 2007a) and myrtle (Temsah et al., 2007b) by light microscopy. Rodríguez-Moreno et al. (2009) performed the first real-time monitoring of Psv disease development and the first illustrated description of the ultrastructure of Psv induced knots. They examined knot sections using a green fluorescent protein tagging a Psv strain, coupled with epifluorescence microscopy and scanning confocal electron microscopy. Additionally, scanning and TEM (transmission electron microscopy) were used for a detailed ultrastructural analysis within

Infection by Psv and subsequent knot formation in young twigs of oleander (*Nerium oleander*) requires vascular cambium activity (Wilson, 1965). The host invasion by the bacterium begins with the colonization of the infection site, followed by the disintegration and breakdown of adjacent plant cells that results in the formation of a large cavity around the area colonized by the bacteria. Curiously, this bacterium produces cell wall degrading enzymes *in vitro* such as cellulase, cellobiase, xylanase and peptinase (Magie, 1963). In a

(upper left), leaf (upper right), branches (lower left) and fruits (lower right).

knot tissues (Rodríguez-Moreno et al., 2009).

2010; Pérez-Martínez et al., 2010).

second phase, intact cells surrounding the pathogen suffer the effect of the hormones that Psv produces and increase in size (hypertrophy) followed by an abnormal cell division (hyperplasia). Finally, there is a differentiation of certain cells of the hyperplastic area, elements of xylem and phloem.

During infection of young olive stems after inoculation, the bacteria multiply by a succession of phases which include a population increase, a stationary phase and a population decline. There is a clear parabolic trend whose maximum value depends on cultivar susceptibility (Varvaro & Surico, 1978). Pathogen multiplication inside tissues of micropropagated olive plants can reach densities of 107 to 108 cfu/ knot (Rodríguez-Moreno et al., 2008), values very similar to those previously described with 1-2 years old seedlings by Penyalver et al. (2006). The first reaction of tissue from the inoculated slit of a young stem is to renew or quickly increase cambium activity, although this depends on whether the inoculation takes place in winter, summer or spring (Surico, 1977). The increased activity of the cambium promotes the formation of two new tissue masses on both sides of the wound, which grow until their junction and form a knot. Differentiation of phloem and xylem elements, which are organized or not in vascular bundles, occurs within the new parenchyma tissue. Light microscopy shows the presence of vascular bundles of new formation in olive knots, connected with the stem vascular cylinder (Rodríguez-Moreno et al., 2009). Psv has been located in cavities formed after the collapse of intercellular plant cells, as well as in peripheral areas close to the epidermis or invading the newly formed xylem bundles (Rodríguez-Moreno et al., 2009). This could be related to the spread of the pathogen and its external output through plant exudates. Formation of bacterial aggregates, microcolonies and multilayer biofilms has been observed in knot sections by scanning electron microscopy. Besides, TEM analysis of knot sections shows the release of outer membrane vesicles from the pathogen surface (Rodríguez-Moreno et al., 2009).

Subsequently, in old knots, plant cells collapse and form cavities containing large numbers of bacteria. Fisures reaching the knot surface develop inside these cavities, allowing bacteria to escape to the external surface of intact knots (Surico, 1977). However, it remains unclear how knot formation in the host benefits Psv. Knots may represent a favorable environment for bacteria to multiply and also protect them against extreme environmental conditions, such as the usually dry and hot summers of the olive-growing areas (Comai & Kosuge, 1980).

The causative agent of olive knot disease is not the only organism living in knots, because there are also white or yellow saprophytic bacteria characterized as *Pantoea agglomerans* (García de los Ríos, 1989), other species of enterobacteria and even putative human pathogenic bacteria (Ouzari et al., 2008). The etiologic agent of olive knot disease has only been isolated from 5 to 10% of olive knots, similarly to that observed in other Psv hosts species, such as oleander and ash (García de los Ríos, 1989). Four new bacterial species belonging to the genus *Pantoea* were proposed in a study of endophytic bacteria from olive knots associated to Psv (Rojas, 1999) and one of them known as *Erwinia toletana* has been accepted as new species (Rojas et al., 2004). These bacteria would be incorporated to the knot subsequently to the infection caused by the etiologic agent, according to García de los Ríos (1989). Indeed, a symbiotic relationship may exist between Psv and *Pantoea* (or may be other bacteria) because they are found together not only in knots but also as epiphytic bacteria in infected plants (Ercolani, 1978, 1991; Quesada et al., 2007). Furthermore, preliminary tests have shown that strains of an uncharacterized *Erwinia*, isolated from Psv-related olive

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

*agglomerans* with 51, 6.7 and 6%, respectively (Ercolani, 1991).

plating serial dilution of the washings.

(Varvaro & Martella, 1993).

among leaves of the same tree.

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 303

(Ercolani, 1971, 1978, 1979, 1983, 1985, 1991, 1993) and southeastern Spain (Quesada et al., 2007; Quesada et al., 2010a; 2010b). In the aforementioned studies, epiphytic Psv populations in the phyllosphere of olive trees were estimated by washing, followed by

Microbial communities of the olive tree phylloplane can grow embedded in a matrix of exopolysaccharides and form biofilms adhered to the leaf surface (Morris et al., 1997). Furthermore, a great diversity of pigmented bacteria colonizing the surface of olive tree was observed by washing olive leaves and plating the washings. In Italy, Ercolani collected bacteria from the leaf surfaces of olive trees for two sampling periods over several years in the 70s and 80s (Ercolani, 1978, 1991). Phenotypic characterization of these isolates allowed Ercolani to record over 20 bacterial species colonizing the leaf surface. The three highest frequency values of occurrence corresponded to Psv, *Xanthomonas campestris* and *Pantoea* 

Spanish studies found that averages of the total bacterial population from leaves and stems were generally significantly higher in Psv-inoculated than in non-inoculated olive trees, suggesting that Psv might have a positive effect on the growth of other epiphytic bacteria or on their ability to colonize olive organs (Quesada et al., 2010a). Populations of *P. agglomerans* could accompany Psv and contribute to the significant differences in total bacterial populations between inoculated and non-inoculated olive trees. Besides, there was a positive correlation between Psv and yellow *P. agglomerans* either on stems or leaf surfaces of naturally infected olive trees (Quesada et al., 2007), and a similar fluctuation of both bacterial populations on the same host. This is of interest because both bacteria produce indoleacetic acid and this can contribute to the epiphytic fitness of Psv in olive trees

The bacterial community composition on the surface of olive leaves is more strongly influenced by the sampling season than by leaf age (Ercolani, 1991). The diversity and size of the total bacterial populations within the olive phyllosphere were lower during the hot dry months and higher during the cold rainy months (Ercolani, 1991). Our observations on one olive orchard showed that seasonal fluctuations of Psv populations felt into the pattern of seasonal shifts described above (Quesada et al., 2007). Interestingly, Psv population sizes in stems and leaf surfaces were correlated (with r2 values of 0.7 and 0.43, respectively) with rainfall, temperature and relative humidity (Quesada et al., 2007). Therefore, these climatic parameters may exert a more or less strong influence on the Psv population values. Epiphytic bacterial communities on olive leaves were more uniform in mature leaves than in young leaves (Ercolani, 1991). In addition, the olive phyllosphere apparently selects specific genotypes of the bacterial community (Lindow & Brandl, 2003). This gives us an idea of the great variability, in terms of epiphytic populations, existing

Over 50% of bacterial isolates collected from olive leaves by Ercolani (1978, 1991) were identified as Psv and this bacterium survived and multiplied on the leaf surfaces of olive trees (Varvaro & Ferrulli, 1983). Abu-Ghorrah (1988) observed maximum Psv population levels of about 107 cfu/cm2 in olive trees and the Psv generation time in this host was 24 to 36 hours. In studies of leaves inoculated by spraying a suspension of Psv, the bacteria

knots, could have a synergistic effect with Psv in the development of typical symptoms of olive knot disease (Fernandes & Marcelo, 2002). Similar results have been reported in olive trees coinoculated with strains of Psv and *P. agglomerans*, which produced larger knots than inoculations with Psv strains alone and this effect could be due to auxin production by *P. agglomerans* (Marchi et al., 2006).

#### **3. General characteristics of phyllosphere habitats**

The surfaces of the aerial parts of plants, including stems, buds, flowers and mainly leaves, can be denominated phyllosphere (Lindow & Brandl, 2003). The leaf surface is a large microbial habitat and Morris & Kinkel (2002) estimated that in total terrestrial surface area there would be about 4x108 km2 of leaf surface area, which could be colonized by 1026 bacteria.

Leaf-borne microbial communities are diverse and include many different genera of bacteria, filamentous fungi, yeasts, algae and, less frequently, protozoa and nematodes (Lindow & Brandl, 2003). The phyllosphere of field plants is a harsh environment for bacteria. This habitat is severe because they are exposed to extreme microclimatic changes in temperature, relative humidity, wind speed, radiation, etc. in time periods as short as hours. Plant nutrient resources are scarce and accordingly plants like olive trees have a thicker cuticle (Lindow & Andersen, 1996). The phyllosphere may also be exposed to long dry periods or, conversely, heavy rains could dramatically alter the microbiota by the "washing" effect (Hirano & Upper, 2000).

The phyllosphere colonizing microorganisms are called epiphytes and Psv is one of them. A bacterium is considered as epiphytic when it lives and multiplies in the phyllosphere and constitutes the main colonizing group of the leaf surface, with average numbers from 106 to 107 cfu/cm2 of leaf (Hirano & Upper, 1983, 2000). Interestingly, not all epiphytic bacteria colonizing the phyllosphere have a strictly commensal relationship with their host plant. This has also been demonstrated in many plant pathogenic bacteria including Psv, which have also an epiphytic resident phase (Ercolani, 1971).

The size and composition of epiphytic bacterial populations vary according to plant characteristics (species, age) and factors related to nutritional and climatic conditions (Hirano & Upper, 2000; Lindow & Brandl, 2003). The size estimation of epiphytic bacterial populations in the laboratory depends on the sampling, bacteriological and statistical procedures used (Hirano & Upper, 2000; Jacques & Morris, 1995). Most studies about epiphytic bacteria have estimated population sizes by washing or sonication to release bacteria from the leaf, followed by plating of serial dilution of the washings (Hirano & Upper, 2000). The estimated population sizes with this technique correspond to a proportion of total microorganisms living in the phyllosphere (Wilson & Lindow, 1992). The error involved in the recovery of epiphytic bacteria on solid medium is not important compared to the high variability of populations in field samples, which could range from 5 to 6 orders of magnitude (Hirano & Upper, 2000).

#### **4. Epiphytic populations of Psv**

There are few studies on the epidemiology of olive knot disease and most data of epiphytic Psv populations on the aerial surface of the tree come from studies done in southern Italy

knots, could have a synergistic effect with Psv in the development of typical symptoms of olive knot disease (Fernandes & Marcelo, 2002). Similar results have been reported in olive trees coinoculated with strains of Psv and *P. agglomerans*, which produced larger knots than inoculations with Psv strains alone and this effect could be due to auxin

The surfaces of the aerial parts of plants, including stems, buds, flowers and mainly leaves, can be denominated phyllosphere (Lindow & Brandl, 2003). The leaf surface is a large microbial habitat and Morris & Kinkel (2002) estimated that in total terrestrial surface area there would

Leaf-borne microbial communities are diverse and include many different genera of bacteria, filamentous fungi, yeasts, algae and, less frequently, protozoa and nematodes (Lindow & Brandl, 2003). The phyllosphere of field plants is a harsh environment for bacteria. This habitat is severe because they are exposed to extreme microclimatic changes in temperature, relative humidity, wind speed, radiation, etc. in time periods as short as hours. Plant nutrient resources are scarce and accordingly plants like olive trees have a thicker cuticle (Lindow & Andersen, 1996). The phyllosphere may also be exposed to long dry periods or, conversely, heavy rains could dramatically alter the microbiota by the "washing"

The phyllosphere colonizing microorganisms are called epiphytes and Psv is one of them. A bacterium is considered as epiphytic when it lives and multiplies in the phyllosphere and constitutes the main colonizing group of the leaf surface, with average numbers from 106 to 107 cfu/cm2 of leaf (Hirano & Upper, 1983, 2000). Interestingly, not all epiphytic bacteria colonizing the phyllosphere have a strictly commensal relationship with their host plant. This has also been demonstrated in many plant pathogenic bacteria including Psv, which

The size and composition of epiphytic bacterial populations vary according to plant characteristics (species, age) and factors related to nutritional and climatic conditions (Hirano & Upper, 2000; Lindow & Brandl, 2003). The size estimation of epiphytic bacterial populations in the laboratory depends on the sampling, bacteriological and statistical procedures used (Hirano & Upper, 2000; Jacques & Morris, 1995). Most studies about epiphytic bacteria have estimated population sizes by washing or sonication to release bacteria from the leaf, followed by plating of serial dilution of the washings (Hirano & Upper, 2000). The estimated population sizes with this technique correspond to a proportion of total microorganisms living in the phyllosphere (Wilson & Lindow, 1992). The error involved in the recovery of epiphytic bacteria on solid medium is not important compared to the high variability of populations in field samples, which could range from 5 to 6 orders

There are few studies on the epidemiology of olive knot disease and most data of epiphytic Psv populations on the aerial surface of the tree come from studies done in southern Italy

be about 4x108 km2 of leaf surface area, which could be colonized by 1026 bacteria.

production by *P. agglomerans* (Marchi et al., 2006).

have also an epiphytic resident phase (Ercolani, 1971).

of magnitude (Hirano & Upper, 2000).

**4. Epiphytic populations of Psv** 

effect (Hirano & Upper, 2000).

**3. General characteristics of phyllosphere habitats** 

(Ercolani, 1971, 1978, 1979, 1983, 1985, 1991, 1993) and southeastern Spain (Quesada et al., 2007; Quesada et al., 2010a; 2010b). In the aforementioned studies, epiphytic Psv populations in the phyllosphere of olive trees were estimated by washing, followed by plating serial dilution of the washings.

Microbial communities of the olive tree phylloplane can grow embedded in a matrix of exopolysaccharides and form biofilms adhered to the leaf surface (Morris et al., 1997). Furthermore, a great diversity of pigmented bacteria colonizing the surface of olive tree was observed by washing olive leaves and plating the washings. In Italy, Ercolani collected bacteria from the leaf surfaces of olive trees for two sampling periods over several years in the 70s and 80s (Ercolani, 1978, 1991). Phenotypic characterization of these isolates allowed Ercolani to record over 20 bacterial species colonizing the leaf surface. The three highest frequency values of occurrence corresponded to Psv, *Xanthomonas campestris* and *Pantoea agglomerans* with 51, 6.7 and 6%, respectively (Ercolani, 1991).

Spanish studies found that averages of the total bacterial population from leaves and stems were generally significantly higher in Psv-inoculated than in non-inoculated olive trees, suggesting that Psv might have a positive effect on the growth of other epiphytic bacteria or on their ability to colonize olive organs (Quesada et al., 2010a). Populations of *P. agglomerans* could accompany Psv and contribute to the significant differences in total bacterial populations between inoculated and non-inoculated olive trees. Besides, there was a positive correlation between Psv and yellow *P. agglomerans* either on stems or leaf surfaces of naturally infected olive trees (Quesada et al., 2007), and a similar fluctuation of both bacterial populations on the same host. This is of interest because both bacteria produce indoleacetic acid and this can contribute to the epiphytic fitness of Psv in olive trees (Varvaro & Martella, 1993).

The bacterial community composition on the surface of olive leaves is more strongly influenced by the sampling season than by leaf age (Ercolani, 1991). The diversity and size of the total bacterial populations within the olive phyllosphere were lower during the hot dry months and higher during the cold rainy months (Ercolani, 1991). Our observations on one olive orchard showed that seasonal fluctuations of Psv populations felt into the pattern of seasonal shifts described above (Quesada et al., 2007). Interestingly, Psv population sizes in stems and leaf surfaces were correlated (with r2 values of 0.7 and 0.43, respectively) with rainfall, temperature and relative humidity (Quesada et al., 2007). Therefore, these climatic parameters may exert a more or less strong influence on the Psv population values. Epiphytic bacterial communities on olive leaves were more uniform in mature leaves than in young leaves (Ercolani, 1991). In addition, the olive phyllosphere apparently selects specific genotypes of the bacterial community (Lindow & Brandl, 2003). This gives us an idea of the great variability, in terms of epiphytic populations, existing among leaves of the same tree.

Over 50% of bacterial isolates collected from olive leaves by Ercolani (1978, 1991) were identified as Psv and this bacterium survived and multiplied on the leaf surfaces of olive trees (Varvaro & Ferrulli, 1983). Abu-Ghorrah (1988) observed maximum Psv population levels of about 107 cfu/cm2 in olive trees and the Psv generation time in this host was 24 to 36 hours. In studies of leaves inoculated by spraying a suspension of Psv, the bacteria

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

October (Ercolani, 1993).

bacterial pathogens.

et al., 2010; Matas, 2010).

**5. Endophytic populations of Psv** 

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 305

of different ages at different times of the year. Most of the Psv isolates obtained by washing leaves of different ages taken at random in April, were less similar to each other in 60 phenotypic characters (they formed a single group at 65% similarity) than Psv isolates obtained from six-month-old knots in October (one group formed 85% similarity). A similar result was obtained when Psv isolates obtained by washing leaves in October were compared with Psv isolates obtained from six-month-old knots in April (Ercolani, 1993). Psv isolation from these knots was performed six months after washing leaves in April and October as above indicated. Almost all isolates from knots reflected the dominant phenotype of the isolates obtained from the phyllosphere six months earlier. Most Psv isolated by washing leaves in April and October were phenotypically classified near to isolates obtained by washing 13-month-old leaves. According to these authors, senescent leaves (13 months old) could be the main source of bacteria for knot formation in April and

Endophytic bacteria are defined as bacteria living in plant tissues without doing substantive harm or gaining benefit other than residency (Kado, 1992). The endophytic colonization of plants is probably fundamental for plant-associated bacteria to develop sustainable epiphytic populations (Manceau & Kasempour, 2002). Although the information about endophytic populations of plant pathogenic bacteria is still scarce, it is very likely that most of the pathogens can undergo an endophytic step during the disease cycle. However, as their numbers are low inside the asymptomatic plants and their distribution is not homogeneous, laborious studies are required to detect them. Consequently, little is known about the real importance of the endophytic phase for most

Psv could also present an endophyte phase spanning a considerable part of its life cycle due to its multiplication in the intercellular spaces, substomatal cavities or in vascular tissues of the plant, without any visible symptoms (Schiff-Giorgini, 1906; Smith 1908, 1920; Wilson & Magie, 1964). Regarding Psv, the bacteria that survive inside and outside the knots could have a greater impact than the bacteria colonizing the olive as a symptomless endophyte. In fact, some studies described the endophytic phase of Psv in olive plants as rare (Wilson & Magie, 1964). According to other authors, Psv could also present an endophytic phase, moving through the intercellular spaces and even in the xylem vessels and infecting areas close to the first infected zone (Penyalver et al., 2006; Schiff-Giorgini, 1906; Smith 1908, 1920; Wilson & Magie, 1964; Wilson & Ogawa, 1979). Further studies are needed to reliably assess the importance of this phase of Psv in olive knot epidemiology. Nowadays, this is relatively easier to address, thanks to an established model system for the study of olive knot disease covering a wide range of aspects. It is formed by a micropropagated olive plant, coming from an *in vitro* germinated seed and a Psv strain (NCPPB 3335) producing the characteristic symptoms of olive knot disease in both woody and micropropagated olive plants (Pérez-Martínez et al., 2007; Rodríguez-Moreno et al., 2008). Besides, Psv strain NCPPB 3335 has been studied in depth and many genetic resources are available because its genome has been sequenced and analyzed using appropriate bioinformatic tools (Rodríguez-Palenzuela

colonized the lower leaf surface better than the upper surface (Surico, 1993). Basically, they sticked to the vein depressions and to specific structures such as the shields of pectate hairs (Surico, 1993).

The seasonal fluctuation of Psv populations on olive leaves in Italy, recorded over three consecutive years by Ercolani (1971, 1978), showed that Psv populations were higher in spring and fall (about 104 cfu/cm2 of leaf) than in winter and summer (about 102-103 cfu/cm2) (Ercolani, 1971, 1978; Varvaro & Surico, 1978). Psv populations on olive leaves in Spain, also recorded over three consecutive years, reached the highest (ca. 103-104 cfu/cm2 of leaf) densities mainly in warm and rainy months (mainly spring season) and the lowest (ca. 0-10 cfu/cm2 of leaf) in hot and dry months (summer season) (Quesada et al., 2007). Significant differences were observed between Psv populations in summer and in the other seasons over the three-year study (Quesada et al., 2007).

Lavermicocca & Surico (1987) simultaneously analyzed Psv populations on olive tree leaves and stems for the first time and during one year, reporting higher frequencies of Psv isolation in stems than in leaves with relatively high epiphytic Psv populations in July (about 105 cfu/cm2) and only 10 cfu/cm2 in September and March. However, in Spain no significant differences were found between either leaves or stems with respect to the number of analyzed samples where Psv was isolated, detected by PCR, or regarding the average Psv populations over several years (Bertolini et al., 2003a; Quesada et al., 2007; Quesada et al., 2010a, 2010b). Given that in such studies the Psv number were evaluated on stems after they were cut into pieces, some endophytic Psv could be also counted (Penyalver et al., 2006). Furthermore, both types of plant material (stems and leaves) should be analyzed from symptomless shoots to make the evaluation of Psv populations in the phyllosphere more accurate (Bertolini et al., 2003a; Quesada et al., 2007). Psv was also isolated from the surface of olive fruits, but at lower frequency than from leaves, reaching a high Psv population size in September (106 bacteria / g fresh weight) (Lavermicocca & Surico, 1987).

Between 70 and 95% of the maximum variance of some microbiological parameters, such as Psv density in the olive tree phyllosphere, was explained by the influence of seven factors, four of which were related with the weather: summer, summer rainfall, winter rainfall and warm fronts (Ercolani, 1985) and the three remaining factors were cambium activity, leaf age and time of flowering. As described for other epiphytic bacteria and hosts (Kinkel, 1997), Psv population densities varied over several orders of magnitude among leaves sampled concurrently from the same shoot, as assessed by the comparison of leaf printing and isolation experiments (Quesada et al., 2007). Due to the low detection level associated with leaf printing (Jacques & Morris, 1995), such results also suggested that Psv probably colonizes low numbers of leaves with high populations, in bulked samples.

The size of Psv populations on each leaf correlated with leaf age, the time when it formed and the time of the year when the sample was taken (Ercolani, 1991). In addition, phenotypically distinct Psv isolates from the phyllosphere, succeed each other in time in the olive tree phyllosphere (Ercolani, 1983). This was discovered because Psv isolates obtained by washing leaves of a certain age at a particular time of the year (over eight years) showed more phenotypic similarity with each other, than with isolates obtained by washing leaves of different ages at different times of the year. Most of the Psv isolates obtained by washing leaves of different ages taken at random in April, were less similar to each other in 60 phenotypic characters (they formed a single group at 65% similarity) than Psv isolates obtained from six-month-old knots in October (one group formed 85% similarity). A similar result was obtained when Psv isolates obtained by washing leaves in October were compared with Psv isolates obtained from six-month-old knots in April (Ercolani, 1993). Psv isolation from these knots was performed six months after washing leaves in April and October as above indicated. Almost all isolates from knots reflected the dominant phenotype of the isolates obtained from the phyllosphere six months earlier. Most Psv isolated by washing leaves in April and October were phenotypically classified near to isolates obtained by washing 13-month-old leaves. According to these authors, senescent leaves (13 months old) could be the main source of bacteria for knot formation in April and October (Ercolani, 1993).

#### **5. Endophytic populations of Psv**

304 Plant Pathology

colonized the lower leaf surface better than the upper surface (Surico, 1993). Basically, they sticked to the vein depressions and to specific structures such as the shields of pectate hairs

The seasonal fluctuation of Psv populations on olive leaves in Italy, recorded over three consecutive years by Ercolani (1971, 1978), showed that Psv populations were higher in spring and fall (about 104 cfu/cm2 of leaf) than in winter and summer (about 102-103 cfu/cm2) (Ercolani, 1971, 1978; Varvaro & Surico, 1978). Psv populations on olive leaves in Spain, also recorded over three consecutive years, reached the highest (ca. 103-104 cfu/cm2 of leaf) densities mainly in warm and rainy months (mainly spring season) and the lowest (ca. 0-10 cfu/cm2 of leaf) in hot and dry months (summer season) (Quesada et al., 2007). Significant differences were observed between Psv populations in summer and in the other

Lavermicocca & Surico (1987) simultaneously analyzed Psv populations on olive tree leaves and stems for the first time and during one year, reporting higher frequencies of Psv isolation in stems than in leaves with relatively high epiphytic Psv populations in July (about 105 cfu/cm2) and only 10 cfu/cm2 in September and March. However, in Spain no significant differences were found between either leaves or stems with respect to the number of analyzed samples where Psv was isolated, detected by PCR, or regarding the average Psv populations over several years (Bertolini et al., 2003a; Quesada et al., 2007; Quesada et al., 2010a, 2010b). Given that in such studies the Psv number were evaluated on stems after they were cut into pieces, some endophytic Psv could be also counted (Penyalver et al., 2006). Furthermore, both types of plant material (stems and leaves) should be analyzed from symptomless shoots to make the evaluation of Psv populations in the phyllosphere more accurate (Bertolini et al., 2003a; Quesada et al., 2007). Psv was also isolated from the surface of olive fruits, but at lower frequency than from leaves, reaching a high Psv population size in September (106 bacteria / g

Between 70 and 95% of the maximum variance of some microbiological parameters, such as Psv density in the olive tree phyllosphere, was explained by the influence of seven factors, four of which were related with the weather: summer, summer rainfall, winter rainfall and warm fronts (Ercolani, 1985) and the three remaining factors were cambium activity, leaf age and time of flowering. As described for other epiphytic bacteria and hosts (Kinkel, 1997), Psv population densities varied over several orders of magnitude among leaves sampled concurrently from the same shoot, as assessed by the comparison of leaf printing and isolation experiments (Quesada et al., 2007). Due to the low detection level associated with leaf printing (Jacques & Morris, 1995), such results also suggested that Psv probably colonizes low numbers of leaves with high populations, in bulked

The size of Psv populations on each leaf correlated with leaf age, the time when it formed and the time of the year when the sample was taken (Ercolani, 1991). In addition, phenotypically distinct Psv isolates from the phyllosphere, succeed each other in time in the olive tree phyllosphere (Ercolani, 1983). This was discovered because Psv isolates obtained by washing leaves of a certain age at a particular time of the year (over eight years) showed more phenotypic similarity with each other, than with isolates obtained by washing leaves

seasons over the three-year study (Quesada et al., 2007).

fresh weight) (Lavermicocca & Surico, 1987).

(Surico, 1993).

samples.

Endophytic bacteria are defined as bacteria living in plant tissues without doing substantive harm or gaining benefit other than residency (Kado, 1992). The endophytic colonization of plants is probably fundamental for plant-associated bacteria to develop sustainable epiphytic populations (Manceau & Kasempour, 2002). Although the information about endophytic populations of plant pathogenic bacteria is still scarce, it is very likely that most of the pathogens can undergo an endophytic step during the disease cycle. However, as their numbers are low inside the asymptomatic plants and their distribution is not homogeneous, laborious studies are required to detect them. Consequently, little is known about the real importance of the endophytic phase for most bacterial pathogens.

Psv could also present an endophyte phase spanning a considerable part of its life cycle due to its multiplication in the intercellular spaces, substomatal cavities or in vascular tissues of the plant, without any visible symptoms (Schiff-Giorgini, 1906; Smith 1908, 1920; Wilson & Magie, 1964). Regarding Psv, the bacteria that survive inside and outside the knots could have a greater impact than the bacteria colonizing the olive as a symptomless endophyte. In fact, some studies described the endophytic phase of Psv in olive plants as rare (Wilson & Magie, 1964). According to other authors, Psv could also present an endophytic phase, moving through the intercellular spaces and even in the xylem vessels and infecting areas close to the first infected zone (Penyalver et al., 2006; Schiff-Giorgini, 1906; Smith 1908, 1920; Wilson & Magie, 1964; Wilson & Ogawa, 1979). Further studies are needed to reliably assess the importance of this phase of Psv in olive knot epidemiology. Nowadays, this is relatively easier to address, thanks to an established model system for the study of olive knot disease covering a wide range of aspects. It is formed by a micropropagated olive plant, coming from an *in vitro* germinated seed and a Psv strain (NCPPB 3335) producing the characteristic symptoms of olive knot disease in both woody and micropropagated olive plants (Pérez-Martínez et al., 2007; Rodríguez-Moreno et al., 2008). Besides, Psv strain NCPPB 3335 has been studied in depth and many genetic resources are available because its genome has been sequenced and analyzed using appropriate bioinformatic tools (Rodríguez-Palenzuela et al., 2010; Matas, 2010).

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

**7. Bacteria survive from one season to the next inside the knots**

as a quarantine organism.

**8. High humidity level favor the plant exudate production with high quantity of bacteria**

> **6. Bacterial spreading to healthy plants by rain, wind and cultural practices**

as red bacilli (kindly provided by E. Bertolini, 2003).

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 307

**4. Bacteria + adequate conditions = new knots are formed**

**5. Bacteria + inadequate conditions = new knots are not formed**

Fig. 2. Disease cycle of olive knot caused by *Pseudomonas savastanoi* pv. *savastanoi* simulated

Although the olive knot disease is widespread throughout most olive-growing areas, there is no accurate estimation of the losses it causes. This is very difficult to measure because many factors can influence the severity of the symptoms. Severe infections can cause death of branches and a progressive weakening, resulting in a loss of tree vigor (Tjamos et al., 1993) and thus of harvest. De Andrés (1991) estimated that Psv-related losses were around 1.3% of national olive production in Spain. Occasionally, this disease has caused the loss of almost the olive local harvest due to the combination of optimal weather conditions for bacterial entry and multiplication, as observed in two Spanish localities in 1987 and 2001 (B. Celada, personal communication) after severe hail storms. Furthermore, this disease is present with variable incidence in many nursery plants, as it limits their commercialization due to the visible symptoms. This is especially important in plants for export because several countries that import plants from the European Union (EU), like Chile, consider Psv

The quantitative effects of olive knot disease on vigor and olive fruit yields are not yet well established because there is only information available from one study in California and another in Spain. In a commercial orchard in California (USA) significant differences were

**7. Effect of olive knot disease on the vigor and yield of olive trees** 

**2. Wounds caused by leaf scars, frost, pruning and harvesting, etc.**

> **3. Bacterial colonization of wounds**

**1. Epiphytic and endophytic phase on leaves, buds and branches**

Endophytic Psv populations could contribute to a dramatic increase in Psv numbers and in olive knots in infected olive groves, when the copper-based control treatments are not applied (Quesada et al., 2010b).

#### **6. Epidemiology and disease cycle**

Disease caused by Psv populations has an epiphytic-pathogen type cycle. The bacteria have an epiphytic phase in which they multiply on the surface of olive tree stems and leaves without developing symptoms (Ercolani, 1978, 1991; Varvaro & Ferrulli, 1983). Interestingly, Psv populations were recovered from symptomless shoots from non-inoculated control trees prior to the appearance of symptoms, suggesting that these epiphytic bacteria were the potential source of inoculum for infection of healthy plants (Quesada et al., 2010a).

The temperature range in which Psv can initiate infection is between 5 and 37 ºC and this would allow the bacteria to cause infections throughout the year. However, optimal conditions for disease development are about 22-25 ºC and the subsequent time periods with high infection probability are fall and spring (Protta, 1995). Psv can infect olive trees at any time of the year and trigger knot formation only when conditions are favorable. Thus, when the bacteria infect an olive tree in the fall, knots will begin to develop several months later, but if the infection occurs during the spring, the time required for knot formation may be only two weeks (Wilson, 1935). Field trials performed in California showed that Psv inoculations of olive trees carried out in April caused higher levels of olive knot disease than Psv inoculations carried out in December (Teviotdale & Krueger, 2004).

Dissemination of Psv bacteria from infected (or inoculated) to non-infected (or noninoculated) trees was suggested (Quesada et al., 2010a). Bacteria could spread over long distances due to the introduction and planting of infected material, or over short distances transported by splashing rain, windblown aerosols, insects and cultural practices (Horne et al., 1912; Wilson, 1935). Currently, bacterial dissemination is facilitated by cultural practices, new plantations with high tree density, frequent severe pruning and with small distance between plants (Tous et al., 2007). Wounds caused by harvesting and pruning, as well as by hail, frost and leaf scars, create niches where infection occurs (Wilson, 1935; Janse, 1982) and olive tree infection by Psv is directly related to the degree of wounding of the trees (Smith et al., 1991). In an assay to evaluate natural dissemination of the bacteria on young plants, knots were not observed in inoculated and non-inoculated trees until 3 and 10 months after inoculations, respectively (Quesada et al., 2010a). The compatible Psv-olive tree interaction facilitates the invasion, infection and multiplication, triggering hypertrophy and hyperplasia of the plant tissues with subsequent knot formation. Bacteria can survive inside the knots from one season to another and when humidity is high enough, exudates containing large amounts of bacteria are emitted in which they can survive as epiphytes (Wilson, 1935). However, the bacteria can only survive in soil for a few days (Wilson & Ogawa, 1979). The disease cycle is summarized in Figure 2.

In 1909, Petri isolated Psv from the intestinal tract and eggs of the olive fly (*Bactrocera oleae*), but there is no other scientific evidence that this, or other insects, can be efficient vectors of olive knot disease. Additionally, there is no conclusive evidence about the role that birds may play as vectors of this disease (Wilson, 1935), although they can transport living bacteria from plant to plant.

Endophytic Psv populations could contribute to a dramatic increase in Psv numbers and in olive knots in infected olive groves, when the copper-based control treatments are not

Disease caused by Psv populations has an epiphytic-pathogen type cycle. The bacteria have an epiphytic phase in which they multiply on the surface of olive tree stems and leaves without developing symptoms (Ercolani, 1978, 1991; Varvaro & Ferrulli, 1983). Interestingly, Psv populations were recovered from symptomless shoots from non-inoculated control trees prior to the appearance of symptoms, suggesting that these epiphytic bacteria were the

The temperature range in which Psv can initiate infection is between 5 and 37 ºC and this would allow the bacteria to cause infections throughout the year. However, optimal conditions for disease development are about 22-25 ºC and the subsequent time periods with high infection probability are fall and spring (Protta, 1995). Psv can infect olive trees at any time of the year and trigger knot formation only when conditions are favorable. Thus, when the bacteria infect an olive tree in the fall, knots will begin to develop several months later, but if the infection occurs during the spring, the time required for knot formation may be only two weeks (Wilson, 1935). Field trials performed in California showed that Psv inoculations of olive trees carried out in April caused higher levels of olive knot disease than

Dissemination of Psv bacteria from infected (or inoculated) to non-infected (or noninoculated) trees was suggested (Quesada et al., 2010a). Bacteria could spread over long distances due to the introduction and planting of infected material, or over short distances transported by splashing rain, windblown aerosols, insects and cultural practices (Horne et al., 1912; Wilson, 1935). Currently, bacterial dissemination is facilitated by cultural practices, new plantations with high tree density, frequent severe pruning and with small distance between plants (Tous et al., 2007). Wounds caused by harvesting and pruning, as well as by hail, frost and leaf scars, create niches where infection occurs (Wilson, 1935; Janse, 1982) and olive tree infection by Psv is directly related to the degree of wounding of the trees (Smith et al., 1991). In an assay to evaluate natural dissemination of the bacteria on young plants, knots were not observed in inoculated and non-inoculated trees until 3 and 10 months after inoculations, respectively (Quesada et al., 2010a). The compatible Psv-olive tree interaction facilitates the invasion, infection and multiplication, triggering hypertrophy and hyperplasia of the plant tissues with subsequent knot formation. Bacteria can survive inside the knots from one season to another and when humidity is high enough, exudates containing large amounts of bacteria are emitted in which they can survive as epiphytes (Wilson, 1935). However, the bacteria can only survive in soil for a few days (Wilson & Ogawa, 1979). The

In 1909, Petri isolated Psv from the intestinal tract and eggs of the olive fly (*Bactrocera oleae*), but there is no other scientific evidence that this, or other insects, can be efficient vectors of olive knot disease. Additionally, there is no conclusive evidence about the role that birds may play as vectors of this disease (Wilson, 1935), although they can transport living

potential source of inoculum for infection of healthy plants (Quesada et al., 2010a).

Psv inoculations carried out in December (Teviotdale & Krueger, 2004).

applied (Quesada et al., 2010b).

**6. Epidemiology and disease cycle** 

disease cycle is summarized in Figure 2.

bacteria from plant to plant.

Fig. 2. Disease cycle of olive knot caused by *Pseudomonas savastanoi* pv. *savastanoi* simulated as red bacilli (kindly provided by E. Bertolini, 2003).

### **7. Effect of olive knot disease on the vigor and yield of olive trees**

Although the olive knot disease is widespread throughout most olive-growing areas, there is no accurate estimation of the losses it causes. This is very difficult to measure because many factors can influence the severity of the symptoms. Severe infections can cause death of branches and a progressive weakening, resulting in a loss of tree vigor (Tjamos et al., 1993) and thus of harvest. De Andrés (1991) estimated that Psv-related losses were around 1.3% of national olive production in Spain. Occasionally, this disease has caused the loss of almost the olive local harvest due to the combination of optimal weather conditions for bacterial entry and multiplication, as observed in two Spanish localities in 1987 and 2001 (B. Celada, personal communication) after severe hail storms. Furthermore, this disease is present with variable incidence in many nursery plants, as it limits their commercialization due to the visible symptoms. This is especially important in plants for export because several countries that import plants from the European Union (EU), like Chile, consider Psv as a quarantine organism.

The quantitative effects of olive knot disease on vigor and olive fruit yields are not yet well established because there is only information available from one study in California and another in Spain. In a commercial orchard in California (USA) significant differences were

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

(EPPO, 2006).

certification programs (Cambra et al., 1998).

in Spain (Chomé, 1998).

are not considered.

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 309

very important that Psv appears in the EU list of pests and diseases that significantly affect plant quality standards, drawn up by the European Commission (Directive Nº 92/34/EU). As advised by the European and Mediterranean Plant Protection Organization (EPPO-OEPP), new olive groves should be established using Psv-free certified plant material

As an example, fifteen years ago, there was scarce reliable information available on the sanitary status of Spanish olive plants with respect to pathogenic bacteria (Bertolini et al., 1998; Padilla, 1997). Although there is now more information and analyses have been performed, there is still a lack of scientific published data available on the status of olive plants in the field or in nurseries, either in Spain or in other olive-growing countries. Government agencies have shown an interest to control the planting material given the increase number of plantations, the notable changes in production technologies and the frequent commercial exchange of olive plants. All these facts emphasize the convenience of providing plant material with certain quality standards and the implementation of

With respect to this issue the EU has, so far, required the minimum conditions for the nursery plants of type Agricultural Conformitas Comunitatis (or CAC). The implementation of certification systems is the responsibility of each member state, but the European and Mediterranean Plant Protection Organization (EPPO) has developed specifications for certification of olive plants. These referred specifically to health although they were based on studies conducted in Italy and Portugal and thus should be contrasted with the situation

Italy was the first country to publish standards for certification of plant material from olive trees in 1993. Certified plants should be free from Psv, *Verticillium dahliae* and six virus (*Olive latent virus 1* (OLV-1), *Olive latent virus 2* (OLV-2), *Cucumber mosaic virus* (CMV), *Arabis mosaic virus* (ArMV), *Cherry leaf roll virus* (CLRV) and Strawberry latent ring spot virus (SLRSV)) (Martelli, 1998). Besides Italy, other countries like Portugal, Israel, Argentina and Spain, have also established certification programs for olive plant material. In the Argentinan Certification program, the mother plants are annually tested and must be free of *P. syringae*, Psv, *Agrobacterium tumefaciens*, *V. dahliae* and *Phytophthora cinnamomi* but viruses

There are more than 260 nurseries registered in Spain, mainly located in Andalusia and Valencia, which produced about 5.5 million olive plants in 1999-2000 for new plantations and also for international trade (Chomé, 1998). Given this significant production, the *Real Decreto 1678/1999* (Anonymous, 1999) established quality control and certification requirements for olive seedlings in the certification program of olive plant material in Spain. Currently, the qualification of certified plant material is the responsibility of the competent institutions in each region. To qualify for certification, plant material must meet certain conditions such as having a known origin and having been submitted to cultivar analyses and sanitary tests. Mother plants of the starting material and base material should be officially inspected to verify that they are free of *V. dahliae*, Psv and viruses OLV-1, OLV-2, CMV, ArMV, CLRV and SLRSV. Each year the plants for certification should be sampled and tested for Psv by the responsible official body using approved techniques, which include isolation, serology and nested multiplex RT-PCR. With this technique, Psv and four

not found in vigor between 40-year-old olive trees lightly (0.10-0.30 knots in 0.3 m of fruit wood) and mildly (0.31-0.50 knots) infected with the disease (Schroth et al., 1973). In contrast, in Spain in a study on non-inoculated and inoculated trees 7-year-old of cv. Arbequina in a high-density grove, vigor was significantly higher in non-inoculated trees (Quesada et al., 2010a). Therefore, vigor was higher in trees of cv. Arbequina where olive knot disease was lower during the study, suggesting a negative effect over time of the disease on plant development. Schroth et al. (1973) in California showed that there was a clear relationship between crop losses and the number of tumors caused by Psv in branches. They observed significant differences in the weight of olive fruits per tree in only one year between lightly and mildly infected olive trees with 121.3 and 94.6 kg, respectively. In the Spanish assays, the different levels of the disease did not significantly affect cumulative olive yield (Quesada et al., 2010a) in young trees.

Furthermore, low oil quality was reported when olive fruits were harvested from olive trees moderately affected with olive knot presenting odors and flavors such as bitter, stale or salty, but the data were lacking of statistical support (Schroth et al., 1968; Tjamos et al., 1993). In another study, olive knot disease incidence did not modify either the chemical or organoleptic characteristics of virgin olive oil extracted from young olive tree fruits in a high-density grove (Quesada et al., unpublished data).

#### **8. Control methods**

The methods used to control plant pathogenic bacteria are based on preventive and curative measures and the combination of the two should be used in the context of an integrated control. The five main goals of an integrated plant disease control program are to eliminate or reduce the initial inoculum, reduce the effectiveness of the initial inoculum, increase host resistance, delay disease onset and slow the secondary cycles (Agrios, 2005). The key of any integrated control program is a question of sustainability at different levels (Caballero & Murillo, 2003). In economic terms, it must ensure farmers' profits and at the environmental level, select control methods that minimize environmental impact. And finally, control methods by themselves should ensure sustainability and they must remain effective over time. The monitoring of a strategy of this type is essential to ensure safe and sustainable agriculture.

Disease management of bacterial pathogens in the field is mainly based on preventive procedures, because it is difficult to eradicate pathogens once established. Due to the economic impact of the olive knot disease, growers require adequate control methods to overcome its negative repercussions on yield and even on olive fruit quality (Quesada et al., 2010a; Schroth et al., 1973). Olive knot control should be based on an integrated control strategy, giving priority to the most effective measures that are of preventive type. These are very diverse and can be grouped into regulatory measures, preventing introduction of the pathogen in protected areas and prophylactic measures to reduce or eliminate the pathogen or hinder its establishment in nurseries or orchards (Montesinos & López, 1996).

#### **8.1 Regulatory measures**

The production, maintenance and use of certified plant material which is pathogen free, is one of the main preventive measures used to control plant pathogens. In this regard, it is

not found in vigor between 40-year-old olive trees lightly (0.10-0.30 knots in 0.3 m of fruit wood) and mildly (0.31-0.50 knots) infected with the disease (Schroth et al., 1973). In contrast, in Spain in a study on non-inoculated and inoculated trees 7-year-old of cv. Arbequina in a high-density grove, vigor was significantly higher in non-inoculated trees (Quesada et al., 2010a). Therefore, vigor was higher in trees of cv. Arbequina where olive knot disease was lower during the study, suggesting a negative effect over time of the disease on plant development. Schroth et al. (1973) in California showed that there was a clear relationship between crop losses and the number of tumors caused by Psv in branches. They observed significant differences in the weight of olive fruits per tree in only one year between lightly and mildly infected olive trees with 121.3 and 94.6 kg, respectively. In the Spanish assays, the different levels of the disease did not significantly affect cumulative

Furthermore, low oil quality was reported when olive fruits were harvested from olive trees moderately affected with olive knot presenting odors and flavors such as bitter, stale or salty, but the data were lacking of statistical support (Schroth et al., 1968; Tjamos et al., 1993). In another study, olive knot disease incidence did not modify either the chemical or organoleptic characteristics of virgin olive oil extracted from young olive tree fruits in a

The methods used to control plant pathogenic bacteria are based on preventive and curative measures and the combination of the two should be used in the context of an integrated control. The five main goals of an integrated plant disease control program are to eliminate or reduce the initial inoculum, reduce the effectiveness of the initial inoculum, increase host resistance, delay disease onset and slow the secondary cycles (Agrios, 2005). The key of any integrated control program is a question of sustainability at different levels (Caballero & Murillo, 2003). In economic terms, it must ensure farmers' profits and at the environmental level, select control methods that minimize environmental impact. And finally, control methods by themselves should ensure sustainability and they must remain effective over time. The monitoring of a strategy of this type is essential to ensure safe and sustainable

Disease management of bacterial pathogens in the field is mainly based on preventive procedures, because it is difficult to eradicate pathogens once established. Due to the economic impact of the olive knot disease, growers require adequate control methods to overcome its negative repercussions on yield and even on olive fruit quality (Quesada et al., 2010a; Schroth et al., 1973). Olive knot control should be based on an integrated control strategy, giving priority to the most effective measures that are of preventive type. These are very diverse and can be grouped into regulatory measures, preventing introduction of the pathogen in protected areas and prophylactic measures to reduce or eliminate the pathogen

The production, maintenance and use of certified plant material which is pathogen free, is one of the main preventive measures used to control plant pathogens. In this regard, it is

or hinder its establishment in nurseries or orchards (Montesinos & López, 1996).

olive yield (Quesada et al., 2010a) in young trees.

high-density grove (Quesada et al., unpublished data).

**8. Control methods** 

agriculture.

**8.1 Regulatory measures** 

very important that Psv appears in the EU list of pests and diseases that significantly affect plant quality standards, drawn up by the European Commission (Directive Nº 92/34/EU). As advised by the European and Mediterranean Plant Protection Organization (EPPO-OEPP), new olive groves should be established using Psv-free certified plant material (EPPO, 2006).

As an example, fifteen years ago, there was scarce reliable information available on the sanitary status of Spanish olive plants with respect to pathogenic bacteria (Bertolini et al., 1998; Padilla, 1997). Although there is now more information and analyses have been performed, there is still a lack of scientific published data available on the status of olive plants in the field or in nurseries, either in Spain or in other olive-growing countries. Government agencies have shown an interest to control the planting material given the increase number of plantations, the notable changes in production technologies and the frequent commercial exchange of olive plants. All these facts emphasize the convenience of providing plant material with certain quality standards and the implementation of certification programs (Cambra et al., 1998).

With respect to this issue the EU has, so far, required the minimum conditions for the nursery plants of type Agricultural Conformitas Comunitatis (or CAC). The implementation of certification systems is the responsibility of each member state, but the European and Mediterranean Plant Protection Organization (EPPO) has developed specifications for certification of olive plants. These referred specifically to health although they were based on studies conducted in Italy and Portugal and thus should be contrasted with the situation in Spain (Chomé, 1998).

Italy was the first country to publish standards for certification of plant material from olive trees in 1993. Certified plants should be free from Psv, *Verticillium dahliae* and six virus (*Olive latent virus 1* (OLV-1), *Olive latent virus 2* (OLV-2), *Cucumber mosaic virus* (CMV), *Arabis mosaic virus* (ArMV), *Cherry leaf roll virus* (CLRV) and Strawberry latent ring spot virus (SLRSV)) (Martelli, 1998). Besides Italy, other countries like Portugal, Israel, Argentina and Spain, have also established certification programs for olive plant material. In the Argentinan Certification program, the mother plants are annually tested and must be free of *P. syringae*, Psv, *Agrobacterium tumefaciens*, *V. dahliae* and *Phytophthora cinnamomi* but viruses are not considered.

There are more than 260 nurseries registered in Spain, mainly located in Andalusia and Valencia, which produced about 5.5 million olive plants in 1999-2000 for new plantations and also for international trade (Chomé, 1998). Given this significant production, the *Real Decreto 1678/1999* (Anonymous, 1999) established quality control and certification requirements for olive seedlings in the certification program of olive plant material in Spain. Currently, the qualification of certified plant material is the responsibility of the competent institutions in each region. To qualify for certification, plant material must meet certain conditions such as having a known origin and having been submitted to cultivar analyses and sanitary tests. Mother plants of the starting material and base material should be officially inspected to verify that they are free of *V. dahliae*, Psv and viruses OLV-1, OLV-2, CMV, ArMV, CLRV and SLRSV. Each year the plants for certification should be sampled and tested for Psv by the responsible official body using approved techniques, which include isolation, serology and nested multiplex RT-PCR. With this technique, Psv and four

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

factors favoring infection can vary in different areas.

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 311

However, in woody crops, such as olive trees, breeders and plant pathologists are hindered by the slow improvement in breeding processes as a result of delayed entry into fruition.

Another drawback is that the information available about cultivar susceptibility to olive knot disease is scarce and mainly comes from field observations, such as those reported in the USA and Spain (Barranco, 1998; Trapero & Blanco, 1998; Wilson, 1935). Very few data are available from comparative inoculation experiments and is limited to several cultivars (five to eight) from Italy, Greece, Morocco, and Portugal (Benjama, 1994; Catara et al., 2005; Hassani et al., 2003; Marcelo et al., 1999; Panagopoulos, 1993; Varvaro & Surico 1978), with the exception of Spain where 29 cultivars were evaluated (Penyalver et al., 2006). Field observations do not always give universally valid information on the intrinsic susceptibility of each cultivar because the initial quantity of bacterial inoculum differs between plants and

Varvaro & Surico (1978) compared the behavior of six Italian olive cultivars inoculated with Psv and found no difference, because more than 95% of the inoculated wounds developed tumors. This was probably due to the high inoculum dose applied (more than 106 bacteria per wound) and because the inoculated plants were only one year old. Different doses of eight Psv isolates were inoculated in six olive cultivars in comparative inoculation experiments from Morocco (Benjama, 1994). The results showed that the cultivar Frantoio was the most susceptible among those tested, followed by Ascolana dura, Manzanilla, Picholine marocaine, Dahbia and Gordal Sevillana, which was the least susceptible, although a statistical analysis of the data was not performed. Marcelo et al. (1999) evaluated six Portuguese cultivars and found they differed in the percentage of knots formed at inoculation points, ranging from 36 to 66%. These authors considered that the cultivars Blanqueta, Cobrancosa, Cordovil de Serpa, Galega Vulgar, Redondil and Santulhana were moderately susceptible to olive knot disease, but their data were not statistically analysed. Hassani et al. (2003) evaluated the Italian cultivars Frantoio, Leccino, Moraiolo and Nostrale di Rigali by inoculation of five Psv strains with an inoculum dose of 5x107 bacteria per wound and subsequently knot weights were compared. Although they did not indicate the percentage of inoculation sites that developed knots, it is likely that this parameter exceeded

90% in the four cultivars because an excessively high inoculum dose was used.

World Olive Germplasm Bank of Spain, located in Cordoba.

Penyalver et al. (2006) developed a methodology for evaluation of cultivar susceptibility to Psv and reported that most of that 21 Psv strains evaluated in virulence tests showed a high degree of aggressiveness but also, in some combinations, cultivar-strain interactions were observed. Consequently, strain selection for inoculation is a pre-requisite to obtaining useful data, and at least two strains should be used for accurate evaluations. The methodology was optimized for the first time with 29 olive cultivars. It was concluded that plant material should be genetically homogeneous, at least two or three years old, inoculated in spring or early summer by wounds made with a sterile scalpel. The use of at least two Psv strains with high degree of virulence was also recommended. They should be inoculated at low inoculum doses (102 bacteria per wound) to differentiate among different cultivars, as well as at a high dose (106 bacteria per wound) to identify the less susceptible cultivars. Ten olive plants should be used per bacterial strain and dose. Five wounds should be performed thus per plant and several measurements of symptoms taken for each combination, although the measurement taken at 90 days was the data included in the analysis of 29 cultivars from the

olive viruses (CMV, CLRV, SLRSV and ArMV) can be detected simultaneously in a sensitive single reaction (Bertolini et al., 2003b). Finally, parent plants of certified nursery stock must be at least free of symptoms of diseases caused by fungi, bacteria and the viruses previously cited.

#### **8.2 Prophylactic measures**

Prophylactic measures are designed to reduce or eliminate the pathogen levels or impede its establishment in a crop and these measures can also be of eradicative nature, based on cultivar susceptibility or by direct protection (Montesinos & López, 1996). In the case of Psv, they include all those performed for disinfecting plants, agricultural machinery, or anything in contact with plants.

#### **8.2.1 Eradication**

The presence of knots in a tree is related to a high level of disease after several years, and this highlights the need of using preventive control methods or eradication methods to maintain olive trees without knots (Quesada et al., 2010a). In affected plantations the main olive knot disease eradication method would be the uprooting of the affected trees or the use of cultural practices to reduce the inoculum source, performing copper treatments, pruning of infected branches and reduction of number of wounds during the growing season and especially at harvest (Beltrá, 1956; Penyalver et al., 1998; Trapero & Blanco, 1998; Wilson, 1935). This is especially relevant in new plantations with high tree density and frequent severe pruning, where control measures should be accurately monitored (Tous et al., 2007).

The removal of knots is very laborious and may not be entirely effective because new wounds are usually done when knots are removed and new knots can develop in these wounds in the following years, even when treated with preventive chemicals (Wilson, 1935). Pruning of infected branches is more effective than knot removal as fewer wounds are caused to the olive tree and the bacterial inoculum load is minimised (Quesada et al., 2010a; Teviotdale & Krueger, 2004; Wilson, 1935). All cut branches should be burned in the same field to prevent the spread of the disease (Trapero & Blanco, 1998).

In the case of partially contaminated olive groves, healthy trees should be harvested and pruned first (Wilson, 1935). Besides, growers should harvest olives in dry weather only and avoid the use of techniques like knocking the olive tree branches with wooden poles (Krueger et al., 1999). Manual harvesting methods, like the "milking" method or the use of mechanical vibration are more suitable. It is important to assess the index of tree damage in terms of broken branches and compare this to the olive fruit harvested. It has been reported that knocking the olive tree branches with wood poles can break from 13 to 18% of branches while for mechanical vibration this is only 6 to 9%, on complete harvesting (Civantos et al., 2008).

#### **8.2.2 Cultivar susceptibility**

The use of resistant cultivars, or low susceptibility cultivars to bacterial plant diseases would be one of the most appropriate disease control methods (Montesinos & López, 1996).

olive viruses (CMV, CLRV, SLRSV and ArMV) can be detected simultaneously in a sensitive single reaction (Bertolini et al., 2003b). Finally, parent plants of certified nursery stock must be at least free of symptoms of diseases caused by fungi, bacteria and the viruses previously

Prophylactic measures are designed to reduce or eliminate the pathogen levels or impede its establishment in a crop and these measures can also be of eradicative nature, based on cultivar susceptibility or by direct protection (Montesinos & López, 1996). In the case of Psv, they include all those performed for disinfecting plants, agricultural machinery, or anything

The presence of knots in a tree is related to a high level of disease after several years, and this highlights the need of using preventive control methods or eradication methods to maintain olive trees without knots (Quesada et al., 2010a). In affected plantations the main olive knot disease eradication method would be the uprooting of the affected trees or the use of cultural practices to reduce the inoculum source, performing copper treatments, pruning of infected branches and reduction of number of wounds during the growing season and especially at harvest (Beltrá, 1956; Penyalver et al., 1998; Trapero & Blanco, 1998; Wilson, 1935). This is especially relevant in new plantations with high tree density and frequent severe pruning, where control measures should be accurately monitored

The removal of knots is very laborious and may not be entirely effective because new wounds are usually done when knots are removed and new knots can develop in these wounds in the following years, even when treated with preventive chemicals (Wilson, 1935). Pruning of infected branches is more effective than knot removal as fewer wounds are caused to the olive tree and the bacterial inoculum load is minimised (Quesada et al., 2010a; Teviotdale & Krueger, 2004; Wilson, 1935). All cut branches should be burned in the same

In the case of partially contaminated olive groves, healthy trees should be harvested and pruned first (Wilson, 1935). Besides, growers should harvest olives in dry weather only and avoid the use of techniques like knocking the olive tree branches with wooden poles (Krueger et al., 1999). Manual harvesting methods, like the "milking" method or the use of mechanical vibration are more suitable. It is important to assess the index of tree damage in terms of broken branches and compare this to the olive fruit harvested. It has been reported that knocking the olive tree branches with wood poles can break from 13 to 18% of branches while for mechanical vibration this is only 6 to 9%, on complete

The use of resistant cultivars, or low susceptibility cultivars to bacterial plant diseases would be one of the most appropriate disease control methods (Montesinos & López, 1996).

field to prevent the spread of the disease (Trapero & Blanco, 1998).

cited.

**8.2 Prophylactic measures** 

in contact with plants.

**8.2.1 Eradication** 

(Tous et al., 2007).

harvesting (Civantos et al., 2008).

**8.2.2 Cultivar susceptibility** 

However, in woody crops, such as olive trees, breeders and plant pathologists are hindered by the slow improvement in breeding processes as a result of delayed entry into fruition.

Another drawback is that the information available about cultivar susceptibility to olive knot disease is scarce and mainly comes from field observations, such as those reported in the USA and Spain (Barranco, 1998; Trapero & Blanco, 1998; Wilson, 1935). Very few data are available from comparative inoculation experiments and is limited to several cultivars (five to eight) from Italy, Greece, Morocco, and Portugal (Benjama, 1994; Catara et al., 2005; Hassani et al., 2003; Marcelo et al., 1999; Panagopoulos, 1993; Varvaro & Surico 1978), with the exception of Spain where 29 cultivars were evaluated (Penyalver et al., 2006). Field observations do not always give universally valid information on the intrinsic susceptibility of each cultivar because the initial quantity of bacterial inoculum differs between plants and factors favoring infection can vary in different areas.

Varvaro & Surico (1978) compared the behavior of six Italian olive cultivars inoculated with Psv and found no difference, because more than 95% of the inoculated wounds developed tumors. This was probably due to the high inoculum dose applied (more than 106 bacteria per wound) and because the inoculated plants were only one year old. Different doses of eight Psv isolates were inoculated in six olive cultivars in comparative inoculation experiments from Morocco (Benjama, 1994). The results showed that the cultivar Frantoio was the most susceptible among those tested, followed by Ascolana dura, Manzanilla, Picholine marocaine, Dahbia and Gordal Sevillana, which was the least susceptible, although a statistical analysis of the data was not performed. Marcelo et al. (1999) evaluated six Portuguese cultivars and found they differed in the percentage of knots formed at inoculation points, ranging from 36 to 66%. These authors considered that the cultivars Blanqueta, Cobrancosa, Cordovil de Serpa, Galega Vulgar, Redondil and Santulhana were moderately susceptible to olive knot disease, but their data were not statistically analysed. Hassani et al. (2003) evaluated the Italian cultivars Frantoio, Leccino, Moraiolo and Nostrale di Rigali by inoculation of five Psv strains with an inoculum dose of 5x107 bacteria per wound and subsequently knot weights were compared. Although they did not indicate the percentage of inoculation sites that developed knots, it is likely that this parameter exceeded 90% in the four cultivars because an excessively high inoculum dose was used.

Penyalver et al. (2006) developed a methodology for evaluation of cultivar susceptibility to Psv and reported that most of that 21 Psv strains evaluated in virulence tests showed a high degree of aggressiveness but also, in some combinations, cultivar-strain interactions were observed. Consequently, strain selection for inoculation is a pre-requisite to obtaining useful data, and at least two strains should be used for accurate evaluations. The methodology was optimized for the first time with 29 olive cultivars. It was concluded that plant material should be genetically homogeneous, at least two or three years old, inoculated in spring or early summer by wounds made with a sterile scalpel. The use of at least two Psv strains with high degree of virulence was also recommended. They should be inoculated at low inoculum doses (102 bacteria per wound) to differentiate among different cultivars, as well as at a high dose (106 bacteria per wound) to identify the less susceptible cultivars. Ten olive plants should be used per bacterial strain and dose. Five wounds should be performed thus per plant and several measurements of symptoms taken for each combination, although the measurement taken at 90 days was the data included in the analysis of 29 cultivars from the World Olive Germplasm Bank of Spain, located in Cordoba.

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

treatment application method, or physiological state of the host plant.

**8.2.3.1 Chemical control** 

infection (Smith et al., 1991).

penetration through the plant wounds.

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 313

Chemical control of bacterial plant diseases is only effective when they are used in preventive strategies before the onset of infection or very early in the bacterial infection process (Montesinos & López, 1996). Specifically, chemical control of olive knot disease has given inconsistent results in field experiments and may also have low efficacy and even show phytotoxicity to some tissues. This variability is due to several factors, such as the amount of inoculum, timing of treatments, climatic conditions, cultivar susceptibility,

Copper compounds are the main preventive chemical treatment recommended against olive knot disease and their use is recommended every year when there is a risk of infection, in spring and fall before the rains, after the leaf fall and especially after hail and frost or other events causing olive injures (Penyalver et al., 1998; Protta, 1995; Smith et al., 1991; Wilson, 1935). A positive correlation has been found between disease incidence and spring rains (Teviotdale & Krueger, 2004) and it was observed that moist winds in coastal areas promote

The copper-based compounds used in olive groves in Spain include various salts and formulations (hydroxides, oxychlorides, oxides or sulfates) as well as their mixture with organic compounds obtained by chemical synthesis. An interesting example is the combination of cuprocalcic sulfate plus mancozeb because it has a synergistic effect against several bacterial diseases (Hausbeck et al., 2000; Jones et al., 1991; Marco & Stall, 1983). Currently copper oxychloride is the copper compound most commonly recommended against olive knot disease by the Spanish extension services. The active ingredient in these products is the divalent copper ion solubilized and both, bacteria and plant exudates, contain compounds which are capable of solubilizing copper. Generally, these products have a toxic or bacteriostatic effect, only preventing the multiplication of bacteria and most bacteria may die due to the toxic effects of Cu++ ions, or enter in the VBNC (Viable But Non Culturable) state in which they are unable to grow on solid medium. This state could be induced by copper ions, as previously reported for several plant pathogenic bacteria (Alexander et al., 1999; Grey & Steck, 2001; Ordax et al., 2005). These preventive chemical treatments are recommended for both to reduce epiphytic Psv populations and prevent their

The effectiveness of chemical control of olive knot has been poorly evaluated, both in the field and in experimental assays under controlled conditions. The effect of the treatments against epiphytic inoculum of Psv or the optimal time of application are not well known. Several studies suggest that the management of epiphytic Psv populations probably reduces the incidence of olive knot disease (Ercolani, 1978, 1991; Lavermicocca & Surico, 1987; Quesada et al., 2007, 2010a). In this context, a chemical control program using copper compounds was proposed, based on field observations in California (Horne et al., 1912; Wilson, 1935). The first field experiments described in the literature were conducted in California where several Bordeaux mixture formulations controlled olive knot disease with minimal phytotoxicity symptoms in commercial olive groves with prevalent Psv infections (Krueger et al., 1999; Teviotdale & Krueger, 2004; Wilson, 1935). Assays performed with copper hydroxide showed that a single post-harvest copper application provided only minimal protection against the disease and subsequently, additional sprays in spring were needed to substantially improve its control (Teviotdale & Krueger, 2004). The efficacy of

Disease severity of a particular cultivar was found to be highly dependent on the pathogen dose applied at the inoculation point. In addition, secondary knot formation in non-inoculated wounds in previously inoculated plants, would suggest pathogen migration in the plant tissues. They also observed a correlation between the number of inoculation sites in which knots developed and the number of secondary knots formed when the initial wounds were inoculated with low bacterial doses. All cultivars developed knots at inoculation points, at least when high inoculum doses were applied. According to the results, six cultivars were classified as highly susceptible (Arbequina, Arróniz, Nevadillo Blanco de Jaén, Pajarero, Picudo and Vallesa). Some cultivars were classified as slightly susceptible (Azapa, Cerezuela, Chemlali, Dulzal de Carmona, Frantoio, FS-17, Gordal de Archidona, Gordal de Hellín, Lechín de Granada, Manzanilla Cacereña, Manzanilla de Sevilla, Nevadillo negro and Villalonga). The remaining cultivars (Changlot Real, Morisca, Gordal sevillana, Lechín de Sevilla, Oblonga, Picual, Ascolana tenera, Royal de Cazorla, Mollar de Cieza and Koroneiki) were classified as moderately susceptible.

So far studies on cultivar susceptibility to olive knot disease suggest that true resistance to this disease is uncommon among cultivated olive cultivars. In contrast, significant differences were observed in the degree of susceptibility among the cultivars tested. In addition, *in vitro* studies of Psv interaction with cell cultures of the cultivar Galega vulgar showed the typical events of a hypersensitive response in inoculated plant cells, such as an increase in reactive oxygen species, the activation of programmed cell death and decreased cell viability (Cruz & Tavares, 2005). High resolution liquid chromatography and mass spectrometry analysis of Psv-related knot extracts from outbreaks in olive trees of cultivar Koroneiki revealed high amounts of phenolic compounds, o-diphenols (oleopurina) and polyamines (spermidine, spermine, putrescine), in addition to auxins (Roussos et al., 2002). These authors postulated that the production of indole-3-acetonitrile and phenolic compounds could be related to the olive tree's defense mechanisms in knots. Cayuela et al. (2006) identified verbascoside as the main phenolic compound produced at significant levels in Psv-related knot extracts in olive trees of the cultivar Picual.

Balanced soil fertilization, avoiding excess nitrogen, may increase plant resistance to infection (Paoletti, 1993). However, in modern olivicultural practices such a balance is hard to maintain because the rapid development of young plants is valued, with early production onset and increased yields from one year to the next. A common mistake made to meet the demands of modern oliviculture is to apply an excess of nitrogen fertilizer, as this increases susceptibility to olive knot disease (Balestra & Varvaro, 1997). It is advisable to perform main fertilization of olive trees in January-February (Baratta & Di Marco, 1981) with low winter temperatures.

#### **8.2.3 Direct control**

Direct protection measures are mainly based on chemical or biological principles and are used by the growers when prophylactic measures have failed to stop disease progression in one zone (Montesinos & López, 1996), or are combined with all the other measures in an integrated control strategy.

#### **8.2.3.1 Chemical control**

312 Plant Pathology

Disease severity of a particular cultivar was found to be highly dependent on the pathogen dose applied at the inoculation point. In addition, secondary knot formation in non-inoculated wounds in previously inoculated plants, would suggest pathogen migration in the plant tissues. They also observed a correlation between the number of inoculation sites in which knots developed and the number of secondary knots formed when the initial wounds were inoculated with low bacterial doses. All cultivars developed knots at inoculation points, at least when high inoculum doses were applied. According to the results, six cultivars were classified as highly susceptible (Arbequina, Arróniz, Nevadillo Blanco de Jaén, Pajarero, Picudo and Vallesa). Some cultivars were classified as slightly susceptible (Azapa, Cerezuela, Chemlali, Dulzal de Carmona, Frantoio, FS-17, Gordal de Archidona, Gordal de Hellín, Lechín de Granada, Manzanilla Cacereña, Manzanilla de Sevilla, Nevadillo negro and Villalonga). The remaining cultivars (Changlot Real, Morisca, Gordal sevillana, Lechín de Sevilla, Oblonga, Picual, Ascolana tenera, Royal de Cazorla, Mollar de Cieza and Koroneiki) were classified as

So far studies on cultivar susceptibility to olive knot disease suggest that true resistance to this disease is uncommon among cultivated olive cultivars. In contrast, significant differences were observed in the degree of susceptibility among the cultivars tested. In addition, *in vitro* studies of Psv interaction with cell cultures of the cultivar Galega vulgar showed the typical events of a hypersensitive response in inoculated plant cells, such as an increase in reactive oxygen species, the activation of programmed cell death and decreased cell viability (Cruz & Tavares, 2005). High resolution liquid chromatography and mass spectrometry analysis of Psv-related knot extracts from outbreaks in olive trees of cultivar Koroneiki revealed high amounts of phenolic compounds, o-diphenols (oleopurina) and polyamines (spermidine, spermine, putrescine), in addition to auxins (Roussos et al., 2002). These authors postulated that the production of indole-3-acetonitrile and phenolic compounds could be related to the olive tree's defense mechanisms in knots. Cayuela et al. (2006) identified verbascoside as the main phenolic compound produced at significant levels

Balanced soil fertilization, avoiding excess nitrogen, may increase plant resistance to infection (Paoletti, 1993). However, in modern olivicultural practices such a balance is hard to maintain because the rapid development of young plants is valued, with early production onset and increased yields from one year to the next. A common mistake made to meet the demands of modern oliviculture is to apply an excess of nitrogen fertilizer, as this increases susceptibility to olive knot disease (Balestra & Varvaro, 1997). It is advisable to perform main fertilization of olive trees in January-February (Baratta & Di Marco, 1981) with low

Direct protection measures are mainly based on chemical or biological principles and are used by the growers when prophylactic measures have failed to stop disease progression in one zone (Montesinos & López, 1996), or are combined with all the other measures in an

in Psv-related knot extracts in olive trees of the cultivar Picual.

moderately susceptible.

winter temperatures.

**8.2.3 Direct control** 

integrated control strategy.

Chemical control of bacterial plant diseases is only effective when they are used in preventive strategies before the onset of infection or very early in the bacterial infection process (Montesinos & López, 1996). Specifically, chemical control of olive knot disease has given inconsistent results in field experiments and may also have low efficacy and even show phytotoxicity to some tissues. This variability is due to several factors, such as the amount of inoculum, timing of treatments, climatic conditions, cultivar susceptibility, treatment application method, or physiological state of the host plant.

Copper compounds are the main preventive chemical treatment recommended against olive knot disease and their use is recommended every year when there is a risk of infection, in spring and fall before the rains, after the leaf fall and especially after hail and frost or other events causing olive injures (Penyalver et al., 1998; Protta, 1995; Smith et al., 1991; Wilson, 1935). A positive correlation has been found between disease incidence and spring rains (Teviotdale & Krueger, 2004) and it was observed that moist winds in coastal areas promote infection (Smith et al., 1991).

The copper-based compounds used in olive groves in Spain include various salts and formulations (hydroxides, oxychlorides, oxides or sulfates) as well as their mixture with organic compounds obtained by chemical synthesis. An interesting example is the combination of cuprocalcic sulfate plus mancozeb because it has a synergistic effect against several bacterial diseases (Hausbeck et al., 2000; Jones et al., 1991; Marco & Stall, 1983). Currently copper oxychloride is the copper compound most commonly recommended against olive knot disease by the Spanish extension services. The active ingredient in these products is the divalent copper ion solubilized and both, bacteria and plant exudates, contain compounds which are capable of solubilizing copper. Generally, these products have a toxic or bacteriostatic effect, only preventing the multiplication of bacteria and most bacteria may die due to the toxic effects of Cu++ ions, or enter in the VBNC (Viable But Non Culturable) state in which they are unable to grow on solid medium. This state could be induced by copper ions, as previously reported for several plant pathogenic bacteria (Alexander et al., 1999; Grey & Steck, 2001; Ordax et al., 2005). These preventive chemical treatments are recommended for both to reduce epiphytic Psv populations and prevent their penetration through the plant wounds.

The effectiveness of chemical control of olive knot has been poorly evaluated, both in the field and in experimental assays under controlled conditions. The effect of the treatments against epiphytic inoculum of Psv or the optimal time of application are not well known. Several studies suggest that the management of epiphytic Psv populations probably reduces the incidence of olive knot disease (Ercolani, 1978, 1991; Lavermicocca & Surico, 1987; Quesada et al., 2007, 2010a). In this context, a chemical control program using copper compounds was proposed, based on field observations in California (Horne et al., 1912; Wilson, 1935). The first field experiments described in the literature were conducted in California where several Bordeaux mixture formulations controlled olive knot disease with minimal phytotoxicity symptoms in commercial olive groves with prevalent Psv infections (Krueger et al., 1999; Teviotdale & Krueger, 2004; Wilson, 1935). Assays performed with copper hydroxide showed that a single post-harvest copper application provided only minimal protection against the disease and subsequently, additional sprays in spring were needed to substantially improve its control (Teviotdale & Krueger, 2004). The efficacy of

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

showed antagonistic *in vitro* activity against Psv (Krid et al., 2010).

quantitative effects of the disease on olive production are insufficient.

work was supported in part by grant CAO00-007 from INIA, Spain.

especially important for high density new plantations.

**8.2.3.2 Biological control** 

advising their commercial registration.

**9. General conclusions** 

effective control.

**10. Acknowledgments** 

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 315

Biological control is another alternative to control olive knot disease, but is seldom tested against Psv. To date, biological control agents have been evaluated using isolates of *P. fluorescens* (Blightban) and Psv mutants producing bacteriocins, but without satisfactory results (Krueger et al., 1999; Varvaro & Martella, 1993). Besides, non pathogenic *Pseudomonas* sp. isolated from olive tree rhizosphere proved antagonistic against Psv (Rokni-Zadeh et al., 2008). Recently, *P. fluorescens* and *Bacillus subtilis* isolates from knots and leaves of olive trees

Bacteriocins are excellent candidates for using in agriculture to control plant pathogenic bacteria due to their high specificity. A bacteriocin produced by *P. syringae* pv. *ciccaronei* was shown to inhibit the proliferation and survival of epiphytic Psv form (Lavermicocca et al., 2002, 2003). Effectiveness of assays with two-year-old olive plants in a culture chamber was equivalent to that of copper hydroxide, although it would be interesting to evaluate its efficacy in nursery or field plants and determine their toxicity and persistence before

Remarkable progress has been made in several aspects of the host-pathogen interaction of the causal agent of olive knot disease, recently. Additionally, studies on the epidemiology of olive knot disease, as well as on its chemical control, have also been reported to add to the scarce information available. However, further studies are needed to assess reliably the importance of the endophytic phase of Psv in the epidemiology of olive knot as well as the effect of different chemical on disease incidence. Furthermore, studies on the qualitative and

The production, maintenance and use of certified and potentially pathogen-free plant material, is one of the main preventive measures used to control plant pathogens and certification schemes based in analytical tests performed on olive plants before leaving the nurseries should be implemented. Although, so far, true resistance to this disease is uncommon among olive cultivars tested, significant differences were observed in the degree of susceptibility to the disease among them. Cultivars tolerant to olive knot and resistant/tolerant to climatic conditions, or avoiding cultural practices which favor olive knot development should be considered for planting in the new commercial fields. This is

The olive knot disease integrated control should combine healthy plant material with appropriate cultural practices and the use, like preventive treatments, of chemical compounds. In such a context, copper treatments should be used regularly to achieve

Authors wish to thank M. Cambra for critical reading and suggestions. We also thank E. Bertolini for generously give us permission to include Figure 2. The research of J.M. Quesada was supported by a predoctoral fellowship from IFAPA, Andalucía, Spain. This

copper hydroxide to control the incidence of knots was higher after three sprays than after two or one single spray. New information has been gathered about the effect of copper compounds on the population dynamics of epiphytic Psv, the possible appearance of copper resistance, or its role in decreasing olive knot incidence under Mediterranean conditions in high-density groves (Quesada et al., 2010b).

The effect of copper oxychloride or cuprocalcic sulfate plus mancozeb treatments on Psv populations and subsequent disease development were evaluated in an olive grove planted with two susceptible cultivars, Arbequina and Picudo, over a four-year period. Unlike the previous studies, to homogenize the knot number per tree before beginning treatments, olive trees were inoculated. The effect of copper on Psv populations was observed after the first application, but the greatest differences between copper-treated and untreated plants were observed in the third year, after five copper applications. Two applications of copper compounds per year, reduced Psv populations effectively. We also found that treatment with copper compounds had a drastic effect on reducing disease incidence (Quesada et al., 2010b). These results for both cultivars, in this high-density grove, supported previous observations by Teviotdale and Krueger (2004) in California, in standard groves. Unlike other plant pathogenic bacteria that develop copper resistance after extensive exposure to copper compounds (Cazorla et al., 2002; Cooksey, 1990; Garret & Schwartz, 1998; Marco & Stall, 1983; Scheck et al., 1996; Sundin et al., 1989, 1994), copper resistance was not detected in the remaining Psv bacteria in copper-treated olives trees.

Chemical treatments based on antibiotics and oil-water emulsion containing hydrocarbons have also been recommended but without encouraging results (Scrivani & Bugiani, 1955; Schroth & Hildebrand, 1968). The use of antibiotics such as streptomycin and terramycin has been successful under experimental conditions (Trapero & Blanco, 1998) but their application against plant pathogenic bacteria is currently forbidden by the EU legislation, although it is permitted in some other countries.

Systemic acquired response, or SAR, is plants' ability to generate defense reactions against external aggression at sites far away from the point of attack. In these distant sites, the genes involved in defense processes are activated (e.g. PR proteins), thereby increasing the resistance of these tissues against possible further attacks (Durrant & Dong, 2004; Kessmann et al., 1994). Some products were recently evaluated for their induction of plant resistance against different pathogens such as acibenzolar-S-methyl (Bion ®), fosetyl-aluminium, calcium prohexadione or harpins. They were assayed to control some bacterial plant diseases like fire blight, citrus canker, apical necrosis of mango, etc. Most of these products do not produce phytotoxicity and their efficacy is sometimes comparable to that of antibiotics or copper-based compounds while others could not control these diseases (Brisset et al., 2000; Cazorla et al., 2006; Graham & Leite, 2004; Scortichini, 2002,). There are reports of acibenzolar-S-methyl-related activation of certain genes involved in defense responses in olive leaves of the cultivar Lechín de Sevilla (Muñoz et al., 2005). However, in one experiment acibenzolar-S-methyl did not reduce either Psv populations or the incidence of olive knot disease after two treatments per year over a four-year period (Quesada et al., 2010b). It is possible that different doses and more product applications could be required for achieve better efficacy. Curiously, vigor of cv. Picudo was significantly higher in olive trees treated with acibenzolar-S-methyl than in untreated trees, although disease incidence was similar in both treated and untreated olive trees (Quesada et al., 2010b).

#### **8.2.3.2 Biological control**

314 Plant Pathology

copper hydroxide to control the incidence of knots was higher after three sprays than after two or one single spray. New information has been gathered about the effect of copper compounds on the population dynamics of epiphytic Psv, the possible appearance of copper resistance, or its role in decreasing olive knot incidence under Mediterranean conditions in

The effect of copper oxychloride or cuprocalcic sulfate plus mancozeb treatments on Psv populations and subsequent disease development were evaluated in an olive grove planted with two susceptible cultivars, Arbequina and Picudo, over a four-year period. Unlike the previous studies, to homogenize the knot number per tree before beginning treatments, olive trees were inoculated. The effect of copper on Psv populations was observed after the first application, but the greatest differences between copper-treated and untreated plants were observed in the third year, after five copper applications. Two applications of copper compounds per year, reduced Psv populations effectively. We also found that treatment with copper compounds had a drastic effect on reducing disease incidence (Quesada et al., 2010b). These results for both cultivars, in this high-density grove, supported previous observations by Teviotdale and Krueger (2004) in California, in standard groves. Unlike other plant pathogenic bacteria that develop copper resistance after extensive exposure to copper compounds (Cazorla et al., 2002; Cooksey, 1990; Garret & Schwartz, 1998; Marco & Stall, 1983; Scheck et al., 1996; Sundin et al., 1989, 1994), copper resistance was not detected

Chemical treatments based on antibiotics and oil-water emulsion containing hydrocarbons have also been recommended but without encouraging results (Scrivani & Bugiani, 1955; Schroth & Hildebrand, 1968). The use of antibiotics such as streptomycin and terramycin has been successful under experimental conditions (Trapero & Blanco, 1998) but their application against plant pathogenic bacteria is currently forbidden by the EU legislation,

Systemic acquired response, or SAR, is plants' ability to generate defense reactions against external aggression at sites far away from the point of attack. In these distant sites, the genes involved in defense processes are activated (e.g. PR proteins), thereby increasing the resistance of these tissues against possible further attacks (Durrant & Dong, 2004; Kessmann et al., 1994). Some products were recently evaluated for their induction of plant resistance against different pathogens such as acibenzolar-S-methyl (Bion ®), fosetyl-aluminium, calcium prohexadione or harpins. They were assayed to control some bacterial plant diseases like fire blight, citrus canker, apical necrosis of mango, etc. Most of these products do not produce phytotoxicity and their efficacy is sometimes comparable to that of antibiotics or copper-based compounds while others could not control these diseases (Brisset et al., 2000; Cazorla et al., 2006; Graham & Leite, 2004; Scortichini, 2002,). There are reports of acibenzolar-S-methyl-related activation of certain genes involved in defense responses in olive leaves of the cultivar Lechín de Sevilla (Muñoz et al., 2005). However, in one experiment acibenzolar-S-methyl did not reduce either Psv populations or the incidence of olive knot disease after two treatments per year over a four-year period (Quesada et al., 2010b). It is possible that different doses and more product applications could be required for achieve better efficacy. Curiously, vigor of cv. Picudo was significantly higher in olive trees treated with acibenzolar-S-methyl than in untreated trees, although disease incidence

was similar in both treated and untreated olive trees (Quesada et al., 2010b).

high-density groves (Quesada et al., 2010b).

in the remaining Psv bacteria in copper-treated olives trees.

although it is permitted in some other countries.

Biological control is another alternative to control olive knot disease, but is seldom tested against Psv. To date, biological control agents have been evaluated using isolates of *P. fluorescens* (Blightban) and Psv mutants producing bacteriocins, but without satisfactory results (Krueger et al., 1999; Varvaro & Martella, 1993). Besides, non pathogenic *Pseudomonas* sp. isolated from olive tree rhizosphere proved antagonistic against Psv (Rokni-Zadeh et al., 2008). Recently, *P. fluorescens* and *Bacillus subtilis* isolates from knots and leaves of olive trees showed antagonistic *in vitro* activity against Psv (Krid et al., 2010).

Bacteriocins are excellent candidates for using in agriculture to control plant pathogenic bacteria due to their high specificity. A bacteriocin produced by *P. syringae* pv. *ciccaronei* was shown to inhibit the proliferation and survival of epiphytic Psv form (Lavermicocca et al., 2002, 2003). Effectiveness of assays with two-year-old olive plants in a culture chamber was equivalent to that of copper hydroxide, although it would be interesting to evaluate its efficacy in nursery or field plants and determine their toxicity and persistence before advising their commercial registration.

### **9. General conclusions**

Remarkable progress has been made in several aspects of the host-pathogen interaction of the causal agent of olive knot disease, recently. Additionally, studies on the epidemiology of olive knot disease, as well as on its chemical control, have also been reported to add to the scarce information available. However, further studies are needed to assess reliably the importance of the endophytic phase of Psv in the epidemiology of olive knot as well as the effect of different chemical on disease incidence. Furthermore, studies on the qualitative and quantitative effects of the disease on olive production are insufficient.

The production, maintenance and use of certified and potentially pathogen-free plant material, is one of the main preventive measures used to control plant pathogens and certification schemes based in analytical tests performed on olive plants before leaving the nurseries should be implemented. Although, so far, true resistance to this disease is uncommon among olive cultivars tested, significant differences were observed in the degree of susceptibility to the disease among them. Cultivars tolerant to olive knot and resistant/tolerant to climatic conditions, or avoiding cultural practices which favor olive knot development should be considered for planting in the new commercial fields. This is especially important for high density new plantations.

The olive knot disease integrated control should combine healthy plant material with appropriate cultural practices and the use, like preventive treatments, of chemical compounds. In such a context, copper treatments should be used regularly to achieve effective control.

#### **10. Acknowledgments**

Authors wish to thank M. Cambra for critical reading and suggestions. We also thank E. Bertolini for generously give us permission to include Figure 2. The research of J.M. Quesada was supported by a predoctoral fellowship from IFAPA, Andalucía, Spain. This work was supported in part by grant CAO00-007 from INIA, Spain.

Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

*y Cooperación*. Vol. 173, pp. 30-31, ISSN 0301-438X

1/2, (January-June 2005), pp. 41-52, ISSN 0371-5124

536, ISSN 0929-1873

ISSN 0021-8561

2002), pp. 909-916, ISSN 0031-949X

2006), pp. 279-288, ISSN 0929-1873

Española, ISBN 978-84-85441-93-8, Madrid, Spain

Available from http://hdl.handle.net/1822/3437

*Phytopathology*, Vol. 42, pp. 185-209, ISSN 0066-4286

Ediciones, ISBN 8486216192, Jaén, Spain

143, No. 2, (August 1980), pp. 950-957, ISSN 0021-9193

*Review of Phytopathology*, Vol. 28, pp. 201-219, ISSN 0066-4286

41-44, ISSN 1131-8988

Spain

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 317

Caballero, P., & Murillo, J. (2003). *Protección de cultivos. Conceptos actuales y fuentes de* 

Cambra, M., López, M.M., Durán-Vila, N., Bertolini, E., Penyalver, R., & Gorris, M.T. (1998).

Catara, V., Colina, P., Bella, P., Tessitori, M., & Tirrò, A. (2005). Variabilità di *Pseudomonas* 

Cayuela, J.A., Rada, M., Ríos, J.J., Albi, T., & Guinda, A. (2006). Changes in phenolic

Cazorla, F.M., Arrebola, E., Sesma, A., Pérez-García, A., Codina, J.C., Murillo, J., & de

Cazorla, F.M., Arrebola, E., Olea, F., Velasco, L., Hermoso, J.M., Pérez-García, A., Torés, J.A.,

Chomé, P.M. (1998). Programa de certificación de plantas de vivero de olivo en España y

Civantos, L. (2008). *Obtención del aceite de oliva virgen*, (Third edition), Editorial Agrícola

Comai, L., & Kosuge, T. (1980). Involvement of plasmid deoxyribonucleic acid in

Cooksey, D.A. (1990). Genetics of bactericide resistance in plant pathogenic bacteria. *Annual* 

Cruz, A. Braga da, & Tavares, R.M. (2005). Evaluation of programmed cell death in *Olea* 

De Andrés, F. (1991). *Enfermedades y plagas del olivo* (second edition), Riquelme y Vargas

Durrant, W.E., & Dong, X. (2004). Systemic Acquired Resistance. *Annual Review of* 

fire blight. *European Journal of Plant Pathology*, Vol. 106, No. 6, (July 2000), pp. 529-

*información* (First edition), Universidad Pública de Navarra, ISBN 84-9769-014-1,

Programa de certificación vegetal en olivo. Incidencia en la producción. *Agricultura* 

*savastanoi* pv. *savastanoi* in un'area olivicola della Sicilia e comportamento di alcune varietà di olivo alle inoculazioni [*Olea europaea* L.]. *Tecnica Agricola*, Vol. 57, No.

composition induced by *Pseudomonas savastanoi* pv. *savastanoi* infection in olive tree: presence of large amounts of verbascoside in nodules of tuberculosis disease. *Journal of Agricultural and Food Chemistry*, Vol. 54, No. 15, (July 2006), pp. 5363-5368,

Vicente, A. (2002). Copper resistance in *Pseudomonas syringae* strains isolated from mango is encoded mainly by plasmids. *Phytopathology*, Vol. 92, No. 8, (August

Farré, J.M., & de Vicente, A. (2006). Field evaluation of treatments for the control of the bacterial apical necrosis of mango (*Mangifera indica*) caused by *Pseudomonas syringae* pv. *syringae*. *European Journal of Plant Pathology*, Vol. 116, No. 4, (December

registro de variedades comerciales. *Phytoma-España*, No. 102, (October 1998), pp.

indoleacetic acid synthesis in *Pseudomonas savastanoi*. *Journal of Bacteriology*, Vol.

*europaea* var. Galega vulgar suspension cell cultures elicited with *Pseudomonas savastanoi*, *IX Congresso Luso-Espanhol de Fisiologia Vegetal*, (September 2005),

#### **11. References**


Alexander, E., Pham, D., & Steck, T.R. (1999). The viable-but-nonculturable condition is

Anonymous. (1999). Ministerio de Agricultura, Pesca y Alimentación. Real Decreto

Abu-Ghorrah, M. (1988). Taxonomie et pouvoir pathogène de *Pseudomonas syringae* pv.

Agrios, G.N. (ed.). (2005). *Plant Pathology*, (5th edition), Elsevier Academic Press, ISBN 0-12-

Balestra, G.M., & Varvaro, L. (1997). Influence of nitrogen fertilization on the colonization of

Barranco, D. (1998). Variedades y patrones, In: *El cultivo del olivo*, Barranco, D., Fernández-

Beltrá, R. (1956). New technique for the identification of *Pseudomonas savastanoi*. *Microbiología* 

Benjama, A. (1994). Étude de la sensibilité variétale de l'olivier au Maroc vis-à-vis de

Bertolini, E. (2003). Virosis y bacteriosis del olivo: detección serológica y molecular. Thesis

Bertolini, E., Penyalver, R., García, A., Olmos, A., Quesada, J.M., Cambra, M., & López,

Bertolini, E., Olmos, A., Lopez, M.M., & Cambra, M. (2003b). Multiplex nested reverse

Brisset, M.N., Cesbron, S., Thomson, S.V., & Paulin, J.P. (2000). Acibenzolar-S-methyl

(Ph. D.). Universidad Politécnica de Valencia, Spain

Vol. 3, No. 6, (November/December 1994), pp. 405-408, ISSN 1166-7699 Bertolini, E., Fadda, Z., García, F., Celada, B., Olmos, A., Gorris, M.T., Del Río, C., Caballero,

*savastanoi*. Thesis (Ph. D.). University of Angers, France

induced by copper in *Agrobacterium tumefaciens* and *Rhizobium leguminosarum*. *Applied and Environmental Microbiology*, Vol. 65, No. 8, (August 1999), pp. 3754-3756,

1678/1999 de 29 de octubre de 1999. BOE 276, 18 de noviembre de 1999, pp. 40077-

olive phylloplane by *Pseudomonas syringae* subsp. *savastanoi*, *Developments in Plant Pathology. Vol. 9:* Pseudomonas syringae *pathovars and related pathogens*, pp. 88-92, Kluwer Academic Publishers, ISBN 0-7923-4601-7, Dordrecht, The Netherlands Baratta, B., & Di Marco, L. (1981). Controllo degli attacchi di rogna nella cultivar Nocellara

del Belice. *Informatore Fitopatologico*. Vol. 31, No. 1-2, (January/February 1981), pp.

Escobar, D., & Rallo, L., pp. 56-79, Junta de Andalucía- Mundi-Prensa, ISBN 978-84-

*Pseudomonas syringae* pv. *savastanoi*, agent de la tuberculose. *Cahiers Agricultures*,

J., Durán-Vila, N., & Cambra, M. (1998). Virosis del olivo detectadas en España. Nuevos métodos de diagnóstico. *Phytoma-España*, No. 102, (October 1998), pp. 191-

M.M. (2003a). Highly sensitive detection of *Pseudomonas savastanoi* pv. *savastanoi* in asymptomatic olive plants by nested-PCR in a single closed tube. *Journal of Microbiological Methods*, Vol. 52, No. 2, (February 2003), pp. 261-266, ISSN 0167-7012

transcription-polymerase chain reaction in a single tube for sensitive and simultaneous detection of four RNA viruses and *Pseudomonas savastanoi* pv. *savastanoi* in olive trees. *Phytopathology*, Vol. 93, No. 3, (March 2003), pp. 286-292,

induces the accumulation of defense-related enzymes in apple and protects from

**11. References** 

ISSN 0099-2240

044565-4, San Diego, CA

115-116, ISSN 0020-0735

8474-234-0, Madrid, Spain

*España*, Vol. 9, pp. 433-504

193, ISSN 1131-8988

ISSN 0031-949X

40079

fire blight. *European Journal of Plant Pathology*, Vol. 106, No. 6, (July 2000), pp. 529- 536, ISSN 0929-1873


Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

0099-2240

0031-949X

2172

USA

ISSN 1439-0434

1466-5026

949X

1-14, ISSN 0168-6496

ISBN 0387972587, New York, USA

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 319

Grey, B.E., & Steck, T.R. (2001). The viable but nonculturable state of *Ralstonia solanacearum*

Hassani, D., Buonaurio, R., & Tombesi, A. (2003). Response of some olive cultivars, hybrid

489-494, Kluwer Academic Publishers, ISBN: 1402012276, Dordrecht, Boston Hausbeck, M.K., Bell, J., Medina-Mora, C., Podolsky, R., & Fulbright, D.W. (2000). Effect of

Hirano, S.S., & Upper, C.D. (1983). Ecology and epidemiology of foliar bacterial plant pathogens. *Annual Review of Phytopathology*, Vol. 21, pp. 243-269, ISSN 0066-4286 Hirano, S.S., & Upper, C.D. (2000). Bacteria in the leaf ecosystem with emphasis on

Horne, T., Parker, B., & Daines, L.L. (1912). The method of spreading of the olive knot

Iacobellis, N.S. (2001). Olive knot. In: *Encyclopedia of Plant Pathology*, Vol. 2, O.C. Maloy &

Iacobellis, N.S., Sisto, A., Surico, G., Evidente, A., & Di Maio, E. (1994). Pathogenicity of

Jacques, M.E., & Morris, C.E. (1995). A review of issues to the quantification of bacteria from

Janse, J.D. (1982). *Pseudomonas syringae* subsp. *savastanoi* ex Smith subsp. nov., nom. rev., the

Jones, J.B., Woltz, S.S., Jones, J.P., & Portier, K.L. (1991). Population dynamics of

Kado, C.I. (1992). Plant pathogenic bacteria, In: *The Prokaryotes: a handbook on the biology of* 

disease. *Phytopathology*. Vol. 2, pp. 101-105, ISSN: 0031-949X

may be involved in long-term survival and plant infection. *Applied and Environmental Microbiology*, Vol. 67, No. 9, (September 2001), pp. 3866-3872, ISSN

and open pollinated seedling to *Pseudomonas savastanoi* pv. *savastanoi*. *International Conference on Pseudomonas syringae Pathovars and Related Pathogens*, In: Pseudomonas syringae *and related pathogens: biology and genetic*, N.S. Iacobellis et al. (Eds.), pp.

bactericides on population sizes and spread of *Clavibacter michiganensis* subsp. *michiganensis* on tomatoes in the greenhouse and on disease development and crop yield in the field. *Phytopathology*, Vol. 90, No. 1, (January 2000), pp. 38-44, ISSN

*Pseudomonas syringae*- a pathogen, ice nucleus, and epiphyte. *Microbiology and Molecular Biology Reviews*, Vol. 64, No. 3, (September 2000), pp. 624-653, ISSN 1092-

T.D Murray (Eds.), pp. 713-715, John Wiley and sons, ISBN 0471298174, New York,

*Pseudomonas syringae* subsp. *savastanoi* mutants defective in phytohormone production. *Journal of Phytopathology*, Vol. 140, No. 3, (March 1994), pp. 238-248,

the phyllosphere. *FEMS Microbiology Ecology*, Vol. 18, No. 1, (September 1995), pp.

bacterium causing excrescences on *Oleaceae* and *Nerium oleander* L. *International Journal of Systematic Bacteriology*, Vol. 32, No. 2, (April 1982), pp. 166-169, ISSN:

*Xanthomonas campestris* pv. *vesicatoria* on tomato leaflets treated with copper bactericides. *Phytopathology*, Vol. 81, No. 7, (July 1991), pp. 714-719, ISSN 0031-

*bacteria: ecophysiology, isolation, identification, applications*, Balows, A., Truper, H. G., Dworkin, M., Harder, W. & Schleifer, K.H., Vol. I, pp. 659-674, Springer-Verlag,


EPPO (2006). Pathogen-tested olive trees and rootstocks. *Bulletin OEPP / EPPO Bulletin*, Vol.

Ercolani, G.L. (1971). Presenza epifitica di *Pseudomonas savastanoi* (E. f. Smith) Stevens

Ercolani, G.L. (1978). *Pseudomonas savastanoi* and other bacteria colonizing the surface of

Ercolani, G.L. (1979). Distribuzione di *Pseudomonas savastanoi* sulle foglie dell'olivo.

Ercolani, G.L. (1983). Variability among isolates of *Pseudomonas syringae* pv. *savastanoi* from

Ercolani, G.L. (1985). Factor analysis of fluctuation in populations of *Pseudomonas syringae* 

Ercolani, G.L. (1991). Distribution of epiphytic bacteria on olive leaves and the influence of

Ercolani, G.L. (1993). Comparison of strains of *Pseudomonas syringae* pv. *savastanoi* from olive

Fernandes, A., & Marcelo, M. (2002). A possible synergistic effect of *Erwinia* sp. on the

García de los Ríos, J.E. (1989). Estudio acerca de la tuberculosis del olivo. Thesis (Ph. D.).

García, A., Penyalver, R., & López, M.M. (2001). Sensibilidad de variedades de olivo a

Gardan, L., Bollet, C., Abu-Ghorrah, M.A., Grimont, F., & Grimont, P.A.D. (1992). DNA

Garret, K.A., & Schwartz, H.F. (1998). Epiphytic *Pseudomonas syringae* on dry beans treated

Graham, J.H., & Leite, R.P.Jr. (2004). Lack of control of citrus canker by induced systemic

*Phytopathologia Mediterranea*, Vol. 18, pp. 85-88, ISSN 0031-9465

sull'Olivo, in Pluglia. *Phytopathologia Mediterranea*, Vol. 10, No. 1, pp. 130-132, ISSN

olive leaves in the field. *Journal of General Microbiology*, Vol. 109, (December 1978),

the pylloplane of the olive. *Journal of General Microbiology*, Vol. 129, (April 1983), pp.

pv. *savastanoi* on the phylloplane of the olive. *Microbial Ecology*, Vol. 11, No. 1,

leaf age and sampling time. *Microbial Ecology*, Vol. 21, No. 1, pp. 35-48, ISSN 0095-

leaves and knots. *Letters in Applied Microbiology*, Vol. 16, No. 4, (April 1993), pp. 199-

development of olive knot symptoms caused by *Pseudomonas syringae* pv. *savastanoi* in *Olea europaea*, In: *Proceedings of the fourth International Symposium on Olive Growing*, Vitagliano, C. & Martelli, G.P., No. 586, pp. 729-731, Acta Horticulturae

*Pseudomonas savastanoi* pv. *savastanoi*, causante de la tuberculosis. *Fruticultura* 

relatedness among the pathovar strains of *Pseudomonas syringae* subsp. *savastanoi* Janse (1982) and proposal of *Pseudomonas savastanoi* sp. nov. *International Journal of Systematic and Evolutionary Microbiology*, Vol. 42, No. 4, (October 1992), pp. 606-612,

with copper-based bactericides. *Plant Disease*, Vol. 82, No. 1, (January 1998), pp. 30-

resistance compounds. *Plant Disease*, Vol. 88, No. 7, (July 2004), pp. 745-750, ISSN

36, No. 1, (April 2006), pp. 77–83, ISSN 1365-2338

0031-9465

3628

pp. 245-257, ISSN 0022-1287

901-916, ISSN 0022-1287

202, ISSN 0266-8254

ISSN 1466 5026

35, ISSN 0191-2917

0191-2917

(March 1985), pp. 41-49, ISSN 0095-3628

(ISHS), ISSN 0567-7572, Valenzano, Italy

Universidad Complutense de Madrid, Spain

*Profesional*, No. 120, pp. 57-58, ISSN 1131-5660


Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

1, (February 2009), pp. 152-158, ISSN 0032-0862

*Disease*, Vol. 67, pp. 779-781, ISSN 0191-2917

624, ISSN 0032-0862

1035, ISSN 0099-2240

6, USA

ISSN 0099-2240

84-921910-0-7, Valencia, Spain

1997), pp. 1570-1576, ISSN 0099-2240

ISBN 848474146X, Madrid, Spain

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 321

Marchi, G., Sisto, A., Cimmino, A., Andolfi, A., Cipriani, M.G., Evidente, A., & Surico, G.

Marchi, G., Mori, B., Pollacci, P., Mencuccini, M., & Surico, G. (2009). Systemic spread of

Marco, G.M., & Stall, R.E. (1983). Control of bacterial spot of pepper initiated by strains of

Martelli, G.P. 1998. Enfermedades infecciosas y certificación del olivo: Panorama general. *Phytoma-España*, No. 102, (October 1998), pp. 180-186, ISSN 1131-8988 Matas, I.M., Pérez-Martínez, I., Quesada, J.M., Rodríguez-Herva, J.J., Penyalver, R., &

Matas, I.M. (2010). Genómica funcional de la interacción *Pseudomonas savastanoi* pv.

Montesinos, E., & López, M.M. (1996). Métodos de control de las bacteriosis, In: *Patología* 

Morris, C.E., Monier, J., & Jacques, M. (1997). Methods for observing microbial biofilms

Morris, C.E., & Kinkel, L.L. (2002). Fifty years of phylosphere microbiology: significant

Muñoz, J., Benítez, Y., Trapero, A., Caballero, J.L., & Dorado, G. (2005). Identificación de

Ordax, M., Marco-Noales, E., López, M.M., & Biosca, E.G. (2005). Survival strategy of

Ouzari, H., Khsairi, A., Raddadi, N., Jaoua, L., Hassen, A., Zarrouk, M., Daffonchio, D., &

*savastanoi*- olivo. Thesis (Ph. D.). Universidad de Málaga, Spain

(2006). Interaction between *Pseudomonas savastanoi* pv. *savastanoi* and *Pantoea agglomerans* in olive knots. *Plant Pathology*, Vol. 55, No. 5, (October 2006), pp. 614-

*Pseudomonas savastanoi* pv. *savastanoi* in olive explants. *Plant Pathology*, Vol. 58, No.

*Xanthomonas campestris* pv. *vesicatoria* that differ in sensitivity to copper. *Plant* 

Ramos, C. (2009). *Pseudomonas savastanoi* pv. *savastanoi* contains two *iaaL* paralogs, one of which exhibits a variable number of a trinucleotide (TAC) tandem repeat. *Applied and Environmental Microbiology*, Vol. 75, No. 4, (February 2009), pp. 1030-

*Vegetal*, Llácer, G., López, M.M., Trapero, A., & Bello, A., pp. 653-678, Sociedad Española de Fitopatología- Phytoma España, S.L.- Grupo Mundi-Prensa, S.A., ISBN

directly on leaf surfaces and recovering them for isolation of culturable microorganisms. *Applied and Environmental Microbiology*, Vol. 63, No. 4, (April

contributions to research in related fields, In: *Phyllosphere Microbiology*, Lindow, S.E., Hecht-Poinar, E.I. & Elliott, V.J., pp. 365-375, APS Press, ISBN 978-0-89054-286-

genes expresados diferencialmente en la interacción entre olivo *Olea europaea* L. y el hongo parásito causante del repilo *Spilocaea oleagina*, In: *Variedades de Olivo en España*, Rallo, L., Barranco, D., Caballero, J.M., Del Río, C., Martín, A., Tous, J., & Trujillo, I., pp. 459-470, Junta de Andalucía, MAPA and Ediciones Mundi- Prensa,

*Erwinia amylovora* against copper: induction of the viable-but-nonculturable state. *Applied and Environmental Microbiology*, Vol. 72, No. 5, (May 2006), pp. 3482-3488,

Boudabous, A. (2008). Diversity of auxin-producing bacteria associated to


Kessmann, H., Staub, T., Hofmann, C., Maetzke, T., Herzog, J., Ward, E., Uknes, S., & Ryals,

Krid, S., Rhouma, A., Mogou, I., Quesada, J.M., Nesme, X., & Gargouri, A. (2010).

Krueger, W.H., Tevitodale, B.L., Scroth, M.N., Metzidakis, I.T., & Voyiaztzis, D.G. (1999).

474, pp. 567-571, Acta Horticulturae (ISHS), ISSN 0567-7572, Chania, Greece Lavermicocca, P., & Surico, G. (1987). Presenza epifitica di *Pseudomonas syringae* pv.

Lavermicocca, P., Lonigro, S.L., Valerio, F., Evidente, A., & Visconti, A. (2002). Reduction of

Lavermicocca, P., Valerio, F., Lonigro, S.L, Lazzaroni, S., Evidente, A., & Visconti, A. (2003).

Lindow, E.L., & Andersen, G.L. (1996). Influence of immigration on epiphytic bacterial

Lindow, E.L., & Brandl, M.T. (2003). Microbiology of the phyllosphere. *Applied and* 

Magie, A.R. (1963). Physiological factors involved in tumor production by the oleander knot

Manceau, C., & Kasempour, M.N. (2002). Endophytic versus epiphytic colonization of

E.I. & Elliott, V.J., pp. 115-123, APS Press, ISBN 978-0-89054-286-6, USA Marcelo, A., Fernández M., Fatima Potes, M., & Serrano, J.F. (1999). Reactions of some

No. 8, (August 1996), pp. 2978-2987, ISSN 0099-2240

*Annual Review of Phytopathology*, Vol. 32, pp. 439-459, ISSN 0066-4286 Kinkel, L.L. (1997). Microbial population dynamics on leaves. *Annual Review of* 

*Pathology*, Vol. 92, No. 2, (July 2010), pp. 335-341, ISSN: 1125-4653

*Phytopathology*, Vol. 35, pp. 327-347, ISSN 0066-4286

Vol. 26, pp. 136-141, ISSN 0031-9465

Dordrecht, The Netherlands

ISSN 0567-7572, Chania, Greece

0099-2240

2240

USA

J. (1994). Induction of systemic acquired disease resistance in plants by chemicals.

*Pseudomonas savastanoi* endophytic bacteria in olive tree knots and antagonistic potential of strains of *Pseudomonas fluorescens* and *Bacillus subtilis*. *Journal of Plant* 

Improvements in the control of olive knot disease, In: *Proceedings of the third International Symposium on Olive Growing*, Metzidakis, I.T. & Voyiaztzis, D.G., No.

*savastanoi* e di altri batteri sull'olivo e sull'oleandro. *Phytopathologia Mediterranea*,

olive knot disease by a bacteriocin from *Pseudomonas syringae* pv. *ciccaronei*. *Applied and Environmental Microbiology*, Vol. 68, No. 3, (March 2002), pp. 1403-1407, ISSN

Control of olive knot disease with a bacteriocin, In: Pseudomonas syringae *and related pathogens: Biology and Genetics*, Iacobellis, N.S., Collmer, A., Hutcheson, S.W., Mansfield, J.W., Morris, C.E., Murillo, J., Schaad, N.W., Stead, D.E., Surico, G. & Ullrich, M.S., pp. 451-457, Kluwer Academic Publishers, ISBN 1-4020-1227-6

populations on navel orange leaves. *Applied and Environmental Microbiology*, Vol. 62,

*Environmental Microbiology*, Vol. 69, No. 4, (April 2003), pp. 1875-1883, ISSN 0099-

pathogen, *Pseudomonas savastanoi*. Thesis (Ph. D.). University of California, Davis,

plants: what comes first?, In: *Phyllosphere Microbiology*, Lindow, S.E., Hecht-Poinar,

cultivars of *Olea europaea* L. to experimental inoculation with *Pseudomonas syringae* pv. *savastanoi*, In: *Proceedings of the third International Symposium on Olive Growing*, Metzidakis, I.T. & Voyiaztzis, D.G., No. 474, pp. 581-584, Acta Horticulturae (ISHS),


Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

(April 2010), pp. 262–269, ISSN 1365-3059

No. 4, (July 2009), pp. 476-488, ISSN 1751-7907

2010), pp. 1604-1620, ISSN 1462 2920

No. 1, pp. 199-203, ISSN 1379 1176

2002), pp. 533-541, ISSN 0168-9452

1413-1420, ISSN 0261-2194

822, ISSN 0031-949X

Spain

2222, ISSN 1466 5026

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 323

Quesada, J.M., Penyalver, R., Pérez-Panadés, J., Salcedo, C.I., Carbonell, E.A., & López,

Quesada, J.M., Penyalver, R., Pérez-Panadés, J., Salcedo, C.I., Carbonell, E.A., & López,

Rodríguez-Moreno, L., Barceló-Muñoz, A., & Ramos, C. (2008). In vitro analysis of the

Rodríguez-Moreno, L., Jiménez, A.J., & Ramos, C. (2009). Endopathogenic lifestyle of

Rodríguez-Palenzuela, P., Matas, I.M., Murillo, J., López-Solanilla, E., Bardaji, L., Pérez-

Rojas, A.M. (1999). Análisis fenotípicos, genéticos y filogenéticos de flora endofítica asociada

Rojas, A.M., García de los Ríos, J.E., Fischer-Le Saux, M., Jiménez, P., Reche, P., Bonneau, S.,

Rokni-Zadeh, H., Khavazi, K., Asgharzadeh, A., Hosseini-Mazinani, M., & De Mot, R.

Roussos, P.A., Pontikis, C.A., & Tsantili, E. (2002). Root promoting compounds detected in

Scheck, H.J., Pscheidt, J.W., & Moore, L.W. (1996). Copper and streptomycin resistance in

83, No. 9, (September 1996), pp. 1034-1039, ISSN 0191-2917

M.M. (2010a). Dissemination of *Pseudomonas savastanoi* pv. *savastanoi* populations and subsequent appearance of olive knot disease. *Plant Pathology*, Vol. 59, No. 2,

M.M. (2010b). Comparison of chemical treatments for reducing epiphytic *Pseudomonas savastanoi* pv. *savastanoi* populations and for improving subsequent control of olive knot disease. *Crop Protection*, Vol. 29, No. 12, (December 2010), pp.

interaction of *Pseudomonas savastanoi* pvs. *savastanoi* and *nerii* with micropropagated olive plants. *Phytopathology*, Vol. 98, No. 7, (July 2008), pp. 815-

*Pseudomonas savastanoi* pv. *savastanoi* in olive knots. *Microbial Biotechnology*, Vol. 2,

Martínez, I., Rodríguez-Moskera, M.E., Penyalver, R., López, M.M., Quesada, J.M., Biehl, B.S., Perna, N.T., Glasner, J.D., Cabot, E.L., Neeno-Eckwall, E., & Ramos, C. (2010). Annotation and overview of the *Pseudomonas savastanoi* pv. *savastanoi* NCPPB 3335 draft genome reveals the virulence gene complement of a tumourinducing pathogen of woody hosts. *Environmental Microbiology*, Vol. 12, No. 6, (June

a *Pseudomonas savastanoi*. Thesis (Ph. D.). Universidad San Pablo CEU, Madrid,

Sutra, L., Mathieu-Daudé, F., & McCelland, M. (2004). *Erwinia toletana* sp. nov., associated with *Pseudomonas savastanoi*-induced tree knots. *International Journal of Systematic and Evolutionary Microbiology*, Vol. 54, No. 6, (November 2004), pp. 2217-

(2008). Biocontrol of *Pseudomonas savastanoi*, causative agent of olive knot disease: antagonistic potential of non-pathogenic rhizosphere isolates of fluorescent *Pseudomonas*. *Communications in Agricultural and Applied Biological Sciences,* Vol. 73,

olive knot extract in high quantities as a response to infection by the bacterium *Pseudomonas savastanoi* pv. *savastanoi*. *Plant Science*, Vol. 163, No. 3, (September

strains of *Pseudomonas syringae* from Pacific Northwest nurseries. *Plant Disease*, Vol.

*Pseudomonas savastanoi* -induced olive knots. *Journal of Basic Microbiology*, Vol. 48, No. 5, (October 2008), pp. 370-377, ISSN 1521-4028


Padilla, V. (1997). Virosis en olivo. Problemática del material vegetal. *Fruticultura Profesional*,

Panagopoulos, C.G. (1993). Olive knot disease in Greece. *Bulletin OEPP / EPPO Bulletin*, Vol.

Paoletti, V. (1933). Osservasioni ed esperimenti orientativi di lotta contro la rogna dell'olivo.

Penyalver, R., García, A., Ferrer, A., & López, M.M. (1998). La tuberculosis del olivo:

Penyalver, R., García, A., Del Río, C., Caballero, J.M., Pinochet, J., Piquer, J., & López, M.M.

Penyalver, R., García, A., Pérez-Panadés, J., Del Río, C., Caballero, J.M., Pinochet, J., Piquer,

MAPA and Ediciones Mundi- Prensa, ISBN 848474146X, Madrid, Spain Penyalver, R., García, A., Ferrer, A., Bertolini, E., Quesada, J.M., Salcedo, C.I., Piquer, J.,

Pérez-Martínez, I., Rodríguez-Moreno, L., Matas, I.M., & Ramos, C. (2007). Strain selection

Pérez-Martínez, I., Zhao, Y., Murillo, J., Sundin, G.W., & Ramos C. (2008). Global genomic

Pérez-Martínez, I., Rodríguez-Moreno, L., Lambertsen, L., Matas, I.M., Murillo, J., Tegli, S.,

Protta, U. (1995). Le malattie dell' olivo. *Informatore Fitopatologico*, No. 12, pp. 16-26, ISSN

Quesada, J.M., García, A., Bertolini, E., López, M.M., & Penyalver, R. (2007). Recovery of

Vol. 190, No. 2, (January 2008), pp. 625-635, ISSN 0021-9193

diagnóstico, epidemiología y control. *Phytoma-España*, No. 102, (October 1998), pp.

(2003). Sensibilidad varietal del olivo a la tuberculosis causada por *Pseudomonas savastanoi* pv. *savastanoi*. *Agrícola vergel: Fruticultura, horticultura, floricultura*, No.

J., Carbonell, E., & López, M.M. (2005). Resistencia y susceptibilidad a la tuberculosis, In: *Variedades de Olivo en España*, Rallo, L., Barranco, D., Caballero, J.M., Del Río, C., Martín, A., Tous, J., & Trujillo, I., pp. 339-346, Junta de Andalucía,

Pérez-Panadés, J., Carbonell, E.A., del Río, C., Caballero, J.M., & López, M.M. (2006). Factors affecting *Pseudomonas savastanoi* pv. *savastanoi* plant inoculations and their use for evaluation of olive cultivar susceptibility. *Phytopathology*, Vol. 96, No.

and improvement of gene transfer for genetic manipulation of *Pseudomonas savastanoi* isolated from olive knots. *Research in Microbiology*, Vol. 158, No. 1,

analisis of *Pseudomonas savastanoi* pv. *savastanoi* plasmids. *Journal of Bacteriology*,

Jiménez, A.J., & Ramos, C. (2010). Fate of a *Pseudomonas savastanoi* pv. *savastanoi* type III secretion system mutant in olive plants (*Olea europaea* L.). *Applied and Environmental Microbiology*, Vol. 76, No. 11, (June 2010), pp. 3611-3619, ISSN 0099-

*Pseudomonas savastanoi* pv. *savastanoi* from symptomless shoots of naturally infected olive trees. *International Microbiology,* Vol. 10, No. 2, (June 2007), pp. 77-84, ISSN

No. 5, (October 2008), pp. 370-377, ISSN 1521-4028

23, No. 3, (September 1993), pp. 417-422, ISSN 1365-2338

*Rivista di patologia vegetale*, 23, pp. 47-50, ISSN 0035-6441

No. 88, pp. 56-58, ISSN 1131-5660

177-179, ISSN 1131-8988

253, pp. 13-17, ISSN 0211-2728

3, (March 2006), pp. 313-319, ISSN 0031-949X

(January 2007), pp. 60-69, ISSN 0923-2508

2240

0020-0735

1139-6709

*Pseudomonas savastanoi* -induced olive knots. *Journal of Basic Microbiology*, Vol. 48,


Epidemiology and Control of Plant Diseases Caused by Phytopathogenic Bacteria:

309-320, ISSN 0885-5765

64–72, ISSN 1439-0329

1365-2338

0031-9465

9, pp. 233-264

pp. 241–249, ISSN: 1125-4653

(May 1993), pp. 31-40, ISSN 1439-0434

The Case of Olive Knot Disease Caused by *Pseudomonas savastanoi* pv. *savastanoi* 325

Surico, G., Iacobellis, N.S., & Sisto, S. (1985). Studies on the role of indole-3-acetic acid and

Surico, G. (1993). Scanning electron microscopy of olive and oleander leaves colonized by

Temsah, M., Hanna, L., & Saad, A.T. (2007a) Anatomical observations of *Pseudomonas*

Temsah, M., Hanna, L., & Saad, A.T. (2007b) Histological pathogenesis of *Pseudomonas* 

Teviotdale, B.L., & Krueger, W.H. (2004). Effects of timing of copper sprays, defoliation,

Tous, J., Romero, A., & Hermoso, J.F. (2007). The hedgerow system for olive growing. *Olea* 

Trapero, A., & Blanco, M.A. (1998). Enfermedades, In: *El cultivo del olivo*, Barranco, D.,

Vallad, G.E., & Goodman, R.M. (2004). Systemic acquired resistance and induced systemic

Varvaro, L., & Ferrulli, M. (1983). Sopravvivenza di *Pseudomonas syringae* pv. *savastanoi*

Varvaro, L., & Martella, L. (1993). Virulent and avirulent isolates of *Pseudomonas syringae*

Varvaro, L., & Surico, G. (1978). Comportamento di diverse cultivars di olivo *Olea europaea*

Vivian, A., & Mansfield, J. (1993). A proposal for a uniform genetic nomenclature for

Wilson, E.E. (1935). The olive knot disease: its inception, development and control. *Hilgardia*,

*Interactions,* Vol. 6, No. 1, (January 1993), pp. 9-10, ISSN 0894-0282

*Disease*, Vol. 88, No. 2, (February 2004), pp. 131-135, ISSN 0191-2917 Tjamos, E.C., Graniti, A., Smith, I.M., & Lamberti, F. (1993). Conference on olive diseases.

*FAO Olive Network,* No. 26, pp. 20-26, ISSN 0214-6614

ISBN 978-84-8474-234-0, Madrid, Spain

2004), pp. 1920-1934, ISSN 0011-183X

(September 1993), pp. 423-427, ISSN 1365-2338

citokinins in the formation of knots on olive and oleander plants by *Pseudomonas syringae* pv. *savastanoi*. *Physiological Plant Pathology*, Vol. 26, No. 3, (May 1985), pp.

*Pseudomonas syringae* subsp. *savastanoi*. *Journal of Phytopathology*, Vo. 138, No. 1,

*savastanoi* on *Rhamnus alaternus*. *Forest Pathology*, Vol. 37, No. 1, (February 2007), pp.

*savastanoi* on *Myrtus communis*. *Journal of Plant Pathology*, Vol. 89, No. 2, (July 2007),

rainfall, and inoculum concentration on incidence of olive knot disease. *Plant* 

*Bulletin OEPP / EPPO Bulletin*, Vol. 23, No. 3, (September 1993), pp. 365-550, ISSN

Fernández-Escobar, D., & Rallo, L., pp. 461-507, Junta de Andalucía- Mundi-Prensa,

resistance in conventional agriculture. *Crop Science*, Vol. 44, No. 6, (November

(Smith) Young et al. sulle foglie di due varietà di olivo (*Olea europea L*.). *Phytopathologia Mediterranea*, Vol. 22, No. 1/2, (April 1983), pp. 1-4, ISSN 0031-9465

subsp. *savastanoi* as colonizers of olive leaves: evaluation of possible biological control of the olive knot pathogen. *Bulletin OEPP / EPPO Bulletin*, Vol. 23, No. 3,

L. alla inoculazione artificiale con *Pseudomonas savastanoi* E. F. Smith Stevens. *Phytopathologia Mediterranea*, Vol. 17, No. 3, (December 1978), pp. 174-178, ISSN

avirulence genes in phytopathogenic pseudomonads. *Molecular Plant Microbe* 


Schiff-Giorgini, R. (1906). Untersuchungen über die tuberkelkrankheit des oelbaumes.

Schroth, M.N., & Hildebrand, D.C. (1968). A chemotherapeutic treatment for selectively

Schroth M.N., Hildbrand, D.C., & Reilly, H.J. (1968). Off-flavor of olives from trees with olive knot tumors. *Phytopathology*, Vol. 58, pp. 524-525, ISSN 0031-949X Schroth, M.N., Osgood, J.W., & Miller, T.D. (1973). Quantitative assessment of the effect of

Scortichini, M. (2002). Bacterial canker and decline of European hazelnut. *Plant Disease*, Vol.

Scrivani, P., & Bugiani, A. (1955). Messa a punto de un metodo per la riproduzione artificiale

Sisto, A., Cipriani, M.G., & Morea, M. (2004). Knot formation caused by *Pseudomonas* 

Sisto, A., & Iacobellis, N.S. (1999). La "Rogna dell' olivo": aspetti patogenetici,

Smidt, M., & Kosuge, T. (1978). The role of indole-3-acetic acid accumulation by

Smith, E.F. (1908). Recent studies on the olive-tubercle organism. U.S. Dept. Agr. Bur. Plant

Smith, E.F. (1920). Pathogenicity of the olive knot organism on hosts related to the olive.

Smith, I.M., Dunez, J., Lelliot, R.A., Phillips, D.H., & Archer, S.A. (1991). *Manual de* 

Sundin, G.W., Jones, A.L., & Fulbright, D.W. (1989). Copper resistance in *Pseudomonas* 

Sundin, G.W., Demezas, D.H., & Bender, C.L. (1994). Genetic and plasmid diversity within

Surico, G. (1977). Histological observations on olive knots. *Phytopathologia Mediterranea*, Vol.

*enfermedades de las plantas*, Editorial Mundi-Prensa, ISBN 84-7114-358-5, Madrid,

*syringae* pv. *syringae* from cherry orchards and its associated transfer in vitro and in planta with a plasmid. *Phytopathology*, Vol. 79, No. 8, (August 1989), pp. 861-865,

natural populations of *Pseudomonas syringae* with various exposures to copper and streptomycin bactericides. *Applied and Environmental Microbiology*, Vol. 60, No. 12,

antibiotiche. *L'Italia Agrícola*, Vol. 92, pp. 361-369, ISSN 0021-275X

eradicating crown gall and olive knot neoplasms. *Phytopathology*, Vol. 58, No. 6,

the olive knot disease on olive yield and quality. *Phytopathology*, Vol. 63, No. 8,

della rogna dell'Olivo e risultati dei primi saggi terapeutici a mezo di sostanze

*syringae* subsp. *savastanoi* on olive plants is hrp-dependent. *Phytopathology*, Vol. 94,

epidemiologici e strategie di lotta. *Olivo & olio*, Vol. 2, No. 12, pp. 32-38, ISSN 1127-

alphamethyl tryptophan-resistant mutants of *Pseudomonas savastanoi* in gall formation on oleanders. *Physiological Plant Pathology*, Vol. 13, No. 2, (September

Centralb. Bakteriol., Parasitenk. Abt. 2. 15, pp. 200-211

(June 1968), pp. 848-854, ISSN 0031-949X

(August 1973), pp. 1064–1065, ISSN 0031-949X

86, No. 7, (July 2002), pp. 704-709, ISSN 0191-2917

No. 5, (May 2004), pp. 484-489, ISSN 0031-949X

*Phytopathology*, Vol. 12, pp. 271-278, ISSN 0031-949X

(December 1994), pp. 4421-4431, ISSN 0099-2240

16, pp. 109-125, ISSN 0031-9465

Smith, E.F., & Rorer, J.B. (1904). The olive tubercule. *Science N.Y.*, 19, pp. 416-417

1978), pp. 203-214, ISSN 0885-5765

Indust. Bull. No. 131 Part, IV.

0713

Spain

ISSN 0031-949X


**1. Introduction** 

**13** 

P.J. Keane

*Australia* 

**Horizontal or Generalized** 

*Department of Botany, La Trobe University, Victoria* 

**Resistance to Pathogens in Plants** 

The threat to world wheat production and the panic among the agricultural science community caused by the emergence of the 'super virulent' wheat stem rust (*Puccinia graminis tritici*) race Ug99 in East Africa (Singh et al., 2011) is a reminder that the name and ideas of the South African J.E. van der Plank should not be forgotten. Based on his long experience with resistance to *Phytophthora infestans* in potatoes, he developed in his seminal books, *Plant Diseases: Epidemics and Control* (1963) and *Disease Resistance in Plants* (1968), the quantitative study of disease epidemics and the associated concepts of 'vertical' and 'horizontal' resistance to emphasize the two contrasting types of resistance to disease in crops. He contended that our preoccupation through the 20th Century with the more scientifically fascinating and precise vertical resistance, controlled by identifiable genes with a major effect, had resulted in the unfortunate neglect of the more mundane and nebulous horizontal resistance, mostly inherited quantitatively, even though it is evident that the former is unstable in the field while the latter is more stable and consistently useful. The saga of the scientific study of disease resistance shows our human tendency to dig where the light shines brightest, not where we know the potatoes are buried. This tendency continues unabated with the preoccupation of molecular biologists with vertical resistance, often

Before their domestication, plants co-evolved with their parasites and underwent natural selection for resistance to them. Since the dawn of agriculture, plants with a degree of resistance to pathogens and insect pests have been selected by farmers, either consciously or unconsciously. In the genetically diverse crops of early agriculture, when plants of particular species began to be crowded together and so made more vulnerable to pest and disease attack, plants with great susceptibility would have been selected against in competition with more resistant types. They would have contributed fewer offspring to the next generation. Traditional farmers would have learned very early that it was better to select seed or vegetative propagating material from the healthiest plants and they still do so today. It is highly likely that they were selecting for partial or quantitative (van der Plank's 'horizontal') resistance. They weren't selecting for resistance to particular pests or diseases but rather for general plant health. They selected for pest and disease resistance as they selected for higher yield and other quantitatively inherited traits such as size and quality of the harvested product and adaptation to the environment. This process has been replicated

discussed currently as if it is the only form of resistance.


## **Horizontal or Generalized Resistance to Pathogens in Plants**

P.J. Keane

*Department of Botany, La Trobe University, Victoria Australia* 

#### **1. Introduction**

326 Plant Pathology

Wilson, E.E. (1965). Pathological histogenesis in oleander tumors induced by *Pseudomonas* 

Wilson, E.E., & Magie, A.R. (1964). Systemic invasion of the host plant by the tumor-

Wilson, E.E., & Ogawa, J.M. (1979). *Fungal, bacterial, and certain nonparasitic diseases of fruit* 

Wilson, M., & Lindow, S.E. (1992). Relationship of total viable and culturable cells in

inducing bacterium, *Pseudomonas savastanoi*. *Phytopathology*, Vol. 54, pp. 576-579,

*and nut crops in California*, Division of Agricultural Sciences, University of

epiphytic populations of *Pseudomonas syringae*. *Applied and Environmental Microbiology,* Vol. 58, No. 12, (December 1992), pp. 3908-3913, ISSN 0099-2240

*savastanoi*. *Phytopathology*, Vol. 55, pp. 1244-1249, ISSN 0031-949X

California, ISBN 093187629X, Berkeley, USA

ISSN 0031-949X

The threat to world wheat production and the panic among the agricultural science community caused by the emergence of the 'super virulent' wheat stem rust (*Puccinia graminis tritici*) race Ug99 in East Africa (Singh et al., 2011) is a reminder that the name and ideas of the South African J.E. van der Plank should not be forgotten. Based on his long experience with resistance to *Phytophthora infestans* in potatoes, he developed in his seminal books, *Plant Diseases: Epidemics and Control* (1963) and *Disease Resistance in Plants* (1968), the quantitative study of disease epidemics and the associated concepts of 'vertical' and 'horizontal' resistance to emphasize the two contrasting types of resistance to disease in crops. He contended that our preoccupation through the 20th Century with the more scientifically fascinating and precise vertical resistance, controlled by identifiable genes with a major effect, had resulted in the unfortunate neglect of the more mundane and nebulous horizontal resistance, mostly inherited quantitatively, even though it is evident that the former is unstable in the field while the latter is more stable and consistently useful. The saga of the scientific study of disease resistance shows our human tendency to dig where the light shines brightest, not where we know the potatoes are buried. This tendency continues unabated with the preoccupation of molecular biologists with vertical resistance, often discussed currently as if it is the only form of resistance.

Before their domestication, plants co-evolved with their parasites and underwent natural selection for resistance to them. Since the dawn of agriculture, plants with a degree of resistance to pathogens and insect pests have been selected by farmers, either consciously or unconsciously. In the genetically diverse crops of early agriculture, when plants of particular species began to be crowded together and so made more vulnerable to pest and disease attack, plants with great susceptibility would have been selected against in competition with more resistant types. They would have contributed fewer offspring to the next generation. Traditional farmers would have learned very early that it was better to select seed or vegetative propagating material from the healthiest plants and they still do so today. It is highly likely that they were selecting for partial or quantitative (van der Plank's 'horizontal') resistance. They weren't selecting for resistance to particular pests or diseases but rather for general plant health. They selected for pest and disease resistance as they selected for higher yield and other quantitatively inherited traits such as size and quality of the harvested product and adaptation to the environment. This process has been replicated

Horizontal or Generalized Resistance to Pathogens in Plants 329

from Mexico was shown to be immune to late blight (Salaman, 1910). In breeding experiments, individuals of this species stood out as completely healthy amongst genotypes of the cultivated potato that were severely diseased. This stimulated collecting expeditions to Central America to discover sources of resistance in wild relatives of the cultivated potato. *Solanum demissum*, a wild potato species from Mexico, was shown to have several resistance (R) genes that conferred immunity to late blight. This immunity was evident as a hypersensitive necrotic response in the leaf tissues invaded by the pathogen. At the time, it was considered to be the solution to the late blight problem in Europe and North America (Reddick, 1934). These R genes were cross-bred into the domesticated potato (*S. tuberosum*  ssp. *tuberosum*) that formerly had no identifiable genes for immunity to late blight. However, with their widespread use in the field these varieties soon succumbed completely to the disease (Thurston, 1971). They were immune to some isolates (races) of the fungus but were completely susceptible to other races that increased in the pathogen population in response to the selective pressure resulting from the widespread use of a particular resistant variety. It was said that their resistance "broke down" with the selection of 'virulent' races of the pathogen that could invade the varieties with R genes. Van der Plank (1963) said that these varieties showed 'vertical resistance', named after the extreme vertical differences evident in the graphic plot of the degree of resistance (or, conversely, the amount of disease) on the Yaxis against a series of races of the pathogen along the X-axis (Figure 1). The sharp contrast between varieties with high levels of disease and those with very low levels and often no disease at all (immunity), shown by the abrupt vertical jumps in van der Plank's bar graphs, allowed identification of Mendelian genes with strong effects on resistance; hence the genes are often referred to as 'major resistance genes' or just 'resistance genes'. Varieties with different resistance genes gave completely different plots of resistance (or amount of

Varieties lacking R genes, or whose R genes were matched by the virulence of the prevailing pathogen races, gave more-or-less similar amounts of disease when inoculated with different races. The amount of disease could be high or low. Van der Plank said that varieties showing low levels of disease had 'horizontal resistance', the plot of degree of resistance or amount of disease against a series of races being more-or-less horizontal, or at least not showing extreme variation from complete resistance to great susceptibility evident with vertical resistance (Figure 1; van der Plank, 1963, Figs. 14.1, 14.2). The plot for two varieties may be displaced up or down, but the more resistant variety is more resistant to all races. The graph may not be completely horizontal; it may show some up and down displacements depending on the relative 'aggressiveness' of the races, but the displacements are the same for different varieties. The fundamental difference between the two types of resistance is that vertical resistance in the host varieties shows a sharp differential interaction (a strong statistical interaction) with the pathogen races; i.e. the amount of vertical resistance is specific for a particular race (very high for one race in Figure 1, very low for the other). It is 'race-specific'. Horizontal resistance is not race-specific to the extreme degree evident in vertical resistance (Figure 1). Because it is race-specific, the effect of vertical resistance is prone to being lost rapidly due to selection of virulent races in the pathogen population. Lacking this sharp interaction, horizontal resistance tends to be more stable, more 'durable'. That is its big advantage. Researchers working with late blight resistance in potato concluded that "R-gene hypersensitivity cannot be relied upon as a permanent protection against *Phytophthora infestans* and so the necessity of providing a

disease) against races (Figure 1).

by Simmonds (1964, 1966) who re-created the domesticated potato common in Europe (*Solanum tuberosum* ssp. *tuberosum*) by repeated mass selection of true seed of the best types from the highly variable wild Andian potato (*S. tuberosum* ssp. *andigena*). The potatoes were exposed to late blight epidemics during the selection process, and it was shown that the selected materials were more resistant to the disease than the standard domesticated potato (Thurston, 1971).

Through the 1800s there were reports of wheat farmers noticing in their fields occasional 'off-types' with complete resistance or immunity (probably 'vertical resistance' in van der Plank's terms) to a prevalent disease, although little practical use was made of this until it was understood that it was inherited (Biffen, 1905, p.40). It would have been possible to spot these completely resistant types and to distinguish them from 'escapes' only when disease levels were very high and practically all plants in a crop were heavily diseased. In the late 1800s, plants that appeared to be resistant in the field were selected, multiplied and promoted for use on a wider scale. A farmer in South Australia, James Ward, planted a South African variety called De Toit and noticed that it was generally "as rusty as a horse nail" except for a few plants that were rust-free (Callaghan & Millington, 1956) and were later thought to have arisen from a contaminant (Farrer, 1898). Ward saved the seed of these plants and increased it to produce a commercial variety named "Ward's Prolific". Because of its rust resistance and other characters, this became the most widely grown wheat in South Australia at the time. Rees et al. (1979) showed that in the 1970s this variety had little horizontal resistance and so it is likely that Ward had selected a type of extreme resistance that has since been overcome by the pathogen. Another South Australian farmer, Daniel Leak, noticed an occasional rust-free plant in a crop of heavily rusted Tuscany wheat, and selected and multiplied this as a commercial variety called 'Leak's Rust-proof' (Williams, 1991). There were many similar attempts at selecting rust resistant wheat, resulting in other varieties like 'Anderson's Rust-proof' and 'Kalm's Rust-proof' for which rust resistance was claimed (Cobb 1890). Farmers were developing a general appreciation of inherited variation in disease resistance. The Australian wheat breeder, William Farrer (1898), was one of the first to declare that resistance to wheat stem rust was inherited (Biffen, 1905). With the discovery of Mendel's work on the genetics of particular traits of the garden pea, his methods were soon applied to the trait of extreme disease resistance that had been observed in certain crops. R.H. Biffen, working at Cambridge University with resistance to stripe rust (*Puccinia striiformis*) in wheat, was the first to take up these studies, beginning in 1902 and summarizing his findings in papers entitled 'Mendel's laws of inheritance and wheat breeding' (Biffen, 1905) and 'Studies in the inheritance of disease resistance' (Biffen, 1907). He showed that a high level of disease resistance or immunity was inherited as a simple Mendelian character, and so could be crossed readily into well adapted varieties. Thus began our enchantment with the use of 'resistance genes' to protect our crops from disease and the search for them in crop varieties and in the centres of evolution and diversification of the crops (Vavilov, 1951).

Potato late blight caused by *Ph. infestans* has been central to modern plant pathology since the catastrophic epidemics in 1845-47 in Western Europe and Ireland that triggered the terrible Irish famine of the period and led to our understanding that a fungus could invade and cause disease in a healthy plant. While no completely resistant 'off-types' were noticed amongst the heavily blighted potato crops of the 1800s, a wild *Solanum* species collected

by Simmonds (1964, 1966) who re-created the domesticated potato common in Europe (*Solanum tuberosum* ssp. *tuberosum*) by repeated mass selection of true seed of the best types from the highly variable wild Andian potato (*S. tuberosum* ssp. *andigena*). The potatoes were exposed to late blight epidemics during the selection process, and it was shown that the selected materials were more resistant to the disease than the standard domesticated potato

Through the 1800s there were reports of wheat farmers noticing in their fields occasional 'off-types' with complete resistance or immunity (probably 'vertical resistance' in van der Plank's terms) to a prevalent disease, although little practical use was made of this until it was understood that it was inherited (Biffen, 1905, p.40). It would have been possible to spot these completely resistant types and to distinguish them from 'escapes' only when disease levels were very high and practically all plants in a crop were heavily diseased. In the late 1800s, plants that appeared to be resistant in the field were selected, multiplied and promoted for use on a wider scale. A farmer in South Australia, James Ward, planted a South African variety called De Toit and noticed that it was generally "as rusty as a horse nail" except for a few plants that were rust-free (Callaghan & Millington, 1956) and were later thought to have arisen from a contaminant (Farrer, 1898). Ward saved the seed of these plants and increased it to produce a commercial variety named "Ward's Prolific". Because of its rust resistance and other characters, this became the most widely grown wheat in South Australia at the time. Rees et al. (1979) showed that in the 1970s this variety had little horizontal resistance and so it is likely that Ward had selected a type of extreme resistance that has since been overcome by the pathogen. Another South Australian farmer, Daniel Leak, noticed an occasional rust-free plant in a crop of heavily rusted Tuscany wheat, and selected and multiplied this as a commercial variety called 'Leak's Rust-proof' (Williams, 1991). There were many similar attempts at selecting rust resistant wheat, resulting in other varieties like 'Anderson's Rust-proof' and 'Kalm's Rust-proof' for which rust resistance was claimed (Cobb 1890). Farmers were developing a general appreciation of inherited variation in disease resistance. The Australian wheat breeder, William Farrer (1898), was one of the first to declare that resistance to wheat stem rust was inherited (Biffen, 1905). With the discovery of Mendel's work on the genetics of particular traits of the garden pea, his methods were soon applied to the trait of extreme disease resistance that had been observed in certain crops. R.H. Biffen, working at Cambridge University with resistance to stripe rust (*Puccinia striiformis*) in wheat, was the first to take up these studies, beginning in 1902 and summarizing his findings in papers entitled 'Mendel's laws of inheritance and wheat breeding' (Biffen, 1905) and 'Studies in the inheritance of disease resistance' (Biffen, 1907). He showed that a high level of disease resistance or immunity was inherited as a simple Mendelian character, and so could be crossed readily into well adapted varieties. Thus began our enchantment with the use of 'resistance genes' to protect our crops from disease and the search for them in crop varieties and in the centres of evolution and diversification

Potato late blight caused by *Ph. infestans* has been central to modern plant pathology since the catastrophic epidemics in 1845-47 in Western Europe and Ireland that triggered the terrible Irish famine of the period and led to our understanding that a fungus could invade and cause disease in a healthy plant. While no completely resistant 'off-types' were noticed amongst the heavily blighted potato crops of the 1800s, a wild *Solanum* species collected

(Thurston, 1971).

of the crops (Vavilov, 1951).

from Mexico was shown to be immune to late blight (Salaman, 1910). In breeding experiments, individuals of this species stood out as completely healthy amongst genotypes of the cultivated potato that were severely diseased. This stimulated collecting expeditions to Central America to discover sources of resistance in wild relatives of the cultivated potato. *Solanum demissum*, a wild potato species from Mexico, was shown to have several resistance (R) genes that conferred immunity to late blight. This immunity was evident as a hypersensitive necrotic response in the leaf tissues invaded by the pathogen. At the time, it was considered to be the solution to the late blight problem in Europe and North America (Reddick, 1934). These R genes were cross-bred into the domesticated potato (*S. tuberosum*  ssp. *tuberosum*) that formerly had no identifiable genes for immunity to late blight. However, with their widespread use in the field these varieties soon succumbed completely to the disease (Thurston, 1971). They were immune to some isolates (races) of the fungus but were completely susceptible to other races that increased in the pathogen population in response to the selective pressure resulting from the widespread use of a particular resistant variety. It was said that their resistance "broke down" with the selection of 'virulent' races of the pathogen that could invade the varieties with R genes. Van der Plank (1963) said that these varieties showed 'vertical resistance', named after the extreme vertical differences evident in the graphic plot of the degree of resistance (or, conversely, the amount of disease) on the Yaxis against a series of races of the pathogen along the X-axis (Figure 1). The sharp contrast between varieties with high levels of disease and those with very low levels and often no disease at all (immunity), shown by the abrupt vertical jumps in van der Plank's bar graphs, allowed identification of Mendelian genes with strong effects on resistance; hence the genes are often referred to as 'major resistance genes' or just 'resistance genes'. Varieties with different resistance genes gave completely different plots of resistance (or amount of disease) against races (Figure 1).

Varieties lacking R genes, or whose R genes were matched by the virulence of the prevailing pathogen races, gave more-or-less similar amounts of disease when inoculated with different races. The amount of disease could be high or low. Van der Plank said that varieties showing low levels of disease had 'horizontal resistance', the plot of degree of resistance or amount of disease against a series of races being more-or-less horizontal, or at least not showing extreme variation from complete resistance to great susceptibility evident with vertical resistance (Figure 1; van der Plank, 1963, Figs. 14.1, 14.2). The plot for two varieties may be displaced up or down, but the more resistant variety is more resistant to all races. The graph may not be completely horizontal; it may show some up and down displacements depending on the relative 'aggressiveness' of the races, but the displacements are the same for different varieties. The fundamental difference between the two types of resistance is that vertical resistance in the host varieties shows a sharp differential interaction (a strong statistical interaction) with the pathogen races; i.e. the amount of vertical resistance is specific for a particular race (very high for one race in Figure 1, very low for the other). It is 'race-specific'. Horizontal resistance is not race-specific to the extreme degree evident in vertical resistance (Figure 1). Because it is race-specific, the effect of vertical resistance is prone to being lost rapidly due to selection of virulent races in the pathogen population. Lacking this sharp interaction, horizontal resistance tends to be more stable, more 'durable'. That is its big advantage. Researchers working with late blight resistance in potato concluded that "R-gene hypersensitivity cannot be relied upon as a permanent protection against *Phytophthora infestans* and so the necessity of providing a

Horizontal or Generalized Resistance to Pathogens in Plants 331

nature of this resistance as highlighted by van der Plank, although at a fine level a degree of race-specificity has been shown to apply to it (Parlevliet, 1995). Field resistance was commonly used for potato varieties lacking R genes, but is not a good general term as the resistance of a crop variety in the field could be due to a combination of vertical and horizontal resistance. 'General resistance' can refer to the resistance of a variety to several pests and diseases and again could be the result of both vertical and horizontal resistance. Sometimes 'tolerance' is used for 'horizontal resistance', but this is certainly incorrect. Tolerance has a special meaning: it refers to plant varieties that suffer less damage for a given degree of infection compared with a disease sensitive variety (Caldwell et al., 1958; Schafer, 1971). This has the same meaning as 'rust-enduring' referred to by N.A. Cobb (1894), one of the earliest students of rust resistance. Certainly, horizontal resistance is usually 'partial' and 'quantitative' and can be expressed in a gradient from very little disease to quite a lot, depending on the host genotype, the aggressiveness of the pathogen and the environment. But, for some diseases it can be complete. Thus, the non-prescriptive term, 'horizontal resistance', is perhaps the best. 'Generalized resistance' as applied to potatoes is also apt. Robinson (1976) favoured the terms 'vertical' and 'horizontal' resistance because they were somewhat abstract, and in fact had application beyond disease resistance; e.g. to fungicide use whereby copper-based fungicides could be regarded as 'horizontal' in effect because they knocked out several enzyme systems and the fungi could not adapt to them, while the new, highly specific fungicides like benomyl are 'vertical' because they knock out only one narrow function and the fungi can adapt to them (they 'break down' in

Until it is matched by a virulent race, vertical resistance tends to be complete or to reduce reproduction of the pathogen to a tiny amount. That is its great attraction and that is how it was first noticed (Callaghan & Millington, 1956; Biffen, 1907; Salaman, 1910). In the 1800s it was observed by farmers as stark 'off-types' in crops of rusted wheat. The domesticated wheat species probably had vertical resistance genes against stem rust, while the domesticated potato in Europe did not have vertical resistance genes against late blight until they were bred into it from a wild relative. Horizontal resistance is harder to detect and measure, although it is likely to be selected unconsciously by observant farmers who collect their seed from their healthiest looking plants. It tends to be partial or quantitative in its expression (i.e. there is some disease and some sporulation of the pathogen) but the rate of development of the disease epidemic is reduced compared with that on a susceptible variety under similar environmental conditions. Van der Plank's interest in disease epidemiology (the quantitative study of populations of pathogens and crops) led to his understanding of the importance of partial resistance. There has been a recent shift in thinking about pests and diseases – we no longer talk about their 'control', which implies their elimination from a crop; rather we now talk about pest and disease 'management' which implies acceptance of some level of their presence as long as they cause little economic loss. Under the modern concept of Integrated Pest Management (IPM), moderate levels of pest/disease resistance in a crop may be sufficient if applied synergistically with cultural control methods and minimal, targeted use of pesticides. Under the IPM approach, resistance does not have to be complete; partial resistance may be all that is required, and horizontal resistance becomes important. Modern plant breeders and pathologists talk about "avoiding high degrees of susceptibility". The idea of IPM was promoted initially by entomologists in response to the phenomenon of 'breakdown of insecticide efficacy' that is analogous to the 'breakdown of

the same way that vertical resistance breaks down).

degree of field protection in new cultivars is generally recognised by potato breeders."(Malcolmson, 1976). The 'field protection' referred to here is horizontal resistance.

Fig. 1. Plot of percent disease in two varieties with vertical resistance (Vr R1 with resistance gene R1; Vr R2 with resistance gene R2) and two varieties lacking vertical resistance (R genes) but expressing some horizontal resistance (Hr a, Hr b) infected with two pathogen races (A1 with avirulence against R1; A2 with avirulence against R2). Vertical resistance shows a strong interaction with the races (i.e. is 'race-specific'). Horizontal resistance does not (i.e. is 'non-race-specific') although it shows significant main effects of Variety (variety Hr a is more susceptible to both races than Hr b) and Race (race A2 is more aggressive than A1 on both varieties, and this is also evident in the vertically resistant varieties where the R gene is ineffective).

Horizontal resistance has had a much longer history in human knowledge than vertical resistance and has had a greater profusion of names. Once, probably all observed resistance was of this type. It is probably the resistance that keeps 'minor pathogens' consistently 'minor' and consequently is not much studied for these pathogens because they are of minor importance. For the 'major' pathogens, farmers would often recognize that certain varieties 'got less disease' than other varieties. For particular diseases, 'slow-rusting', 'slowmildewing' (for powdery mildews), 'slow-blighting' (for potato late blight) or 'slowblasting' (for rice blast) are older terms that accurately describe horizontal resistance, an essential feature of which is the slowing down of epidemics. In natural plant communities or crops with a diversity of resistance genes (e.g. traditional mixed crops), vertical resistance will also slow down epidemics, but in current monocultures vertical resistance tends to prevent epidemics until such time as it is matched by virulence in a large proportion of the pathogen population. Several terms have been used to distinguish this general resistance from the race-specific resistance discovered in the early 1900s. These have included 'partial resistance', 'quantitative resistance', 'generalized resistance', 'field resistance', 'adult-plant resistance', 'durable resistance', and 'tolerance'. All have their problems. 'Race-non-specific resistance', usefully abbreviated to 'non-specific resistance', best captures the essential

degree of field protection in new cultivars is generally recognised by potato breeders."(Malcolmson, 1976). The 'field protection' referred to here is horizontal resistance.

Fig. 1. Plot of percent disease in two varieties with vertical resistance (Vr R1 with resistance gene R1; Vr R2 with resistance gene R2) and two varieties lacking vertical resistance (R genes) but expressing some horizontal resistance (Hr a, Hr b) infected with two pathogen races (A1 with avirulence against R1; A2 with avirulence against R2). Vertical resistance shows a strong interaction with the races (i.e. is 'race-specific'). Horizontal resistance does not (i.e. is 'non-race-specific') although it shows significant main effects of Variety (variety Hr a is more susceptible to both races than Hr b) and Race (race A2 is more aggressive than A1 on both varieties, and this is also evident in the vertically resistant varieties where the R

**Variety Vr R2**

**Variet**y **Hr a**

**Variety Hr b**

Race A1 Race A2

**Percent Disease**

> **Variety Vr R1**

Horizontal resistance has had a much longer history in human knowledge than vertical resistance and has had a greater profusion of names. Once, probably all observed resistance was of this type. It is probably the resistance that keeps 'minor pathogens' consistently 'minor' and consequently is not much studied for these pathogens because they are of minor importance. For the 'major' pathogens, farmers would often recognize that certain varieties 'got less disease' than other varieties. For particular diseases, 'slow-rusting', 'slowmildewing' (for powdery mildews), 'slow-blighting' (for potato late blight) or 'slowblasting' (for rice blast) are older terms that accurately describe horizontal resistance, an essential feature of which is the slowing down of epidemics. In natural plant communities or crops with a diversity of resistance genes (e.g. traditional mixed crops), vertical resistance will also slow down epidemics, but in current monocultures vertical resistance tends to prevent epidemics until such time as it is matched by virulence in a large proportion of the pathogen population. Several terms have been used to distinguish this general resistance from the race-specific resistance discovered in the early 1900s. These have included 'partial resistance', 'quantitative resistance', 'generalized resistance', 'field resistance', 'adult-plant resistance', 'durable resistance', and 'tolerance'. All have their problems. 'Race-non-specific resistance', usefully abbreviated to 'non-specific resistance', best captures the essential

gene is ineffective).

nature of this resistance as highlighted by van der Plank, although at a fine level a degree of race-specificity has been shown to apply to it (Parlevliet, 1995). Field resistance was commonly used for potato varieties lacking R genes, but is not a good general term as the resistance of a crop variety in the field could be due to a combination of vertical and horizontal resistance. 'General resistance' can refer to the resistance of a variety to several pests and diseases and again could be the result of both vertical and horizontal resistance. Sometimes 'tolerance' is used for 'horizontal resistance', but this is certainly incorrect. Tolerance has a special meaning: it refers to plant varieties that suffer less damage for a given degree of infection compared with a disease sensitive variety (Caldwell et al., 1958; Schafer, 1971). This has the same meaning as 'rust-enduring' referred to by N.A. Cobb (1894), one of the earliest students of rust resistance. Certainly, horizontal resistance is usually 'partial' and 'quantitative' and can be expressed in a gradient from very little disease to quite a lot, depending on the host genotype, the aggressiveness of the pathogen and the environment. But, for some diseases it can be complete. Thus, the non-prescriptive term, 'horizontal resistance', is perhaps the best. 'Generalized resistance' as applied to potatoes is also apt. Robinson (1976) favoured the terms 'vertical' and 'horizontal' resistance because they were somewhat abstract, and in fact had application beyond disease resistance; e.g. to fungicide use whereby copper-based fungicides could be regarded as 'horizontal' in effect because they knocked out several enzyme systems and the fungi could not adapt to them, while the new, highly specific fungicides like benomyl are 'vertical' because they knock out only one narrow function and the fungi can adapt to them (they 'break down' in the same way that vertical resistance breaks down).

Until it is matched by a virulent race, vertical resistance tends to be complete or to reduce reproduction of the pathogen to a tiny amount. That is its great attraction and that is how it was first noticed (Callaghan & Millington, 1956; Biffen, 1907; Salaman, 1910). In the 1800s it was observed by farmers as stark 'off-types' in crops of rusted wheat. The domesticated wheat species probably had vertical resistance genes against stem rust, while the domesticated potato in Europe did not have vertical resistance genes against late blight until they were bred into it from a wild relative. Horizontal resistance is harder to detect and measure, although it is likely to be selected unconsciously by observant farmers who collect their seed from their healthiest looking plants. It tends to be partial or quantitative in its expression (i.e. there is some disease and some sporulation of the pathogen) but the rate of development of the disease epidemic is reduced compared with that on a susceptible variety under similar environmental conditions. Van der Plank's interest in disease epidemiology (the quantitative study of populations of pathogens and crops) led to his understanding of the importance of partial resistance. There has been a recent shift in thinking about pests and diseases – we no longer talk about their 'control', which implies their elimination from a crop; rather we now talk about pest and disease 'management' which implies acceptance of some level of their presence as long as they cause little economic loss. Under the modern concept of Integrated Pest Management (IPM), moderate levels of pest/disease resistance in a crop may be sufficient if applied synergistically with cultural control methods and minimal, targeted use of pesticides. Under the IPM approach, resistance does not have to be complete; partial resistance may be all that is required, and horizontal resistance becomes important. Modern plant breeders and pathologists talk about "avoiding high degrees of susceptibility". The idea of IPM was promoted initially by entomologists in response to the phenomenon of 'breakdown of insecticide efficacy' that is analogous to the 'breakdown of

Horizontal or Generalized Resistance to Pathogens in Plants 333

rust with virulence on Sr11 increased from nil to 91% by 1950 and the resistance of Gabo was broken. The period of the 'boom and bust' cycle of resistance breeding had begun and

**Disease/Resistance situation** 

**in the field** 

broken down

broken down

broken down

broken down

the fascinating phenomenon of pathogen-resistance specificity was revealed.

**Dominant stem rust race** 

1938 Eureka (Sr6) released 126-Avirulent on Sr6 No disease/Resistance effective <sup>1942</sup> 126-Virulent on Sr6 Disease widespread/Resistance

1942 Gabo types (Sr11) released 222-Avriulent on Sr11 No disease/Resistance effective <sup>1948</sup> 222-Virulent on Sr11 Disease widespread/Resistance

1950 Festival (Sr9b) released 21-Avirulent on Sr9b No disease/Resistance effective <sup>1959</sup> 21-Virulent on Sr9b Disease widespread/Resistance

1958 Mengavi (Sr36) released 34-Avirulent on Sr34 No disease/Resistance effective <sup>1960</sup> 34-Virulent on Sr34 Disease widespread/Resistance

Table 1. The interaction between the wheat cultivars Eureka, Gabo, Festival and Mengavi and wheat stem rust (*Puccinia graminis tritici*) races in Australia, showing the repeated breakdown of vertical resistance as new races evolved (Watson and Luig, 1963; after Knott,

Many researchers prefer the term 'race-specific resistance', which accurately describes the statistical interaction which is the essential feature van der Plank sought to highlight in his definition of 'vertical resistance' and is the basis of resistance breakdown. Races that can specifically invade (i.e. are virulent on) varieties with certain resistance genes cause the breakdown of that resistance when they increase to a high proportion of the total rust population in the field. 'Specific resistance' is a neat abbreviation as long as it is understood as 'race-specificity' not 'species-specificity'. The use of the term 'virulence' in plant pathology, where it is used to describe the ability of a pathogen race to invade a plant with a particular resistance gene, is different from its use in medicine, where it is used to describe what in plant pathology would be called the aggressiveness of a pathogen. In plant pathology, the pathogenicity of an organism consists of its virulence (ability to infect varieties with particular vertical resistance genes) and its aggressiveness (the amount of disease it causes on varieties it is able to infect). 'Virulence' is the ability of a pathogen to overcome vertical resistance, while 'aggressiveness' is the ability to overcome horizontal resistance. In much current plant pathology writing, there is a tendency to revert to the medical meaning of 'virulence', which requires the development of another term for the

Another set of terms is used to describe the interaction of a specialized pathogen and its host. If a pathogen can infect and sporulate more-or-less normally on a plant, the interaction

ability of a pathogen to match vertical resistance.

**Year Variety** 

1989).

**(Resistance gene)** 

disease resistance'. Entomologists have realized the futility of the 'scorched earth' approach to the use of insecticides in an attempt to eliminate pests from crops. They now accept that there is a threshold level of pest infestation below which little economic damage is caused. Entomologists are now emphasizing crop resistance (mostly of a horizontal nature) to insect pests after a long period of total reliance on insecticides (and consequent neglect and decline of resistance to insect pests in crops), while the repeated problem of the breakdown of disease resistance in many crops, exemplified by the emergence of wheat stem rust race Ug99, has led to a re-awakening of plant pathologists to the merits of horizontal resistance after a long period of pre-occupation with vertical resistance. It appears that both entomologists and plant pathologists are emerging from a period of bedazzlement by scientific and technical 'revolutions' (the 'insecticide revolution' and the 'resistance gene revolution') that showed great promise in the laboratory but lost their effectiveness following their widespread use as 'stand-alone' control measures in the farmers' fields. It is now realized that these brilliant technical developments, that offered an illusion of a simple and complete 'revolution' in control of pests and diseases, have to be incorporated into the ecological complexity of crop growth and production in the field. Rather than 'revolutions', a steady evolution of stable IPM methods, based on a foundation of steady evolution of horizontal resistance, is required. Of course, in addition to cultural methods, IPM methods may include targeted use of pesticides and vertical resistance, which, in IPM, are supported by the other methods that prolong and enhance their value. This approach is far from new. In the 1890s, Australia and North America produced vast quantities of very cheap wheat that flooded the European markets. It is obvious that the production was highly successful in most years. The potentially devastating wheat stem rust was managed effectively for long periods by a combination of use of early maturing varieties (that avoided the worst of the epidemics late in the growing season and allowed disease escape), drought adapted varieties (that allowed wheat to be grown in drier climates not conducive to the rust), the use of varieties that had probably been selected unconsciously over the years for a degree of horizontal resistance, and later, the addition of vertical resistance (e.g. in a variety like Thatcher in North America) and the deliberate incorporation of horizontal resistance by cross-breeding with tetraploid wheats.

#### **2. Vertical or specific resistance**

Researchers working to develop and deploy stem rust resistance genes (Sr genes) in wheat varieties in North America (Stakman & Levine, 1922) and Australia (Waterhouse, 1936, 1952; Watson, 1958; Watson and Luig, 1963) had exactly the same experience of 'breakdown of resistance' controlled by single resistance genes (Sr genes) as the researchers working with R genes in potato. This is well documented for several countries by Person (1967). The situation was starkest in the spring wheat crops in Australia (Table 1). Eureka, the first commercial wheat variety bred with a vertical resistance gene (Sr6) against stem rust in Australia, was released in 1938 and because of its resistance and other qualities was very popular, increasing to constitute nearly 20% of the wheat area in northern New South Wales by 1945 (Watson, 1958). However, races of stem rust able to attack the variety (i.e. virulent on Sr6) increased from practically nil in 1938 to make up 72% of the rust isolates collected in the area in 1945. The resistance of Eureka was seen to have 'broken down', and the variety rapidly lost popularity. The wheat variety Gabo and some others with resistance gene Sr11 were released in 1942 and by 1950 made up 62% of the same wheat area. But races of stem

disease resistance'. Entomologists have realized the futility of the 'scorched earth' approach to the use of insecticides in an attempt to eliminate pests from crops. They now accept that there is a threshold level of pest infestation below which little economic damage is caused. Entomologists are now emphasizing crop resistance (mostly of a horizontal nature) to insect pests after a long period of total reliance on insecticides (and consequent neglect and decline of resistance to insect pests in crops), while the repeated problem of the breakdown of disease resistance in many crops, exemplified by the emergence of wheat stem rust race Ug99, has led to a re-awakening of plant pathologists to the merits of horizontal resistance after a long period of pre-occupation with vertical resistance. It appears that both entomologists and plant pathologists are emerging from a period of bedazzlement by scientific and technical 'revolutions' (the 'insecticide revolution' and the 'resistance gene revolution') that showed great promise in the laboratory but lost their effectiveness following their widespread use as 'stand-alone' control measures in the farmers' fields. It is now realized that these brilliant technical developments, that offered an illusion of a simple and complete 'revolution' in control of pests and diseases, have to be incorporated into the ecological complexity of crop growth and production in the field. Rather than 'revolutions', a steady evolution of stable IPM methods, based on a foundation of steady evolution of horizontal resistance, is required. Of course, in addition to cultural methods, IPM methods may include targeted use of pesticides and vertical resistance, which, in IPM, are supported by the other methods that prolong and enhance their value. This approach is far from new. In the 1890s, Australia and North America produced vast quantities of very cheap wheat that flooded the European markets. It is obvious that the production was highly successful in most years. The potentially devastating wheat stem rust was managed effectively for long periods by a combination of use of early maturing varieties (that avoided the worst of the epidemics late in the growing season and allowed disease escape), drought adapted varieties (that allowed wheat to be grown in drier climates not conducive to the rust), the use of varieties that had probably been selected unconsciously over the years for a degree of horizontal resistance, and later, the addition of vertical resistance (e.g. in a variety like Thatcher in North America) and the deliberate incorporation of horizontal resistance by

Researchers working to develop and deploy stem rust resistance genes (Sr genes) in wheat varieties in North America (Stakman & Levine, 1922) and Australia (Waterhouse, 1936, 1952; Watson, 1958; Watson and Luig, 1963) had exactly the same experience of 'breakdown of resistance' controlled by single resistance genes (Sr genes) as the researchers working with R genes in potato. This is well documented for several countries by Person (1967). The situation was starkest in the spring wheat crops in Australia (Table 1). Eureka, the first commercial wheat variety bred with a vertical resistance gene (Sr6) against stem rust in Australia, was released in 1938 and because of its resistance and other qualities was very popular, increasing to constitute nearly 20% of the wheat area in northern New South Wales by 1945 (Watson, 1958). However, races of stem rust able to attack the variety (i.e. virulent on Sr6) increased from practically nil in 1938 to make up 72% of the rust isolates collected in the area in 1945. The resistance of Eureka was seen to have 'broken down', and the variety rapidly lost popularity. The wheat variety Gabo and some others with resistance gene Sr11 were released in 1942 and by 1950 made up 62% of the same wheat area. But races of stem

cross-breeding with tetraploid wheats.

**2. Vertical or specific resistance** 


rust with virulence on Sr11 increased from nil to 91% by 1950 and the resistance of Gabo was broken. The period of the 'boom and bust' cycle of resistance breeding had begun and the fascinating phenomenon of pathogen-resistance specificity was revealed.

Table 1. The interaction between the wheat cultivars Eureka, Gabo, Festival and Mengavi and wheat stem rust (*Puccinia graminis tritici*) races in Australia, showing the repeated breakdown of vertical resistance as new races evolved (Watson and Luig, 1963; after Knott, 1989).

broken down

Many researchers prefer the term 'race-specific resistance', which accurately describes the statistical interaction which is the essential feature van der Plank sought to highlight in his definition of 'vertical resistance' and is the basis of resistance breakdown. Races that can specifically invade (i.e. are virulent on) varieties with certain resistance genes cause the breakdown of that resistance when they increase to a high proportion of the total rust population in the field. 'Specific resistance' is a neat abbreviation as long as it is understood as 'race-specificity' not 'species-specificity'. The use of the term 'virulence' in plant pathology, where it is used to describe the ability of a pathogen race to invade a plant with a particular resistance gene, is different from its use in medicine, where it is used to describe what in plant pathology would be called the aggressiveness of a pathogen. In plant pathology, the pathogenicity of an organism consists of its virulence (ability to infect varieties with particular vertical resistance genes) and its aggressiveness (the amount of disease it causes on varieties it is able to infect). 'Virulence' is the ability of a pathogen to overcome vertical resistance, while 'aggressiveness' is the ability to overcome horizontal resistance. In much current plant pathology writing, there is a tendency to revert to the medical meaning of 'virulence', which requires the development of another term for the ability of a pathogen to match vertical resistance.

Another set of terms is used to describe the interaction of a specialized pathogen and its host. If a pathogen can infect and sporulate more-or-less normally on a plant, the interaction

Horizontal or Generalized Resistance to Pathogens in Plants 335

resistance gene in flax there is a corresponding gene for virulence (actually, avirulence if genes are named after their dominant allele) in flax rust (Table 2). Resistance is usually dominant to susceptibility and resistance genes occur as multiple alleles at a restricted number of loci in the plant while avirulence is usually dominant to virulence and avirulence genes occur at separate loci in the rust. That is, the number of resistance genes that can be expressed in the plant is restricted while there is no such restriction on the number of possible virulences that can be expressed in the rust. This ensures that the pathogen will always be able to overcome the resistance expressed in the host. While the meticulous and exhaustive work of Flor with flax rust has been repeated for very few other diseases, there is evidence that the relationship occurs in many highly specialized parasitic relationships (Sidhu, 1975), including several insect-plant relationships (Broekgaarden et al., 2011). It is usually expressed after the formation of haustoria in the highly specialized biotrophic parasites such as the rusts and powdery mildews and so appears to require intimate molecular contact between the pathogen and host. Rust resistance genes have been cloned from four of the five resistance loci in flax and they all appear to code for similar proteins, in the Nucleotide Binding Site-Leucine Rich Repeat (NBS-LRR) class (Ellis et al., 2007). Many resistance genes for a wide range of pathogens (including *Phytophthora,* rusts, powdery mildew, downy mildew, viruses, nematodes and bacteria) in a wide range of hosts (including potato, lettuce, tomato, barley, maize and *Arabidopsis*) have been found to fit into the same or similar class (Martin et al., 2003; Nimchuk et al., 2003). The resistance genes against the highly specialized phloem-feeding insect parasites of plants also appear to fall into this class (Broekgaarden et al., 2011). Thus, the recent molecular studies appear to confirm that vertical resistance involves a particular molecular system for recognition and rapid response to an invading parasite. On the other hand, the avirulence genes cloned from flax rust code for small secreted proteins ('effector proteins') that show no similarity between loci, providing evidence that their main function is probably something to do with the normal metabolism of the fungus and not to make them 'avirulent' on their host. Their 'avirulence' arises from the fact that they happen to be recognized by the resistance-coded proteins, leading to inhibition of the fungus. If avirulence genes code for proteins with a function in the normal life of the pathogen, this would explain the commonly observed phenomenon of stabilizing selection (Flor, 1956; van der Plank, 1968), whereby races expressing virulence (recessive mutants of A genes, often assumed to be non-functional) at one or more loci tend to be less fit than avirulent races on hosts that have no resistance genes. If avirulence genes code for a variety of normal functions in a pathogen, they could vary in the likelihood of their virulent (double recessive) mutant rising to prominence in the pathogen population, via mutation and selection for fitness, and so overcoming the particular resistance in the plant population (Luig, 1983). For example, if an avirulence gene (AvrX) coded for an essential function in the pathogen, then loss of this function in the homozygous virulent mutant (avrX avrX) may mitigate against the selection and buildup of this mutant, even if it matches the resistance RX in the host population. This could help explain the phenomenon of 'weak' and 'strong' vertical resistance genes as proposed by van der Plank (1968) and observed commonly in the field – i.e. some vertical resistance genes break down much more rapidly than others. The genes that are overcome rapidly are matched by races in which the mutation to virulence has little cost in fitness of the pathogen. If the mutation to virulence against a particular gene imposes a high cost in general fitness of the pathogen, the virulent race will not build up rapidly and the resistance gene will not

be rapidly overcome; it will be seen to be 'strong'.

is said to be 'compatible'; if the interaction results in hypersensitive necrosis that largely excludes the pathogen, it is said to be 'incompatible', a term used very early in describing the effect of vertical resistance (see Hayes et al., 1925)(Table 2). These terms are particularly apt for biotrophic pathogens such as rusts and powdery mildews, where there is an intimate parasitic symbiosis between the pathogen and its host. In the same sense, the normal interactions of a plant and its mycorrhiza and endophyte symbionts would be said to be 'compatible'. The importance of the 'basic compatibility' required for a symbiont to live in its host has been well explored by Heath (1981). In fact, the copious molecular investigations of incompatibility would be better directed to trying to understand the mechanisms of compatibility – how does a biotrophic symbiont like a rust or a powdery mildew obtain nutrients from its host plant and why can't this feeding relationship be replicated in a Petri dish?


Table 2. Summary of the gene-for-gene interaction involved in vertical resistance. Resistance is usually dominant to susceptibility in the plant. Avirulence is usually dominant to virulence in the pathogen. The only starkly unique interaction occurs when an A gene matches an R gene. Arrow 1 indicates the change induced by breeding an R gene into a plant variety; arrow 2 indicates the change associated with the breakdown of resistance. Note that for wheat stem rust, the R gene is given the symbol Sr, for wheat stripe (yellow) rust it is given the symbol Yr etc.

Our knowledge of vertical resistance has a short history, beginning with the discovery of Mendel's genetics and the subsequent work of R.H. Biffen at Cambridge University in 1902. It has been shown to be expressed almost universally as a hypersensitive necrosis of host cells contacted by the pathogen during the early stages of infection, in some cases occurring rapidly in the first few cells contacted and so evident only under a microscope (infection type 0 in the scheme of Stakman and Levine, 1922, for cereal rusts), and in others occurring only after the pathogen has invaded a large patch of cells which dies and so is evident to the naked eye as a necrotic fleck (infection types 0; and 1 in cereal rusts). In some cases, the pathogen may develop to the extent of a small amount of sporulation before the lesion becomes necrotic (infection type 2). It has been shown to be controlled by a 'gene-for-gene' interaction between the pathogen and the host (Flor, 1956). Flor worked with flax rust (*Melampsora lini*), which completes its sexual reproductive cycle on flax (*Linum usitatissimum*), rather than with wheat stem rust which requires two hosts (wheat and barberry) to complete its sexual cycle. In a brilliant study, he showed that for every rust

is said to be 'compatible'; if the interaction results in hypersensitive necrosis that largely excludes the pathogen, it is said to be 'incompatible', a term used very early in describing the effect of vertical resistance (see Hayes et al., 1925)(Table 2). These terms are particularly apt for biotrophic pathogens such as rusts and powdery mildews, where there is an intimate parasitic symbiosis between the pathogen and its host. In the same sense, the normal interactions of a plant and its mycorrhiza and endophyte symbionts would be said to be 'compatible'. The importance of the 'basic compatibility' required for a symbiont to live in its host has been well explored by Heath (1981). In fact, the copious molecular investigations of incompatibility would be better directed to trying to understand the mechanisms of compatibility – how does a biotrophic symbiont like a rust or a powdery mildew obtain nutrients from its host plant and

Resistance gene RR or Rr Double mutant rr

(Susceptible regardless of A/a)

**Horizontal resistance evident** 

**Horizontal resistance evident**

**Compatible interaction**  Pathogen infects and completes its life cycle

**Compatible interaction**  Pathogen infects and completes its life cycle

why can't this feeding relationship be replicated in a Petri dish?

Avirulence gene AA or Aa

Double mutant

(Gives virulence against R)

rust it is given the symbol Yr etc.

aa

Pathogen Plant host

**Incompatible interaction** 

**Compatible interaction** 

its life cycle

Hypersensitive necrosis; pathogen does not complete its life-cycle **1**

**Horizontal resistance is masked** 

Pathogen infects and completes **2**

Table 2. Summary of the gene-for-gene interaction involved in vertical resistance. Resistance

Our knowledge of vertical resistance has a short history, beginning with the discovery of Mendel's genetics and the subsequent work of R.H. Biffen at Cambridge University in 1902. It has been shown to be expressed almost universally as a hypersensitive necrosis of host cells contacted by the pathogen during the early stages of infection, in some cases occurring rapidly in the first few cells contacted and so evident only under a microscope (infection type 0 in the scheme of Stakman and Levine, 1922, for cereal rusts), and in others occurring only after the pathogen has invaded a large patch of cells which dies and so is evident to the naked eye as a necrotic fleck (infection types 0; and 1 in cereal rusts). In some cases, the pathogen may develop to the extent of a small amount of sporulation before the lesion becomes necrotic (infection type 2). It has been shown to be controlled by a 'gene-for-gene' interaction between the pathogen and the host (Flor, 1956). Flor worked with flax rust (*Melampsora lini*), which completes its sexual reproductive cycle on flax (*Linum usitatissimum*), rather than with wheat stem rust which requires two hosts (wheat and barberry) to complete its sexual cycle. In a brilliant study, he showed that for every rust

is usually dominant to susceptibility in the plant. Avirulence is usually dominant to virulence in the pathogen. The only starkly unique interaction occurs when an A gene matches an R gene. Arrow 1 indicates the change induced by breeding an R gene into a plant variety; arrow 2 indicates the change associated with the breakdown of resistance. Note that for wheat stem rust, the R gene is given the symbol Sr, for wheat stripe (yellow)

**Horizontal resistance evident** 

resistance gene in flax there is a corresponding gene for virulence (actually, avirulence if genes are named after their dominant allele) in flax rust (Table 2). Resistance is usually dominant to susceptibility and resistance genes occur as multiple alleles at a restricted number of loci in the plant while avirulence is usually dominant to virulence and avirulence genes occur at separate loci in the rust. That is, the number of resistance genes that can be expressed in the plant is restricted while there is no such restriction on the number of possible virulences that can be expressed in the rust. This ensures that the pathogen will always be able to overcome the resistance expressed in the host. While the meticulous and exhaustive work of Flor with flax rust has been repeated for very few other diseases, there is evidence that the relationship occurs in many highly specialized parasitic relationships (Sidhu, 1975), including several insect-plant relationships (Broekgaarden et al., 2011). It is usually expressed after the formation of haustoria in the highly specialized biotrophic parasites such as the rusts and powdery mildews and so appears to require intimate molecular contact between the pathogen and host. Rust resistance genes have been cloned from four of the five resistance loci in flax and they all appear to code for similar proteins, in the Nucleotide Binding Site-Leucine Rich Repeat (NBS-LRR) class (Ellis et al., 2007). Many resistance genes for a wide range of pathogens (including *Phytophthora,* rusts, powdery mildew, downy mildew, viruses, nematodes and bacteria) in a wide range of hosts (including potato, lettuce, tomato, barley, maize and *Arabidopsis*) have been found to fit into the same or similar class (Martin et al., 2003; Nimchuk et al., 2003). The resistance genes against the highly specialized phloem-feeding insect parasites of plants also appear to fall into this class (Broekgaarden et al., 2011). Thus, the recent molecular studies appear to confirm that vertical resistance involves a particular molecular system for recognition and rapid response to an invading parasite. On the other hand, the avirulence genes cloned from flax rust code for small secreted proteins ('effector proteins') that show no similarity between loci, providing evidence that their main function is probably something to do with the normal metabolism of the fungus and not to make them 'avirulent' on their host. Their 'avirulence' arises from the fact that they happen to be recognized by the resistance-coded proteins, leading to inhibition of the fungus. If avirulence genes code for proteins with a function in the normal life of the pathogen, this would explain the commonly observed phenomenon of stabilizing selection (Flor, 1956; van der Plank, 1968), whereby races expressing virulence (recessive mutants of A genes, often assumed to be non-functional) at one or more loci tend to be less fit than avirulent races on hosts that have no resistance genes. If avirulence genes code for a variety of normal functions in a pathogen, they could vary in the likelihood of their virulent (double recessive) mutant rising to prominence in the pathogen population, via mutation and selection for fitness, and so overcoming the particular resistance in the plant population (Luig, 1983). For example, if an avirulence gene (AvrX) coded for an essential function in the pathogen, then loss of this function in the homozygous virulent mutant (avrX avrX) may mitigate against the selection and buildup of this mutant, even if it matches the resistance RX in the host population. This could help explain the phenomenon of 'weak' and 'strong' vertical resistance genes as proposed by van der Plank (1968) and observed commonly in the field – i.e. some vertical resistance genes break down much more rapidly than others. The genes that are overcome rapidly are matched by races in which the mutation to virulence has little cost in fitness of the pathogen. If the mutation to virulence against a particular gene imposes a high cost in general fitness of the pathogen, the virulent race will not build up rapidly and the resistance gene will not be rapidly overcome; it will be seen to be 'strong'.

Horizontal or Generalized Resistance to Pathogens in Plants 337

Many words have been written and much heated argument generated in trying to define horizontal resistance precisely. The diversity of terminology applied to disease resistance has been summarized by Robinson (1969, 1976). Much has been said above about vertical resistance. This is necessary in a chapter about horizontal resistance if we define horizontal resistance as any resistance that is not vertical, as originally proposed by Black and Gallegly (1957) who defined field resistance (i.e. horizontal resistance) in the potato as "all forms of inherited resistance that plants possess with the exception of hypersensitivity as controlled by R-genes". Black restated this view in 1970 – "Field resistance to blight may be defined as the degree of resistance exhibited by a plant to all races of the fungus to which it is not hypersensitive." Such a definition is clear when 'specific resistance' and 'non-specific resistance' are substituted for 'vertical resistance' and 'horizontal resistance', respectively, as many like to do. Race-non-specific (horizontal) resistance is any resistance that is not racespecific, i.e. that does not operate on the gene-for-gene recognition system of Flor (1956) involving a hypersensitive necrosis response in the plants, and, on initial evidence, a particular molecular interaction as described by Ellis et al. (2007). It is the resistance expressed when there are no genes for vertical resistance in the plant or when the resistance has been overcome. Hayes et al. (1925) and Stakman and Levine (1922), in describing the infection types in wheat stem rust, considered that the presence or absence of hypersensitive necrosis marked the divide between resistance and susceptibility; it was then recognized that there are "different levels of susceptibility" (Parlevliet, 1995). Such a definition opens up a pandora's box of possible phenotypes, genotypes and mechanisms of horizontal resistance, which is why its definition has been so difficult and contentious. Just about any attempt at precision in defining it raises exceptions that defy the particular definition

Vertical resistance determines the basic compatibility or incompatibility of an interaction between a plant and its parasite (Table 2). Disease develops normally or it does not. However, once a parasite establishes basic compatibility with its host (i.e. is able to invade and reproduce normally) it is logical to suppose that there are very many points in the subsequent compatible symbiotic infection process that may determine whether the invasion and reproduction is fast and prolific or slow and limited. This is especially so in the highly specialized biotrophic pathogens that depend for their nutrition on an intimate physical and physiological association with live host cells, usually occurring through highly specialized haustoria formed within the cells. Its spores have to germinate on the leaf surface, germ tubes have to locate stomata and form appressoria and penetration pegs through stomatal pores (in rusts) or grow over the surface and directly penetrate the cuticle (in powdery mildews), then the infection hyphae have to penetrate cell walls, form haustoria and establish the active metabolic process of deriving nutrients from the host and becoming a sink for nutrients within the plant. It then has to invade further, forming many more haustoria, and eventually sporulate on the surface of the plant (for powdery mildews) or break through the epidermis to form a pustule of spores (for rusts). During all of these interactions there are opportunities for the physiological processes or morphological structures of the plant to hinder or slow down the interaction, and this could depend on very many genes that play a part in normal plant metabolism and structure. The pathogen will invade fast and sporulate prolifically, allowing it to create a destructive epidemic in the

**3. Horizontal or non-specific resistance** 

(Robinson, 1976).

The ecological significance of the gene-for-gene relationship is that the rust is always able to match the resistance in the host – it can have unrestricted expression of virulence genes able to match any resistance genes that may occur in the plant population. The evolutionary significance is that the rust and the flax can co-exist and co-evolve. The evidence for this is that the host and the pathogen still exist: the plant has not driven the rust to extinction and the rust has not driven the plant to extinction. Mathematical studies have shown that the gene-for-gene system as described by Flor and elucidated further by Person et al. (1962) can be the basis of co-evolution when both the plant and the parasite are genetically variable and adaptive over time (i.e. are outbreeding) and genetically diverse in space (i.e. occur in populations of the species consisting of several different genotypes)(Mode, 1958). Geneticists call this 'balanced polymorphism' (Person, 1967). The gene-for-gene relationship (vertical resistance) probably evolved as a system that protected natural, genetically diverse, outbreeding and adaptive plant populations from excessive disease on the basis of the well documented 'mixture' or 'multiline' effect; pathogen races sporulating on a particular plant would not have been able to attack the immediate neighbors which had other resistance genes. This controls the pathogen population so that it doesn't overly reduce the fitness of the host population (otherwise the host could be outcompeted by other species and become extinct) and the parasite is also able to survive. This system functions as long as the pathogen does not build up a 'super race' with virulence against all the genes in the plant population. The stabilizing selection first observed by Flor (1956) and referred to above would tend to reduce the chance of such a race developing.

Thus, we can hypothesize that vertical resistance evolved in the outbreeding, genetically diverse, wild ancestors of crop species before their domestication, and that, as Mode (1958) said, the systems of vertical resistance we see in crop species today "are the relics of ancient systems of polymorphism, stemming from the time when wheat, barley and flax reproduced by outbreeding." (i.e. before their complete domestication). Evidence for this is the fact that the regions of evolution and diversification of crops (the Vavilov Regions) are the repositories of the vertical resistance genes in those crops (Leppik, 1970); that is where plant breeders, inspired by Vavilov (1951), have gone to find new resistance genes. It is possible that vertical resistance continued to play a role in stabilization of disease in traditional agriculture, where crop diversity was maintained. The rapid breakdown of vertical resistance in modern agriculture is due to the fact that we are now using the genes in planting systems that lack the genetic diversity in space and time of the ancestral wild and the early domesticated plant communities (Browning, 1974; Simmonds, 1979). It is a fascinating fact of agricultural botany that domestication has transformed many of our most important crop species from outbreeders in the wild to inbreeders in agriculture, and we are increasingly transforming our agricultural systems from polycultures to monocultures. The constant trend in modern industrial agriculture, driven, against all ecological wisdom, by global economics and centralized, powerful agricultural institutions, has been the steady elimination of this diversity in crop populations, including the critically important repositories of diversity in the regions of crop evolution (the Vavilov Centres). Thus, in modern agriculture the use of vertical resistance has coincided with a tendency to remove the genetic diversity that probably underpinned it in the wild pathosystems and in traditional mixed agricultural systems. In the deployment of resistance genes, crops are in fact becoming global monocultures, hence the problem and the panic created by Ug99.

#### **3. Horizontal or non-specific resistance**

336 Plant Pathology

The ecological significance of the gene-for-gene relationship is that the rust is always able to match the resistance in the host – it can have unrestricted expression of virulence genes able to match any resistance genes that may occur in the plant population. The evolutionary significance is that the rust and the flax can co-exist and co-evolve. The evidence for this is that the host and the pathogen still exist: the plant has not driven the rust to extinction and the rust has not driven the plant to extinction. Mathematical studies have shown that the gene-for-gene system as described by Flor and elucidated further by Person et al. (1962) can be the basis of co-evolution when both the plant and the parasite are genetically variable and adaptive over time (i.e. are outbreeding) and genetically diverse in space (i.e. occur in populations of the species consisting of several different genotypes)(Mode, 1958). Geneticists call this 'balanced polymorphism' (Person, 1967). The gene-for-gene relationship (vertical resistance) probably evolved as a system that protected natural, genetically diverse, outbreeding and adaptive plant populations from excessive disease on the basis of the well documented 'mixture' or 'multiline' effect; pathogen races sporulating on a particular plant would not have been able to attack the immediate neighbors which had other resistance genes. This controls the pathogen population so that it doesn't overly reduce the fitness of the host population (otherwise the host could be outcompeted by other species and become extinct) and the parasite is also able to survive. This system functions as long as the pathogen does not build up a 'super race' with virulence against all the genes in the plant population. The stabilizing selection first observed by Flor (1956) and referred to above

Thus, we can hypothesize that vertical resistance evolved in the outbreeding, genetically diverse, wild ancestors of crop species before their domestication, and that, as Mode (1958) said, the systems of vertical resistance we see in crop species today "are the relics of ancient systems of polymorphism, stemming from the time when wheat, barley and flax reproduced by outbreeding." (i.e. before their complete domestication). Evidence for this is the fact that the regions of evolution and diversification of crops (the Vavilov Regions) are the repositories of the vertical resistance genes in those crops (Leppik, 1970); that is where plant breeders, inspired by Vavilov (1951), have gone to find new resistance genes. It is possible that vertical resistance continued to play a role in stabilization of disease in traditional agriculture, where crop diversity was maintained. The rapid breakdown of vertical resistance in modern agriculture is due to the fact that we are now using the genes in planting systems that lack the genetic diversity in space and time of the ancestral wild and the early domesticated plant communities (Browning, 1974; Simmonds, 1979). It is a fascinating fact of agricultural botany that domestication has transformed many of our most important crop species from outbreeders in the wild to inbreeders in agriculture, and we are increasingly transforming our agricultural systems from polycultures to monocultures. The constant trend in modern industrial agriculture, driven, against all ecological wisdom, by global economics and centralized, powerful agricultural institutions, has been the steady elimination of this diversity in crop populations, including the critically important repositories of diversity in the regions of crop evolution (the Vavilov Centres). Thus, in modern agriculture the use of vertical resistance has coincided with a tendency to remove the genetic diversity that probably underpinned it in the wild pathosystems and in traditional mixed agricultural systems. In the deployment of resistance genes, crops are in fact becoming global monocultures, hence the problem and the panic created by Ug99.

would tend to reduce the chance of such a race developing.

Many words have been written and much heated argument generated in trying to define horizontal resistance precisely. The diversity of terminology applied to disease resistance has been summarized by Robinson (1969, 1976). Much has been said above about vertical resistance. This is necessary in a chapter about horizontal resistance if we define horizontal resistance as any resistance that is not vertical, as originally proposed by Black and Gallegly (1957) who defined field resistance (i.e. horizontal resistance) in the potato as "all forms of inherited resistance that plants possess with the exception of hypersensitivity as controlled by R-genes". Black restated this view in 1970 – "Field resistance to blight may be defined as the degree of resistance exhibited by a plant to all races of the fungus to which it is not hypersensitive." Such a definition is clear when 'specific resistance' and 'non-specific resistance' are substituted for 'vertical resistance' and 'horizontal resistance', respectively, as many like to do. Race-non-specific (horizontal) resistance is any resistance that is not racespecific, i.e. that does not operate on the gene-for-gene recognition system of Flor (1956) involving a hypersensitive necrosis response in the plants, and, on initial evidence, a particular molecular interaction as described by Ellis et al. (2007). It is the resistance expressed when there are no genes for vertical resistance in the plant or when the resistance has been overcome. Hayes et al. (1925) and Stakman and Levine (1922), in describing the infection types in wheat stem rust, considered that the presence or absence of hypersensitive necrosis marked the divide between resistance and susceptibility; it was then recognized that there are "different levels of susceptibility" (Parlevliet, 1995). Such a definition opens up a pandora's box of possible phenotypes, genotypes and mechanisms of horizontal resistance, which is why its definition has been so difficult and contentious. Just about any attempt at precision in defining it raises exceptions that defy the particular definition (Robinson, 1976).

Vertical resistance determines the basic compatibility or incompatibility of an interaction between a plant and its parasite (Table 2). Disease develops normally or it does not. However, once a parasite establishes basic compatibility with its host (i.e. is able to invade and reproduce normally) it is logical to suppose that there are very many points in the subsequent compatible symbiotic infection process that may determine whether the invasion and reproduction is fast and prolific or slow and limited. This is especially so in the highly specialized biotrophic pathogens that depend for their nutrition on an intimate physical and physiological association with live host cells, usually occurring through highly specialized haustoria formed within the cells. Its spores have to germinate on the leaf surface, germ tubes have to locate stomata and form appressoria and penetration pegs through stomatal pores (in rusts) or grow over the surface and directly penetrate the cuticle (in powdery mildews), then the infection hyphae have to penetrate cell walls, form haustoria and establish the active metabolic process of deriving nutrients from the host and becoming a sink for nutrients within the plant. It then has to invade further, forming many more haustoria, and eventually sporulate on the surface of the plant (for powdery mildews) or break through the epidermis to form a pustule of spores (for rusts). During all of these interactions there are opportunities for the physiological processes or morphological structures of the plant to hinder or slow down the interaction, and this could depend on very many genes that play a part in normal plant metabolism and structure. The pathogen will invade fast and sporulate prolifically, allowing it to create a destructive epidemic in the

Horizontal or Generalized Resistance to Pathogens in Plants 339

controlled by 'polygenes' or inherited 'polygenically'. It is entirely possible that factors in the host that fortuitously inhibit the pathogen may be overcome by adaptation in the pathogen population (Parlevliet, 1995). That is, there may be variants of the pathogen that are not inhibited as much as other variants, and these variants may have a selective advantage because of their slightly greater fitness. They may sporulate more than other isolates and so contribute more offspring to the next generation. However, this adaptive process is not expected to be as rapid as that involved in the breakdown of vertical resistance. When it occurs, it is more likely to be expressed as a slow 'erosion' rather than a rapid 'breakdown' of resistance, as noted by Toxopeus (1956) and Niederhauser (1962) for late blight in potatoes. The difference between the more inhibited and less inhibited pathogen phenotypes is likely to be a matter of degree, not the extreme differences evident in vertical resistance, and so the selective pressures changing the pathogen population are likely to be far less. There are commonly several points of inhibition; being part of the normal functioning of the plant, they are likely to function independently in resistance, and so the adaptation of a pathogen variant at one point of inhibition is not likely to affect other

Horizontal resistance can be accumulated by continually crossing and selecting varieties with resistance with little detailed understanding of the genes involved, in the same way that yield and environmental adaptation of a crop have been built up steadily from generation to generation with little understanding of the many genes involved. This is shown in some of the examples given below. Resistance to vascular streak dieback in cocoa was selected in Papua New Guinea even before the cause of the disease was known. Because the genes controlling horizontal resistance are not primarily 'resistance genes' but just the genes involved in the normal processes of the plant, van der Plank (1968) has suggested that 'there may be large untapped reserves of horizontal resistance in many crops'. Parlevliet (1995) concludes that the search for quantitative (horizontal) resistance in alien species is unlikely to be fruitful and advises that "Fortunately, there is in most crop-pathogen systems no need for these procedures, as quantitative resistance appears to be present sufficiently within the crop species whenever scientists look for it." There are many examples where crossing of susceptible or resistant varieties results in transgressive segregation of resistance, whereby some progeny are more resistant than either parent (and some are more susceptible)(Skovmand et al., 1978). Robinson (1979) suggests that 'good sources' of resistance are not necessary for breeding for horizontal resistance, which, because it is not based on R genes but rather on the normal processes of a plant, can be built up from the normal range of genetic resources in a species. In fact, the preoccupation with genes and gene-transfer (by crossing and back-crossing) in conventional breeding (and now in genetic engineering using recombinant DNA methods) is inimical to the development of horizontal resistance, which usually requires the accumulation of many unknown genes, better served

The early students of vertical resistance were well aware of horizontal resistance and greatly valued it, probably because they were the first witnesses of the catastrophic breakdown of vertical resistance. Hayes et al. (1925), based on observations of wheat stem rust, described the difference between vertical and horizontal resistance very early in the development of our understanding of disease resistance – "It is known definitely that there are two types of resistance: (1) a true protoplasmic resistance which varies very little, and (2) a morphological resistance which varies with the age of the host and the conditions under

points of inhibition.

by recurrent selection methods (Robinson, 1979).

host population, if it encounters no great physical or physiological obstructions during the process of obtaining nutrients and colonizing the plant tissue and sporulating on the surface of the plant. It will invade more slowly if it encounters any physical or physiological obstructions during the parasitism. These obstructions are likely to be fortuitous, related to normal functions in the plant that, primarily, have nothing to do with resistance; they will exist whether or not the pathogen is present. For example, the proportion of peduncle tissue occupied by sclerenchyma in a wheat variety may restrict invasion and sporulation by stem rust (Hursh, 1924; Hart, 1931). A plant may just have tougher structures that are not damaged by the invading pathogen. This is especially important in stem pathogens, where invasion of a weak stem may result in the collapse of the whole plant. This is clearly evident in all damping-off diseases caused by *Pythium* species. Pythiums can only invade soft, immature hypocotyls, causing collapse (damping-off) of the plants. Once the hypocotyls become lignified they are resistant. This is horizontal resistance. A variety in which lignification is delayed could have less resistance than a variety that is lignified early. It is now possible to alter or reduce the lignification of pasture grasses in order to improve their digestibility to livestock; it has been observed that plants altered in this way become highly susceptible to rusts and insect attack, indicating that lignification of cell walls may be linked to the horizontal or generalized resistance of plants to parasites and herbivores (P. Dracatos, pers. comm.). It is important to note that impediments apart from the basic determination of compatibility may occur also in the pre-penetration and penetration phases of infection, as noted below in potato varieties expressing horizontal resistance prior to the incorporation of R genes. For example, the waxiness of the leaf surface may determine the proportion of spores landing on a leaf that are able to locate stomata and penetrate the leaf. Partial resistance to *Puccinia hordei* in barley has been shown to act before haustoria are formed (Niks, 1988). The cuticle thickness and the rate of vacuolization of epidermal cells may determine the proportion of powdery mildew spores that can establish infections (Schlosser, 1980). These are all expressions of horizontal resistance. It is to this multitude of processes that molecular biologists might profitably look for ways to enhance the resistance in plants, rather than perpetuating the preoccupation with vertical resistance genes whose effects are always likely to be overcome by mutations and selection in the pathogen.

The inheritance of horizontal resistance is best discussed in contrast to the inheritance of vertical resistance. Vertical resistance is invariably controlled by easily identifiable Mendelian genes with strong effects and is very well understood, now even down to the molecular expression of some of the genes involved (Ellis et al., 2007). In most cases, the inheritance of horizontal resistance is complex, different in different diseases and poorly understood, except to say that it is mostly additive and quantitative; it is about as well understood as the genetics of any other quantitative character such as 'yield'. This is not surprising given that horizontal resistance is likely to consist of any aspect of a plant's biology that slows down the growth and sporulation of a pathogen invading the plant in a basically compatible interaction. The obstructions the parasite may encounter are numerous and varied, and so their modes of inheritance will be numerous and varied. A single mechanism may be of great importance in the inhibition, and so the resistance may be dominated by the single gene that controls that mechanism (which may be called a 'resistance gene', e.g. the gene Rpg1 for durable resistance to stem rust in barley; Steffenson, 1992). Or it may be due to many aspects of the interaction, in which case it would be recognized as having 'quantitative' or 'additive' or 'complex' inheritance, and said to be

host population, if it encounters no great physical or physiological obstructions during the process of obtaining nutrients and colonizing the plant tissue and sporulating on the surface of the plant. It will invade more slowly if it encounters any physical or physiological obstructions during the parasitism. These obstructions are likely to be fortuitous, related to normal functions in the plant that, primarily, have nothing to do with resistance; they will exist whether or not the pathogen is present. For example, the proportion of peduncle tissue occupied by sclerenchyma in a wheat variety may restrict invasion and sporulation by stem rust (Hursh, 1924; Hart, 1931). A plant may just have tougher structures that are not damaged by the invading pathogen. This is especially important in stem pathogens, where invasion of a weak stem may result in the collapse of the whole plant. This is clearly evident in all damping-off diseases caused by *Pythium* species. Pythiums can only invade soft, immature hypocotyls, causing collapse (damping-off) of the plants. Once the hypocotyls become lignified they are resistant. This is horizontal resistance. A variety in which lignification is delayed could have less resistance than a variety that is lignified early. It is now possible to alter or reduce the lignification of pasture grasses in order to improve their digestibility to livestock; it has been observed that plants altered in this way become highly susceptible to rusts and insect attack, indicating that lignification of cell walls may be linked to the horizontal or generalized resistance of plants to parasites and herbivores (P. Dracatos, pers. comm.). It is important to note that impediments apart from the basic determination of compatibility may occur also in the pre-penetration and penetration phases of infection, as noted below in potato varieties expressing horizontal resistance prior to the incorporation of R genes. For example, the waxiness of the leaf surface may determine the proportion of spores landing on a leaf that are able to locate stomata and penetrate the leaf. Partial resistance to *Puccinia hordei* in barley has been shown to act before haustoria are formed (Niks, 1988). The cuticle thickness and the rate of vacuolization of epidermal cells may determine the proportion of powdery mildew spores that can establish infections (Schlosser, 1980). These are all expressions of horizontal resistance. It is to this multitude of processes that molecular biologists might profitably look for ways to enhance the resistance in plants, rather than perpetuating the preoccupation with vertical resistance genes whose effects are

always likely to be overcome by mutations and selection in the pathogen.

The inheritance of horizontal resistance is best discussed in contrast to the inheritance of vertical resistance. Vertical resistance is invariably controlled by easily identifiable Mendelian genes with strong effects and is very well understood, now even down to the molecular expression of some of the genes involved (Ellis et al., 2007). In most cases, the inheritance of horizontal resistance is complex, different in different diseases and poorly understood, except to say that it is mostly additive and quantitative; it is about as well understood as the genetics of any other quantitative character such as 'yield'. This is not surprising given that horizontal resistance is likely to consist of any aspect of a plant's biology that slows down the growth and sporulation of a pathogen invading the plant in a basically compatible interaction. The obstructions the parasite may encounter are numerous and varied, and so their modes of inheritance will be numerous and varied. A single mechanism may be of great importance in the inhibition, and so the resistance may be dominated by the single gene that controls that mechanism (which may be called a 'resistance gene', e.g. the gene Rpg1 for durable resistance to stem rust in barley; Steffenson, 1992). Or it may be due to many aspects of the interaction, in which case it would be recognized as having 'quantitative' or 'additive' or 'complex' inheritance, and said to be controlled by 'polygenes' or inherited 'polygenically'. It is entirely possible that factors in the host that fortuitously inhibit the pathogen may be overcome by adaptation in the pathogen population (Parlevliet, 1995). That is, there may be variants of the pathogen that are not inhibited as much as other variants, and these variants may have a selective advantage because of their slightly greater fitness. They may sporulate more than other isolates and so contribute more offspring to the next generation. However, this adaptive process is not expected to be as rapid as that involved in the breakdown of vertical resistance. When it occurs, it is more likely to be expressed as a slow 'erosion' rather than a rapid 'breakdown' of resistance, as noted by Toxopeus (1956) and Niederhauser (1962) for late blight in potatoes. The difference between the more inhibited and less inhibited pathogen phenotypes is likely to be a matter of degree, not the extreme differences evident in vertical resistance, and so the selective pressures changing the pathogen population are likely to be far less. There are commonly several points of inhibition; being part of the normal functioning of the plant, they are likely to function independently in resistance, and so the adaptation of a pathogen variant at one point of inhibition is not likely to affect other points of inhibition.

Horizontal resistance can be accumulated by continually crossing and selecting varieties with resistance with little detailed understanding of the genes involved, in the same way that yield and environmental adaptation of a crop have been built up steadily from generation to generation with little understanding of the many genes involved. This is shown in some of the examples given below. Resistance to vascular streak dieback in cocoa was selected in Papua New Guinea even before the cause of the disease was known. Because the genes controlling horizontal resistance are not primarily 'resistance genes' but just the genes involved in the normal processes of the plant, van der Plank (1968) has suggested that 'there may be large untapped reserves of horizontal resistance in many crops'. Parlevliet (1995) concludes that the search for quantitative (horizontal) resistance in alien species is unlikely to be fruitful and advises that "Fortunately, there is in most crop-pathogen systems no need for these procedures, as quantitative resistance appears to be present sufficiently within the crop species whenever scientists look for it." There are many examples where crossing of susceptible or resistant varieties results in transgressive segregation of resistance, whereby some progeny are more resistant than either parent (and some are more susceptible)(Skovmand et al., 1978). Robinson (1979) suggests that 'good sources' of resistance are not necessary for breeding for horizontal resistance, which, because it is not based on R genes but rather on the normal processes of a plant, can be built up from the normal range of genetic resources in a species. In fact, the preoccupation with genes and gene-transfer (by crossing and back-crossing) in conventional breeding (and now in genetic engineering using recombinant DNA methods) is inimical to the development of horizontal resistance, which usually requires the accumulation of many unknown genes, better served by recurrent selection methods (Robinson, 1979).

The early students of vertical resistance were well aware of horizontal resistance and greatly valued it, probably because they were the first witnesses of the catastrophic breakdown of vertical resistance. Hayes et al. (1925), based on observations of wheat stem rust, described the difference between vertical and horizontal resistance very early in the development of our understanding of disease resistance – "It is known definitely that there are two types of resistance: (1) a true protoplasmic resistance which varies very little, and (2) a morphological resistance which varies with the age of the host and the conditions under

Horizontal or Generalized Resistance to Pathogens in Plants 341

rapidly, decimated yield and rotted even the few tubers that were formed. Beginning in 1912, many observers documented the occurrence of degrees of resistance to late blight in the field before the time when R genes were bred into the potato (Thurston, 1971). This was often referred to as 'general resistance'. It was noted that inhibition of disease in these early resistant varieties could be due to inhibition at several stages of the process of pathogenesis, most notably resulting in (i) a reduced number of infections for a given inoculum dose (i.e. inhibition acting prior to penetration of the leaf by the fungus), (ii) a reduced rate of growth of mycelium in the plant tissues, (iii) a delay in sporulation, and (iv) a reduced number of sporangia produced per unit area of lesion. While vertical resistance is invariably associated with the sudden collapse and death of host cells during the initial establishment of parasitism (especially establishment of haustoria), horizontal resistance can be associated with death of invaded host tissue much later in the parasitic process. Van der Plank (1968, p.185) described how necrosis often occurs in the centres of developing lesions on potato varieties with horizontal resistance to *Ph. infestans*, resulting in a narrower zone of sporulation on the lesion than in a susceptible variety; he considered that it was possible to judge the degree of horizontal resistance of a variety by the amount of necrosis evident in sporulating lesions. Necrosis that appears to reduce the amount of sporulation on lesions is also evident in wheat varieties with horizontal (adultplant) resistance to stripe rust (*Puccinia striiformis*) and Robusta coffee with horizontal

Recent molecular studies have found that factors controlling general (horizontal) resistance to potato late blight occur on almost every potato chromosome and have confirmed that this resistance is, indeed, polygenic (Gebhardt & Valkonen, 2001). Several Quantitative Trait Loci (QTL) for use in marker assisted breeding for horizontal resistance have been located. The degree of general resistance observed in the early varieties was often seen to be affected by environmental factors and the developmental stage of the plant. The well documented history of general resistance in potatoes allows the conclusion that this resistance has, indeed, been durable. For example, Thurston (1971) documents the history of the variety Champion, which was first widely grown in Ireland in 1877 and was clearly popular because of its resistance to late blight, constituting 70% of the potato plantings in 1898. In 1953 Muller & Haigh reported that Champion still had a very high level of resistance. However, following the discovery of the R genes in *S. demissum* and their transfer into *S. tuberosum* ssp. *tuberosum,* as Thurston (1971) commented, *"*For several decades, almost all potato breeders dropped their work on general resistance and concentrated on obtaining commercial potato varieties with R-genes." Following the failure of R genes to provide longterm resistance, potato breeders turned back to horizontal resistance (Toxopeus, 1964). Van der Plank (1971) documented the fact that six potato varieties released without R genes in the 1920s and 1930s maintained their resistance rating of 6-9 (on a scale of 3=very susceptible to 10=very resistant) over a 30-year period from 1938 to 1968. Black (1970) showed that such resistance could be accumulated rapidly through crossing and selecting appropriate resistant material. In fact, Black turned back to *S. demissum* as a source of horizontal resistance, maintaining that it was mainly horizontal resistance that protected the wild potato species from *Ph. infestans* in Mexico. He showed that by crossing and selecting agronomically useful potato varieties in the presence of late blight, high levels of horizontal resistance could be accumulated. He established that the crossing of two moderately resistant varieties could result in some highly resistant progeny (due to transgressive

resistance to leaf rust (*Hemileia vastatrix*).

which it is grown." They considered that the former was due to a "real physiological incompatibility between the resistant plants and the invading fungus" and that "the struggle between host and parasite was short and decisive and involved only a few cells in the most resistant plants ---- In susceptible varieties, however, the fungus apparently does not injure the host cells immediately but actually seems to stimulate them to increase physiological activity." Stakman and Harrar (1957) in their important textbook *Principles of Plant Pathology* recognized that "There are various types of resistance in plants. The more kinds a variety has, the more likely it is to be generally resistant. The high degree of specificity between certain physiological races of pathogens and certain varieties of plants has been emphasized repeatedly. A variety may be immune from one race but completely susceptible to another. If more can be learned about the kinds of resistance that are effective against all physiological races, however, it might be possible to breed varieties that have at least some resistance to all races. --- For example, some varieties of wheat have physiological resistance to many races of the stem rust fungus. If it is possible to add resistance to entrance because of stomatal characters, to extension in the tissues because of tough cells and barriers of sclerenchyma, and to the rupture of the epidermis by the sporulating mycelium, the variety should be much more resistant than those which have only one or a few of the many characters that can contribute to resistance. Even though the specific contribution of each character might be relatively slight, the combination of all of them might be effective under a wide range of conditions." The insights of these early students of disease resistance have often been forgotten.

It is worth discussing resistance to late blight in potato in more detail as this clearly shows the contrast between vertical and horizontal resistance, as thoroughly reviewed by Thurston (1971). The potato now widely grown throughout the world, *S. tuberosum* ssp. *tuberosum*, but thought to have originated in the Andian region of South America, had no vertical resistance to *Ph. infestans* until resistance genes were bred into it in Europe by crossing with a wild relative, *S. demissum*, which is native to Central America and clearly had a co-evolved vertical resistance pathosystem with *Ph. infestans* (controlled by R genes, eleven of which have been transferred into the potato in the 20th Century; Malcolmson & Black, 1966). It appears that *Ph. infestans* evolved as a parasite on *S. demissum* and other wild species in Central America and not on *S. tuberosum* ssp. *tuberosum* and *S. tuberosum* ssp. *andigena* in the Andes, and is in fact a 'new-encounter' pathogen on *S. tuberosum* ssp. *tuberosum* (Leppik, 1970). Varieties like Maritta and Kennebec that have the R1 resistance gene transferred from *S. demissum* show complete resistance expressed as hypersensitive necrosis to pathogen races with avirulence on R1, but are susceptible to races that have virulence against R1 (Table 2); however, Kennebec is more susceptible than Maritta to these virulent races (van der Plank 1963). Maritta has more background horizontal resistance than Kennebec to races that can infect both varieties. Many potato varieties lack R genes, and these vary in horizontal resistance to *Ph. infestans.* The variety Capella has a very high degree of horizontal resistance: it can become infected (i.e. it can be said to be 'compatible' with *Ph. infestans* – the fungus can grow and reproduce in the variety), but the fungus takes longer to produce lesions, the lesions are smaller, the fungus sporulates sparsely on the lesions, the plants remain green overall, the epidemic develops slowly, and the variety still yields well (van der Plank, 1963). This is in sharp contrast to the very susceptible varieties being grown in Ireland at the time of the great Irish Potato Famine in 1845-47, where the disease developed very fast, spread throughout western Europe in a matter of months, killed plants

which it is grown." They considered that the former was due to a "real physiological incompatibility between the resistant plants and the invading fungus" and that "the struggle between host and parasite was short and decisive and involved only a few cells in the most resistant plants ---- In susceptible varieties, however, the fungus apparently does not injure the host cells immediately but actually seems to stimulate them to increase physiological activity." Stakman and Harrar (1957) in their important textbook *Principles of Plant Pathology* recognized that "There are various types of resistance in plants. The more kinds a variety has, the more likely it is to be generally resistant. The high degree of specificity between certain physiological races of pathogens and certain varieties of plants has been emphasized repeatedly. A variety may be immune from one race but completely susceptible to another. If more can be learned about the kinds of resistance that are effective against all physiological races, however, it might be possible to breed varieties that have at least some resistance to all races. --- For example, some varieties of wheat have physiological resistance to many races of the stem rust fungus. If it is possible to add resistance to entrance because of stomatal characters, to extension in the tissues because of tough cells and barriers of sclerenchyma, and to the rupture of the epidermis by the sporulating mycelium, the variety should be much more resistant than those which have only one or a few of the many characters that can contribute to resistance. Even though the specific contribution of each character might be relatively slight, the combination of all of them might be effective under a wide range of conditions." The insights of these early students of disease resistance have

It is worth discussing resistance to late blight in potato in more detail as this clearly shows the contrast between vertical and horizontal resistance, as thoroughly reviewed by Thurston (1971). The potato now widely grown throughout the world, *S. tuberosum* ssp. *tuberosum*, but thought to have originated in the Andian region of South America, had no vertical resistance to *Ph. infestans* until resistance genes were bred into it in Europe by crossing with a wild relative, *S. demissum*, which is native to Central America and clearly had a co-evolved vertical resistance pathosystem with *Ph. infestans* (controlled by R genes, eleven of which have been transferred into the potato in the 20th Century; Malcolmson & Black, 1966). It appears that *Ph. infestans* evolved as a parasite on *S. demissum* and other wild species in Central America and not on *S. tuberosum* ssp. *tuberosum* and *S. tuberosum* ssp. *andigena* in the Andes, and is in fact a 'new-encounter' pathogen on *S. tuberosum* ssp. *tuberosum* (Leppik, 1970). Varieties like Maritta and Kennebec that have the R1 resistance gene transferred from *S. demissum* show complete resistance expressed as hypersensitive necrosis to pathogen races with avirulence on R1, but are susceptible to races that have virulence against R1 (Table 2); however, Kennebec is more susceptible than Maritta to these virulent races (van der Plank 1963). Maritta has more background horizontal resistance than Kennebec to races that can infect both varieties. Many potato varieties lack R genes, and these vary in horizontal resistance to *Ph. infestans.* The variety Capella has a very high degree of horizontal resistance: it can become infected (i.e. it can be said to be 'compatible' with *Ph. infestans* – the fungus can grow and reproduce in the variety), but the fungus takes longer to produce lesions, the lesions are smaller, the fungus sporulates sparsely on the lesions, the plants remain green overall, the epidemic develops slowly, and the variety still yields well (van der Plank, 1963). This is in sharp contrast to the very susceptible varieties being grown in Ireland at the time of the great Irish Potato Famine in 1845-47, where the disease developed very fast, spread throughout western Europe in a matter of months, killed plants

often been forgotten.

rapidly, decimated yield and rotted even the few tubers that were formed. Beginning in 1912, many observers documented the occurrence of degrees of resistance to late blight in the field before the time when R genes were bred into the potato (Thurston, 1971). This was often referred to as 'general resistance'. It was noted that inhibition of disease in these early resistant varieties could be due to inhibition at several stages of the process of pathogenesis, most notably resulting in (i) a reduced number of infections for a given inoculum dose (i.e. inhibition acting prior to penetration of the leaf by the fungus), (ii) a reduced rate of growth of mycelium in the plant tissues, (iii) a delay in sporulation, and (iv) a reduced number of sporangia produced per unit area of lesion. While vertical resistance is invariably associated with the sudden collapse and death of host cells during the initial establishment of parasitism (especially establishment of haustoria), horizontal resistance can be associated with death of invaded host tissue much later in the parasitic process. Van der Plank (1968, p.185) described how necrosis often occurs in the centres of developing lesions on potato varieties with horizontal resistance to *Ph. infestans*, resulting in a narrower zone of sporulation on the lesion than in a susceptible variety; he considered that it was possible to judge the degree of horizontal resistance of a variety by the amount of necrosis evident in sporulating lesions. Necrosis that appears to reduce the amount of sporulation on lesions is also evident in wheat varieties with horizontal (adultplant) resistance to stripe rust (*Puccinia striiformis*) and Robusta coffee with horizontal resistance to leaf rust (*Hemileia vastatrix*).

Recent molecular studies have found that factors controlling general (horizontal) resistance to potato late blight occur on almost every potato chromosome and have confirmed that this resistance is, indeed, polygenic (Gebhardt & Valkonen, 2001). Several Quantitative Trait Loci (QTL) for use in marker assisted breeding for horizontal resistance have been located. The degree of general resistance observed in the early varieties was often seen to be affected by environmental factors and the developmental stage of the plant. The well documented history of general resistance in potatoes allows the conclusion that this resistance has, indeed, been durable. For example, Thurston (1971) documents the history of the variety Champion, which was first widely grown in Ireland in 1877 and was clearly popular because of its resistance to late blight, constituting 70% of the potato plantings in 1898. In 1953 Muller & Haigh reported that Champion still had a very high level of resistance. However, following the discovery of the R genes in *S. demissum* and their transfer into *S. tuberosum* ssp. *tuberosum,* as Thurston (1971) commented, *"*For several decades, almost all potato breeders dropped their work on general resistance and concentrated on obtaining commercial potato varieties with R-genes." Following the failure of R genes to provide longterm resistance, potato breeders turned back to horizontal resistance (Toxopeus, 1964). Van der Plank (1971) documented the fact that six potato varieties released without R genes in the 1920s and 1930s maintained their resistance rating of 6-9 (on a scale of 3=very susceptible to 10=very resistant) over a 30-year period from 1938 to 1968. Black (1970) showed that such resistance could be accumulated rapidly through crossing and selecting appropriate resistant material. In fact, Black turned back to *S. demissum* as a source of horizontal resistance, maintaining that it was mainly horizontal resistance that protected the wild potato species from *Ph. infestans* in Mexico. He showed that by crossing and selecting agronomically useful potato varieties in the presence of late blight, high levels of horizontal resistance could be accumulated. He established that the crossing of two moderately resistant varieties could result in some highly resistant progeny (due to transgressive

Horizontal or Generalized Resistance to Pathogens in Plants 343

400-year separation from the rust, resulting in a destructive epidemic when the pathogen was eventually introduced. In fact, *P. polysora* was barely mentioned in the plant pathology literature before it became destructive on maize in Africa. The pathogen spread eastward across Africa and into Southeast Asia and Melanesia. However, the destructiveness of the disease declined after the initial epidemic (Cammack, 1960), and now throughout this extended range it is regarded as of little importance although it can be found on most maize plants. It is evident that selection by farmers of resistant types from genetically variable populations, as has been done since time immemorial in America, resulted in rapid accumulation of horizontal resistance to *P. polysora* in African and Asian maize populations. Farmers would have selected seed preferentially from the resistant survivors of the epidemic; often they would have had no choice since highly susceptible genotypes were killed (Harlan, 1976) or would not have produced much seed. Van der Plank (1968) and Robinson (1976) argued that this experience showed how rapidly and effectively adequate levels of horizontal resistance could be accumulated by bulk selection from genetically diverse crop populations. In contrast to what happened on the farms, researchers conducting seedling tests concluded that there was no resistance in African maize. They were looking for vertical resistance. They were looking for 'genes for resistance' (Stanton &

The contrast between vertical and horizontal resistance has been evident in the quest to control leaf rust (*Hemileia vastatrix*) on Arabica coffee (*Coffea arabica*), the species grown in the highlands of many tropical countries to produce high-flavor coffee. Leaf rust has long been a devastating disease on Arabica coffee. It destroyed the plantations in Ceylon (Sri Lanka) in the period 1870 - 1890, reducing the industry from the world's major supplier of coffee to nil (Large 1962), and has caused serious problems since its spread to all coffeeproducing countries, including the Americas following its introduction from Africa in 1970. Much of the damage results from the premature defoliation of leaves with even moderate amounts of infection. Severe defoliation eventually kills the coffee bushes. Beginning in 1911 with Kent's selection in India, a succession of resistance genes (SH genes 1 to 6) was used in an attempt to control the disease, but with their widespread use in the field these all succumbed rapidly to selection of virulent rust races (having virulence genes 1 to

Quantitative resistance to leaf rust has been found in Arabica coffee, for example in Ethiopia and in particular material from eastern Sudan and Kenya (van der Graaff, 1986). This was expressed as differences in latent period, number of lesions, and period of leaf retention after infection. Transgressive segregation for resistance was observed in some crosses and there was no doubt that the resistance was inherited quantitatively. However, most interest has centred on the resistance of a less important commercial species of coffee, *Coffea canephora* (especially the varieties known as 'Kouillou' and 'Robusta'). This species is adapted to the tropical lowlands, where it has become commercially important (e.g. in Brazil and Indonesia) although it is regarded as having inferior flavor to Arabica coffee. Leaf rust commonly infects Kouillou coffee in Brazil but it is not regarded as a serious problem (Eskes 1983), despite the fact that the warm, humid lowland environment appears ideal for the activity of the rust, which is more damaging on Arabica coffee at lower than at higher

6)(Rodrigues, 1984). This was the typical expression of vertical resistance.

Cammak, 1953).

**4.2 Leaf rust (***Hemileia vastatrix***) of coffee** 

segregation). As he said, "it is possible for two resisters --- to possess different resistance factors, and thereby to produce on hybridization a proportion of seedlings of greater resistance than either parent." In fact, Black's 1970 paper is a compact manual for breeding for horizontal resistance. It shows how easy breeding for a quantitative character can be, involving steady accumulation of resistance rather than the game of snakes and ladders associated with breeding for vertical resistance. It avoids the bewildering work of collecting, identifying and naming new pathogen races, and involves working on races only to the extent that inoculation of test plots must be done with pathogen races that have virulence on all the vertical resistance genes that occur in the parent plants.

#### **4. Examples of disease management with horizontal resistance**

#### **4.1 Rusts of maize**

In the Americas, the two co-evolved rust pathogens of maize, common rust (*Puccinia sorghi*) and tropical rust (*P. polysora*), are regarded as minor diseases. At least one of them is found infecting nearly every maize plant throughout its natural range in Central America, but there is no report of serious rust epidemics on maize in the region (Borlaug, 1972). Certainly, the maize rusts "have been much less important in limiting corn production in the tropics and subtropics than has *Puccinia graminis tritici* on wheat under similar conditions." (Borlaug, 1965). In the extensive and productive corn belt of the United States, the common species, *P. sorghi*, has caused little damage even though the conditions of vast areas of intensive cultivation, continuous presence of the pathogen and environmental conditions conducive to the rust are ideal for epidemic development (Hooker, 1967). There are vertical resistance genes against the maize rusts but these have been unimportant, and the minor status of the rusts has been maintained by horizontal resistance. The fact that maize is outbreeding has facilitated the continuous bulk selection in maize for horizontal resistance, whereas the inbreeding small grain cereals have not allowed this process except following conscious cross-breeding to create the genetic diversity required for selection of improved types. Experience with the maize rusts is evidence that the long list of 'minor diseases' observed for each crop species and listed in the various compendia are kept to their 'minor' status by horizontal resistance (Hooker, 1967; Simmonds, 1991).

Van der Plank (1968, p. 155) has described the local, on-farm and highly effective selection for horizontal resistance to tropical rust (*P. polysora*) in maize in Africa. Maize was probably first introduced to Africa soon after Columbus crossed the Atlantic in 1492 and began the introduction to the Old World of American crops. Given the amount of shipping contact between Africa and America over the centuries, there were undoubtedly numerous introductions, resulting in great genetic diversity of the crop in Africa. The fact that maize is outbreeding would also have ensured its genetic diversity. *Puccinia sorghi* was introduced very early and remained of no importance, as in its centre of evolution (Harlan, 1976). Maize became a staple crop and thrived in Africa for at least four centuries in the absence of the tropical rust with which it had co-evolved in America. When *P. polysora* eventually found its way to Africa in 1949 (Schieber, 1971), it caused devastating epidemics, killed plants and massively reduced maize yields, and swept across the continent in a way that suggested a grand epidemic and great susceptibility in the maize populations, the like of which has never been reported in America (Borlaug, 1972). Van der Plank (1968) presented evidence that horizontal resistance to tropical rust had declined greatly in maize in Africa during its

segregation). As he said, "it is possible for two resisters --- to possess different resistance factors, and thereby to produce on hybridization a proportion of seedlings of greater resistance than either parent." In fact, Black's 1970 paper is a compact manual for breeding for horizontal resistance. It shows how easy breeding for a quantitative character can be, involving steady accumulation of resistance rather than the game of snakes and ladders associated with breeding for vertical resistance. It avoids the bewildering work of collecting, identifying and naming new pathogen races, and involves working on races only to the extent that inoculation of test plots must be done with pathogen races that have virulence on

In the Americas, the two co-evolved rust pathogens of maize, common rust (*Puccinia sorghi*) and tropical rust (*P. polysora*), are regarded as minor diseases. At least one of them is found infecting nearly every maize plant throughout its natural range in Central America, but there is no report of serious rust epidemics on maize in the region (Borlaug, 1972). Certainly, the maize rusts "have been much less important in limiting corn production in the tropics and subtropics than has *Puccinia graminis tritici* on wheat under similar conditions." (Borlaug, 1965). In the extensive and productive corn belt of the United States, the common species, *P. sorghi*, has caused little damage even though the conditions of vast areas of intensive cultivation, continuous presence of the pathogen and environmental conditions conducive to the rust are ideal for epidemic development (Hooker, 1967). There are vertical resistance genes against the maize rusts but these have been unimportant, and the minor status of the rusts has been maintained by horizontal resistance. The fact that maize is outbreeding has facilitated the continuous bulk selection in maize for horizontal resistance, whereas the inbreeding small grain cereals have not allowed this process except following conscious cross-breeding to create the genetic diversity required for selection of improved types. Experience with the maize rusts is evidence that the long list of 'minor diseases' observed for each crop species and listed in the various compendia are kept to their 'minor'

Van der Plank (1968, p. 155) has described the local, on-farm and highly effective selection for horizontal resistance to tropical rust (*P. polysora*) in maize in Africa. Maize was probably first introduced to Africa soon after Columbus crossed the Atlantic in 1492 and began the introduction to the Old World of American crops. Given the amount of shipping contact between Africa and America over the centuries, there were undoubtedly numerous introductions, resulting in great genetic diversity of the crop in Africa. The fact that maize is outbreeding would also have ensured its genetic diversity. *Puccinia sorghi* was introduced very early and remained of no importance, as in its centre of evolution (Harlan, 1976). Maize became a staple crop and thrived in Africa for at least four centuries in the absence of the tropical rust with which it had co-evolved in America. When *P. polysora* eventually found its way to Africa in 1949 (Schieber, 1971), it caused devastating epidemics, killed plants and massively reduced maize yields, and swept across the continent in a way that suggested a grand epidemic and great susceptibility in the maize populations, the like of which has never been reported in America (Borlaug, 1972). Van der Plank (1968) presented evidence that horizontal resistance to tropical rust had declined greatly in maize in Africa during its

all the vertical resistance genes that occur in the parent plants.

status by horizontal resistance (Hooker, 1967; Simmonds, 1991).

**4.1 Rusts of maize** 

**4. Examples of disease management with horizontal resistance** 

400-year separation from the rust, resulting in a destructive epidemic when the pathogen was eventually introduced. In fact, *P. polysora* was barely mentioned in the plant pathology literature before it became destructive on maize in Africa. The pathogen spread eastward across Africa and into Southeast Asia and Melanesia. However, the destructiveness of the disease declined after the initial epidemic (Cammack, 1960), and now throughout this extended range it is regarded as of little importance although it can be found on most maize plants. It is evident that selection by farmers of resistant types from genetically variable populations, as has been done since time immemorial in America, resulted in rapid accumulation of horizontal resistance to *P. polysora* in African and Asian maize populations. Farmers would have selected seed preferentially from the resistant survivors of the epidemic; often they would have had no choice since highly susceptible genotypes were killed (Harlan, 1976) or would not have produced much seed. Van der Plank (1968) and Robinson (1976) argued that this experience showed how rapidly and effectively adequate levels of horizontal resistance could be accumulated by bulk selection from genetically diverse crop populations. In contrast to what happened on the farms, researchers conducting seedling tests concluded that there was no resistance in African maize. They were looking for vertical resistance. They were looking for 'genes for resistance' (Stanton & Cammak, 1953).

#### **4.2 Leaf rust (***Hemileia vastatrix***) of coffee**

The contrast between vertical and horizontal resistance has been evident in the quest to control leaf rust (*Hemileia vastatrix*) on Arabica coffee (*Coffea arabica*), the species grown in the highlands of many tropical countries to produce high-flavor coffee. Leaf rust has long been a devastating disease on Arabica coffee. It destroyed the plantations in Ceylon (Sri Lanka) in the period 1870 - 1890, reducing the industry from the world's major supplier of coffee to nil (Large 1962), and has caused serious problems since its spread to all coffeeproducing countries, including the Americas following its introduction from Africa in 1970. Much of the damage results from the premature defoliation of leaves with even moderate amounts of infection. Severe defoliation eventually kills the coffee bushes. Beginning in 1911 with Kent's selection in India, a succession of resistance genes (SH genes 1 to 6) was used in an attempt to control the disease, but with their widespread use in the field these all succumbed rapidly to selection of virulent rust races (having virulence genes 1 to 6)(Rodrigues, 1984). This was the typical expression of vertical resistance.

Quantitative resistance to leaf rust has been found in Arabica coffee, for example in Ethiopia and in particular material from eastern Sudan and Kenya (van der Graaff, 1986). This was expressed as differences in latent period, number of lesions, and period of leaf retention after infection. Transgressive segregation for resistance was observed in some crosses and there was no doubt that the resistance was inherited quantitatively. However, most interest has centred on the resistance of a less important commercial species of coffee, *Coffea canephora* (especially the varieties known as 'Kouillou' and 'Robusta'). This species is adapted to the tropical lowlands, where it has become commercially important (e.g. in Brazil and Indonesia) although it is regarded as having inferior flavor to Arabica coffee. Leaf rust commonly infects Kouillou coffee in Brazil but it is not regarded as a serious problem (Eskes 1983), despite the fact that the warm, humid lowland environment appears ideal for the activity of the rust, which is more damaging on Arabica coffee at lower than at higher

Horizontal or Generalized Resistance to Pathogens in Plants 345

the plant tops may collapse and die before maturity. There is variation in the pathogenicity of isolates of the pathogen; some weakly pathogenic types can form lesions on cotyledons and leaves but not stem cankers while highly pathogenic types progress to form damaging stem cankers. The latter predominate in Australia and selection of disease resistance was

Salisbury and co-workers selected horizontal resistance to blackleg by exposing a wide range of canola genotypes to the pathogen in nursery plots heavily contaminated with infested crop residues. The more resistant types survived the blackleg epidemics that developed and were selected for further breeding work. These had mature plant resistance which was evidently inherited polygenically. The resistance was partial: the pathogen invaded and colonized the cotyledons and leaves of resistant types but stem cankering was reduced or eliminated, although under heavy disease pressure, the resistant types could still suffer significant amounts of disease. Continued improvement in resistance was achieved by crossing of partially resistant types and further selection using the same method of field testing (Salisbury, 1988), with the result that the canola varieties produced had "the highest levels of blackleg resistance of any spring canola varieties in the world" and when these were grown with appropriate cultural control measures, losses were negligible. In this initial work little attention appears to have been paid to pathogen races even though it was known that the fungus reproduced sexually and was highly variable. It was not necessary to do so. In a disease such as blackleg, it is possible to imagine that any plant characters associated with strengthening of the stem base may well contribute to resistance or tolerance to the disease; a stronger stem may be less liable to invasion by the fungus, and, if invaded, may be less liable to collapse leading to the death of the plant. This is the sort of resistance seen in many plant species to weak pathogens such as *Pythium*: as tissues of the hypocotyl and lower stem mature, they become completely resistant simply by dint of their increased

There are many lessons to be learned from this work. Firstly, inoculations of seedlings in a glasshouse gave different results than field tests. Later Salisbury & Ballinger (1996) showed that the resistance of seedlings tested in a glasshouse and of developing plants tested in the nursery plots were under different genetic control. The resistance evident in the blackleg nurseries was effective in the field. The basis for the success of this work was the field testing of resistance against the prevailing races of the highly variable fungus. There is evidence that this resistance can be eroded over time under severe disease pressure (Salisbury et al., 1995), but it has been relatively stable and subject to steady improvement in breeding programs. There has been no spectacular breakdown. In stark contrast, when in a separate program several varieties with vertical resistance (immunity expressed as hypersensitive necrosis) to the disease, controlled by a single dominant resistance gene bred into canola from a related species, *Brassica rapa* ssp. *sylvestris*, were released, the resistance broke down within three years (Sprague et al., 2006). Races of the pathogen virulent on these resistant varieties were selected from among the highly variable *L. maculans* population. Researchers then had to worry about the races of the pathogen and its high variability.

It appears that these vertically resistant varieties had been developed without a background of the horizontal resistance selected by Salisbury. When the resistance broke down, disease severity (measured as percent of the stem cross section blackened) was very high in the varieties with vertical resistance compared with nearby older varieties with only horizontal

essential for the survival of the crop.

mechanical strength.

altitudes. Following the introduction of *C. canephora* to Java in 1900 after the devastation of Arabica coffee by rust, the Robusta variety showed high levels of resistance, which has been maintained and even increased by selection and breeding to the present time (Kushalappa & Eskes, 1989). On trees in Indonesia, older leaves commonly have some lesions, but these never cover the leaves and they never appear to cause premature defoliation. No one seems concerned about the disease. The resistance is partial, is quantitatively inherited, and has been stable over a very long time; there are no reports of a sudden destructive upsurge of rust on the lowland species. It is horizontal resistance. Like the horizontal resistance of potato to *P. infestans*, it is associated with necrosis of large areas of tissue which appears to limit sporulation. This resistance has played a big part in the management of rust in Arabica coffee in recent decades (Kushalappa & Eskes, 1989). The horizontal resistance of Robusta coffee was incorporated into Arabica coffee in a rare hybridization between the tetraploid, self-compatible *C. arabica* and the diploid, cross-pollinated *C. canephora*, discovered in 1927 in East Timor (Rodrigues, 1984) where plantings of highlands Arabica coffee overlapped with plantings of the lowlands Robusta coffee. Tetraploid progeny of this hybridization were selected as 'Hibrido de Timor' and planted widely as a rust resistant Arabica flavor type in East Timor. Later in Brazil, the compact high quality variety Caturra was crossed with the Timor hybrid to produce the agronomically acceptable Catimor lines of Arabica coffee with the flavor of Arabica and the rust resistance of Robusta (Rodrigues, 1984). Catimor lines are now grown widely around the world to manage rust, apparently without any catastrophic loss of resistance. Similar types of full flavoured, rust-resistant coffee, known as 'Arabusta coffee', presumably of a similar origin, are now grown commercially in Indonesia (e.g. in the Toraja region of South Sulawesi). Moreno-Ruiz & Castillo-Zapata (1990) have described in detail the development of the rust resistant, compact variety 'Colombia' from 'Hibrido de Timor' in Colombia.

Arabica coffee can still be found growing wild and semi-domesticated in the highlands of Ethiopia, where it and the rust co-evolved. Here we can see the ecology of a crop species and its co-evolved pathogen in wild, ancestral communities of the species. It is evident that the wild coffee forests consist of a mixture of genotypes with different resistance genes, and probably types with a moderate degree of horizontal resistance, such that coffee rust is not seen as being epidemic there, certainly not to the extent seen in commercial Arabica coffee plantations in various countries since the 1870s (van der Graaff, 1986).

#### **4.3 Blackleg disease of canola caused by** *Leptosphaeria maculans*

The value of horizontal resistance was shown by its straightforward selection in the oil-seed crop canola (*Brassica napus*, bred for seed with low levels of toxic erucic acid and glucosinolates) to control blackleg disease (caused by the ascomycete *Leptosphaeria maculans*) that had practically destroyed the crop in southern Australia in the 1970s (Salisbury et al., 1995). This brilliant work enabled the establishment of a highly productive canola industry and added a crucial crop to the wheat–legume rotation that has sustained dryland cropping in Australia. The fungus invades the laminae of cotyledons and initial leaves as a biotroph but tissues behind the hyphal front die and the fungus eventually sporulates on the dead tissue. The fungus grows down the petiole and into the stem where it invades and eventually kills tissues of the stem cortex. Stem cankering ('blackleg') is the main cause of damage to the plants; in the most susceptible types stems may be completely girdled and

altitudes. Following the introduction of *C. canephora* to Java in 1900 after the devastation of Arabica coffee by rust, the Robusta variety showed high levels of resistance, which has been maintained and even increased by selection and breeding to the present time (Kushalappa & Eskes, 1989). On trees in Indonesia, older leaves commonly have some lesions, but these never cover the leaves and they never appear to cause premature defoliation. No one seems concerned about the disease. The resistance is partial, is quantitatively inherited, and has been stable over a very long time; there are no reports of a sudden destructive upsurge of rust on the lowland species. It is horizontal resistance. Like the horizontal resistance of potato to *P. infestans*, it is associated with necrosis of large areas of tissue which appears to limit sporulation. This resistance has played a big part in the management of rust in Arabica coffee in recent decades (Kushalappa & Eskes, 1989). The horizontal resistance of Robusta coffee was incorporated into Arabica coffee in a rare hybridization between the tetraploid, self-compatible *C. arabica* and the diploid, cross-pollinated *C. canephora*, discovered in 1927 in East Timor (Rodrigues, 1984) where plantings of highlands Arabica coffee overlapped with plantings of the lowlands Robusta coffee. Tetraploid progeny of this hybridization were selected as 'Hibrido de Timor' and planted widely as a rust resistant Arabica flavor type in East Timor. Later in Brazil, the compact high quality variety Caturra was crossed with the Timor hybrid to produce the agronomically acceptable Catimor lines of Arabica coffee with the flavor of Arabica and the rust resistance of Robusta (Rodrigues, 1984). Catimor lines are now grown widely around the world to manage rust, apparently without any catastrophic loss of resistance. Similar types of full flavoured, rust-resistant coffee, known as 'Arabusta coffee', presumably of a similar origin, are now grown commercially in Indonesia (e.g. in the Toraja region of South Sulawesi). Moreno-Ruiz & Castillo-Zapata (1990) have described in detail the development of the rust resistant, compact variety

Arabica coffee can still be found growing wild and semi-domesticated in the highlands of Ethiopia, where it and the rust co-evolved. Here we can see the ecology of a crop species and its co-evolved pathogen in wild, ancestral communities of the species. It is evident that the wild coffee forests consist of a mixture of genotypes with different resistance genes, and probably types with a moderate degree of horizontal resistance, such that coffee rust is not seen as being epidemic there, certainly not to the extent seen in commercial Arabica coffee

The value of horizontal resistance was shown by its straightforward selection in the oil-seed crop canola (*Brassica napus*, bred for seed with low levels of toxic erucic acid and glucosinolates) to control blackleg disease (caused by the ascomycete *Leptosphaeria maculans*) that had practically destroyed the crop in southern Australia in the 1970s (Salisbury et al., 1995). This brilliant work enabled the establishment of a highly productive canola industry and added a crucial crop to the wheat–legume rotation that has sustained dryland cropping in Australia. The fungus invades the laminae of cotyledons and initial leaves as a biotroph but tissues behind the hyphal front die and the fungus eventually sporulates on the dead tissue. The fungus grows down the petiole and into the stem where it invades and eventually kills tissues of the stem cortex. Stem cankering ('blackleg') is the main cause of damage to the plants; in the most susceptible types stems may be completely girdled and

'Colombia' from 'Hibrido de Timor' in Colombia.

plantations in various countries since the 1870s (van der Graaff, 1986).

**4.3 Blackleg disease of canola caused by** *Leptosphaeria maculans* 

the plant tops may collapse and die before maturity. There is variation in the pathogenicity of isolates of the pathogen; some weakly pathogenic types can form lesions on cotyledons and leaves but not stem cankers while highly pathogenic types progress to form damaging stem cankers. The latter predominate in Australia and selection of disease resistance was essential for the survival of the crop.

Salisbury and co-workers selected horizontal resistance to blackleg by exposing a wide range of canola genotypes to the pathogen in nursery plots heavily contaminated with infested crop residues. The more resistant types survived the blackleg epidemics that developed and were selected for further breeding work. These had mature plant resistance which was evidently inherited polygenically. The resistance was partial: the pathogen invaded and colonized the cotyledons and leaves of resistant types but stem cankering was reduced or eliminated, although under heavy disease pressure, the resistant types could still suffer significant amounts of disease. Continued improvement in resistance was achieved by crossing of partially resistant types and further selection using the same method of field testing (Salisbury, 1988), with the result that the canola varieties produced had "the highest levels of blackleg resistance of any spring canola varieties in the world" and when these were grown with appropriate cultural control measures, losses were negligible. In this initial work little attention appears to have been paid to pathogen races even though it was known that the fungus reproduced sexually and was highly variable. It was not necessary to do so. In a disease such as blackleg, it is possible to imagine that any plant characters associated with strengthening of the stem base may well contribute to resistance or tolerance to the disease; a stronger stem may be less liable to invasion by the fungus, and, if invaded, may be less liable to collapse leading to the death of the plant. This is the sort of resistance seen in many plant species to weak pathogens such as *Pythium*: as tissues of the hypocotyl and lower stem mature, they become completely resistant simply by dint of their increased mechanical strength.

There are many lessons to be learned from this work. Firstly, inoculations of seedlings in a glasshouse gave different results than field tests. Later Salisbury & Ballinger (1996) showed that the resistance of seedlings tested in a glasshouse and of developing plants tested in the nursery plots were under different genetic control. The resistance evident in the blackleg nurseries was effective in the field. The basis for the success of this work was the field testing of resistance against the prevailing races of the highly variable fungus. There is evidence that this resistance can be eroded over time under severe disease pressure (Salisbury et al., 1995), but it has been relatively stable and subject to steady improvement in breeding programs. There has been no spectacular breakdown. In stark contrast, when in a separate program several varieties with vertical resistance (immunity expressed as hypersensitive necrosis) to the disease, controlled by a single dominant resistance gene bred into canola from a related species, *Brassica rapa* ssp. *sylvestris*, were released, the resistance broke down within three years (Sprague et al., 2006). Races of the pathogen virulent on these resistant varieties were selected from among the highly variable *L. maculans* population. Researchers then had to worry about the races of the pathogen and its high variability.

It appears that these vertically resistant varieties had been developed without a background of the horizontal resistance selected by Salisbury. When the resistance broke down, disease severity (measured as percent of the stem cross section blackened) was very high in the varieties with vertical resistance compared with nearby older varieties with only horizontal

Horizontal or Generalized Resistance to Pathogens in Plants 347

*Eucalyptus globulus* (blue gum) is undergoing a rapid process of domestication. It is fast becoming one of the few indigenous Australian species to be added to the pantheon of the world's domesticated plants and is now one of the most widely planted tree species in the temperate zones. While foliar diseases are of little concern in native forests, they can be destructive in plantations of a single species such as blue gum (Park et al., 2000). One of the most serious diseases has been Mycosphaerella leaf blight caused by species of the ascomycete *Mycosphaerella*, which have co-evolved with *Eucalyptus*. The fungi initially invade the leaf tissues biotrophically and then cause sudden death of the invaded area to produce a necrotic blight on which the ascocarps are formed. Young, soft, expanding leaves are much more susceptible to infection than older, fully expanded, harder leaves. When collections of blue gum provenances from throughout its natural range were compared at one location favourable for Mycosphaerella leaf blight, significantly different degrees of disease incidence and severity occurred on the different provenances (Carnegie et al., 1994). Provenances from cold locations where the disease was likely to be less active tended to be very susceptible, while those from warmer areas with more summer rainfall where the disease was likely to be active were much more resistant. There had apparently been greater selection for disease resistance in locations where it was of more benefit to the host. This is horizontal resistance. It is partial, being assessed on a continuous scale from low to high percent leaf area affected, and is quantitatively inherited (Dungey et al., 1997). It has not been seen to be associated with hypersensitive necrosis of leaf tissue. It can be readily selected for in breeding programs and will be important in the development of improved

Some history of the early selection of 'off-types' of wheat with apparent high levels of resistance to stem rust during severe epidemics in Australia is referred to above. In 1894, a farmer, H.J. Gluyas, from the northern wheat belt of South Australia selected from Ward's Prolific an 'off-type' which he called "Early Gluyas"; from 1910 to about 1940 this was an important variety in the drier areas of Australia and became an important parent in the wheat breeding programs that developed from the turn of the 20th Century (Callaghan & Millington, 1956). An early contribution to rust control in Australia was the selection of early maturing varieties of wheat by William Farrer (Callaghan & Millington, 1956). These tended to escape stem rust, which built up and did most of its damage late in the growing season, and could be grown in drier areas where the disease was less of a problem. As well as aiming to produce early maturing varieties, Farrer (1898) also aimed to produce rust resistant varieties and this was one of his selection criteria. It has since been shown that some of his varieties did indeed have resistance to some of the rust races common up until 1926 (Waterhouse, 1936). Farrer's most famous variety was Federation, derived from crosses between Indian varieties, Canadian Fife wheats, and a high yielding commercial variety of the time, Purple Straw. This had stiff, short straw, was a good yielder, and matured early. It was first released in 1901, and from 1910 to 1925 was the most widely grown wheat in Australia. Dundee, a variety derived from Federation with similar characteristics, was in the top two or three most popular varieties in New South Wales and Victoria in 1938, on the dawn of the era of breeding and deployment of varieties with

**4.5 Foliar disease of** *Eucalyptus* **in Australia** 

varieties of blue gum for places where the disease is serious.

**5. Resistance to stem rust and stripe rust in wheat** 

resistance. The availability of varieties with immunity gave the farmers a false sense of security and encouraged them to plant the crop more intensively than previously, placing immense selective pressure on the pathogen. It is clear that the management of this disease in the future should rely on the horizontal resistance selected in the 1970s and since built up by regular crossing and selection among resistant varieties, combined with cultural control measures such as crop rotation and separation of new plantings from the previous crops (Marcroft et al., 2004). If vertical resistance is used, it must be added to a background of horizontal resistance. If there is evidence of erosion of horizontal resistance, this can be addressed by a program of steady improvement in resistance as practiced during the 1980s.

#### **4.4 Vascular streak dieback of cocoa caused by** *Oncobasidium theobromae* **in Southeast Asia and Melanesia**

It is now rare to see complete susceptibility to a pathogen in the field. Historical records sometimes give an indication of it (as in the Irish Potato Famine of 1845-47, or the coffee leaf rust epidemics in Southeast Asia in the 1890s), or we can glimpse it when a very susceptible variety is inoculated in a glasshouse, but in general we grow up seeing only crops that have been selected for a relatively high degree of horizontal resistance. These are the survivors of epidemics past. In 1969 in Papua New Guinea the author witnessed the extreme susceptibility of cocoa (*Theobroma cacao*) to a dieback disease later shown to be caused by the indigenous basidiomycete, *Oncobasidium* (*Ceratobasidium*) *theobromae*, which invades only the xylem and causes vascular streaking after which the disease was named (Keane & Prior, 1991). This new-encounter pathogen killed a large proportion of the genetically diverse cocoa plantings established in Papua New Guinea in the 1950s and 60s, leaving only the types with a degree of resistance that enabled them to survive the destructive epidemic. Farmers in the field and agronomists at the Lowlands Agricultural Experiment Station, Keravat, East New Britain Province, had no choice but to propagate from the survivors and, in so doing, selected types with disease resistance that has ever since sustained the industry in Papua New Guinea and throughout Southeast Asia where cocoa has become a major crop despite the presence of the disease (Indonesia is now the third largest producer of cocoa in the world). In fact, this resistance was selected by farmers and agronomists even before the cause of the disease was known, following the fundamental process of natural selection that has undoubtedly sustained wild plant species through evolutionary time and domesticated species since the dawn of agriculture. Some cocoa clones being tested on the Experiment Station were highly susceptible and became extinct - the fungus grew through their xylem so rapidly that it penetrated into the lower stems and roots, completely blocked the xylem, and killed the trees. Others were only slightly affected. Resistant genotypes become infected but the disease progresses more slowly in the xylem, doing less damage to the trees, and the fungus sporulates less. Resistance is quantitatively inherited and has high heritability (Tan & Tan, 1988). It has been relatively easy to select for in breeding programs. The epidemics are much reduced compared with those seen in the 1960s and the resistance is adequate to control the disease as part of an IPM program that includes heavy pruning of cocoa and shade trees to remove infected branches and maintain an open, drier canopy. It has been durable for over 50 years and is still important wherever cocoa is grown throughout the region. Vertical resistance has not been found for this disease, and it is postulated that it is unlikely to occur in this newencounter pathogen that has had, at most, a history of 300 years of contact with cocoa since the first introduction of the crop to Southeast Asia from Central America.

#### **4.5 Foliar disease of** *Eucalyptus* **in Australia**

346 Plant Pathology

resistance. The availability of varieties with immunity gave the farmers a false sense of security and encouraged them to plant the crop more intensively than previously, placing immense selective pressure on the pathogen. It is clear that the management of this disease in the future should rely on the horizontal resistance selected in the 1970s and since built up by regular crossing and selection among resistant varieties, combined with cultural control measures such as crop rotation and separation of new plantings from the previous crops (Marcroft et al., 2004). If vertical resistance is used, it must be added to a background of horizontal resistance. If there is evidence of erosion of horizontal resistance, this can be addressed by a program of steady improvement in resistance as practiced during the 1980s.

**4.4 Vascular streak dieback of cocoa caused by** *Oncobasidium theobromae* **in** 

first introduction of the crop to Southeast Asia from Central America.

It is now rare to see complete susceptibility to a pathogen in the field. Historical records sometimes give an indication of it (as in the Irish Potato Famine of 1845-47, or the coffee leaf rust epidemics in Southeast Asia in the 1890s), or we can glimpse it when a very susceptible variety is inoculated in a glasshouse, but in general we grow up seeing only crops that have been selected for a relatively high degree of horizontal resistance. These are the survivors of epidemics past. In 1969 in Papua New Guinea the author witnessed the extreme susceptibility of cocoa (*Theobroma cacao*) to a dieback disease later shown to be caused by the indigenous basidiomycete, *Oncobasidium* (*Ceratobasidium*) *theobromae*, which invades only the xylem and causes vascular streaking after which the disease was named (Keane & Prior, 1991). This new-encounter pathogen killed a large proportion of the genetically diverse cocoa plantings established in Papua New Guinea in the 1950s and 60s, leaving only the types with a degree of resistance that enabled them to survive the destructive epidemic. Farmers in the field and agronomists at the Lowlands Agricultural Experiment Station, Keravat, East New Britain Province, had no choice but to propagate from the survivors and, in so doing, selected types with disease resistance that has ever since sustained the industry in Papua New Guinea and throughout Southeast Asia where cocoa has become a major crop despite the presence of the disease (Indonesia is now the third largest producer of cocoa in the world). In fact, this resistance was selected by farmers and agronomists even before the cause of the disease was known, following the fundamental process of natural selection that has undoubtedly sustained wild plant species through evolutionary time and domesticated species since the dawn of agriculture. Some cocoa clones being tested on the Experiment Station were highly susceptible and became extinct - the fungus grew through their xylem so rapidly that it penetrated into the lower stems and roots, completely blocked the xylem, and killed the trees. Others were only slightly affected. Resistant genotypes become infected but the disease progresses more slowly in the xylem, doing less damage to the trees, and the fungus sporulates less. Resistance is quantitatively inherited and has high heritability (Tan & Tan, 1988). It has been relatively easy to select for in breeding programs. The epidemics are much reduced compared with those seen in the 1960s and the resistance is adequate to control the disease as part of an IPM program that includes heavy pruning of cocoa and shade trees to remove infected branches and maintain an open, drier canopy. It has been durable for over 50 years and is still important wherever cocoa is grown throughout the region. Vertical resistance has not been found for this disease, and it is postulated that it is unlikely to occur in this newencounter pathogen that has had, at most, a history of 300 years of contact with cocoa since the

**Southeast Asia and Melanesia** 

*Eucalyptus globulus* (blue gum) is undergoing a rapid process of domestication. It is fast becoming one of the few indigenous Australian species to be added to the pantheon of the world's domesticated plants and is now one of the most widely planted tree species in the temperate zones. While foliar diseases are of little concern in native forests, they can be destructive in plantations of a single species such as blue gum (Park et al., 2000). One of the most serious diseases has been Mycosphaerella leaf blight caused by species of the ascomycete *Mycosphaerella*, which have co-evolved with *Eucalyptus*. The fungi initially invade the leaf tissues biotrophically and then cause sudden death of the invaded area to produce a necrotic blight on which the ascocarps are formed. Young, soft, expanding leaves are much more susceptible to infection than older, fully expanded, harder leaves. When collections of blue gum provenances from throughout its natural range were compared at one location favourable for Mycosphaerella leaf blight, significantly different degrees of disease incidence and severity occurred on the different provenances (Carnegie et al., 1994). Provenances from cold locations where the disease was likely to be less active tended to be very susceptible, while those from warmer areas with more summer rainfall where the disease was likely to be active were much more resistant. There had apparently been greater selection for disease resistance in locations where it was of more benefit to the host. This is horizontal resistance. It is partial, being assessed on a continuous scale from low to high percent leaf area affected, and is quantitatively inherited (Dungey et al., 1997). It has not been seen to be associated with hypersensitive necrosis of leaf tissue. It can be readily selected for in breeding programs and will be important in the development of improved varieties of blue gum for places where the disease is serious.

#### **5. Resistance to stem rust and stripe rust in wheat**

Some history of the early selection of 'off-types' of wheat with apparent high levels of resistance to stem rust during severe epidemics in Australia is referred to above. In 1894, a farmer, H.J. Gluyas, from the northern wheat belt of South Australia selected from Ward's Prolific an 'off-type' which he called "Early Gluyas"; from 1910 to about 1940 this was an important variety in the drier areas of Australia and became an important parent in the wheat breeding programs that developed from the turn of the 20th Century (Callaghan & Millington, 1956). An early contribution to rust control in Australia was the selection of early maturing varieties of wheat by William Farrer (Callaghan & Millington, 1956). These tended to escape stem rust, which built up and did most of its damage late in the growing season, and could be grown in drier areas where the disease was less of a problem. As well as aiming to produce early maturing varieties, Farrer (1898) also aimed to produce rust resistant varieties and this was one of his selection criteria. It has since been shown that some of his varieties did indeed have resistance to some of the rust races common up until 1926 (Waterhouse, 1936). Farrer's most famous variety was Federation, derived from crosses between Indian varieties, Canadian Fife wheats, and a high yielding commercial variety of the time, Purple Straw. This had stiff, short straw, was a good yielder, and matured early. It was first released in 1901, and from 1910 to 1925 was the most widely grown wheat in Australia. Dundee, a variety derived from Federation with similar characteristics, was in the top two or three most popular varieties in New South Wales and Victoria in 1938, on the dawn of the era of breeding and deployment of varieties with

Horizontal or Generalized Resistance to Pathogens in Plants 349

their aggressiveness as well as their virulence (Katsuya & Green, 1967). On varieties on which both races were virulent, race 56 gave a higher number of infections per unit amount of inoculum, especially at higher temperatures (20-25oC), and had a 2-day shorter latent period than race 15B. Uredinia of race 56 expanded faster although race 15B ultimately had larger uredinia, and race 56 produced more spores per uredinium than race 15B. These differences are important characteristics of the races. They indicate variation in the ability of the races to invade a plant after basic compatibility has been established, equivalent to

In Australia, relatively stable control of the rust in the most rust-prone areas of northern New South Wales and southern Queensland was achieved after about 1960 by assembling combinations of several resistance genes (up to five) in particular varieties so that mutants virulent for one or two genes were still blocked by other unmatched resistance genes (Watson, 1970; McIntosh, 1976; Park, 2007). It was also considered that races with multiple virulences were likely to be less fit than races with simple virulence (Flor, 1956; van der Plank, 1968; Leonard, 1969), and so were unlikely to build up rapidly in the rust population (Watson, 1970). The release of varieties with just one or two resistance genes was avoided so that the rust was denied possible stepping stones for developing full virulence on the varieties with several resistance genes. However, even some of the multiple resistances broke down - e.g. Sr7a, Sr11, Sr17, Sr36 in the variety Mendos (Luig, 1983; Park, 2007), and Sr5, Sr6, Sr8, Sr12 in the variety Oxley (Luig, 1983). However, the strategy was largely successful and McIntosh (1976) was able to conclude that "The sacrifice for almost 35 years of rust resistance has been a regular turnover of cultivars and the loss of effectiveness of a number of resistance genes." Park (2007) considered that particular combinations of genes such as Sr2, Sr24 and Sr26 had been particularly effective. The success was built on a constant effort in surveying the occurrence of rust races and breeding of new varieties with

There was a general view among wheat pathologists and breeders that horizontal resistance to stem rust was "uncommon in bread wheats" (Watson, 1974) or "yet to be clearly demonstrated" (Knott, 1971), and that it was less likely to have been accumulated over time in the inbreeding crops like wheat than in an outbreeding crop like maize (van der Plank, 1968; Knott 1971). However, Knott (1968) acknowledged the importance of the resistance of Hope and H-44 which can be considered to show horizontal resistance. Scraps of evidence of horizontal resistance can be seen in the early preoccupation with vertical resistance. For example, of the group of varieties with Sr11 involved in the second breakdown of resistance noted in Australia (Table 1), Waterhouse (1952) ranked Gabo as being more resistant than Yalta, both of which had Sr11. After the breakdown of the Sr11 resistance, Yalta was rapidly eliminated from the rust liable areas of New South Wales while Gabo, "being somewhat less susceptible" was still grown successfully (Watson, 1958). It is likely that Gabo had a greater degree of horizontal resistance than Yalta. It was generally known that there were potentially useful forms of resistance other than the vertical resistance expressed as hypersensitivity. Watson (1958) recognized the potential of what he called the "morphological resistance" in Webster that had been transferred into the variety Fedweb and had remained effective against all local races of stem rust. In fact, the resistance of Fedweb lasted from 1938 to 1964 (Park, 2007). Watson (1974) recognized that there were two known types of non-specific resistance that had been transferred into bread wheat (*Triticum aestivum*) from other wheat species. Resistance from *T. turgidum* var. *dicoccum* (Yaroslav

variation in horizontal resistance in the plants.

appropriate resistance genes.

identified vertical resistance genes. The relatively long-lived popularity of the Federationtype wheats may indicate that, although they were regarded as being susceptible to stem rust, they may have had a degree of horizontal resistance that enabled them to continue to yield well and remain popular with farmers. The fact that they had short, stiff straw could have contributed to this. Farrer had been aware that varieties with erect, stiffer leaves tended to suffer less rust infection. He attempted to combine the (partial) rust resistance of late maturing varieties with earliness (Guthrie, 1922). His methods showed an awareness of quantitative genetics and what is now called 'transgressive segregation'. While it is often stated that he did not develop rust-resistant varieties, this assessment is usually made through the lens of vertical resistance that came to dominate breeding for stem rust resistance in Australia after his death. One of his varieties, Bomen, was still regarded as a valuable variety in the rust-prone northern districts of New South Wales in the early 1920s, and in fact won a Royal Agricultural Society prize for the best crop in 1921 when stem rust was a serious problem (Guthrie, 1922). It is possible that, given his breeding intentions and his methods of selecting for quantitative characters, Farrer did select a degree of horizontal resistance which underpinned the evident longevity of some of his varieties. After his death, his methods which favored the selection of horizontal resistance were replaced by the selection of the Mendelian genes for vertical resistance which has continued to dominate wheat breeding to the present time.

As discussed above for Australia (Watson and Luig, 1963; Table 1) and summarized for North America and Kenya by Person (1967), the use of vertical resistance to control wheat stem rust up until about 1960 resulted in the rapid breakdown of resistance as new races in the rust population adapted to the successive deployment of one or two resistance genes in particular varieties of wheat. This resistance broke down rapidly in Australia and Kenya (within about 5 years), and was also lost in North America, although there it was longerlived (being effective for more than 10 years in some of the most important varieties). The breeding and deployment of vertical resistance involved a massive effort in surveying the races in the rust populations as the researchers attempted to keep track of the adaptation of the rust to the new varieties, producing bewildering lists of races. In fact, trying to review the race changes in the rust populations is truly confusing, as noted by Waterhouse (1952). The race names bear no relationship to the resistance genes they are matching, but rather are named after the pattern of virulence shown on sets of 'differential' varieties with different resistance genes (McIntosh et al., 1995). Only the fully initiated can easily keep track of the virulence genes that are being expressed in the rust populations. Van der Plank (1983) criticized the current concept of a pathogen race as stretching the bounds of taxonomic practice. If a new resistance gene is found in a host, the number of possible pathogen races increases exponentially (following Flor's gene-for-gene hypothesis, the potential number of races is 2 to the power of the number of resistance genes) and the previously described races have to be re-described to include their virulence on the new gene. Each race has a unique set of characteristics which consists of its virulence, its aggressiveness, and its overall fitness to survive in the environment (Luig, 1983); together these characteristics contribute to its ability to cause epidemics and so each race has to have a taxonomic identifier of some sort. For example, the most common races in Canada during the 1920s had a longer uredinial period on standard varieties than the less common races (Newton et al., 1932), and this trait would have contributed to their survival in the rust population. The two major races of stem rust in North America from the 1930s to the 1960s, race 56 and race 15B, differed greatly in

identified vertical resistance genes. The relatively long-lived popularity of the Federationtype wheats may indicate that, although they were regarded as being susceptible to stem rust, they may have had a degree of horizontal resistance that enabled them to continue to yield well and remain popular with farmers. The fact that they had short, stiff straw could have contributed to this. Farrer had been aware that varieties with erect, stiffer leaves tended to suffer less rust infection. He attempted to combine the (partial) rust resistance of late maturing varieties with earliness (Guthrie, 1922). His methods showed an awareness of quantitative genetics and what is now called 'transgressive segregation'. While it is often stated that he did not develop rust-resistant varieties, this assessment is usually made through the lens of vertical resistance that came to dominate breeding for stem rust resistance in Australia after his death. One of his varieties, Bomen, was still regarded as a valuable variety in the rust-prone northern districts of New South Wales in the early 1920s, and in fact won a Royal Agricultural Society prize for the best crop in 1921 when stem rust was a serious problem (Guthrie, 1922). It is possible that, given his breeding intentions and his methods of selecting for quantitative characters, Farrer did select a degree of horizontal resistance which underpinned the evident longevity of some of his varieties. After his death, his methods which favored the selection of horizontal resistance were replaced by the selection of the Mendelian genes for vertical resistance which has continued to dominate

As discussed above for Australia (Watson and Luig, 1963; Table 1) and summarized for North America and Kenya by Person (1967), the use of vertical resistance to control wheat stem rust up until about 1960 resulted in the rapid breakdown of resistance as new races in the rust population adapted to the successive deployment of one or two resistance genes in particular varieties of wheat. This resistance broke down rapidly in Australia and Kenya (within about 5 years), and was also lost in North America, although there it was longerlived (being effective for more than 10 years in some of the most important varieties). The breeding and deployment of vertical resistance involved a massive effort in surveying the races in the rust populations as the researchers attempted to keep track of the adaptation of the rust to the new varieties, producing bewildering lists of races. In fact, trying to review the race changes in the rust populations is truly confusing, as noted by Waterhouse (1952). The race names bear no relationship to the resistance genes they are matching, but rather are named after the pattern of virulence shown on sets of 'differential' varieties with different resistance genes (McIntosh et al., 1995). Only the fully initiated can easily keep track of the virulence genes that are being expressed in the rust populations. Van der Plank (1983) criticized the current concept of a pathogen race as stretching the bounds of taxonomic practice. If a new resistance gene is found in a host, the number of possible pathogen races increases exponentially (following Flor's gene-for-gene hypothesis, the potential number of races is 2 to the power of the number of resistance genes) and the previously described races have to be re-described to include their virulence on the new gene. Each race has a unique set of characteristics which consists of its virulence, its aggressiveness, and its overall fitness to survive in the environment (Luig, 1983); together these characteristics contribute to its ability to cause epidemics and so each race has to have a taxonomic identifier of some sort. For example, the most common races in Canada during the 1920s had a longer uredinial period on standard varieties than the less common races (Newton et al., 1932), and this trait would have contributed to their survival in the rust population. The two major races of stem rust in North America from the 1930s to the 1960s, race 56 and race 15B, differed greatly in

wheat breeding to the present time.

their aggressiveness as well as their virulence (Katsuya & Green, 1967). On varieties on which both races were virulent, race 56 gave a higher number of infections per unit amount of inoculum, especially at higher temperatures (20-25oC), and had a 2-day shorter latent period than race 15B. Uredinia of race 56 expanded faster although race 15B ultimately had larger uredinia, and race 56 produced more spores per uredinium than race 15B. These differences are important characteristics of the races. They indicate variation in the ability of the races to invade a plant after basic compatibility has been established, equivalent to variation in horizontal resistance in the plants.

In Australia, relatively stable control of the rust in the most rust-prone areas of northern New South Wales and southern Queensland was achieved after about 1960 by assembling combinations of several resistance genes (up to five) in particular varieties so that mutants virulent for one or two genes were still blocked by other unmatched resistance genes (Watson, 1970; McIntosh, 1976; Park, 2007). It was also considered that races with multiple virulences were likely to be less fit than races with simple virulence (Flor, 1956; van der Plank, 1968; Leonard, 1969), and so were unlikely to build up rapidly in the rust population (Watson, 1970). The release of varieties with just one or two resistance genes was avoided so that the rust was denied possible stepping stones for developing full virulence on the varieties with several resistance genes. However, even some of the multiple resistances broke down - e.g. Sr7a, Sr11, Sr17, Sr36 in the variety Mendos (Luig, 1983; Park, 2007), and Sr5, Sr6, Sr8, Sr12 in the variety Oxley (Luig, 1983). However, the strategy was largely successful and McIntosh (1976) was able to conclude that "The sacrifice for almost 35 years of rust resistance has been a regular turnover of cultivars and the loss of effectiveness of a number of resistance genes." Park (2007) considered that particular combinations of genes such as Sr2, Sr24 and Sr26 had been particularly effective. The success was built on a constant effort in surveying the occurrence of rust races and breeding of new varieties with appropriate resistance genes.

There was a general view among wheat pathologists and breeders that horizontal resistance to stem rust was "uncommon in bread wheats" (Watson, 1974) or "yet to be clearly demonstrated" (Knott, 1971), and that it was less likely to have been accumulated over time in the inbreeding crops like wheat than in an outbreeding crop like maize (van der Plank, 1968; Knott 1971). However, Knott (1968) acknowledged the importance of the resistance of Hope and H-44 which can be considered to show horizontal resistance. Scraps of evidence of horizontal resistance can be seen in the early preoccupation with vertical resistance. For example, of the group of varieties with Sr11 involved in the second breakdown of resistance noted in Australia (Table 1), Waterhouse (1952) ranked Gabo as being more resistant than Yalta, both of which had Sr11. After the breakdown of the Sr11 resistance, Yalta was rapidly eliminated from the rust liable areas of New South Wales while Gabo, "being somewhat less susceptible" was still grown successfully (Watson, 1958). It is likely that Gabo had a greater degree of horizontal resistance than Yalta. It was generally known that there were potentially useful forms of resistance other than the vertical resistance expressed as hypersensitivity. Watson (1958) recognized the potential of what he called the "morphological resistance" in Webster that had been transferred into the variety Fedweb and had remained effective against all local races of stem rust. In fact, the resistance of Fedweb lasted from 1938 to 1964 (Park, 2007). Watson (1974) recognized that there were two known types of non-specific resistance that had been transferred into bread wheat (*Triticum aestivum*) from other wheat species. Resistance from *T. turgidum* var. *dicoccum* (Yaroslav

Horizontal or Generalized Resistance to Pathogens in Plants 351

McMurachy, Kenya 58, Thatcher and Idaed 59. In crosses between fast- and slow-rusting varieties, Skovmand et al. (1978) showed that transgressive segregation occurred in all crosses, and that slow-rusting was quantitatively inherited with a narrow-sense heritability of 80%. In Europe, farmers and plant breeders over many years used a satisfactory level of horizontal (partial) resistance to protect spring barley against leaf rust (*Puccinia hordei*)(Parlevliet, 1981). Slow rusting in cereals generally involved decreased frequency of penetration, slower invasion of host tissue, longer latent period, smaller pustules, lower sporulation rate, and shorter period of sporulation, singly or, more commonly, in combination (Kuhn et al., 1978; Parlevliet, 1979). The detailed physiology of these effects is not understood, but early workers determined that some physical features of cereals could affect rust development. For example, Hursh (1924) found evidence that the proportion of sclerenchyma to collenchyma in the upper peduncle was correlated with the resistance to stem rust of Sonem Emmer and Kota wheat compared with Little Club. Horizontal resistance, being partial, is strongly affected by environmental factors. It has long been known that excessive nitrogenous fertilization makes cereals more susceptible to rusts (Hursh, 1924). Hart (1931) showed that Webster had several morphological features that increased its resistance to stem rust compared with very susceptible varieties. These included a higher proportion of sclerenchyma in the peduncle and the degree of lignification and relative toughness of the epidermis which often prevented uredinia breaking through to the surface. The resistance in Webster has since been attributed to the gene Sr30, which has been overcome in the Australian variety Festiguay (Knott & McIntosh, 1978). However, it is unlikely that the set of morphological features that inhibit rust infection in Webster (Hart,

The known sources of horizontal resistance (often referred to as 'adult plant resistance' or 'durable resistance') against stem rust in wheat are very narrow, consisting mainly of the two tetraploid wheats, *T. turgidum* var. *dicoccum* (cv. Yaroslav Emmer) and *T. turgidum*  var. *durum* (cv. Iumillo) and the bread wheat variety Webster (McIntosh et al., 1995). Leppik (1970) lists a range of wild wheat species discovered through the activities of the Russian collecting expeditions that are possible sources of resistance. An unfortunate downside of the spread of dwarf wheat varieties around the world from the 1960s has been the loss of the genetic diversity in the local land races that had probably undergone selection for horizontal resistance. But building up horizontal resistance in a crop does not necessarily involve just searching for sources of resistance, but rather crossing and selection of existing varieties in such as way that genes involved in the normal functioning of the plant that happen to interfere with pathogen growth and development

Stripe (yellow) rust (*Puccinia striiformis*) was first detected in Australia in 1979 and its history of control by resistance has been very different from that of stem rust. The first response to the incursion, which caused heavy losses in some of the most widely grown varieties such as Zenith, was to deploy vertical resistance genes (YrA and Yr6). However, the effectiveness of these genes was lost very rapidly (Wellings & McIntosh, 1990). It was observed that some wheat varieties such as Condor, Egret and Olympic, although clearly susceptible at the seedling stage, showed varying degrees of adult-plant resistance and suffered much lower losses than the very susceptible varieties such as Zenith (McIntosh & Wellings, 1986). These had similar temperature-sensitive, partial, adult-plant resistance to that observed in some prominent varieties such as Cappelle-Desprez in Europe (Johnson, 1978), and Gaines,

1931) is controlled by only one gene.

are accumulated and resistance is built up.

Emmer) had been transferred into the varieties Hope and Renown, and from *T. turgidum*  var. *durum* (Iumillo) into the famous variety Thatcher. These resistances appeared to be controlled mainly by single genes, but Watson thought there were other unidentified genes involved. Watson (1974) thought non-specific resistance (presumably from the above sources) had performed well in the cultivars Warigo and Selkirk in the 1973-74 wheat stem rust epidemic in Australia. Warigo had been an exceptional variety during the period of repeated release and breakdown of varieties with single resistance genes (1938 – 1960; Table 1). Released in 1943 and known to have Sr17 (a recessive resistance; McIntosh et al., 1995), its resistance lasted an exceptional 16 years until 1959 (Park, 2007). It had Yaroslav Emmer in its parentage and Watson (1974) assumed that this was part of its success. Now it is known also to contain Sr2, another recessive gene from Yaroslav Emmer that has conferred durable resistance on many varieties (McIntosh et al., 1995). Rees et al. (1979) documented a wide range of horizontal resistance in wheat varieties including some of the older ones used in Australia.

In North America there were two spectacular resistance breakdowns leading to major epidemics in 1935 (associated with the coming to dominance in the rust population of race 56) and 1954 (associated with the dominance of race 15B) (Person, 1967), but it appears that stem rust was generally controlled, except for these two major epidemics, through the use of multiple resistance genes (e.g. Thatcher had Sr5, Sr9g, Sr12 and Sr16; Kolmer et al., 2011; Luig, 1983), and incorporation into the background of many varieties of the horizontal resistance (referred to as 'adult plant resistance') derived from tetraploid wheats (Hare & McIntosh, 1979). The resistance of Hope (with resistance from Yaroslav Emmer) and Thatcher (with resistance from Iumillo) may have helped to partly protect the spring wheat crops in North America during the severe epidemic of 1954. Selkirk, which became the leading spring wheat variety after the 1954 epidemic, had six identified resistance genes (Sr2, Sr6, Sr7b, Sr9d, Sr17, Sr23; Luig, 1983), including Sr2 and Sr17, the two identified recessive genes from Yaroslav Emmer. The evidence that the main North American varieties may have had a degree of horizontal resistance could also account for the longevity of the vertical resistance of these varieties from 1935 through to the 1960s. Certainly, Stakman & Christensen (1960) recognized the occurrence of important levels of resistance in wheat varieties that were susceptible at the seedling stage. It was noted during the stem rust epidemic of 1954 that the amount of damage on the durum wheat variety Stewart was three times that on the bread wheat variety Lee, and this was attributed to the earliness and nonspecific resistance of Lee (Loegering et al., 1967).

The ability of cereals to slow down the development of rusts, even though the plants were considered basically susceptible, was recognized by pre-eminent breeders and pathologists many years ago (Farrer, 1898; Stakman & Harrar, 1957). A famous early example was the oat species *Avena byzantina* known as Red Rustproof, which was introduced to the southern United States and recognized as being resistant to crown rust (*Puccinia coronata*) in the 1860s (Luke et al., 1972). It was partially resistant, not immune, and has remained so for over 100 years. It is 'late-rusting'. It is also 'slow-rusting', expressed as a low percent of leaf area infected during the growing season. The degree of resistance varies between varieties of the species. Luke et al. (1972) had no hesitation in recognizing this as 'horizontal resistance'. They were also inclined to call it 'generalized resistance'. Wilcoxson (1981) comprehensively reviewed the biology of slow rusting in cereals and discussed the evidence for long-lived, slow-rusting against stem rust in some well known wheat varieties such as Lee,

Emmer) had been transferred into the varieties Hope and Renown, and from *T. turgidum*  var. *durum* (Iumillo) into the famous variety Thatcher. These resistances appeared to be controlled mainly by single genes, but Watson thought there were other unidentified genes involved. Watson (1974) thought non-specific resistance (presumably from the above sources) had performed well in the cultivars Warigo and Selkirk in the 1973-74 wheat stem rust epidemic in Australia. Warigo had been an exceptional variety during the period of repeated release and breakdown of varieties with single resistance genes (1938 – 1960; Table 1). Released in 1943 and known to have Sr17 (a recessive resistance; McIntosh et al., 1995), its resistance lasted an exceptional 16 years until 1959 (Park, 2007). It had Yaroslav Emmer in its parentage and Watson (1974) assumed that this was part of its success. Now it is known also to contain Sr2, another recessive gene from Yaroslav Emmer that has conferred durable resistance on many varieties (McIntosh et al., 1995). Rees et al. (1979) documented a wide range of horizontal resistance in wheat varieties including some of

In North America there were two spectacular resistance breakdowns leading to major epidemics in 1935 (associated with the coming to dominance in the rust population of race 56) and 1954 (associated with the dominance of race 15B) (Person, 1967), but it appears that stem rust was generally controlled, except for these two major epidemics, through the use of multiple resistance genes (e.g. Thatcher had Sr5, Sr9g, Sr12 and Sr16; Kolmer et al., 2011; Luig, 1983), and incorporation into the background of many varieties of the horizontal resistance (referred to as 'adult plant resistance') derived from tetraploid wheats (Hare & McIntosh, 1979). The resistance of Hope (with resistance from Yaroslav Emmer) and Thatcher (with resistance from Iumillo) may have helped to partly protect the spring wheat crops in North America during the severe epidemic of 1954. Selkirk, which became the leading spring wheat variety after the 1954 epidemic, had six identified resistance genes (Sr2, Sr6, Sr7b, Sr9d, Sr17, Sr23; Luig, 1983), including Sr2 and Sr17, the two identified recessive genes from Yaroslav Emmer. The evidence that the main North American varieties may have had a degree of horizontal resistance could also account for the longevity of the vertical resistance of these varieties from 1935 through to the 1960s. Certainly, Stakman & Christensen (1960) recognized the occurrence of important levels of resistance in wheat varieties that were susceptible at the seedling stage. It was noted during the stem rust epidemic of 1954 that the amount of damage on the durum wheat variety Stewart was three times that on the bread wheat variety Lee, and this was attributed to the earliness and non-

The ability of cereals to slow down the development of rusts, even though the plants were considered basically susceptible, was recognized by pre-eminent breeders and pathologists many years ago (Farrer, 1898; Stakman & Harrar, 1957). A famous early example was the oat species *Avena byzantina* known as Red Rustproof, which was introduced to the southern United States and recognized as being resistant to crown rust (*Puccinia coronata*) in the 1860s (Luke et al., 1972). It was partially resistant, not immune, and has remained so for over 100 years. It is 'late-rusting'. It is also 'slow-rusting', expressed as a low percent of leaf area infected during the growing season. The degree of resistance varies between varieties of the species. Luke et al. (1972) had no hesitation in recognizing this as 'horizontal resistance'. They were also inclined to call it 'generalized resistance'. Wilcoxson (1981) comprehensively reviewed the biology of slow rusting in cereals and discussed the evidence for long-lived, slow-rusting against stem rust in some well known wheat varieties such as Lee,

the older ones used in Australia.

specific resistance of Lee (Loegering et al., 1967).

McMurachy, Kenya 58, Thatcher and Idaed 59. In crosses between fast- and slow-rusting varieties, Skovmand et al. (1978) showed that transgressive segregation occurred in all crosses, and that slow-rusting was quantitatively inherited with a narrow-sense heritability of 80%. In Europe, farmers and plant breeders over many years used a satisfactory level of horizontal (partial) resistance to protect spring barley against leaf rust (*Puccinia hordei*)(Parlevliet, 1981). Slow rusting in cereals generally involved decreased frequency of penetration, slower invasion of host tissue, longer latent period, smaller pustules, lower sporulation rate, and shorter period of sporulation, singly or, more commonly, in combination (Kuhn et al., 1978; Parlevliet, 1979). The detailed physiology of these effects is not understood, but early workers determined that some physical features of cereals could affect rust development. For example, Hursh (1924) found evidence that the proportion of sclerenchyma to collenchyma in the upper peduncle was correlated with the resistance to stem rust of Sonem Emmer and Kota wheat compared with Little Club. Horizontal resistance, being partial, is strongly affected by environmental factors. It has long been known that excessive nitrogenous fertilization makes cereals more susceptible to rusts (Hursh, 1924). Hart (1931) showed that Webster had several morphological features that increased its resistance to stem rust compared with very susceptible varieties. These included a higher proportion of sclerenchyma in the peduncle and the degree of lignification and relative toughness of the epidermis which often prevented uredinia breaking through to the surface. The resistance in Webster has since been attributed to the gene Sr30, which has been overcome in the Australian variety Festiguay (Knott & McIntosh, 1978). However, it is unlikely that the set of morphological features that inhibit rust infection in Webster (Hart, 1931) is controlled by only one gene.

The known sources of horizontal resistance (often referred to as 'adult plant resistance' or 'durable resistance') against stem rust in wheat are very narrow, consisting mainly of the two tetraploid wheats, *T. turgidum* var. *dicoccum* (cv. Yaroslav Emmer) and *T. turgidum*  var. *durum* (cv. Iumillo) and the bread wheat variety Webster (McIntosh et al., 1995). Leppik (1970) lists a range of wild wheat species discovered through the activities of the Russian collecting expeditions that are possible sources of resistance. An unfortunate downside of the spread of dwarf wheat varieties around the world from the 1960s has been the loss of the genetic diversity in the local land races that had probably undergone selection for horizontal resistance. But building up horizontal resistance in a crop does not necessarily involve just searching for sources of resistance, but rather crossing and selection of existing varieties in such as way that genes involved in the normal functioning of the plant that happen to interfere with pathogen growth and development are accumulated and resistance is built up.

Stripe (yellow) rust (*Puccinia striiformis*) was first detected in Australia in 1979 and its history of control by resistance has been very different from that of stem rust. The first response to the incursion, which caused heavy losses in some of the most widely grown varieties such as Zenith, was to deploy vertical resistance genes (YrA and Yr6). However, the effectiveness of these genes was lost very rapidly (Wellings & McIntosh, 1990). It was observed that some wheat varieties such as Condor, Egret and Olympic, although clearly susceptible at the seedling stage, showed varying degrees of adult-plant resistance and suffered much lower losses than the very susceptible varieties such as Zenith (McIntosh & Wellings, 1986). These had similar temperature-sensitive, partial, adult-plant resistance to that observed in some prominent varieties such as Cappelle-Desprez in Europe (Johnson, 1978), and Gaines,

Horizontal or Generalized Resistance to Pathogens in Plants 353

as important in breeding for resistance as mechanisms of antiobiosis. However, it is based on the behavior of the insects and has no equivalent in pathogens. More recently, an indirect mechanism of protection of plants from insects has been recognized (Broekgaarden et al., 2011). This involves the attraction of predator and parasitoid insects to plants releasing volatile chemicals as a result of attack by herbivorous insects. The predation and parasitism then reduces the populations of the pest, effectively protecting the plant. Again, there is no equivalent in plant pathology. Some plants like *Acacia* species produce extra-floral nectaries that attract ants that in turn protect the plant from insect herbivores. These indirect mechanisms are amenable to selection in order to improve the

Antiobiosis refers to properties of plants that directly reduce the amount of insect infestation on the plant (equivalent to reducing the amount of pathogen infection). This is similar to resistance against plant pathogens, and can include both vertical (race-specific) and horizontal (race-non-specific) resistance as defined for pathogens by van der Plank (1963, 1968). There is strong evidence that plants have vertical resistance to some of the highly specialized insect pests such as phloem feeding aphids. This resistance has the same attributes as vertical resistance to pathogens. It is often controlled by single, identifiable genes and shows a gene-for-gene interaction as in pathogens, in fact involving the same family of resistance proteins in the plants (NBS-LRRs)(reviewed in Broekgaarden et al., 2011). Long ago, hessian fly was considered to have a gene-for-gene interaction with wheat (Sidhu, 1975; Gallun et al., 1975). Wheat-hessian fly and medicago-bluegreen aphid interactions involve a hypersensitive necrosis reaction like that in plant diseases. And, as with plant diseases, there is ample evidence that this vertical resistance to insects breaks

Most of the resistance of plants to insects discussed in the literature fits the category of horizontal resistance (reviewed in Yencho et al., 2000). This can be explained by the fact that most insect pests have a far less intimate association with their host than the microbial pathogens: most insects just eat the host tissue. Most resistance in plants against insect attack is partial, inherited quantitatively, and is relatively stable. In fact, horizontal resistance to insects is easier to understand than horizontal resistance to pathogens because the mechanisms are more obvious and easily observed. If an insect is attracted to feed on a plant, and there is no immediate hypersensitive response that prevents it, then there are likely to be a multitude of constitutive or induced factors involved in the insect-plant interaction that either allow the insect to feed unimpeded and rapidly build up its population to damaging levels or restrict its feeding and so control its population on the plant. In reviewing the topic, Beck (1965) concluded that "It is doubtful that any example of resistance can be explained on the basis of a single simple biological characteristic of the plant. The multiplicity of factors exerting influences on the insect-plant relationship precludes the formulation of meaningful all-inclusive generalizations." Plants produce a wide range of secondary plant compounds such as alkaloids, tannins, essential oils, flavones and phenolics that can inhibit the build-up of insect populations, while not necessarily making the plants immune to attack (Beck, 1965; Levin, 1976). Many morphological and physical properties of plants such as density of sticky secretory glandular trichomes, density of hooked trichomes, and tissue toughness due to silica or lignin content may reduce herbivory and/or digestibility and consequently the build-up of insect populations on the

protection of plants from insect pests.

down with its widespread deployment in the field.

Nugaines and Luke in the Pacific Northwest of the United States (Milus & Line, 1986). The degree of resistance increased as the plants matured, and was greater at higher than lower temperatures (Qayoum & Line, 1985). The resistance was quantitatively inherited, and some crosses showed transgressive segregation (Milus & Line, 1986). This resistance has been long-lived. In Australia, it has been incorporated into many varieties (e.g. Meering and its successors, developed from Condor) and has proved to give long-lasting resistance to stripe rust (Park & Rees, 1989). While it has been identified with Yr18 (McIntosh et al., 1995), additive genes have also been found (Park, 2008) and it has been attributed to a 'Y18 complex' (Ma & Singh, 1996). It is therefore evident that control of stripe rust has relied largely on horizontal resistance.

With the emergence of race Ug99 and its derivatives, virulent on several important Sr genes (Sr24, Sr36, Sr21, Sr31, Sr38) that have been widely distributed around the world from the CIMMYT program in Mexico, wheat breeders are considering turning back to horizontal resistance to control stem rust (Schumann & Leonard, 2011). The emergence of this race has shown the dangers of relying on vertical resistance while steadily eroding the diversity of the global genetic resources of a crop. Before central agencies distributed and promoted particular wheat genotypes around the world, there would have been much greater genetic diversity in the crops. From country to country, from valley to valley and from farmer to farmer there would have been variation in the planting material, including variation in the deployment of resistance genes. An epidemic in one area would not necessarily have threatened another area. With the centralization of breeding for rust resistance in crops such as wheat and the influence of central agencies like CIMMYT in distributing resistance genes, there has been a global narrowing in the base of vertical resistance, such that new races originating in East Africa can threaten the wheat crops of many countries. In fact, it is likely that the global varieties have steadily replaced the local landraces that, in the absence of vertical resistance, probably relied on horizontal resistance to survive and yield in the face of rust infection. Further, the destructive nature of race Ug99 in East Africa suggests that the varieties it is attacking have little horizontal resistance. From the emphasis placed on vertical resistance in wheat breeding (the perpetual search for resistance genes), it is highly likely that horizontal resistance has not been maintained at a high level in the major wheat varieties, in stark contrast to the situation in maize.

#### **6. Resistance of plants to insects**

Resistance of plants to insect attack has many of the same characteristics as resistance to microbial pathogens, except that the plant-insect interaction is complicated by the behavioral biology of insects that is lacking in pathogens. Thus, in many reviews, resistance to insects is divided into three components: (i) non-preference, (ii) antibiosis, and (iii) tolerance (Painter, 1958). Tolerance is used here in precisely the same sense as in plant pathology – it is the ability of a plant to survive a certain level of insect attack without suffering significant loss of yield. Entomologists have developed the concept of a threshold level of infestation, below which the insect does not cause significant loss of yield and is not worth worrying about. Non-preference involves the avoidance by insects of particular plants and attraction to others for oviposition or feeding. This involves inherited traits of the plants (e.g. chemical stimuli, colours, morphologies) that can be just

Nugaines and Luke in the Pacific Northwest of the United States (Milus & Line, 1986). The degree of resistance increased as the plants matured, and was greater at higher than lower temperatures (Qayoum & Line, 1985). The resistance was quantitatively inherited, and some crosses showed transgressive segregation (Milus & Line, 1986). This resistance has been long-lived. In Australia, it has been incorporated into many varieties (e.g. Meering and its successors, developed from Condor) and has proved to give long-lasting resistance to stripe rust (Park & Rees, 1989). While it has been identified with Yr18 (McIntosh et al., 1995), additive genes have also been found (Park, 2008) and it has been attributed to a 'Y18 complex' (Ma & Singh, 1996). It is therefore evident that control of stripe rust has relied

With the emergence of race Ug99 and its derivatives, virulent on several important Sr genes (Sr24, Sr36, Sr21, Sr31, Sr38) that have been widely distributed around the world from the CIMMYT program in Mexico, wheat breeders are considering turning back to horizontal resistance to control stem rust (Schumann & Leonard, 2011). The emergence of this race has shown the dangers of relying on vertical resistance while steadily eroding the diversity of the global genetic resources of a crop. Before central agencies distributed and promoted particular wheat genotypes around the world, there would have been much greater genetic diversity in the crops. From country to country, from valley to valley and from farmer to farmer there would have been variation in the planting material, including variation in the deployment of resistance genes. An epidemic in one area would not necessarily have threatened another area. With the centralization of breeding for rust resistance in crops such as wheat and the influence of central agencies like CIMMYT in distributing resistance genes, there has been a global narrowing in the base of vertical resistance, such that new races originating in East Africa can threaten the wheat crops of many countries. In fact, it is likely that the global varieties have steadily replaced the local landraces that, in the absence of vertical resistance, probably relied on horizontal resistance to survive and yield in the face of rust infection. Further, the destructive nature of race Ug99 in East Africa suggests that the varieties it is attacking have little horizontal resistance. From the emphasis placed on vertical resistance in wheat breeding (the perpetual search for resistance genes), it is highly likely that horizontal resistance has not been maintained at a high level in the major wheat varieties, in stark

Resistance of plants to insect attack has many of the same characteristics as resistance to microbial pathogens, except that the plant-insect interaction is complicated by the behavioral biology of insects that is lacking in pathogens. Thus, in many reviews, resistance to insects is divided into three components: (i) non-preference, (ii) antibiosis, and (iii) tolerance (Painter, 1958). Tolerance is used here in precisely the same sense as in plant pathology – it is the ability of a plant to survive a certain level of insect attack without suffering significant loss of yield. Entomologists have developed the concept of a threshold level of infestation, below which the insect does not cause significant loss of yield and is not worth worrying about. Non-preference involves the avoidance by insects of particular plants and attraction to others for oviposition or feeding. This involves inherited traits of the plants (e.g. chemical stimuli, colours, morphologies) that can be just

largely on horizontal resistance.

contrast to the situation in maize.

**6. Resistance of plants to insects** 

as important in breeding for resistance as mechanisms of antiobiosis. However, it is based on the behavior of the insects and has no equivalent in pathogens. More recently, an indirect mechanism of protection of plants from insects has been recognized (Broekgaarden et al., 2011). This involves the attraction of predator and parasitoid insects to plants releasing volatile chemicals as a result of attack by herbivorous insects. The predation and parasitism then reduces the populations of the pest, effectively protecting the plant. Again, there is no equivalent in plant pathology. Some plants like *Acacia* species produce extra-floral nectaries that attract ants that in turn protect the plant from insect herbivores. These indirect mechanisms are amenable to selection in order to improve the protection of plants from insect pests.

Antiobiosis refers to properties of plants that directly reduce the amount of insect infestation on the plant (equivalent to reducing the amount of pathogen infection). This is similar to resistance against plant pathogens, and can include both vertical (race-specific) and horizontal (race-non-specific) resistance as defined for pathogens by van der Plank (1963, 1968). There is strong evidence that plants have vertical resistance to some of the highly specialized insect pests such as phloem feeding aphids. This resistance has the same attributes as vertical resistance to pathogens. It is often controlled by single, identifiable genes and shows a gene-for-gene interaction as in pathogens, in fact involving the same family of resistance proteins in the plants (NBS-LRRs)(reviewed in Broekgaarden et al., 2011). Long ago, hessian fly was considered to have a gene-for-gene interaction with wheat (Sidhu, 1975; Gallun et al., 1975). Wheat-hessian fly and medicago-bluegreen aphid interactions involve a hypersensitive necrosis reaction like that in plant diseases. And, as with plant diseases, there is ample evidence that this vertical resistance to insects breaks down with its widespread deployment in the field.

Most of the resistance of plants to insects discussed in the literature fits the category of horizontal resistance (reviewed in Yencho et al., 2000). This can be explained by the fact that most insect pests have a far less intimate association with their host than the microbial pathogens: most insects just eat the host tissue. Most resistance in plants against insect attack is partial, inherited quantitatively, and is relatively stable. In fact, horizontal resistance to insects is easier to understand than horizontal resistance to pathogens because the mechanisms are more obvious and easily observed. If an insect is attracted to feed on a plant, and there is no immediate hypersensitive response that prevents it, then there are likely to be a multitude of constitutive or induced factors involved in the insect-plant interaction that either allow the insect to feed unimpeded and rapidly build up its population to damaging levels or restrict its feeding and so control its population on the plant. In reviewing the topic, Beck (1965) concluded that "It is doubtful that any example of resistance can be explained on the basis of a single simple biological characteristic of the plant. The multiplicity of factors exerting influences on the insect-plant relationship precludes the formulation of meaningful all-inclusive generalizations." Plants produce a wide range of secondary plant compounds such as alkaloids, tannins, essential oils, flavones and phenolics that can inhibit the build-up of insect populations, while not necessarily making the plants immune to attack (Beck, 1965; Levin, 1976). Many morphological and physical properties of plants such as density of sticky secretory glandular trichomes, density of hooked trichomes, and tissue toughness due to silica or lignin content may reduce herbivory and/or digestibility and consequently the build-up of insect populations on the

Horizontal or Generalized Resistance to Pathogens in Plants 355

Our personal experience conditions how we see the world, including the world of science with which we work. van der Plank (1963) developed his ideas from life-long experience of breeding for resistance to late blight in potato, in which R genes were not effective. They broke down rapidly. Breeding for resistance to late blight relied more on horizontal resistance, and van der Plank was impressed by horizontal resistance. Workers with wheat stem rust grew up with direct personal experience of one of the great biological phenomena discovered in our time, vertical resistance involving the gene-for-gene recognition of a plant species and its co-evolved parasite. This resistance was genetically simple and made a spectacular difference; addition of a single Mendelian gene could convert a very susceptible variety into an immune one and completely protect a crop that had suffered regular devastating epidemics down through history. It is no wonder that researchers were excited by it and worked so hard to exploit it. Even the breakdown of resistance with which they had to contend was such a striking phenomenon, with incredible practical importance on the farms, that this only added to the excitement of the endeavor; researchers not only had to track the resistance genes in the host but also the virulence genes in the pathogen. All the emphasis was placed on making vertical resistance work, with considerable success in the case of wheat stem rust through breeding several resistance genes into each variety, keeping track of virulence changes in the rust populations, and continuously breeding varieties with new resistance genes. Horizontal resistance to wheat rusts was paid little attention during the grand quest to make vertical resistance work in practice. The inbreeding nature of wheat and the other small grain cereals made it harder to accumulate horizontal resistance as occurred in some outbreeding crops such as maize and cocoa. As a consequence, the researchers involved were not enthusiastic about van der Plank's synthesis and many continue to ignore his insights. The present author, introduced to plant pathology through study of vascular streak dieback of cocoa in Papua New Guinea and Southeast Asia, saw the functioning and value of horizontal resistance at first hand. It was easily selected for in an outbreeding, genetically diverse crop exposed to severe epidemics. Indeed, resistance was selected by farmers and agronomists before the cause of the disease was known. Although the pathogen reproduces only sexually and is therefore likely to be highly variable, it has not been necessary, and indeed the biology of the fungus has made it impossible, to be concerned about 'races'. It has proved durable, protecting cocoa from a potentially devastating pathogen and allowing the region to develop over 50 years into the second most important cocoa producing region after West Africa. As a result, this author has been

impressed by van der Plank's concept of horizontal resistance.

Long ago, the father of the early work on vertical resistance in the cereals, E.C. Stakman (1957, 1958, 1964), called for greater use of horizontal resistance against cereal rusts. Hooker (1967) suggested that, in developing disease resistance in crops, "perhaps man did not properly assess the resources at his disposal or employed the wrong tactics in their usage." In the light of his experience with the maize rusts he concluded that "If the system prevailing in maize and maize rust is applicable to other host-pathogen systems, then genes for specific hypersensitive-based resistance should be avoided or used only as a minor supplement to a high level of generalized resistance. As many modes of generalized resistance as possible should be combined to produce multimodal resistant varieties." In van der Plank's (1968) and Robinson's (1979) terms, horizontal resistance should be built up in crops as a primary objective and as the foundation of disease management, with vertical

**8. Conclusion** 

plants (e.g. Tingey, 1979). In response to insect attack, solanaceous plants produce proteinase inhibitors that enhance their resistance to insects (Heath et al., 1997). Most of these traits are likely to be inherited quantitatively (Yencho et al., 2000). Also, like horizontal resistance to plant pathogens, they are liable to erosion if the insect population is able to adapt to particular mechanisms. Thus, many secondary plant compounds that probably initially conditioned resistance to particular insects have become specific attractants for insects that have adapted to their presence. This process has been especially evident in relation to the glucosinolates in the Brassicaceae (Hopkins et al., 2009).

#### **7. The contribution of molecular biology**

Several studies of the molecular basis of vertical resistance against several types of parasites (bacteria, fungi and insects) have provided evidence that the gene-for-gene interaction involves a specific molecular system, and that the different R genes, both within and across species, fall within similar gene families (Ellis et al., 2007; Broekgaarden et al., 2011). In other words, the basic incompatibility process involves the expression of variants of the same interactive molecular system. This is a special molecular recognition system that could only have developed through a long period of co-evolution between the host and parasite in a situation where the two types of dominant genes (R-resistance, A-avirulence) had a selective advantage when interacting in genetically diverse populations of host and parasite in the region of evolution of the crop. There is evidence that this specific recognition system does not occur in new–encounter diseases in which there has been insufficient time for such a system to develop. This accounts for the fact that in such diseases (e.g. vascular streak dieback of cocoa, possibly late blight of *S. tuberosum* ssp. *tuberosum*) only horizontal and not vertical resistance has been found naturally.

Another exciting development from the use of recombinant DNA technology is the use of DNA molecular markers for important plant traits such as yield and resistance (Young 1996; Yencho et al., 2000). Mapping and development of DNA markers for Quantitative Trait Loci linked to horizontal resistance could enable the Vertifolia Effect of van der Plank (1963, 1968) and referred to by Black (1970) to be avoided; that is, it could enable the selection of vertical resistance while ensuring that the background horizontal resistance is not lost. While R genes can be added to a variety, and combined as in the effective strategy for wheat stem rust in Australia (McIntosh, 1976), the varieties can be monitored using DNA analysis to ensure that the underlying horizontal resistance is not lost. This is an important development as the presence of a high level of horizontal resistance in vertically resistant varieties reduces the chances of rapid breakdown of vertical resistance, and it reduces the damage done if the resistance does break down. If the vertical resistance is underpinned by good horizontal resistance, breakdown of vertical resistance, as seen with the emergence of a race like Ug99, is not likely to be catastrophic.

It is hypothesized that horizontal resistance is due to any aspect of plant biology that happens to slow down the invasion and sporulation of a pathogen in a basically compatible interaction. This understanding opens up a vast array of functions that could be altered by recombinant DNA techniques in a subtle way that may confer partial resistance on the host, rather than continuing the preoccupation with vertical resistance which we know the pathogen can overcome.

#### **8. Conclusion**

354 Plant Pathology

plants (e.g. Tingey, 1979). In response to insect attack, solanaceous plants produce proteinase inhibitors that enhance their resistance to insects (Heath et al., 1997). Most of these traits are likely to be inherited quantitatively (Yencho et al., 2000). Also, like horizontal resistance to plant pathogens, they are liable to erosion if the insect population is able to adapt to particular mechanisms. Thus, many secondary plant compounds that probably initially conditioned resistance to particular insects have become specific attractants for insects that have adapted to their presence. This process has been especially evident in

Several studies of the molecular basis of vertical resistance against several types of parasites (bacteria, fungi and insects) have provided evidence that the gene-for-gene interaction involves a specific molecular system, and that the different R genes, both within and across species, fall within similar gene families (Ellis et al., 2007; Broekgaarden et al., 2011). In other words, the basic incompatibility process involves the expression of variants of the same interactive molecular system. This is a special molecular recognition system that could only have developed through a long period of co-evolution between the host and parasite in a situation where the two types of dominant genes (R-resistance, A-avirulence) had a selective advantage when interacting in genetically diverse populations of host and parasite in the region of evolution of the crop. There is evidence that this specific recognition system does not occur in new–encounter diseases in which there has been insufficient time for such a system to develop. This accounts for the fact that in such diseases (e.g. vascular streak dieback of cocoa, possibly late blight of *S. tuberosum* ssp. *tuberosum*) only horizontal and not

Another exciting development from the use of recombinant DNA technology is the use of DNA molecular markers for important plant traits such as yield and resistance (Young 1996; Yencho et al., 2000). Mapping and development of DNA markers for Quantitative Trait Loci linked to horizontal resistance could enable the Vertifolia Effect of van der Plank (1963, 1968) and referred to by Black (1970) to be avoided; that is, it could enable the selection of vertical resistance while ensuring that the background horizontal resistance is not lost. While R genes can be added to a variety, and combined as in the effective strategy for wheat stem rust in Australia (McIntosh, 1976), the varieties can be monitored using DNA analysis to ensure that the underlying horizontal resistance is not lost. This is an important development as the presence of a high level of horizontal resistance in vertically resistant varieties reduces the chances of rapid breakdown of vertical resistance, and it reduces the damage done if the resistance does break down. If the vertical resistance is underpinned by good horizontal resistance, breakdown of vertical resistance, as seen with the emergence of a

It is hypothesized that horizontal resistance is due to any aspect of plant biology that happens to slow down the invasion and sporulation of a pathogen in a basically compatible interaction. This understanding opens up a vast array of functions that could be altered by recombinant DNA techniques in a subtle way that may confer partial resistance on the host, rather than continuing the preoccupation with vertical resistance

relation to the glucosinolates in the Brassicaceae (Hopkins et al., 2009).

**7. The contribution of molecular biology** 

vertical resistance has been found naturally.

race like Ug99, is not likely to be catastrophic.

which we know the pathogen can overcome.

Our personal experience conditions how we see the world, including the world of science with which we work. van der Plank (1963) developed his ideas from life-long experience of breeding for resistance to late blight in potato, in which R genes were not effective. They broke down rapidly. Breeding for resistance to late blight relied more on horizontal resistance, and van der Plank was impressed by horizontal resistance. Workers with wheat stem rust grew up with direct personal experience of one of the great biological phenomena discovered in our time, vertical resistance involving the gene-for-gene recognition of a plant species and its co-evolved parasite. This resistance was genetically simple and made a spectacular difference; addition of a single Mendelian gene could convert a very susceptible variety into an immune one and completely protect a crop that had suffered regular devastating epidemics down through history. It is no wonder that researchers were excited by it and worked so hard to exploit it. Even the breakdown of resistance with which they had to contend was such a striking phenomenon, with incredible practical importance on the farms, that this only added to the excitement of the endeavor; researchers not only had to track the resistance genes in the host but also the virulence genes in the pathogen. All the emphasis was placed on making vertical resistance work, with considerable success in the case of wheat stem rust through breeding several resistance genes into each variety, keeping track of virulence changes in the rust populations, and continuously breeding varieties with new resistance genes. Horizontal resistance to wheat rusts was paid little attention during the grand quest to make vertical resistance work in practice. The inbreeding nature of wheat and the other small grain cereals made it harder to accumulate horizontal resistance as occurred in some outbreeding crops such as maize and cocoa. As a consequence, the researchers involved were not enthusiastic about van der Plank's synthesis and many continue to ignore his insights. The present author, introduced to plant pathology through study of vascular streak dieback of cocoa in Papua New Guinea and Southeast Asia, saw the functioning and value of horizontal resistance at first hand. It was easily selected for in an outbreeding, genetically diverse crop exposed to severe epidemics. Indeed, resistance was selected by farmers and agronomists before the cause of the disease was known. Although the pathogen reproduces only sexually and is therefore likely to be highly variable, it has not been necessary, and indeed the biology of the fungus has made it impossible, to be concerned about 'races'. It has proved durable, protecting cocoa from a potentially devastating pathogen and allowing the region to develop over 50 years into the second most important cocoa producing region after West Africa. As a result, this author has been impressed by van der Plank's concept of horizontal resistance.

Long ago, the father of the early work on vertical resistance in the cereals, E.C. Stakman (1957, 1958, 1964), called for greater use of horizontal resistance against cereal rusts. Hooker (1967) suggested that, in developing disease resistance in crops, "perhaps man did not properly assess the resources at his disposal or employed the wrong tactics in their usage." In the light of his experience with the maize rusts he concluded that "If the system prevailing in maize and maize rust is applicable to other host-pathogen systems, then genes for specific hypersensitive-based resistance should be avoided or used only as a minor supplement to a high level of generalized resistance. As many modes of generalized resistance as possible should be combined to produce multimodal resistant varieties." In van der Plank's (1968) and Robinson's (1979) terms, horizontal resistance should be built up in crops as a primary objective and as the foundation of disease management, with vertical

Horizontal or Generalized Resistance to Pathogens in Plants 357

Flor, H.H. (1956). The complementary genic systems in flax and flax rust. Advances in

Gallun, R.L., Starks, K.J. & Guthrie, W.D. (1975). Plant resistance to insects attacking cereals.

Gebhardt, C. & Valkonen, J.P.T. (2001). Organization of genes controlling disease resistance

Guthrie, F.B. (1922). *William J. Farrer and the Results of his Work. Science Bulletin No. 22.* 

Hare, R.A. & McIntosh, R.A. (1979). Genetic and cytogenetic studies of durable adult-plant

Harlan, J.R. (1976). Diseases as a factor in plant evolution. *Annual Review of Phytopathology* 

Hart, H. (1931). *Morphologic and Physiologic Studies on Stem-Rust Resistance in Cereals.*

Hayes, H.K., Stakman, E.C. & Aamodt, O.S. (1925). Inheritance in wheat of resistance to

Heath, M.C. (1981). A generalized concept of host-parasite specificity. *Phytopathology* 71,

Heath, R.L., McDonald, G., Christeller, J.T., Lee, M., Bateman, K., West, J., van Heeswijck, R.

Hooker, A.L. (1967). The genetics and expression of resistance in plants to rusts of the genus

Hopkins, R.J., van Dam, N.M. & van Loon, J.J.A. (2009). Role of glucosinolates in insect-

Hursh, C.R. (1924). Morphological and physiological studies on the resistance of wheat to *Puccinia gaminis tritici* Erikss. and Henn. *Journal of Agricultural Research* 27, 381-411.

Johnson, R. (1978). Practical breeding for durable resistance to rust diseases in self-

Katsuya, K. & Green, G.J. (1967). Reproductive potentials of races 15B and 56 of wheat stem

Keane, P.J. & Prior, C. (1991). *Vascular-Streak Dieback of Cocoa.* Phytopathological papers No. 33, International Mycological Institute, Wallingford, Oxon. ISBN 0-85198-733-8 Knott, D.R. (1968). The inheritance of resistance to stem rust races 56 and 15B-1L (Can.) in

Knott, D.R. (1971). Can losses from wheat stem rust be eliminated in North America? *Crop* 

Knott, D.R. & McIntosh, R.A. (1978). The inheritance of stem rust resistance in the common

Knott, D.R. (1989). *The Wheat Rusts-Breeding for Resistance.* Springer-Verlag, Berlin.

the wheat varieties Hope and H-44. *Canadian Journal of Genetics and Cytology* 10, 311-

resistance to insect pests. *Journal of Insect Physiology* 43, 833-842.

*Puccinia. Annual Review of Phytopathology* 5, 163-182.

resistances in 'Hope' and related varieties to wheat rusts. *Zeitschrift* 

Technical Bulletin No. 266, United States Department of Agriculture, Washington

& Anderson, M.A. (1997). Proteinase inhibitors from *Nicotiana alata* enhance plant

plant relationships and multitrophic interactions. *Annual Review of Entomology* 54,

in the potato genome. *Annual Review of Phytopathology* 39, 79-102.

Department of Agriculture, New South Wales, Sydney.

Genetics 8, 29-54.

14, 31-51.

1121-1123.

57-83.

320.

*Science* 11, 97-99.

D.C.

*Annual Review of Entomology* 20, 337-357.

black stem rust. *Phytopathology* 15, 371-386.

pollinating cereals. *Euphytica* 27, 529-540.

rust. *Canadian Journal of Botany* 45, 1077-1091.

wheat cultivar Webster. *Crop Science* 17, 365-369.

*Pflanzenzuchtung* 83, 350-367.

resistance being added as necessary, along with cultural control measures and targeted use of pesticides, as part of an IPM strategy.

#### **9. References**

Beck, S.D. (1965). Resistance of plants to insects. *Annual Review of Entomology* 10, 207-232.


resistance being added as necessary, along with cultural control measures and targeted use

Beck, S.D. (1965). Resistance of plants to insects. *Annual Review of Entomology* 10, 207-232. Biffen, R.H. (1905). Mendel's laws of inheritance and wheat breeding. *Journal of Agricultural* 

Biffen, R.H. (1907). Studies in the inheritance of disease-resistance. *Journal of Agricultural* 

Black, W. (1970). The nature and inheritance of field resistance to late blight (*Phytophthora* 

Black, W. & Gallegly, M.E. (1957). Screening of *Solanum* species for resistance to physiological races of *Phytophthora infestans. American Potato Journal* 34, 273-281.

Borlaug, N.E. (1972). A cereal breeder and ex-forester's evaluation of the progress and

Broekgaarden, C., Snoeren, T.A.L., Dicke, M. & Vosman, B. (2011). Exploiting natural variation to identify insect-resistance genes. *Plant Biotechnology Journal* 2011, 1-7. Browning, J.A. (1974). Relevance of knowledge about natural ecosystems to development of

Caldwell, R.M.; Schafer, J.F.; Compton LE. & Patterson, F.L. (1958). Tolerance to cereal leaf

Callaghan, A.R. & Millington, A.J. (1956). *The Wheat Industry in Australia.* Angus and

Cammack, R.H. (1960). *Puccinia polysora*: a review of some factors affecting the epiphytotic in West Africa. *Report of 5th. Commonwealth Mycological Conference, 196*, pp. 134-138. Carnegie, A.J, Keane, P.J., Ades, P.K. & Smith, I.W. (1994). Variation in susceptibility of

Cobb, N.A. (1890). Contributions to an economic knowledge of the Australian rusts

Dungey, H.S., Potts, B.M., Carnegie, A.J. & Ades, P.K. (1997). *Mycosphaerella* leaf disease:

Ellis, J.G.; Dodds, P.N. & Lawrence, G.J. (2007). Flax rust resistance gene specificity is based

Eskes, A.B. (1983). Incomplete resistance to coffee leaf rust. In: *Durable Resistance in Crops*, F.

Farrer, W. (1898). The making and improvement of wheats for Australian conditions.

(*Uredineae*). *Agricultural Gazette, New South Wales* 1(3), 185-214.

*Agricultural Gazette, New South Wales* 9, 131-168, 241-260.

*Canadian Journal of Forest Research* 27, 750-759.

problems involved in breeding rust resistant forest trees: moderator's summary. *US Department of Agriculture Miscellaneous Publication* 1221, pp. 615-642, Washington

pest management programs for agroecosystems. *Proceedings of the American* 

*Eucalyptus globulus* provenances to Mycosphaerella leaf disease. *Canadian Journal of* 

genetic variation in damage to *Eucalyptus nitens, E. globulus* and their F1 hybrids.

on direct resistance-avirulence protein interactions. *Annual Review of Phytopathology*

Lamberti, J.M. Waller and N.A. van der Graaff (eds.), pp. 291-315, Plenum Press,

*infestans*) in potatoes. *American Potato Journal* 47, 279-288.

Borlaug, N.E. (1965). Wheat, rust, and people. *Phytopathology* 55, 1088-1098.

of pesticides, as part of an IPM strategy.

*Science* 1, 4-48.

*Science* 2, 109-127.

*Phytopathological Society* 1, 191-199.

rusts. *Science* 128, 714-715.

Robertson, Sydney, Australia.

*Forest Research* 24, 1751-1757.

45, 289-306.

New York.

**9. References** 

DC.


Horizontal or Generalized Resistance to Pathogens in Plants 359

Moreno-Ruiz, G. & Castillo-Zapata, J. (1990). *The Variety Colombia: a Variety of Coffee with* 

Muller, K.O. & Haigh, J.C. (1953). Nature of "field resistance" of the potato to *Phytophthora* 

Newton, M., Johnson, T. & Gussow, H.T. (1932). *Studies in Cereal Diseases VIII. Specialization* 

Niks, R.E. (1988). Failure of haustorial development as a factor in slow growth and

Nimchuk, Z., Eulgem, T., Holt, B.F.III & Dangl, J.L. (2003). Recognition and response in the

Painter, R.H. (1958). Resistance of plants to insects. *Annual Review of Entomology* 3, 267-290. Park, R.F. (2007). Stem rust of wheat in Australia. *Australian Journal of Agricultural Research* 

Park, R.F. (2008). Breeding cereals for rust resistance in Australia. *Plant Pathology* 57, 591-602. Park, R.F. & Rees, R.G. (1989). Expression of adult plant resistance and its effect on the

Park, R.F., Keane, P.J., Wingfield, M.J & Crous, P.W. (2000). Fungal diseases of eucalypt

Parlevliet, J.E. (1979). Components of resistance that reduce the rate of epidemic

Parlevliet, J.E. (1981). Race-non-specific disease resistance. In: *Strategies for the Control of* 

Parlevliet, J.E. (1995). Present problems in and aspects of breeding for disease resistance. In:

Qayoum, A. & Line, R.F. (1985). High-temperature, adult-plant resistance to stripe rust of

Reddick, D. (1934). Elimination of potato late blight from North America. *Phytopathology* 24,

Rees, R.G., Thompson, J.P. & Mayer, R.J. (1979). Slow rusting and tolerance to rusts in

wheat cultivars. *Australian Journal of Agricultural Research* 30, 403-419. Robinson, R.A. (1969). Disease resistance terminology. *Review of Applied Mycology* 48, 593-

wheat. I The progress and effects of epidemics of *Puccinia graminis tritici* in selected

Person, C. (1967). Genetic aspects of parasitism. *Canadian Journal of Botany* 45, 1193-1204. Person, C., Samborski, D.J. & Rohringer, R. (1962). The gene-for-gene concept. *Nature* 194,

*Cereal Diseas,* Jenkyn, J.F. & Plumb, R.T. (eds.), Blackwell, London.

plant immune system. *Annual Review of Genetics* 37, 579-609.

development. *Annual Review of Phytopathology* 17, 203-222.

Publishers, Boca Raton, London, Tokyo.

wheat. *Phytopathology* 75, 1121-1125.

No. 160, New Series, Department of Agriculture, Dominion of Canada. Niederhauser, J.S. (1962). Evaluation of multigenic "field resistance" of the potato to

Nacional de Investigacioes de Café, Chinchina, Caldes, Colombia.

*infestans* de Bary. *Nature* 171, 781-783.

*and Molecular Plant Pathology* 28, 309-322.

(Abstr.).

58, 558-566.

7

561-562.

555-557.

606.

*Plant Pathology* 38, 200-208.

*Resistance to Rust (*Hemileia vastatrix *(Berk. & Br.).* Technical Bulletin No. 9, Centro

*and Hybridization of Wheat Stem Rust,* Puccinia graminis tritici*, in Canada.* Bulletin

*Phytophthora infestans* in 10 years of trials at Toluca, Mexico. *Phytopathology* 52, 746

development of *Puccinia hordei* in partially resistant barley seedlings. *Physiological* 

development of *Puccinia striiformis* f.sp. *tritici* in some Australian wheat cultivars.

foliage. In: *Diseases and Pathogens of Eucalypts,* Keane, P.J., Kile, G.A., Podger, F.D. & Brown, B.N. (eds.), pp. 153-239, CSIRO Publishing, Collingwood. ISBN 0 643 06523

*Molecular methods in Plant Pathology*, Singh, R.P. & Singh, U.S. (eds.), CRC, Lewis


Kolmer, J.A., Garvin, D.F. & Jin, Y. (2011). Expression of a Thatcher wheat adult plant stem

Kuhn, R.C., Ohm, H.W. & Shaner, G.E. (1978). Slow leaf-rusting resistance in wheat against twenty-two isolates of *Puccinia recondita. Phytopathology* 68, 651-656.

Kushalappa, A.C. & Eskes, A.B. (1989). *Coffee Rust: Epidemiology, Resistance, and Management.* 

Leonard, K.J. (1969). Selection in heterogeneous populations of *Puccinia graminis* f.sp. *avenae.* 

Leppik, E.E. (1970). Gene centers of plants as sources of disease resistance. *Annual Review of* 

Levin, D.A. (1976). The chemical defenses of plants to pathogens and herbivores. *Annual* 

Loegering, W.Q., Hendrix, J.W. & Browder, L.E. (1967). *The Rust Diseases of Wheat.* 

Luig, N.H. (1983). *A Survey of Virulence Genes in Wheat Stem Rust,* Puccinia graminis *f.sp.* 

Luke, H.H., Chapman, W.H. & Barnett, R.D. (1972). Horizontal resistance of Red Rustproof

Ma, H. & Singh, R.P. (1996). Contribution of adult plant resistance gene *Yr18* in protecting

Malcolmson, J.F. (1976). Assessment of field resistance to blight (*Phytophthora infestans*) in

Malcolmson, J.F. & Black, W. (1966). New R genes in *Solanum demissum* Lindl. And their

Marcroft, S.J., Sprague, S.J., Pymer, S.J., Salisbury, P.A. & Howlett, B.J. (2004). Crop isolation,

Martin, G.B., Bogdanove, A.J. & Sessa, G. (2003). Understanding the functions of plant

McIntosh, R.A. (1976). Genetics of wheat and wheat rusts since Farrer. *The Journal of the* 

McIntosh, R.A. & Wellings, C.R. (1986). Wheat rust resistance – the continuing challenge.

McIntosh, R.A., Wellings, C.R. and Park, R.F. (1995). *Wheat Rusts: an Atlas of Resistance Genes.* 

Milus, E.A. & Line, R.F. (1986). Number of genes controlling high-temperature, adult-plant

Mode, C.J. (1958). A mathematical model for the co-evolution of obligate parasites and their

disease resistance proteins. *Annual Review of Plant Biology* 54, 23-61.

*Australian Institute of Agricultural Science* 42, 203-216.

CSIRO Publications, Melbourne. ISBN 0 643 05428 6

resistance to stripe rust in wheat. *Phytopathology* 76, 93-96.

complementary races of *Phytophthora infestans* (Mont.) de Bary. *Euphytica* 15, 199-

not extended rotation length, reduces blackleg (*Leptosphaeria maculans*) severity of canola (*Brassica napus*) in south-eastern Australia. *Australian Journal of Experimental* 

potatoes. *Transactions of the British Mycological Society* 67, 321-325.

Agriculture Handbook No. 334, United States Department of Agriculture,

Large, E.C. (1940). *The Advance of the Fungi.* Dover Publications, New York.

526-533.

CRC Press, Florida.

Washington, D.C.

*Agriculture* 44, 601-606.

*Australasian Plant Pathology* 15, 1-8.

hosts. *Evolution* 12, 158-165.

203.

*Phytopathology* 59, 1851-57.

*Phytopathology* 8, 323-344.

*Review of Ecology and Systematics* 7, 121-159.

tritici. Verlag Paul Parey, Berlin and Hamburg.

oats to crown rust. *Phytopathology* 62, 414-417.

wheat from yellow rust. *Plant Disease* 80, 66-69.

rust resistance QTL on chromosome arm 2BL is enhanced by *Lr34*. *Crop Science* 51,


Horizontal or Generalized Resistance to Pathogens in Plants 361

Stakman, E.C. & Levine, M.N. (1922). The determination of biologic forms of *Puccinia* 

Stakman, E.C. & Harrar, J.G. (1957). *Principles of Plant Pathology.* The Ronald Press Co., New

Stakman, E.C. & Rodenhiser, H.A. (1958) Race 15B of wheat stem rust – what it is and what

Stakman, E.C. Christensen, J.J. (1960). The problem of breeding resistant varieties. In

Steffenson, B.J. (1992). Analysis of durable resistance to stem rust in barley. *Euphytica* 63,

Tan, G.Y. & Tan, W.K. (1988). Genetic variation in resistance to vascular-streak dieback in

Thurston, H.D. (1971). Relationship of general resistance: late blight of potato. *Phytopathology* 

Tingey, W.M. (1979). Breeding for arthropod resistance in vegetables. pp. 495-522 in *Biology* 

Toxopeus, H.J. (1956). Reflections on the origin of new physiological races of *Phytophthora infestans* and the breeding of resistance in potatoes. *Euphytica* 5, 221-237. Toxopeus, H.J. (1964). Treasure-digging for blight resistance in potatoes. *Euphytica* 13, 206-

van der Graaff, N.A. (1986). Coffees, *Coffea* spp. Chapter 6 in *Breeding for Durable Resistance in Perennial Crops.* FAO Plant Production and Protection Paper 70, Fao, Rome.

van der Plank, J.E. (1971). Stability of resistance to *Phytophthora infestans* in cultivars without

van der Plank, J.E. (1983). Durable resistance in crops: should the concept of physiological

Vavilov, N.I. (1951). *The Origin, Variation, Immunity and Breeding of Cultivated Plants.*  Translatedfrom Russian by K. S. Chester, The Ronald Press Co., New York. Waterhouse, W.L. (1936). Some observations on cereal rust problems in Australia.

Waterhouse, W.L. (1952). Australian rust studies. IX. Physiologic race determinations and

Watson, I.A. (1958). The present status of breeding disease resistant wheats in Australia. *Agriculture Gazette, New South Wales Department of Agriculture* 69 (12), 1-31. Watson, I.A. (1970). Changes in virulence and population shifts in plant pathogens. *Annual* 

Watson, I.A. (1974). Losses from wheat stem rust in Australia – are they inevitable?

races die? In: *Durable Resistance in Crops,* F. Lamberti, J.M. Waller and N.A. van der

surveys of cereal rusts. *Proceedings of the Linnean Society of New South Wales* 61, v-

*and Breeding for Resistance to Arthropods and Pathogens in Agricultural Plants.* M.K.

cocoa (*Theobroma cacao*). *Theoretical and Applied Genetics* 75, 761-766.

Harris (ed.), Texas A & M University, College Station, Texas.

van der Plank, J.E. (1963). *Plant Disease: Epidemics and Control.* Academic Press.

*Proceedings of the Linnean Society of New South Wales* 77, 209-258.

van der Plank, J.E. (1968). *Disease Resistance in Plants.* Academic Press.

Graaff (eds.), pp. 41-44, Plenum Press, New York.

*Australian Plant Pathology Society Newsletter* 3(3), 64-65.

R genes. *Potato Research* 14, 263-270.

*Review of Phytopathology* 8, 209-230.

Horsfall, J.G. & Dimond, A.E., *Plant Pathology* Vol.3, 567-624, Academic Press, N.Y.

*Technical Bulletin* 8.

it means. *Advances in Agronomy* 10, 143-165.

York.

153-167.

61, 620-626.

222.

xxxviii.

*graminis* on *Triticum* spp. *Minnesota University Agricultural Experiment Station* 

Robinson, R.A. (1976). *Plant Pathosystems.* Springer Verlag, Berlin.


Robinson, R.A. (1979). Permanent and impermanent resistance to crop parasites; a re-

Rodrigues, C.J. Jr. (1984).Coffee rust races and resistance. In: *Coffee Rust in the Americas,* R.H

Salaman, R.N. (1910). The inheritance of colour and other characters in the potato. *Journal of* 

Salisbury, P.A. & Ballinger, D.J. (1996). Seedling and adult plant evaluation of race

Salisbury, P.A., Ballinger, D.J, Wratten, N., Plummer, K.M. & Howlett, B.J. (1995). Blackleg

Schlosser, E.W. (1980). Preformed internal chemical defenses. . In Horsfall, J.G. & Cowling, E.B., *Plant Disease. An Advanced Treatise* Vol.V, 161-177, Academic Press, N.Y. Schumann, G.L. & Leonard, K.J. (2000). Stem rust of wheat (black rust). *The Plant health* 

Sidhu, G.S. (1975). Gene-for-gene relationships in plant parasitic systems. *Scientific Progress,* 

Simmonds, N.W. (1964). Studies of the tetraploid potatoes. II. Factors in the evolution of the Tuberosum group. *Journal of the Linnaean Society of London (Botany)* 59, 43-56. Simmonds, N.W. (1966). Studies of the tetraploid potatoes. III. Progress in the experimental

Simmonds, N.W. (1979). *Principles of Crop Improvement.* Longman, ISBN 0-582-44630-9, New

Simmonds, N.W. (1991). Genetics of horizontal resistance to diseases of crops. *Biological* 

Singh, R.P.; Hodson, D.P.; Huerta-Espino, J.; Jin, Y.; Bhavani, S.; Njau, P.; Herrera-Foessel, S.;

Skovmand, B., Wilcoxson, R.D., Shearer, B.L. & Stucker, R.E. (1978). Inheritance of slow

Sprague, S.J., Marcroft, S.J., Hayden, H.L. & Howlett, B.J. (2006). Major gene resistance to

Stakman, E.C. (1964).Will the fight against wheat rust ever end? *Zeitschrift* 

rusting to stem rust in wheat. *Euphytica* 27, 95-107.

in southeastern Australia. *Plant Disease* 90, 190-198.

*Pflanzenkrankheiten Plantzenschutz* 71, 67-73.

re-creation of the Tuberosum group. *Journal of the Linnaean Society of London* 

Singh, P.K.; Singh, S. & Govindan, V. (2011). The emergence of Ug99 races of the stem rust fungus is a threat to world wheat production. *Annual Review of* 

blackleg in *Brassica napus* overcome within three years of commercial production

*Instructor.* DOI:10.1094/PHI-I-2000-0721-01. Updated 2011.

Schafer, J.F. (1971). Tolerance to plant disease. *Annual Review of Phytopathology* 9, 235-252. Schieber, E. (1971). Distribution of *Puccinia polysora* and *P. sorghi* in Africa and their

Salisbury, P.A. (1988). Blackleg resistance in rapeseed. *Plant Protection Quarterly* 3, 47.

examination of the pathosystem concept with special reference to rice blast.

Fulton (ed.), pp. 41-58, The American Phytopathological Society, St. Paul,

variability in *Leptosphaeria maculans* on *Brassica* species in Australia. *Australian* 

disease on oilseed *Brassica* in Australia: a review. *Australian Journal of Experimental* 

pathogenicity on Latin American maize germ plasm. *FAO Plant Protection Bulletin* 

Robinson, R.A. (1976). *Plant Pathosystems.* Springer Verlag, Berlin.

*Journal of Experimental Agriculture* 36, 485-488.

*Zeitschrift Pflanzenzuechtung* 83, 1-39.

Minnesota.

19, 25-31.

*Genetics* 1, 7-46.

*Agriculture* 35, 665-672.

*Oxford* 62, 467-485.

*(Botany)* 59, 279-288.

*Reviews* 66, 189-241.

*Phytopathology* 49, 1-17.

York.


Watson, I.A. & Luig, N.H. (1963). The classification of *Puccinia graminis* var. *tritici* in relation

Williams, R.F. (1991). *To Find the Way. History of the Western Fleurieu Peninsula.* The

Wellings, C.R. & McIntosh, R.A. (1990). *Puccinia striiformis* f.sp. *tritici* in Australasia: pathogenic changes during the first 10 years. *Plant Pathology* 39, 316-325. Wilcoxson, R.D. (1981). Genetics of slow rusting in cereals. *Phytopathology* 71, 989-993. Yencho, G.C., Cohen, M.B. & Byrne, P.F. (2000). Applications of tagging and mapping insect

Young, N.D. (1996). QTL mapping and quantitative disease resistance in plants. *Annual* 

Yankalilla and District Historical Society Inc. ISBN 0646062565

resistance loci in plants. *Annual Review of Entomology* 45, 393-422.

*Review of Phytopathology* 34, 479-501.

88, 235-258.

to breeding resistant varieties. *Proceedings of the Linnean Society of New South Wales* 

### *Edited by Christian Joseph R. Cumagun*

Plant pathology is an applied science that deals with the nature, causes and control of plant diseases in agriculture and forestry. The vital role of plant pathology in attaining food security and food safety for the world cannot be overemphasized.

Photo by Jurgute / iStock

Plant Pathology

Plant Pathology