Management of Plant Disease

*Plant Diseases-Current Threats and Management Trends*

[68] Arencibia AD, Vinagre F, Estevez Y, Bernal A, Perez J, Cavalcanti J, et al. *Gluconacetobacter diazotrophicus* elicitate a sugarcane defense response against a pathogenic bacteria *Xanthomonas albilineans*. Plant Signaling and Behavior. 2006;**1**(5):265-273

[69] Keymanesh K, Soltani S, Sardari S. Application of antimicrobial peptides in agriculture and food industry. World Journal of Microbiology and Biotechnology. 2009;**25**(6):933-944

and Environmental Microbiology.

[62] Weng LX, Xu JL, Li O, Birch RG, Zhang LH. Identification of the essential histidine residue for highaffinity binding of AlbA protein to albicidin antibiotics. Microbiology.

[63] Costet L, Cunff L, Royaert S, Raboin LM, Hervouet C, Toubi L, et al. Haplotype structure around Bru1 reveals a narrow genetic basis for brown rust resistance in modern sugarcane cultivars. Theoretical and Applied Genetics. 2012;**125**:825-836

[64] Birch R, Bower R, Elliott A, Hansom S, Basnayake S, Zhang L. Regulation of transgene expression: Progress towards practical development in sugarcane, and implications for other plant species. Plant genetic engineering: towards the third millennium. In: Proceedings of the International Symposium on Plant Genetic Engineering; 6-10 December; Havana. Cuba: Elsevier Science

2005;**71**:1445-1452

2003;**149**:451-457

Publishers; 1999, 2000

2005;**99**(4):366-371

2002;**153**(6):345-351

[65] Viswanathan R, Padmanaban P. Hand Book on Sugarcane Diseases and their Management. Coimbatore: Sugarcane Breeding Institute; 2008

[66] Blanco Y, Blanch M, Piñón D, Legaz ME, Vicente C. Antagonism of *Gluconacetobacter diazotrophicus* (a sugarcane endosymbiont) against *Xanthomonas albilineans* (pathogen) studied in alginate-immobilized sugarcane stalk tissues. Journal of Bioscience and Bioengineering.

[67] Piñón D, Casas M, Blanch MA, Fontaniella B, Blanco Y, Vicente C, et al. *Gluconacetobacter diazotrophicus*,

*Xanthomonas albilineans*, a sugar cane pathogen. Research in Microbiology.

a sugar cane endosymbiont, produces a bacteriocin against

**130**

**Chapter 9**

*amylovora*

*and Isabel A. Munck*

**Abstract**

**1. Introduction**

**133**

Choosing an Adequate Pesticide

Pathogens with Difficult Biologies:

Case Studies on *Diplodia corticola,*

With the challenges that negatively impact tree-based agriculture, landscapes and forests, such as climate change, plant pathogen and insect range expansion, invasive species and limited new pesticides, it is important to introduce new and effective tree protection options. In the last 20 years, pathogens that invade wood i.e. vascular tissues of trees causing wilt, yellowing, premature defoliation, cankers and tree death, have been on the rise. *Diplodia corticola* causes Bot canker of oak species which can kill trees diminishing the valuable ecological services they provide and reducing profits from wood and cork production. Since this and similar pathogens have difficult biologies because they reside in wood and cause severe internal damage and tree death, their management is difficult or inefficient with classical pesticide application methods that cannot reach and distribute the active ingredient in vascular wood tissues. As practical management options for this and other vascular tissue pathogens of trees are limited, we evaluated efficacy of several trunk injected fungicides in control of *D. corticola* and compared it with the efficacy of trunk injection of similar compounds for control of *Venturia inaequalis* and *Erwinia amylovora*, as two well-studied apple tree pathogens with different or

*Venturia inaequalis* and *Erwinia*

*Srđan G. Aćimović, Danielle K.H. Martin,*

partially similar lifestyles to *D. corticola*, respectively*.*

*Diplodia corticola*, *Venturia inaequalis*, *Erwinia amylovora*

**Keywords:** trunk injection of pesticides, tree disease management,

Agricultural, urban, and natural tree stands have been the focus of extensive plant

pathogen diagnostic and disease management research in recent decades [1–16]

*Richard M. Turcotte, Christopher L. Meredith*

Delivery System for Managing

#### **Chapter 9**

Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies: Case Studies on *Diplodia corticola, Venturia inaequalis* and *Erwinia amylovora*

*Srđan G. Aćimović, Danielle K.H. Martin, Richard M. Turcotte, Christopher L. Meredith and Isabel A. Munck*

#### **Abstract**

With the challenges that negatively impact tree-based agriculture, landscapes and forests, such as climate change, plant pathogen and insect range expansion, invasive species and limited new pesticides, it is important to introduce new and effective tree protection options. In the last 20 years, pathogens that invade wood i.e. vascular tissues of trees causing wilt, yellowing, premature defoliation, cankers and tree death, have been on the rise. *Diplodia corticola* causes Bot canker of oak species which can kill trees diminishing the valuable ecological services they provide and reducing profits from wood and cork production. Since this and similar pathogens have difficult biologies because they reside in wood and cause severe internal damage and tree death, their management is difficult or inefficient with classical pesticide application methods that cannot reach and distribute the active ingredient in vascular wood tissues. As practical management options for this and other vascular tissue pathogens of trees are limited, we evaluated efficacy of several trunk injected fungicides in control of *D. corticola* and compared it with the efficacy of trunk injection of similar compounds for control of *Venturia inaequalis* and *Erwinia amylovora*, as two well-studied apple tree pathogens with different or partially similar lifestyles to *D. corticola*, respectively*.*

**Keywords:** trunk injection of pesticides, tree disease management, *Diplodia corticola*, *Venturia inaequalis*, *Erwinia amylovora*

#### **1. Introduction**

Agricultural, urban, and natural tree stands have been the focus of extensive plant pathogen diagnostic and disease management research in recent decades [1–16]

which recorded an increase in the number of new fungal and bacterial pathogens and their detrimental impact on agroecosystems, ecosystems, and the human society. The economic effects of these pathogens are reflected in lost fresh fruit produce [17–19], reduced yields and quality of fruit or wood and cork products [20, 21], diminished ecological tree services, and death of whole trees, stands, and forest regions or decimation of fruit industries [19, 22].

and pathogens or to provide tree nutrition and/or correction of micronutrient deficiencies. It primarily harnesses the transport capacity of the tree's vascular system driven by transpiration stream of water in these tissues to translocate and distribute the active compounds into the trunk, branches, canopy and roots where protection or nutrition is needed. Tree injection as a plant protection method is viewed as environmentally safer alternative for pesticide application because it secures significant reduction of non-target exposure of water, soil, air and wildlife to pesticides and fertilizers in landscapes and urban greening areas. The active ingredients are delivered within the tree, thus providing selective exposure to plant pests, with limited negative effect of weather conditions like rain or sun radiation on the injected compound and with creating no immediate pesticide residue losses outside the tree.

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

Trunk injection relies significantly on tree physiology processes related to water

transport, xylem and phloem tissue functions, and the weather conditions that influence these specific plant processes. To achieve delivery of an effective pesticide dose, its distribution and expected management of plant detrimental organism or nutrient deficiency, there are several key factors which should be monitored by an applicator as they influence success of trunk injection for these purposes. Besides the plant pathogen biology, ecology, and epidemiology, several factors play a key role in success of trunk injection efficacy: the time of application in relation to detrimental organism establishment and symptom occurrence [11], the season and time of the day of application [41], the chemical properties of pesticide active ingredient and its formulation [42], the injected volume or dose of a pesticide, and the type of tree injection device or technology. For example, a more effective management of plant disease or insect infestation can be achieved by the preventive injections of pesticides in comparison to the therapeutic pesticide applications after the disease or infestation has already occurred. Tree injection of active compounds is much faster and easier during spring and early summer months in comparison to the late or mid-summer, late fall and winter, because water in the soil is abundant and the green leaf canopy is facilitating intensive transpiration pull and flow of water through the xylem tissue in hardwood trees, starting from the roots and branches to the leaves [41]. The three key properties of injected active ingredient that determine its mobility or binding in xylem of the tree are organic carbon-water partitioning coefficient (ml/g or μg/g) or carbon adsorption coefficient (Ko/c), water solubility, and formulation type. Ko/c expresses the level of adhesion or adsorption of pesticide active ingredient to the carbon rich compounds in certain environments such as soil or xylem and is defined as a ratio of mass of a chemical that is adsorbed in a certain environment per unit mass of organic carbon in that environment per the equilibrium chemical concentration in a solution. Active ingredients that have high Ko/c values will strongly bind to the organic compounds present in soil, sap or xylem and reduce their systemic movement i.e. translocation, accumulation and distribution in the canopy, thus reducing the efficacy in pathogen or pest control. In contrast, the ingredients with low or moderate Ko/c values move and distribute fast after tree injection and distribute well in the canopy, securing good pest or pathogen control. Pesticide formulation is a form of a pesticide active ingredient ready for use or which quite often requires dilution in water prior to application. Formulation is made by adding different inactive ingredients with the aim to improve the properties of an active ingredient such as solubility, surface adhesion, distribution, effectiveness, shelf life, stability, handling or application (e.g. solvents, emulsifiers, surfactants and other adjuvants). Formulation of a pesticide or a fertilizer determines the properties and residue stability of an active ingredient and can modulate its mobility in xylem of phloem after tree injection for pest or plant management [12, 42]. Finally, trunk injection devices can loosely be divided into the ones using drill- or needle-based technology [43]. In case of the

**135**

If left unmanaged, apple scab fungus *Venturia inaequalis*, a subcuticular leaf and fruit pathogen can cause 70–100% reduction in marketable fruit yield in each year [23, 24]. A pathogenic fungus *Diplodia corticola*, the causal agent of Bot canker of oak which invades tree xylem is the most widely distributed and virulent fungal pathogen causing canker and decline of cork oak (*Quercus suber*) forests in Europe [25, 26] and of southern live oak (*Q. virginiana*), coast live oak (*Q. agrifolia*), and canyon live oak (*Q. chrysolepis*) in the United States [3, 27]. Recently, there has been a rising incidence of this pathogen in the Unites States on northern red oak, *Q. rubra* [5, 8], black oak, *Q. velutina* [7], white oak, *Q. alba* [28], and bur oak (*Q. macrocarpa*) [29]. Other Botryosphaeriaceae, like *Neofusicoccum australe*, *N. luteum*, *N. parvum* and *N. mediterraneum* infect woody and green plant tissues and are destructive canker pathogens of avocado [1], grapevine, olive, pistachio and ash [30–33] and are recently reported as very virulent pathogens of coast redwood leading to severe decline in urban stands of California [9]. The Blue stain fungi *Grosmannia clavigera* and *Leptographium longiclavatum* are the plant pathogenic insect symbionts vectored by the mountain pine beetle *Dendroctonus ponderosae* [13]. They infect xylem and have led to death of millions of pine trees from 1990 to 2013 and decimation of pine forests in western North America [22, 34, 35]. Under favorable weather conditions during apple bloom and shoot growth, a destructive fire blight bacterium *Erwinia amylovora* inhabits and infects apple flowers and shoots and after spreading through xylem and causing cankers on wood it can kill whole trees. The resulting losses can range from \$3.8 to 100 million due to removal of as much as 450,000 apple trees in only one or two years [17, 18, 36, 37]. Fire blight severely reduced both pear and quince production primarily in continental climate regions of the world. Asiatic citrus canker caused by *Xanthomonas axonopodis* pv. *citri* invades leaf mesophyll tissue and is estimated to cause yield losses and cost of disease management of \$342 million per year [38]. The citrus greening or Huanglongbing disease is caused by a bacterium *Candidatus Liberibacter asiaticus* which proliferates and is limited to phloem vascular tissue of citrus trees. This pathogen led to \$4.5 billion negative economic impact in just 5 years after introduction in the United States [19]. These devastating internal tree pathogens further create a barrier in international trade of fresh fruit and wood products in an attempt to prevent their introduction to new regions [39, 40].

The biology of majority of these microorganisms, excluding *V. inaequalis* and *X. axonopodis* pv. *citri*, has three key shared traits: (1) they invade, reside, and spread in xylem or phloem of woody host tissues, where a significant part of pathogenesis and internal host damage is taking place, and (2) due to specific lifestyle depicted in impacting the internal wood tissues, these pathogens successfully evade exposure to the contact, local-systemic, and green-tissue systemic pesticides applied to plant surfaces, and (3) their management is extremely difficult or inefficient with the classical pesticide delivery methods. Therefore, any pesticide active ingredient in a formulated form needs to distribute in these vascular tissues to reach pathogen propagules at an effective concentration and move systemically to all the uninfected or infected tissue parts to achieve an efficient preventive or curative control, respectively.

Tree injection, often referred to as trunk or stem injection, is a method of target precise delivery or application of pesticides, plant resistance activators and fertilizers into the xylem vascular tissue of a tree with the aim to protect trees from insect pests

#### *Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

and pathogens or to provide tree nutrition and/or correction of micronutrient deficiencies. It primarily harnesses the transport capacity of the tree's vascular system driven by transpiration stream of water in these tissues to translocate and distribute the active compounds into the trunk, branches, canopy and roots where protection or nutrition is needed. Tree injection as a plant protection method is viewed as environmentally safer alternative for pesticide application because it secures significant reduction of non-target exposure of water, soil, air and wildlife to pesticides and fertilizers in landscapes and urban greening areas. The active ingredients are delivered within the tree, thus providing selective exposure to plant pests, with limited negative effect of weather conditions like rain or sun radiation on the injected compound and with creating no immediate pesticide residue losses outside the tree.

Trunk injection relies significantly on tree physiology processes related to water transport, xylem and phloem tissue functions, and the weather conditions that influence these specific plant processes. To achieve delivery of an effective pesticide dose, its distribution and expected management of plant detrimental organism or nutrient deficiency, there are several key factors which should be monitored by an applicator as they influence success of trunk injection for these purposes. Besides the plant pathogen biology, ecology, and epidemiology, several factors play a key role in success of trunk injection efficacy: the time of application in relation to detrimental organism establishment and symptom occurrence [11], the season and time of the day of application [41], the chemical properties of pesticide active ingredient and its formulation [42], the injected volume or dose of a pesticide, and the type of tree injection device or technology. For example, a more effective management of plant disease or insect infestation can be achieved by the preventive injections of pesticides in comparison to the therapeutic pesticide applications after the disease or infestation has already occurred. Tree injection of active compounds is much faster and easier during spring and early summer months in comparison to the late or mid-summer, late fall and winter, because water in the soil is abundant and the green leaf canopy is facilitating intensive transpiration pull and flow of water through the xylem tissue in hardwood trees, starting from the roots and branches to the leaves [41]. The three key properties of injected active ingredient that determine its mobility or binding in xylem of the tree are organic carbon-water partitioning coefficient (ml/g or μg/g) or carbon adsorption coefficient (Ko/c), water solubility, and formulation type. Ko/c expresses the level of adhesion or adsorption of pesticide active ingredient to the carbon rich compounds in certain environments such as soil or xylem and is defined as a ratio of mass of a chemical that is adsorbed in a certain environment per unit mass of organic carbon in that environment per the equilibrium chemical concentration in a solution. Active ingredients that have high Ko/c values will strongly bind to the organic compounds present in soil, sap or xylem and reduce their systemic movement i.e. translocation, accumulation and distribution in the canopy, thus reducing the efficacy in pathogen or pest control. In contrast, the ingredients with low or moderate Ko/c values move and distribute fast after tree injection and distribute well in the canopy, securing good pest or pathogen control. Pesticide formulation is a form of a pesticide active ingredient ready for use or which quite often requires dilution in water prior to application. Formulation is made by adding different inactive ingredients with the aim to improve the properties of an active ingredient such as solubility, surface adhesion, distribution, effectiveness, shelf life, stability, handling or application (e.g. solvents, emulsifiers, surfactants and other adjuvants). Formulation of a pesticide or a fertilizer determines the properties and residue stability of an active ingredient and can modulate its mobility in xylem of phloem after tree injection for pest or plant management [12, 42]. Finally, trunk injection devices can loosely be divided into the ones using drill- or needle-based technology [43]. In case of the

which recorded an increase in the number of new fungal and bacterial pathogens and their detrimental impact on agroecosystems, ecosystems, and the human society. The economic effects of these pathogens are reflected in lost fresh fruit produce [17–19], reduced yields and quality of fruit or wood and cork products [20, 21], diminished ecological tree services, and death of whole trees, stands, and forest regions or

If left unmanaged, apple scab fungus *Venturia inaequalis*, a subcuticular leaf and fruit pathogen can cause 70–100% reduction in marketable fruit yield in each year [23, 24]. A pathogenic fungus *Diplodia corticola*, the causal agent of Bot canker of oak which invades tree xylem is the most widely distributed and virulent fungal pathogen causing canker and decline of cork oak (*Quercus suber*) forests in Europe [25, 26] and of southern live oak (*Q. virginiana*), coast live oak (*Q. agrifolia*), and canyon live oak (*Q. chrysolepis*) in the United States [3, 27]. Recently, there has been a rising incidence of this pathogen in the Unites States on northern red oak, *Q. rubra*

[5, 8], black oak, *Q. velutina* [7], white oak, *Q. alba* [28], and bur oak (*Q. macrocarpa*) [29]. Other Botryosphaeriaceae, like *Neofusicoccum australe*, *N. luteum*, *N. parvum* and *N. mediterraneum* infect woody and green plant tissues and are destructive canker pathogens of avocado [1], grapevine, olive, pistachio and ash [30–33] and are recently reported as very virulent pathogens of coast redwood leading to severe decline in urban stands of California [9]. The Blue stain fungi *Grosmannia clavigera* and *Leptographium longiclavatum* are the plant pathogenic insect symbionts vectored by the mountain pine beetle *Dendroctonus ponderosae* [13]. They infect xylem and have led to death of millions of pine trees from 1990 to 2013 and decimation of pine forests in western North America [22, 34, 35]. Under favorable weather conditions during apple bloom and shoot growth, a destructive fire blight bacterium *Erwinia amylovora* inhabits and infects apple flowers and shoots and after spreading through xylem and causing cankers on wood it can kill whole trees. The resulting losses can range from \$3.8 to 100 million due to removal of as much as 450,000 apple trees in only one or two years [17, 18, 36, 37]. Fire blight severely reduced both pear and quince production primarily in continental climate regions of the world. Asiatic citrus canker caused by *Xanthomonas axonopodis* pv. *citri* invades leaf mesophyll tissue and is estimated to cause yield losses and cost of disease management of \$342 million per year [38]. The citrus greening or Huanglongbing disease is caused by a bacterium *Candidatus Liberibacter asiaticus* which proliferates and is limited to phloem vascular tissue of citrus trees. This pathogen led to \$4.5 billion negative economic impact in just 5 years after introduction in the United States [19]. These devastating internal tree pathogens further create a barrier in international trade of fresh fruit and wood products in an

attempt to prevent their introduction to new regions [39, 40].

parts to achieve an efficient preventive or curative control, respectively.

**134**

The biology of majority of these microorganisms, excluding *V. inaequalis* and *X. axonopodis* pv. *citri*, has three key shared traits: (1) they invade, reside, and spread in xylem or phloem of woody host tissues, where a significant part of pathogenesis and internal host damage is taking place, and (2) due to specific lifestyle depicted in impacting the internal wood tissues, these pathogens successfully evade exposure to the contact, local-systemic, and green-tissue systemic pesticides applied to plant surfaces, and (3) their management is extremely difficult or inefficient with the classical pesticide delivery methods. Therefore, any pesticide active ingredient in a formulated form needs to distribute in these vascular tissues to reach pathogen propagules at an effective concentration and move systemically to all the uninfected or infected tissue

Tree injection, often referred to as trunk or stem injection, is a method of target precise delivery or application of pesticides, plant resistance activators and fertilizers into the xylem vascular tissue of a tree with the aim to protect trees from insect pests

decimation of fruit industries [19, 22].

*Plant Diseases-Current Threats and Management Trends*

first one, access to xylem for pesticide delivery device is enabled by drilling into the trunk or root flare wood, removing a small part of the wood, and sealing of the opened injection port with an inserted plastic plug or not (plug contains an injection valve with a one-way silicone septum) [44]. For the injection application to be faster and hence economical in urban tree care, this system uses compressed air or hydraulic pressure to force-inject the pesticide solution into the wood. The second technology uses a knife-like or a flat, screwdriver-like needle with a lenticular profile, which is inserted into the wood by a hammer thus separating the wood fibers and creating a crevice while the delivery of a pesticide solution is conducted through the needle and infused into the wood [45]. This system can use force of hydraulic or compressed air pressure to deliver the pesticide solution into the xylem or is solely relying on the Venturi effect (vacuum) created by a transpiration stream in xylem to infuse the pesticide solution into the wood [45–47]. This injection technology requires longer time for injection solution delivery, especially when transpiration is limited, and thus is often less economical in urban tree care.

timing optimization for season- and two-seasons-long control [12, 50, 59], trunk wounding by injection ports i.e. points [43], pesticide residue accumulation in fruit, nectar, and leaves [12, 59, 60] and their spatial and temporal distribution in the tree [61]. Even though tree injection originated from widely present needs for pest and disease control and plant nutrition in urban forestry, it holds an important potential for use in tree fruit agriculture where in the last 30 or so years there has been an increase of public pressure on apple producers to reduce pesticide use, while maintaining a high level of fruit quality [62]. Since the tree injection of pesticides and nutrients as a delivery approach is currently gaining more popularity in urban greening, landscapes and forestry management [13, 48, 63–65], we predict intensification of research for insect pest and pathogen control in tree-based agriculture

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

While trunk injection for pesticide delivery is a relatively new technology investigated in tree-based agriculture for managing diseases like citrus greening [14–16, 66–68] or fire blight [11, 44], research in agricultural engineering will first need to design or invent an application system/s that allow scalability, i.e. achieving simultaneous trunk injection of large number of trees in a short period of time. Besides this end goal many other key questions arising from research outlined above will need to be addressed through experimental work before tree injection is used in agriculture, even in limited fashion. The first steps are providing enough evidence i.e. providing proof of concept that injected pesticides are effective in tree pathogen and insect management and that injected materials have minimal negative effect on fresh fruit consumer and beneficial orchard fauna. Because effective management options for Bot canker and decline of different *Quercus* species caused by *D. corticola* and other aforementioned plant pathogens of vascular tissues are limited or lacking, we evaluated the efficacy of trunk injected fungicides for *D. corticola* control and compared it to the efficacy of similar active ingredients for management of *V. inaequalis* and *E. amylovora* which are more intensively studied models in continental climate. Our goal was to present new disease management data that argues in favor of a hypothesis that for plant pathogens with difficult biologies, i.e. for those that impact internal wood tissues, it is necessary to select appropriate pesticide delivery system/s such as trunk injection to achieve the maximum disease control through increasing pathogen exposure and thus efficiency of applied pesticides. We present efficacy data of trunk-injected pesticides in management of these three woody plant pathogens with different or partially similar lifestyles to elucidate and promote the translation of tree injection as a target precise delivery system for plant

and silviculture in the near future.

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

protection in agriculture and silviculture of the future.

*2.1.1 Diplodia corticola*

**137**

**important plant pathogens in continental climate**

**2. Trunk injection delivery of pesticides for management of three**

**2.1 Biology of** *Diplodia corticola***,** *Venturia inaequalis* **and** *Erwinia amylovora*

In the binomial nomenclature of fungi, Bot canker pathogen *D. corticola* is a commonly found asexual stage of an ascomycete *Botryosphaeria corticola*, a sexual stage of this fungus [69]. Asexual stage forms white to dark olive-green aerial mycelium with a dark green to black underside [5]. During 24 weeks after the host plant infection, the fungus forms dark brown to black, circular or flask-shaped fruiting bodies called pycnidia, that are up to 1 mm in diameter and emerge through the dead bark of oak. Pycnidia can form on all above ground tree parts and are

Tree injection was initially developed for pesticide and fertilizer application on large size trees in proximity of urban areas where ground- and air-spray applications are impractical due to substantial pesticide losses through drift, lack of proper canopy coverage, or are prohibited due to possible human and domestic animal exposure. The second driver for development of tree injection and its more frequent use in recent decades has been the destructive nature and an increasing need for effective management options for invasive tree pathogens like *Ophiostoma* fungi that cause Dutch elm disease, *Bretziella fagacearum* fungus that causes oak wilt, and insects pests like Emerald ash borer, *Agrilus planipennis* and Hemlock Wooly Adelgid, *Adelges tsugae*. Because of the unique biology of these organisms which leads to severe internal damage of wood and ultimately causes tree death, their management is extremely difficult or inefficient with classical pesticide application methods like topical spraying. Therefore, the goal of trunk injection to deliver the plant protective materials into the xylem or phloem vascular tissues of trees matches the specific pathogen or pest biologies and pesticide exposure requirements for the most effective management of these detrimental organisms.

Due to a demonstrated ability of single trunk injection to increase the efficacy of injected pesticides over multiple years, a possibility to reduce the of number of topical spray applications [10, 12, 48] and a rising incidence of woody plant pathogens and insect pests in the environment [31, 33, 49–51], this approach has recently been investigated in agriculture where topical pesticide applications for plant food production is intensive. The most investigated tree fruit crops and their pathogens and insect pests are citrus (e.g. *Candidatus* Liberibacter asiaticus) [14, 15], avocado (e.g. avocado thrips, *Scirtothrips perseae*; *Phytophthora* root rot, *Phytophthora cinnamomi*) [49, 50], apple (e.g. fire blight, *Erwinia amylovora*; apple scab, *V. inaequalis* [11, 12]; oblique banded leaf roller, *Choristoneura rosaceana* [10, 52]), and grapevine (e.g. grapevine downy mildew, *Plasmopara viticola* [53]; powdery mildew, *Uncinula necator* [54]). Domesticated apple, *Malus pumila* is an important research model in continental climate because management of *V. inaequalis* in humid regions requires intensive spray programs with as many as 15–22 spray applications of fungicides in one growing season [55]. Since this research is novel for tree disease management in contemporary agriculture, the proof of concept experiments on cultivated trees are conducted by using the trunk injection devices primarily designed for delivering pesticides and fertilizers for tree protection purposes in landscapes and urban forestry [10, 44, 52, 56]. Besides the smaller tree sizes in orchards in comparison to the urban landscapes as an obvious advantage driving the investigation of efficacy of pesticides delivered with this method [52, 57, 58], some of the key researched topics are efficacy and its lasting [10, 11], application

#### *Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

timing optimization for season- and two-seasons-long control [12, 50, 59], trunk wounding by injection ports i.e. points [43], pesticide residue accumulation in fruit, nectar, and leaves [12, 59, 60] and their spatial and temporal distribution in the tree [61]. Even though tree injection originated from widely present needs for pest and disease control and plant nutrition in urban forestry, it holds an important potential for use in tree fruit agriculture where in the last 30 or so years there has been an increase of public pressure on apple producers to reduce pesticide use, while maintaining a high level of fruit quality [62]. Since the tree injection of pesticides and nutrients as a delivery approach is currently gaining more popularity in urban greening, landscapes and forestry management [13, 48, 63–65], we predict intensification of research for insect pest and pathogen control in tree-based agriculture and silviculture in the near future.

While trunk injection for pesticide delivery is a relatively new technology investigated in tree-based agriculture for managing diseases like citrus greening [14–16, 66–68] or fire blight [11, 44], research in agricultural engineering will first need to design or invent an application system/s that allow scalability, i.e. achieving simultaneous trunk injection of large number of trees in a short period of time. Besides this end goal many other key questions arising from research outlined above will need to be addressed through experimental work before tree injection is used in agriculture, even in limited fashion. The first steps are providing enough evidence i.e. providing proof of concept that injected pesticides are effective in tree pathogen and insect management and that injected materials have minimal negative effect on fresh fruit consumer and beneficial orchard fauna. Because effective management options for Bot canker and decline of different *Quercus* species caused by *D. corticola* and other aforementioned plant pathogens of vascular tissues are limited or lacking, we evaluated the efficacy of trunk injected fungicides for *D. corticola* control and compared it to the efficacy of similar active ingredients for management of *V. inaequalis* and *E. amylovora* which are more intensively studied models in continental climate. Our goal was to present new disease management data that argues in favor of a hypothesis that for plant pathogens with difficult biologies, i.e. for those that impact internal wood tissues, it is necessary to select appropriate pesticide delivery system/s such as trunk injection to achieve the maximum disease control through increasing pathogen exposure and thus efficiency of applied pesticides. We present efficacy data of trunk-injected pesticides in management of these three woody plant pathogens with different or partially similar lifestyles to elucidate and promote the translation of tree injection as a target precise delivery system for plant protection in agriculture and silviculture of the future.

#### **2. Trunk injection delivery of pesticides for management of three important plant pathogens in continental climate**

#### **2.1 Biology of** *Diplodia corticola***,** *Venturia inaequalis* **and** *Erwinia amylovora*

#### *2.1.1 Diplodia corticola*

In the binomial nomenclature of fungi, Bot canker pathogen *D. corticola* is a commonly found asexual stage of an ascomycete *Botryosphaeria corticola*, a sexual stage of this fungus [69]. Asexual stage forms white to dark olive-green aerial mycelium with a dark green to black underside [5]. During 24 weeks after the host plant infection, the fungus forms dark brown to black, circular or flask-shaped fruiting bodies called pycnidia, that are up to 1 mm in diameter and emerge through the dead bark of oak. Pycnidia can form on all above ground tree parts and are

first one, access to xylem for pesticide delivery device is enabled by drilling into the trunk or root flare wood, removing a small part of the wood, and sealing of the opened injection port with an inserted plastic plug or not (plug contains an injection valve with a one-way silicone septum) [44]. For the injection application to be faster and hence economical in urban tree care, this system uses compressed air or hydraulic pressure to force-inject the pesticide solution into the wood. The second technology uses a knife-like or a flat, screwdriver-like needle with a lenticular profile, which is inserted into the wood by a hammer thus separating the wood fibers and creating a crevice while the delivery of a pesticide solution is conducted through the needle and infused into the wood [45]. This system can use force of hydraulic or compressed air pressure to deliver the pesticide solution into the xylem or is solely relying on the Venturi effect (vacuum) created by a transpiration stream in xylem to infuse the pesticide solution into the wood [45–47]. This injection technology requires longer time for injection solution delivery, especially when transpiration is limited, and thus is often less economical in urban tree care.

*Plant Diseases-Current Threats and Management Trends*

Tree injection was initially developed for pesticide and fertilizer application on large size trees in proximity of urban areas where ground- and air-spray applications are impractical due to substantial pesticide losses through drift, lack of proper canopy coverage, or are prohibited due to possible human and domestic animal exposure. The second driver for development of tree injection and its more frequent use in recent decades has been the destructive nature and an increasing need for effective management options for invasive tree pathogens like *Ophiostoma* fungi that cause Dutch elm disease, *Bretziella fagacearum* fungus that causes oak wilt, and insects pests like Emerald ash borer, *Agrilus planipennis* and Hemlock Wooly Adelgid, *Adelges tsugae*. Because of the unique biology of these organisms which leads to severe internal damage of wood and ultimately causes tree death, their management is extremely difficult or inefficient with classical pesticide application methods like topical spraying. Therefore, the goal of trunk injection to deliver the plant protective materials into the xylem or phloem vascular tissues of trees matches the specific pathogen or pest biologies and pesticide exposure requirements for the most effective management of these detrimental organisms.

Due to a demonstrated ability of single trunk injection to increase the efficacy of

injected pesticides over multiple years, a possibility to reduce the of number of topical spray applications [10, 12, 48] and a rising incidence of woody plant pathogens and insect pests in the environment [31, 33, 49–51], this approach has recently been investigated in agriculture where topical pesticide applications for plant food production is intensive. The most investigated tree fruit crops and their pathogens and insect pests are citrus (e.g. *Candidatus* Liberibacter asiaticus) [14, 15], avocado (e.g. avocado thrips, *Scirtothrips perseae*; *Phytophthora* root rot, *Phytophthora cinnamomi*) [49, 50], apple (e.g. fire blight, *Erwinia amylovora*; apple scab, *V. inaequalis* [11, 12]; oblique banded leaf roller, *Choristoneura rosaceana* [10, 52]), and grapevine (e.g. grapevine downy mildew, *Plasmopara viticola* [53]; powdery mildew, *Uncinula necator* [54]). Domesticated apple, *Malus pumila* is an important research model in continental climate because management of *V. inaequalis* in humid regions requires intensive spray programs with as many as 15–22 spray applications of fungicides in one growing season [55]. Since this research is novel for tree disease management in contemporary agriculture, the proof of concept experiments on cultivated trees are conducted by using the trunk injection devices primarily designed for delivering pesticides and fertilizers for tree protection purposes in landscapes and urban forestry [10, 44, 52, 56]. Besides the smaller tree sizes in orchards in comparison to the urban landscapes as an obvious advantage driving the investigation of efficacy of pesticides delivered with this method [52, 57, 58], some of the key researched topics are efficacy and its lasting [10, 11], application

**136**

visible on bark as black masses of fungal tissue called stromata. Pycnidia have multiple chambers or locules in them, each 200–300 μm in diameter, in which spores called conidia are produced for around 2 years and serve as source of inoculum for new infections [69]. When pycnidia are mature they are partially erumpent through the host bark and form a circular opening on the top called ostiole serving for conidia release. Conidia are oval-shaped to cylindrical, straight, with both ends rounded, usually translucent and single-celled, but with aging rarely become brown and with multiple cells [69]. Inside, conidia are usually without an oil vesicle called guttule but can form one in the center. The sexual stage *B. corticola* forms spores called ascospores. Eight ascospores arranged in two rows form in one sac called ascus and multiple asci are formed in the dark brown to black fruiting bodies called pseudothecia [69]. Pseudothecia are up to 1 mm in diameter, circular and partially erumpent on the host bark when mature. Each pseudothecium has multiple chambers or locules 200–300 μm in diameter [69]. For release of ascospores, pseudothecia form a circular opening on the top called ostiole. Ascospores are spindle-shaped to rhomboid and translucent, one-celled, or rarely becoming light brown and with age two- or three-celled [69].

support of the true pathogen lifestyle, the inoculation tests with isolated *D. corticola* strains conducted on young, healthy, naturally established trees of black oak clearly demonstrated the pathogenicity of *D. corticola*. In a similar case in California, closely related Botryosphaeriaceae fungi *Neofusicoccum australe*, *N. luteum*, *N. parvum*, *N. mediterraneum* and *Botryosphaeria dothidea* have been isolated from declining coast redwood trees (*Sequoia sempervirens*) in the urban stands, which were severely drought stressed [9]. However, the inoculation tests with the isolates of these fungal species on potted healthy young trees of coast redwood clearly showed that *Neofusicoccum* species were true and very virulent pathogens, while *B. dothidea* was an opportunistic pathogen that did not cause severe infection on healthy trees [9]. The other members of Botryosphaeriaceae family, such as *B. dothidea* [23, 74, 75] or *Diplodia sapinea* [76], are well-known opportunistic pathogens that dwell on the tree asymptomatically for months or even years, until the plant becomes weakened through any number of abiotic or biotic stresses (e.g. drought or insect infestation), and then infect it [9, 77–81]. The stressed plant host is the key conducive condition for these pathogens to establish infection and express the disease symptoms. Future experiments, similar to the drought contribution studies done for *B. dothidea* infection [9, 74, 77–79]*,* should demonstrate which role the different plant stresses play in predisposing the oak species to *D. corticola* infection in continental and other climate types of the world where this pathogen is

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

The sexual stage of an ascomycete fungus *V. inaequalis* (Cooke) Winter, 1875, a cause of apple scab disease, starts by sexual reproduction in fall which results in formation of round initials of fungal fruiting bodies called pseudothecia. These bodies are embedded in a stroma or dense mat of fungal mycelia inside the mesophyll tissue of dead apple leaves. Late in winter and the beginning of spring, pseudothecia mature, gain pear-like shape and form sexual spores called ascospores. There are eight ascospores arranged linearly in each of the 50–100 elongated cylindrical sacs called asci that form in each pseudothecium. Ascospores are translucent to brown, two-celled, with one cell always larger than the other giving them a typical shape of a shoe "footprint". Mature pseudothecia form a circular opening or ostiole at the top of the pseudothecium, which protrudes through the surface of the dead apple leaves and allows ascospore release. There is only one cycle of ascospore production in spring of each year and they cause primary scab infections. The asexual stage *Spilocaea pomi* (Fr.) (syn. *Fusicladium dendriticum*) forms a translucent mycelium below the cuticle of the infected green apple tissues. With time, mycelia become dark gray to black, forming a dense mycelial mat that gives rise to asexual spores called conidia. Conidia are dark green, teardrop-shaped, i.e. pointed at one end and rounded on the other, single-celled or rarely two-celled. Conidia cause secondary infections during the apple growing season. There can be many cycles of conidia production and thus secondary infections, sometimes even more

Depending on the substrate it colonizes over the year, the life cycle of apple scab fungus has the saprophytic and the parasitic phase in its development. The saprophytic phase starts with apple leaf drop in autumn. *V. inaequalis*, the sexual stage of apple scab fungus, overwinters in the dead fallen leaves and fruit debris of apple on the orchard floor as initials of fruiting bodies called pseudothecia. Fungus rarely overwinters as *S. pomi* in the form of mycelium on twig lesions or in the inner bud tissues [23, 83, 84]. After winter rest, pseudothecia mature in early spring and release ascospores that enable first or the primary infections on newly developing

also widely present.

than 20 [82].

**139**

*2.1.2 Venturia inaequalis*

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

Because *D. corticola* has been described only recently [69], its life cycle describing how this pathogen reaches and colonizes oak species as a primary host is not fully known. Based on the other pathogenic and opportunistic pathogen members of the family Botryosphaeriaceae, *D. corticola* can probably infect through wounds and maybe plant natural openings. Once infection is established it spreads within the host via xylem to large distances with xylem sections exhibiting black streaking and cankers can form intermittently on the bark of trunk or branches [5, 8, 28, 29]. Some trees with xylem necrosis do not always exhibit visible external cankers [7]. With time, crown sections of infected trees show wilting and eventually host can die [5, 8]. It is assumed that conidia and ascospore production on cankers and germination are favored by moisture and high relative air humidity. Recently, in a study analyzing aerial fungal spore samples collected by the air spore traps and passive rain collectors, *D. corticola* was detected in 16 of the 32 sampled locations in Canada by a highly specific molecular detection method which targets a pathogenspecific DNA region [70]. This data indicates that *D. corticola* has aerial disseminated spores. It is reported that conidia of *D. corticola* are dispersed by wind [71], water and/or insect vectors [26]. A pest of oak wood, oak pinhole borer (*Platypus cylindrus*), acts as a vector of *D. corticola* spores as this fungus was detected in the insects gut and mycangia (structures in the insect body adapted for transport of spores of insect-symbiotic fungi) [72]. The invasive insects *Xyleborus affinis* and *Xylosandrus crassiusculus* in Florida have also been found to vector *D. corticola* as this fungus was frequently isolated from their mycangia [73]. Even though the knowledge on timing of seasonal spore release and relative inoculum abundance is limited [71], under natural infection pressure originating from a declining cork oak forest, *D. corticola* was found to infect cork oak seedlings at two infections peaks, in May and in September [71]. The role of *B. corticola* ascospores and their importance in pathogen dissemination have not been discussed in literature so far and much about epidemiology of this pathogen is unknown.

Majority of evidence indicates that *D. corticola* is a true pathogen of *Quercus* species [5, 8, 28, 29]. *D. corticola* was isolated from northern red oak trees in an urban stand where no obvious signs of environmental or other biotic stresses were visible, aside of the Bot canker occurrence [5]. It is not clear how environmental and biotic factors that can stress oak trees favor *D. corticola* infections and their severity, but it is possible they could worsen the disease. In the northeastern USA, this fungus has been isolated from infected black oak trees (*Quercus velutina*) which were at the same time infested with a damaging gall wasp, *Zapatella davisae* [7]. However, in

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

support of the true pathogen lifestyle, the inoculation tests with isolated *D. corticola* strains conducted on young, healthy, naturally established trees of black oak clearly demonstrated the pathogenicity of *D. corticola*. In a similar case in California, closely related Botryosphaeriaceae fungi *Neofusicoccum australe*, *N. luteum*, *N. parvum*, *N. mediterraneum* and *Botryosphaeria dothidea* have been isolated from declining coast redwood trees (*Sequoia sempervirens*) in the urban stands, which were severely drought stressed [9]. However, the inoculation tests with the isolates of these fungal species on potted healthy young trees of coast redwood clearly showed that *Neofusicoccum* species were true and very virulent pathogens, while *B. dothidea* was an opportunistic pathogen that did not cause severe infection on healthy trees [9]. The other members of Botryosphaeriaceae family, such as *B. dothidea* [23, 74, 75] or *Diplodia sapinea* [76], are well-known opportunistic pathogens that dwell on the tree asymptomatically for months or even years, until the plant becomes weakened through any number of abiotic or biotic stresses (e.g. drought or insect infestation), and then infect it [9, 77–81]. The stressed plant host is the key conducive condition for these pathogens to establish infection and express the disease symptoms. Future experiments, similar to the drought contribution studies done for *B. dothidea* infection [9, 74, 77–79]*,* should demonstrate which role the different plant stresses play in predisposing the oak species to *D. corticola* infection in continental and other climate types of the world where this pathogen is also widely present.

#### *2.1.2 Venturia inaequalis*

visible on bark as black masses of fungal tissue called stromata. Pycnidia have multiple chambers or locules in them, each 200–300 μm in diameter, in which spores called conidia are produced for around 2 years and serve as source of inoculum for new infections [69]. When pycnidia are mature they are partially erumpent through the host bark and form a circular opening on the top called ostiole serving for conidia release. Conidia are oval-shaped to cylindrical, straight, with both ends rounded, usually translucent and single-celled, but with aging rarely become brown and with multiple cells [69]. Inside, conidia are usually without an oil vesicle called guttule but can form one in the center. The sexual stage *B. corticola* forms spores called ascospores. Eight ascospores arranged in two rows form in one sac called ascus and multiple asci are formed in the dark brown to black fruiting bodies called pseudothecia [69]. Pseudothecia are up to 1 mm in diameter, circular and partially erumpent on the host bark when mature. Each pseudothecium has multiple cham-

bers or locules 200–300 μm in diameter [69]. For release of ascospores, pseudothecia form a circular opening on the top called ostiole. Ascospores are spindle-shaped to rhomboid and translucent, one-celled, or rarely becoming light

Because *D. corticola* has been described only recently [69], its life cycle describing how this pathogen reaches and colonizes oak species as a primary host is not fully known. Based on the other pathogenic and opportunistic pathogen members of the family Botryosphaeriaceae, *D. corticola* can probably infect through wounds and maybe plant natural openings. Once infection is established it spreads within the host via xylem to large distances with xylem sections exhibiting black streaking and cankers can form intermittently on the bark of trunk or branches [5, 8, 28, 29]. Some trees with xylem necrosis do not always exhibit visible external cankers [7]. With time, crown sections of infected trees show wilting and eventually host can die [5, 8]. It is assumed that conidia and ascospore production on cankers and germination are favored by moisture and high relative air humidity. Recently, in a study analyzing aerial fungal spore samples collected by the air spore traps and passive rain collectors, *D. corticola* was detected in 16 of the 32 sampled locations in Canada by a highly specific molecular detection method which targets a pathogenspecific DNA region [70]. This data indicates that *D. corticola* has aerial disseminated spores. It is reported that conidia of *D. corticola* are dispersed by wind [71], water and/or insect vectors [26]. A pest of oak wood, oak pinhole borer (*Platypus cylindrus*), acts as a vector of *D. corticola* spores as this fungus was detected in the insects gut and mycangia (structures in the insect body adapted for transport of spores of insect-symbiotic fungi) [72]. The invasive insects *Xyleborus affinis* and *Xylosandrus crassiusculus* in Florida have also been found to vector *D. corticola* as this fungus was frequently isolated from their mycangia [73]. Even though the knowledge on timing of seasonal spore release and relative inoculum abundance is limited [71], under natural infection pressure originating from a declining cork oak forest, *D. corticola* was found to infect cork oak seedlings at two infections peaks, in May and in September [71]. The role of *B. corticola* ascospores and their importance in pathogen dissemination have not been discussed in literature so far and much about

Majority of evidence indicates that *D. corticola* is a true pathogen of *Quercus* species [5, 8, 28, 29]. *D. corticola* was isolated from northern red oak trees in an urban stand where no obvious signs of environmental or other biotic stresses were visible, aside of the Bot canker occurrence [5]. It is not clear how environmental and biotic factors that can stress oak trees favor *D. corticola* infections and their severity, but it is possible they could worsen the disease. In the northeastern USA, this fungus has been isolated from infected black oak trees (*Quercus velutina*) which were at the same time infested with a damaging gall wasp, *Zapatella davisae* [7]. However, in

brown and with age two- or three-celled [69].

*Plant Diseases-Current Threats and Management Trends*

epidemiology of this pathogen is unknown.

**138**

The sexual stage of an ascomycete fungus *V. inaequalis* (Cooke) Winter, 1875, a cause of apple scab disease, starts by sexual reproduction in fall which results in formation of round initials of fungal fruiting bodies called pseudothecia. These bodies are embedded in a stroma or dense mat of fungal mycelia inside the mesophyll tissue of dead apple leaves. Late in winter and the beginning of spring, pseudothecia mature, gain pear-like shape and form sexual spores called ascospores. There are eight ascospores arranged linearly in each of the 50–100 elongated cylindrical sacs called asci that form in each pseudothecium. Ascospores are translucent to brown, two-celled, with one cell always larger than the other giving them a typical shape of a shoe "footprint". Mature pseudothecia form a circular opening or ostiole at the top of the pseudothecium, which protrudes through the surface of the dead apple leaves and allows ascospore release. There is only one cycle of ascospore production in spring of each year and they cause primary scab infections. The asexual stage *Spilocaea pomi* (Fr.) (syn. *Fusicladium dendriticum*) forms a translucent mycelium below the cuticle of the infected green apple tissues. With time, mycelia become dark gray to black, forming a dense mycelial mat that gives rise to asexual spores called conidia. Conidia are dark green, teardrop-shaped, i.e. pointed at one end and rounded on the other, single-celled or rarely two-celled. Conidia cause secondary infections during the apple growing season. There can be many cycles of conidia production and thus secondary infections, sometimes even more than 20 [82].

Depending on the substrate it colonizes over the year, the life cycle of apple scab fungus has the saprophytic and the parasitic phase in its development. The saprophytic phase starts with apple leaf drop in autumn. *V. inaequalis*, the sexual stage of apple scab fungus, overwinters in the dead fallen leaves and fruit debris of apple on the orchard floor as initials of fruiting bodies called pseudothecia. Fungus rarely overwinters as *S. pomi* in the form of mycelium on twig lesions or in the inner bud tissues [23, 83, 84]. After winter rest, pseudothecia mature in early spring and release ascospores that enable first or the primary infections on newly developing

green apple tissues. Hence, ascospores in the leaf litter and debris are prime inoculum sources in spring. With each wetting from rain of heavy dew [85], pseudothecia absorb water, swell and asci expand through the ostiole, allowing forcible ascospore discharge. Ejected ascospores can reach a height of about 5–30 mm above the ostiole and are further disseminated by air currents, wind and rain aerosol [86]. They germinate only in water droplets or film coating the plant surfaces and their germination ends the saprophytic phase of pathogen's life cycle. Depending on region, the period of ascospore discharge triggered by wetting events can last from 3 to 9 weeks during late March and over April, May and mid-June. Ascospores are airborne and can reach at least 45 m away from the inoculum source [87]. *Spilocaea pomi* as the asexual stage of this fungus begins with ascospore germination and infection of the newly developing green tissues of apple leaves, flowers and fruit. The infection of current season's apple growth starts the parasitic phase of the pathogen's life cycle. Fungal mycelium penetrates below the waxy cuticle and grows between the outer cell wall of host's epidermal cells and the cuticle covering them. If the primary infections with ascospores are successful, after incubation period the plant cuticle of infected organ ruptures due to pressure created by thickening mycelium and masses of asexual spores of *S. pomi* called conidia. The infections are first visible as light chlorotic spots on leaves against the light, then gradually turn into pale olive to dark gray and ultimately black, velvety apple scab lesions. Infections on flower pedicel lead to flower drop. Infected leaves senesce and drop, while young fruit become deformed and fall off. Conidia cause new or often known secondary infections during the season and are dislodged and dispersed primarily with the help of rain water and dew. Their dispersal allowing more new infections is occurring primarily within the tree because very few conidia can reach 10 m or more from the inoculum source [88].

form of droplets or smears of bacterial ooze which are opaque white, amber or orange. Ooze is a sticky mixture of bacterial cells and exopolysaccharides which plays a major role in pathogen dissemination within and between the apple trees and orchards [95, 96]. In humid conditions, ooze exudes from the cracks on infected wood tissues with fire blight cankers and through stomata and lenticels on green, succulent parts of infected flowers, immature fruit, and shoots. In low humidity conditions, fire blight bacteria can survive in dry ooze for more than a year [97]. In the laboratory, *E. amylovora* forms domed, circular, mucoid colonies on microbiological media that contain sucrose nutrient agar [98, 99] which differ in color depending on specific contents of agar medium, ranging from red to orange, yellow white, and light blue opalescent [100]. This pathogen has a wide range of cultivated, landscape or forest plant hosts from Rosaceae family: apple, crabapple (*Malus*), pear, Asian pear, Callery pear (*Pyrus*), quince (*Cydonia*), raspberry (*Rubus*), as well as hawthorn (*Crataegus*), firethorn (*Pyracantha*), mountain ash (*Sorbus*), serviceberry (*Amelanchier*), *Cotoneaster*, loquat (*Eriobotrya*), flowering

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

*E. amylovora* survives through the winter in the bark around the canker edge and below the fire blight cankers initiated after pathogen progression from flower and shoot infections established in the previous years. Bacterial cells can also overwinter in asymptomatic host buds or as latent infections in asymptomatic wood [101]. It is possible that cells of this pathogen are in a viable but non-culturable physiological state in asymptomatic tissues [102, 103]. With warm spring weather, pathogen cells multiply and emerge in bacterial ooze exuding on the edges of overwintered cankers. On apple, this process usually occurs during late bloom and petal fall growth stages. Every single droplet of ooze may contain up to 1 billion cells of *E. amylovora* [95]. The ooze protects bacteria from unfavorable weather conditions and bacteria are disseminated from ooze to flowers, shoots and injured tissues by rain, wind, birds and presumably insects that touch or feed on ooze [95, 96, 104, 105]. For example, in experimental conditions, *E. amylovora* was found to survive and can be transmitted for up to 8 days in the digestive tract of Mediterranean fruit fly *Ceratitis*

*capitata* and up to 28 days on its surface [106]. Insects that might vector

*E. amylovora* are still investigated, but it is probable that the insect vectors will vary depending on the region of the world. Recent investigation shows that flies are attracted to feed on ooze and can acquire from 100 to 100,000 viable *E. amylovora* cells per fly individual [96]. It appears that honeybees do not visit ooze droplets on cankers in spring and might not spread the bacteria from cankers to flowers. It is assumed that in low humidity conditions, dry ooze strings or particles exuded through lenticels or stomata on the bark can break off and reach or settle on nearby susceptible flowers or shoots after carried by wind or insects. The closer the fire blight cankers are to any open flowers or shoots, the higher is the chance for *E. amylovora* cells to reach the flower surfaces by previously mentioned pathways. The period of apple susceptibility to *E. amylovora* infection lasts from the day when the first flowers open in the orchard and ends when the last terminal buds set on shoots. However, risk for severe infections becomes lower after flowering ends because the number of flowers as entry points for the pathogen significantly declines. Shoots initiate growth just before flowering ends and are susceptible until their growth stops. Terminal bud set is an apple growth stage when the current year vegetative growth stops, and a bud is formed at the top of the shoot. In continental climate, this usually occurs during July, varying somewhat among different apple cultivars. After this stage, risk from fire blight infections is negligent due to age-related resistance, unless a hail injury occurs on trees providing new entry points for fire blight infections (trauma blight). Once *E. amylovora* bacteria reach the apple flower surfaces, they multiply rapidly on the surfaces of nutrient-rich flower stigmas if temperatures are favorably

quince (*Chaenomeles*), etc.

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

**141**

During the time after spores of apple scab fungus land on the susceptible plant surface, while they germinate, and up to 72 h after they penetrate below the cuticle on green tissue, they are vulnerable to spray-applied contact and systemic fungicides, respectively. However, once the infection is established by formation of mycelium under the cuticle, almost all fungicides applied to green plant surfaces have no efficacy in eradicating these infections and eventually lesions with conidia, or their effect is minimal. Furthermore, continued post-infection scab management with spray applications of fungicides that aim to prevent new infections on green tissues is complicated by large populations of conidia, which if exposed to fungicides with specific modes of action might increases the potential for fungicide resistance selection in this devastating pathogen. Because of the specific lifestyle of *S. pomi* to reside in subcuticular spaces of green tissues during the parasitic phase of the life cycle, successful management of the apple scab is crucially dependent on preventive fungicide applications that are delivered to leaves and fruit before the major infection periods occur. To time fungicide spray applications, several epidemiological models based on pathogen ecology and biology have been developed and can predict discharge of *V. inaequalis* ascospores and the infection occurrence by using the weather forecast for up to 10 days (NEWA, RIMpro) [89–92]. They are used in all the major apple growing regions.

#### *2.1.3 Erwinia amylovora*

Fire blight is caused by a Gram-negative bacterium *Erwinia amylovora* (Burrill 1882) Winslow et al. [93] in the family Enterobacteriaceae. The bacterial cells are rod-shaped, 0.3 1–3 μm in size and occur as single cells or pairs and sometimes short chains. They are motile by 2–7 peritrichous flagella per cell [94]. The pathogen cells are not visible to the naked eye. On infected hosts, pathogen is visible in the

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

form of droplets or smears of bacterial ooze which are opaque white, amber or orange. Ooze is a sticky mixture of bacterial cells and exopolysaccharides which plays a major role in pathogen dissemination within and between the apple trees and orchards [95, 96]. In humid conditions, ooze exudes from the cracks on infected wood tissues with fire blight cankers and through stomata and lenticels on green, succulent parts of infected flowers, immature fruit, and shoots. In low humidity conditions, fire blight bacteria can survive in dry ooze for more than a year [97]. In the laboratory, *E. amylovora* forms domed, circular, mucoid colonies on microbiological media that contain sucrose nutrient agar [98, 99] which differ in color depending on specific contents of agar medium, ranging from red to orange, yellow white, and light blue opalescent [100]. This pathogen has a wide range of cultivated, landscape or forest plant hosts from Rosaceae family: apple, crabapple (*Malus*), pear, Asian pear, Callery pear (*Pyrus*), quince (*Cydonia*), raspberry (*Rubus*), as well as hawthorn (*Crataegus*), firethorn (*Pyracantha*), mountain ash (*Sorbus*), serviceberry (*Amelanchier*), *Cotoneaster*, loquat (*Eriobotrya*), flowering quince (*Chaenomeles*), etc.

*E. amylovora* survives through the winter in the bark around the canker edge and below the fire blight cankers initiated after pathogen progression from flower and shoot infections established in the previous years. Bacterial cells can also overwinter in asymptomatic host buds or as latent infections in asymptomatic wood [101]. It is possible that cells of this pathogen are in a viable but non-culturable physiological state in asymptomatic tissues [102, 103]. With warm spring weather, pathogen cells multiply and emerge in bacterial ooze exuding on the edges of overwintered cankers. On apple, this process usually occurs during late bloom and petal fall growth stages. Every single droplet of ooze may contain up to 1 billion cells of *E. amylovora* [95]. The ooze protects bacteria from unfavorable weather conditions and bacteria are disseminated from ooze to flowers, shoots and injured tissues by rain, wind, birds and presumably insects that touch or feed on ooze [95, 96, 104, 105]. For example, in experimental conditions, *E. amylovora* was found to survive and can be transmitted for up to 8 days in the digestive tract of Mediterranean fruit fly *Ceratitis capitata* and up to 28 days on its surface [106]. Insects that might vector *E. amylovora* are still investigated, but it is probable that the insect vectors will vary depending on the region of the world. Recent investigation shows that flies are attracted to feed on ooze and can acquire from 100 to 100,000 viable *E. amylovora* cells per fly individual [96]. It appears that honeybees do not visit ooze droplets on cankers in spring and might not spread the bacteria from cankers to flowers. It is assumed that in low humidity conditions, dry ooze strings or particles exuded through lenticels or stomata on the bark can break off and reach or settle on nearby susceptible flowers or shoots after carried by wind or insects. The closer the fire blight cankers are to any open flowers or shoots, the higher is the chance for *E. amylovora* cells to reach the flower surfaces by previously mentioned pathways. The period of apple susceptibility to *E. amylovora* infection lasts from the day when the first flowers open in the orchard and ends when the last terminal buds set on shoots. However, risk for severe infections becomes lower after flowering ends because the number of flowers as entry points for the pathogen significantly declines. Shoots initiate growth just before flowering ends and are susceptible until their growth stops. Terminal bud set is an apple growth stage when the current year vegetative growth stops, and a bud is formed at the top of the shoot. In continental climate, this usually occurs during July, varying somewhat among different apple cultivars. After this stage, risk from fire blight infections is negligent due to age-related resistance, unless a hail injury occurs on trees providing new entry points for fire blight infections (trauma blight).

Once *E. amylovora* bacteria reach the apple flower surfaces, they multiply rapidly on the surfaces of nutrient-rich flower stigmas if temperatures are favorably

green apple tissues. Hence, ascospores in the leaf litter and debris are prime inoculum sources in spring. With each wetting from rain of heavy dew [85], pseudothecia absorb water, swell and asci expand through the ostiole, allowing forcible ascospore discharge. Ejected ascospores can reach a height of about 5–30 mm above the ostiole and are further disseminated by air currents, wind and rain aerosol [86]. They germinate only in water droplets or film coating the plant surfaces and their germination ends the saprophytic phase of pathogen's life cycle. Depending on region, the period of ascospore discharge triggered by wetting events can last from 3 to 9 weeks during late March and over April, May and mid-June. Ascospores are airborne and can reach at least 45 m away from the inoculum source [87]. *Spilocaea pomi* as the asexual stage of this fungus begins with ascospore germination and infection of the newly developing green tissues of apple leaves, flowers and fruit. The infection of current season's apple growth starts the parasitic phase of the pathogen's life cycle. Fungal mycelium penetrates below the waxy cuticle and grows between the outer cell wall of host's epidermal cells and the cuticle covering them. If the primary infections with ascospores are successful, after incubation period the plant cuticle of infected organ ruptures due to pressure created by thickening mycelium and masses of asexual spores of *S. pomi* called conidia. The infections are first visible as light chlorotic spots on leaves against the light, then gradually turn into pale olive to dark gray and ultimately black, velvety apple scab lesions. Infections on flower pedicel lead to flower drop. Infected leaves senesce and drop, while young fruit become deformed and fall off. Conidia cause new or often known secondary infections during the season and are dislodged and dispersed primarily with the help of rain water and dew. Their dispersal allowing more new infections is occurring primarily within the tree because very few conidia can reach 10 m or more from the inoculum

*Plant Diseases-Current Threats and Management Trends*

During the time after spores of apple scab fungus land on the susceptible plant surface, while they germinate, and up to 72 h after they penetrate below the cuticle on green tissue, they are vulnerable to spray-applied contact and systemic fungicides, respectively. However, once the infection is established by formation of mycelium under the cuticle, almost all fungicides applied to green plant surfaces have no efficacy in eradicating these infections and eventually lesions with conidia, or their effect is minimal. Furthermore, continued post-infection scab management with spray applications of fungicides that aim to prevent new infections on green tissues is complicated by large populations of conidia, which if exposed to fungicides with specific modes of action might increases the potential for fungicide resistance selection in this devastating pathogen. Because of the specific lifestyle of *S. pomi* to reside in subcuticular spaces of green tissues during the parasitic phase of the life cycle, successful management of the apple scab is crucially dependent on preventive fungicide applications that are delivered to leaves and fruit before the major infection periods occur. To time fungicide spray applications, several epidemiological models based on pathogen ecology and biology have been developed and can predict discharge of *V. inaequalis* ascospores and the infection occurrence by using the weather forecast for up to 10 days (NEWA, RIMpro) [89–92]. They are

Fire blight is caused by a Gram-negative bacterium *Erwinia amylovora* (Burrill 1882) Winslow et al. [93] in the family Enterobacteriaceae. The bacterial cells are rod-shaped, 0.3 1–3 μm in size and occur as single cells or pairs and sometimes short chains. They are motile by 2–7 peritrichous flagella per cell [94]. The pathogen cells are not visible to the naked eye. On infected hosts, pathogen is visible in the

source [88].

used in all the major apple growing regions.

*2.1.3 Erwinia amylovora*

**140**

warm [107]. After colonizing young, just opened flowers [108], bacteria require achieving necessary population size and presence of moisture for infection establishment. The bacteria only have few days to grow their numbers on young flowers to reach at least 100,000 and up to 1 million live cells before a possible infection event can be triggered by rain, dew, or hail. During this time and before the moisture becomes available to allow infection, honeybees could spread the bacteria from contaminated flowers to newly opened flowers [109, 101]. This pollinator-facilitated spreading continues the necessary pathogen population increase. Flower surfaces of many other species of Rosacease family, except European plum *Prunus domestica*, which are not susceptible to fire blight were found to be potential sites for population increase of *E. amylovora* during their periods of bloom [110]. With a wetting event in the form of rain, dew or hail, the pathogen is washed down from stigmas to the nectar glands located in the floral cup where pathogen enters the host and causes the infection. Infection of succulent green shoots occurs either via (1) internal pathogen spread through green tissues from the infected flowers to the base of the nearby shoots, (2) direct transfer of the pathogen from cankers or contaminated plant or tool surfaces to the shoots, or (3) by pathogen dispersal from contaminated or infected flowers to the shoot tips and leaves. Insects might play a vector role in these three pathways. For limited amount of time, *E. amylovora* cells can survive on other healthy surfaces of leaves and branches. However, population growth on these surfaces does not occur. Pathogen enters and colonizes the cortical parenchyma through stomata on the leaves or green stem, or through the microinjuries i.e. punctures and tears caused by wind, wind-carried soil particles, hail, friction of plant parts, or sucking or chewing insects [111]. During the time before infections take place, the lifestyle of *E. amylovora* involves inhabiting and growing on the plant surfaces and is influenced by the temperature and moisture from the environment and by nutrients on the plant host. This is known as epiphytic phase of *E. amylovora* biology during which successful management of fire blight is achieved by preventive spray applications of antibiotics that are delivered to flowers, before or up to 24 h after the predicted rain event/s that would trigger the infection/s. Several fire blight epidemiological models have been designed based on environmental and biological requirements of *E. amylovora* and can predict infection events by using the weather forecast for up to 10 days in advance to calculate the near-future infection risks (NEWA's EIP, Maryblyt, RIMpro, Cougarblight).

organs. During this phase, pathogen colonizes the cortical parenchyma tissue and xylem vessels and can continue with systemic migration and distribution in the plant [112–114]. Fire blight bacteria migrate internally via xylem of symptomless branch and trunk tissues and ahead of the visible blight or canker symptoms, thus reaching uninfected plant parts and apple rootstocks and causing infection far from the visible infections in the canopy [115]. On susceptible rootstocks, the resulting infections can express as cankers, often causing tree death due to trunk or rootstock girdling, or can remain latent i.e. as a symptomless infections of trunk or rootstock. Rootstock or trunk infections can also be initiated by *E. amylovora* migration from the externally infected rootstock suckers (shoot growth from the root system or rootstock stem) or water sprouts (shoot growth from the trunk or thick branches). When *E. amylovora* resides as an endophyte in an apparently healthy plant tissues of branches, rootstock or budwood, this lifestyle is referred to as an endophytic phase of its biology. Finally, in nurseries, *E. amylovora* cells which survive on bark surfaces can infect rootstock or scion when either are bruised or injured during the

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

processes of vegetative material harvest, transport, or grafting.

*2.2.1 Trunk injection of pesticides for Diplodia corticola management*

resistance (SAR) [11] for reduction of Bot canker caused by *D. corticola* we conducted experiments on potted northern red oak trees (*Q. rubra*) with fully developed canopy. We evaluated trunk-injected fungicides and application rates listed in **Table 1**, which were selected based on the EPA labels of pesticides for landscape use (**Table 1**) and the preliminary fungicide screenings *in vitro* for suppression of *D. corticola* colonies on fungicide-amended Petri of plates with potato dextrose agar medium (Aćimović et al. unpublished data). Since in year one of the experiment Phosphojet at 1.5 ml dose caused phytotoxicity on tree trunks, we reduced the dose to 0.75 ml in year two repetition of the experiment (**Table 1**).

To test the effect of injected fungicides and activators of plant systemic acquired

*Trunk injected fungicide treatments evaluated for management of Bot canker fungus Diplodia corticola on*

**2.2 Materials and methods**

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

**Table 1.**

**143**

*northern red oak trees, Quercus rubra.*

During the incubation, i.e. usually sometime around 10 – 14 days before the first conspicuous blossom or shoot blight symptoms are visible, small white, amber or orange droplets of bacterial ooze can emerge and drip from the infected green tissues (flower pedicels, floral cup, sepals, immature fruit and shoots). With more wetting events and insect activity, ooze can spread to new flowers and actively growing shoots across the whole orchard. Since blossom and shoot blight symptoms are not yet visible, this dissemination of ooze allows secondary infections and can propel a fire blight outbreak into an epidemic, especially if the antibiotic spray application/s were not conducted during bloom. Once incubation is over, blossom blight is visible as dead, black or brown flower clusters with more droplets of bacterial ooze developing if weather conditions are humid. Shoot blight and immature fruit infections are visible as black or brown "flags" or "strikes" and brown to black shriveled fruitlets, respectively. Blighted shoot tips often bend in the typical shape of Shepherd's crook. Fire blight cankers on branches, trunk and rootstock are formed by pathogen's progress via xylem or the cortical parenchyma from the established infections on flowers, shoots and suckers, into the wood bark tissues.

When *E. amylovora* enters the succulent tissues of flowers or shoots, it begins the pathogenic phase of its lifestyle when it causes the disease and acquires moisture and nutrients from the host and can migrate to other close or far host tissues and

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

organs. During this phase, pathogen colonizes the cortical parenchyma tissue and xylem vessels and can continue with systemic migration and distribution in the plant [112–114]. Fire blight bacteria migrate internally via xylem of symptomless branch and trunk tissues and ahead of the visible blight or canker symptoms, thus reaching uninfected plant parts and apple rootstocks and causing infection far from the visible infections in the canopy [115]. On susceptible rootstocks, the resulting infections can express as cankers, often causing tree death due to trunk or rootstock girdling, or can remain latent i.e. as a symptomless infections of trunk or rootstock. Rootstock or trunk infections can also be initiated by *E. amylovora* migration from the externally infected rootstock suckers (shoot growth from the root system or rootstock stem) or water sprouts (shoot growth from the trunk or thick branches). When *E. amylovora* resides as an endophyte in an apparently healthy plant tissues of branches, rootstock or budwood, this lifestyle is referred to as an endophytic phase of its biology. Finally, in nurseries, *E. amylovora* cells which survive on bark surfaces can infect rootstock or scion when either are bruised or injured during the processes of vegetative material harvest, transport, or grafting.

#### **2.2 Materials and methods**

warm [107]. After colonizing young, just opened flowers [108], bacteria require achieving necessary population size and presence of moisture for infection establishment. The bacteria only have few days to grow their numbers on young flowers to reach at least 100,000 and up to 1 million live cells before a possible infection event can be triggered by rain, dew, or hail. During this time and before the moisture becomes available to allow infection, honeybees could spread the bacteria from contaminated flowers to newly opened flowers [109, 101]. This pollinator-facilitated spreading continues the necessary pathogen population increase. Flower surfaces of many other species of Rosacease family, except European plum *Prunus domestica*, which are not susceptible to fire blight were found to be potential sites for population increase of *E. amylovora* during their periods of bloom [110]. With a wetting event in the form of rain, dew or hail, the pathogen is washed down from stigmas to the nectar glands located in the floral cup where pathogen enters the host and causes the infection. Infection of succulent green shoots occurs either via (1) internal pathogen spread through green tissues from the infected flowers to the base of the nearby shoots, (2) direct transfer of the pathogen from cankers or contaminated plant or tool surfaces to the shoots, or (3) by pathogen dispersal from contaminated or infected flowers to the shoot tips and leaves. Insects might play a vector role in these three pathways. For limited amount of time, *E. amylovora* cells can survive on other healthy surfaces of leaves and branches. However, population growth on these surfaces does not occur. Pathogen enters and colonizes the cortical parenchyma through stomata on the leaves or green stem, or through the microinjuries i.e. punctures and tears caused by wind, wind-carried soil particles, hail, friction of plant parts, or sucking or chewing insects [111]. During the time before infections take place, the lifestyle of *E. amylovora* involves inhabiting and growing on the plant surfaces and is influenced by the temperature and moisture from the environment and by nutrients on the plant host. This is known as epiphytic phase of *E. amylovora* biology during which successful management of fire blight is achieved by preventive spray applications of antibiotics that are delivered to flowers, before or up to 24 h after the predicted rain event/s that would trigger the infection/s. Several fire blight epidemiological models have been designed based on environmental and biological requirements of *E. amylovora* and can predict infection events by using the weather forecast for up to 10 days in advance to calculate the near-future infection risks (NEWA's EIP, Maryblyt, RIMpro,

*Plant Diseases-Current Threats and Management Trends*

During the incubation, i.e. usually sometime around 10 – 14 days before the first conspicuous blossom or shoot blight symptoms are visible, small white, amber or orange droplets of bacterial ooze can emerge and drip from the infected green tissues (flower pedicels, floral cup, sepals, immature fruit and shoots). With more wetting events and insect activity, ooze can spread to new flowers and actively growing shoots across the whole orchard. Since blossom and shoot blight symptoms are not yet visible, this dissemination of ooze allows secondary infections and can propel a fire blight outbreak into an epidemic, especially if the antibiotic spray application/s were not conducted during bloom. Once incubation is over, blossom blight is visible as dead, black or brown flower clusters with more droplets of bacterial ooze developing if weather conditions are humid. Shoot blight and immature fruit infections are visible as black or brown "flags" or "strikes" and brown to black shriveled fruitlets, respectively. Blighted shoot tips often bend in the typical shape of Shepherd's crook. Fire blight cankers on branches, trunk and rootstock are formed by pathogen's progress via xylem or the cortical parenchyma from the established infections on flowers, shoots and suckers, into the wood bark tissues. When *E. amylovora* enters the succulent tissues of flowers or shoots, it begins the pathogenic phase of its lifestyle when it causes the disease and acquires moisture and nutrients from the host and can migrate to other close or far host tissues and

Cougarblight).

**142**

#### *2.2.1 Trunk injection of pesticides for Diplodia corticola management*

To test the effect of injected fungicides and activators of plant systemic acquired resistance (SAR) [11] for reduction of Bot canker caused by *D. corticola* we conducted experiments on potted northern red oak trees (*Q. rubra*) with fully developed canopy. We evaluated trunk-injected fungicides and application rates listed in **Table 1**, which were selected based on the EPA labels of pesticides for landscape use (**Table 1**) and the preliminary fungicide screenings *in vitro* for suppression of *D. corticola* colonies on fungicide-amended Petri of plates with potato dextrose agar medium (Aćimović et al. unpublished data). Since in year one of the experiment Phosphojet at 1.5 ml dose caused phytotoxicity on tree trunks, we reduced the dose to 0.75 ml in year two repetition of the experiment (**Table 1**).


#### **Table 1.**

*Trunk injected fungicide treatments evaluated for management of Bot canker fungus Diplodia corticola on northern red oak trees, Quercus rubra.*

One injection point i.e. port per trunk of each potted tree, positioned ca. 5–7 cm above the ground level, was created by drilling 7–10 mm into the xylem tissue with a 4.3 mm diameter drill attached to a cordless drill. To inject the protective liquid solutions listed in **Table 1**, we used a Stinger needle for plugless trunk injection assembled on an individual feed line attached to the Tree IV air/hydraulic microinjection system, which operated at 60 psi air pressure (Arborjet Inc., Woburn, MA). The Stinger needles are used for injection of trees when trunk injection ports of large diameter (9.5 mm) are of concern or should be avoided and for injection of trunks with small diameters. The diameter of injection port for inserting a Stinger needle is smaller and does not require sealing with an Arborplug. In year one, the injected potted oak trees had trunk diameter at 5 cm height averaging 1.3 cm and ranging from 1 to 2.1 cm. In year two, a new set of injected trees had the diameter at 5 cm height averaging 1.5 cm and ranging from 1.1 to 2.2 cm. Trunk injection were conducted on 12 June in year one and on 16 August in year two.

Trees were inoculated with *D. corticola* on 21 June and on 25 August, i.e. 9 days after injection of fungicides. Trunk bark on the opposite side from the injection port and 10 cm above the port was cut at three sides of rectangle to create a sleeve which was peeled longitudinally. A PDA plug 5 mm in diameter from 10-day-old colony of *D. corticola* isolate from our previous work [8] was placed in the sleeve on each injected tree and wrapped with parafilm (**Table 1**). Once the first symptoms of canopy wilt were observed, trees were destructively examined by stripping the bark off above and below the inoculation point. The necrosis length (cm) and width (cm) of Bot canker in xylem of the oak trunks were measured on 10 July in year one and on 27 September in year two. Xylem necrosis area (cm2 ) was calculated by multiplying the length and width for each individual tree and treatment mean was calculated from six replicate trees.

Statistical analysis was done with MIXED procedure in SAS Studio software (SAS Institute Inc. 2017, Cary, NC) using the xylem necrosis areas (cm2 ). If the fungicide effect was found to be statistically significant (*p* < 0.1 in year one; *p* < 0.05 in year two), treatment comparisons were done with LSD test. We presented the fungicide management results as percent reduction of Bot canker necrosis area, also known as percent disease control, calculated as: percent reduction of necrosis area = [percent necrosis area in water control – percent necrosis area in specific treatment] 100/percent necrosis area in water control.

Inc., Woburn, MA) positioned just below the bark level to allow port closure with cambium callus [44]. To inject the fungicides, we used the Quik-jet microinjection system (Arborjet Inc.) operating at hand-generated hydraulic pressure to deliver low volumes of liquid for injection, thus allowing faster application times, and the Tree IV air/hydraulic microinjection system (Arborjet Inc.) operating at up to 60 psi of air pressure to deliver large solution volumes of liquid for injection (≥600 ml). In the experiment 1 (**Table 2**), we injected all the treatments listed for 15 October in year one with Quik-jet (**Table 2**). On 11 April in year two, we injected propiconazole using the Viper air/hydraulic microinjection system set at 90 psi air pressure (Arborjet Inc.). At the later dates, we injected Alamo using the Tree IV and Phosphojet using the Quik-jet.

*Fungicide treatments trunk-injected across two seasons and sprayed for management of apple scab fungus*

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

**Table 2.**

**145**

*Venturia inaequalis on 'Mac Spur' apple trees.*

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

In the experiment 2, we injected Phosphojet with Quik-jet and cyprodinil +

injection time, to be divided and delivered equally among the four ports.

difenoconazole with Tree IV. The needle/s of each of the used injection devices was inserted through the Arborplugs allowing the total liquid volume per tree, at one

All the experiments were conducted under naturally high infection pressure during the primary season of *V. inaequalis* ascospore release in spring. In the experiment 1, we rated percent incidence of apple scab only on leaves, since fruits were lost due to spring frosts. In the experiment 2, we rated percent incidence of scab on leaves and fruit. A total of chose 20 spurs and 20 terminal shoots per tree were selected, with about five from each crown quadrant, and rated for leaf scab incidence. The fruit scab incidence was rated by selecting and rating 100 fruits per tree, with about 25 per crown quadrant, and if less was found we rated all the fruits per tree. The data were analyzed using MIXED procedure in SAS 9.3 (SAS Institute, Cary, NC). The tree was the subject of repeated measures. If the main effects or their interactions were found to be statistically significant (*p <* 0.05),

#### *2.2.2 Trunk injection of pesticides for Venturia inaequalis management*

With the goal to optimize timing and number of fungicide injections for management of apple scab fungus *V. inaequalis*, we conducted two experiments. In the experiment 1, we trunk-injected fungicides listed in **Table 2** on 29-year-old 'Mac Spur' apple trees four times, with the first injection applied in the fall of year one and the next three injections conducted in the spring of next, year two. In the experiment 1, the injection on 11 April was conducted at 50% apple bloom (**Table 2**). In the experiment 2, we injected 29-year-old 'Mac Spur' apple trees with fungicides only one to two times in total, but by delivering them at different seasons i.e. in fall or spring, as per schedule listed in **Table 2**. In the experiment 2, the injection on 21 April was conducted at the silver tip growth stage of apple (**Table 2**).

On each trunk injection date with fungicides listed in **Table 2**, a separate set of four cardinally-oriented trunk injection ports per each tree of 'Mac Spur' was created by drilling 25 mm into the xylem with a 9.5 mm diameter drill bit attached to a cordless drill. The first set of four injection ports was positioned ca. 25 cm above the ground level. The subsequent sets of four injection port were positioned ca. 5 cm above and between the lower four-port sets. Every port was sealed with Arborplug no. 4 (Arborjet *Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*


#### **Table 2.**

One injection point i.e. port per trunk of each potted tree, positioned ca. 5–7 cm above the ground level, was created by drilling 7–10 mm into the xylem tissue with a 4.3 mm diameter drill attached to a cordless drill. To inject the protective liquid solutions listed in **Table 1**, we used a Stinger needle for plugless trunk injection assembled on an individual feed line attached to the Tree IV air/hydraulic microinjection system, which operated at 60 psi air pressure (Arborjet Inc., Woburn, MA). The Stinger needles are used for injection of trees when trunk injection ports of large diameter (9.5 mm) are of concern or should be avoided and for injection of trunks with small diameters. The diameter of injection port for inserting a Stinger needle is smaller and does not require sealing with an Arborplug. In year one, the injected potted oak trees had trunk diameter at 5 cm height averaging 1.3 cm and ranging from 1 to 2.1 cm. In year two, a new set of injected trees had the diameter at 5 cm height averaging 1.5 cm and ranging from 1.1 to 2.2 cm. Trunk injection were

Trees were inoculated with *D. corticola* on 21 June and on 25 August, i.e. 9 days after injection of fungicides. Trunk bark on the opposite side from the injection port and 10 cm above the port was cut at three sides of rectangle to create a sleeve which was peeled longitudinally. A PDA plug 5 mm in diameter from 10-day-old colony of *D. corticola* isolate from our previous work [8] was placed in the sleeve on each injected tree and wrapped with parafilm (**Table 1**). Once the first symptoms of canopy wilt were observed, trees were destructively examined by stripping the bark off above and below the inoculation point. The necrosis length (cm) and width (cm) of Bot canker in xylem of the oak trunks were measured on 10 July in year one

multiplying the length and width for each individual tree and treatment mean was

Statistical analysis was done with MIXED procedure in SAS Studio software

With the goal to optimize timing and number of fungicide injections for management of apple scab fungus *V. inaequalis*, we conducted two experiments. In the experiment 1, we trunk-injected fungicides listed in **Table 2** on 29-year-old 'Mac Spur' apple trees four times, with the first injection applied in the fall of year one and the next three injections conducted in the spring of next, year two. In the experiment 1, the injection on 11 April was conducted at 50% apple bloom

(**Table 2**). In the experiment 2, we injected 29-year-old 'Mac Spur' apple trees with fungicides only one to two times in total, but by delivering them at different seasons i.e. in fall or spring, as per schedule listed in **Table 2**. In the experiment 2, the injection on 21 April was conducted at the silver tip growth stage of apple (**Table 2**). On each trunk injection date with fungicides listed in **Table 2**, a separate set of four cardinally-oriented trunk injection ports per each tree of 'Mac Spur' was created by drilling 25 mm into the xylem with a 9.5 mm diameter drill bit attached to a cordless drill. The first set of four injection ports was positioned ca. 25 cm above the ground level. The subsequent sets of four injection port were positioned ca. 5 cm above and between the lower four-port sets. Every port was sealed with Arborplug no. 4 (Arborjet

fungicide effect was found to be statistically significant (*p* < 0.1 in year one; *p* < 0.05 in year two), treatment comparisons were done with LSD test. We presented the fungicide management results as percent reduction of Bot canker necrosis area, also known as percent disease control, calculated as: percent reduction of necrosis area = [percent necrosis area in water control – percent necrosis area in specific

(SAS Institute Inc. 2017, Cary, NC) using the xylem necrosis areas (cm2

) was calculated by

). If the

conducted on 12 June in year one and on 16 August in year two.

*Plant Diseases-Current Threats and Management Trends*

and on 27 September in year two. Xylem necrosis area (cm2

treatment] 100/percent necrosis area in water control.

*2.2.2 Trunk injection of pesticides for Venturia inaequalis management*

calculated from six replicate trees.

**144**

*Fungicide treatments trunk-injected across two seasons and sprayed for management of apple scab fungus Venturia inaequalis on 'Mac Spur' apple trees.*

Inc., Woburn, MA) positioned just below the bark level to allow port closure with cambium callus [44]. To inject the fungicides, we used the Quik-jet microinjection system (Arborjet Inc.) operating at hand-generated hydraulic pressure to deliver low volumes of liquid for injection, thus allowing faster application times, and the Tree IV air/hydraulic microinjection system (Arborjet Inc.) operating at up to 60 psi of air pressure to deliver large solution volumes of liquid for injection (≥600 ml). In the experiment 1 (**Table 2**), we injected all the treatments listed for 15 October in year one with Quik-jet (**Table 2**). On 11 April in year two, we injected propiconazole using the Viper air/hydraulic microinjection system set at 90 psi air pressure (Arborjet Inc.). At the later dates, we injected Alamo using the Tree IV and Phosphojet using the Quik-jet. In the experiment 2, we injected Phosphojet with Quik-jet and cyprodinil + difenoconazole with Tree IV. The needle/s of each of the used injection devices was inserted through the Arborplugs allowing the total liquid volume per tree, at one injection time, to be divided and delivered equally among the four ports.

All the experiments were conducted under naturally high infection pressure during the primary season of *V. inaequalis* ascospore release in spring. In the experiment 1, we rated percent incidence of apple scab only on leaves, since fruits were lost due to spring frosts. In the experiment 2, we rated percent incidence of scab on leaves and fruit. A total of chose 20 spurs and 20 terminal shoots per tree were selected, with about five from each crown quadrant, and rated for leaf scab incidence. The fruit scab incidence was rated by selecting and rating 100 fruits per tree, with about 25 per crown quadrant, and if less was found we rated all the fruits per tree. The data were analyzed using MIXED procedure in SAS 9.3 (SAS Institute, Cary, NC). The tree was the subject of repeated measures. If the main effects or their interactions were found to be statistically significant (*p <* 0.05),


Actigard 1 was injected only on the first date in both years (**Table 3**). Each dose in every treatment, except the Phosphojet, was diluted and injected with 520 ml of water per tree. The doses per tree were chosen according to the four rules: (1) the dose was equivalent to the US EPA pesticide label rate for a maximum amount per 0.405 ha with 250 planted apple trees; (2) the dose was one half of the maximum US EPA label rate allowed per one season; (3) the dose was equal to a rate delivered in one spray application treatment per 0.405 ha with 250 apple trees; or (4) the dose was selected based on previous research with trunk injection of similar pesticides [116]. Trees injected with water and the non-injected non-inoculated trees served as negative controls for efficacy comparisons. In year one, each treatment was replicated on four trees arranged in a randomized complete block design, where blocking controlled the variable crown tree sizes (large, medium, medium-small, and small) [117]. In the year two, we used the same number of replicate trees per

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

In year one, on 16 April at 80% bloom, apple flowers of all experimental trees were inoculated with a suspension of *E. amylovora* strain in distilled water using a hand-sprayer (5.4 <sup>10</sup><sup>6</sup> CFU/ml; CFU—colony forming units). In year two, on 14 May at 80% bloom, flowers were inoculated with *E. amylovora* (0.7 <sup>10</sup><sup>6</sup> CFU/ml). In year one, we evaluated blossom blight incidence 22, 29 May and 5 June, and in year two on 11, 18, and 25 June. Rating of blossom blight incidence consisted of random selection of flower clusters to form a 100-cluster sample per tree and calculating the percent of diseased and healthy blossom clusters in that sample. Shoot blight incidence was evaluated on 29 May and 5 June in year one and in year two on 11, 18, and 25 June. After randomly selecting enough shoots to form a sample of 100-shoots per tree, shoot blight incidence percent was calculated for each tree from the number of blighted and healthy shoots. Mean percent of blossom and shoot blight incidences for each treatment were calculated from the disease incidences on four replicate trees. For clarity, presented means consist of four replicate trees averaged across two or three time points i.e. dates listed above when fire blight

We analyzed the data with MIXED procedure in SAS 9.3 (SAS Institute, 2012). The main effect of treatments on blossom and shoot blight incidence were analyzed using *F* test (α = 0.05) and if found significant, pairwise treatment comparisons were done using *t*-tests (*α* = 0.05). We presented the fire blight management results as percent of disease reduction, also known as percent disease control, calculated as: percent reduction of blossom or shoot blight incidence = [percent blossom or shoot blight incidence in water control – percent blossom or shoot blight incidence in specific treatment] 100/percent blossom or shoot blight incidence in water control.

To test the reduction of shoot blight severity with bactericide oxytetracycline hydrochloride (Arborbiotic, MFG Scientific Inc., EPA Reg. No 88482-1; Arbor-OTC® Injectable Tree Antibiotic, Arborjet Inc., Reg No. 74578-7), apple trunk injections were performed in a similar fashion described above, but by using a Quik-jet® micro-injection system instead (Arborjet Inc., Woburn, MA, USA). This device relies solely on hand-generated hydraulic pressure to inject the necessary pesticide solution volume in each port. The injection ports were created and sealed with Arborplugs (Arborjet Inc.) in the same way as described above and injected volume per tree was divided equally among the four ports. The experiments were conducted in 2 years. In year one, at petal fall growth stage (23 April) mature 12-year-old apple trees of cv. 'Jonathan' were trunk-injected with Arborbiotic using dose in **Table 3** diluted at 10% in water. The total dose per tree was calculated based

treatment but arranged in a completely randomized design (CRD).

incidences on flowers or shoots were rated.

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

*2.2.3.2 Treatments for reducing shoot blight severity*

**147**

#### **Table 3.**

*Trunk-injected treatments of bactericides and SAR-activators for management of fire blight bacterium Erwinia amylovora on flowers and shoots of 'Gala' and 'Jonathan' apple trees.*

examination, i.e. slicing of interactions within main effects was performed, *F*-tests conducted and pairwise or specific time or treatment comparisons were done with *t*-tests (*α* = 0.05). We presented the fungicide management results as percent of disease reduction, also known as percent disease control, calculated as: percent reduction of disease incidence = [percent disease incidence in water control – percent disease incidence in specific treatment] 100/percent disease incidence in water control.

#### *2.2.3 Trunk injection of pesticides for Erwinia amylovora management*

#### *2.2.3.1 Treatments for reducing blossom and shoot blight incidence*

To test the effect of injected bactericides and activators of plant systemic acquired resistance (SAR) for blossom and shoot blight incidence reduction, the orchard experiments were conducted over 2 years (**Table 3**). The early spring injections in year one (26 March) were conducted with Viper air/hydraulic microinjection system® at under 110 psi of air pressure and late spring injections (23 April) were done with Tree IV® air/hydraulic micro-injection system, at 60 psi air pressure (Arborjet Inc., Woburn, MA). In the year two, trunk injections on 1 and 22 May were applied using Tree IV® air/hydraulic micro-injection system at 60 psi of air pressure. The injection needles of these devices were inserted through the oneway valve silicone septum in the Arborplugs® which allowed delivery of protective solutions into he drilled injection ports. In each injection, the total injected volume per tree was divided equally among the four ports (**Table 3**). Four injection ports per each apple per tree, positioned ca. 10–15 cm above the ground level, were cardinally oriented and created by drilling 25 mm into the xylem tissue using a 9.5 mm diameter drill bit attached to a cordless drill. Each port was sealed with Arborplug® no. 4, by pushing the plug with a specialized screwdriver-like tapper hit with a hammer (Arborjet Inc., Woburn, MA, USA). The plug was positioned just below the bark level to allow port closure with cambium callus.

In the year one, we used 14-year-old 'Gala' apple trees which were trunkinjected using the compounds and dosages listed in **Table 3**. Injections were performed at the tight cluster growth stage in apples (26 March), or 21 days before 80% bloom, and at petal fall growth stage (23 April). In the year two, experiments were conducted on a new set of 21-year-old 'Gala' apple trees, injected with the same doses in **Table 3**. Injections were applied at early tight cluster growth stage (1 May) or 13 days before 80% bloom and at petal fall (22 May). The treatment

#### *Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

Actigard 1 was injected only on the first date in both years (**Table 3**). Each dose in every treatment, except the Phosphojet, was diluted and injected with 520 ml of water per tree. The doses per tree were chosen according to the four rules: (1) the dose was equivalent to the US EPA pesticide label rate for a maximum amount per 0.405 ha with 250 planted apple trees; (2) the dose was one half of the maximum US EPA label rate allowed per one season; (3) the dose was equal to a rate delivered in one spray application treatment per 0.405 ha with 250 apple trees; or (4) the dose was selected based on previous research with trunk injection of similar pesticides [116]. Trees injected with water and the non-injected non-inoculated trees served as negative controls for efficacy comparisons. In year one, each treatment was replicated on four trees arranged in a randomized complete block design, where blocking controlled the variable crown tree sizes (large, medium, medium-small, and small) [117]. In the year two, we used the same number of replicate trees per treatment but arranged in a completely randomized design (CRD).

In year one, on 16 April at 80% bloom, apple flowers of all experimental trees were inoculated with a suspension of *E. amylovora* strain in distilled water using a hand-sprayer (5.4 <sup>10</sup><sup>6</sup> CFU/ml; CFU—colony forming units). In year two, on 14 May at 80% bloom, flowers were inoculated with *E. amylovora* (0.7 <sup>10</sup><sup>6</sup> CFU/ml). In year one, we evaluated blossom blight incidence 22, 29 May and 5 June, and in year two on 11, 18, and 25 June. Rating of blossom blight incidence consisted of random selection of flower clusters to form a 100-cluster sample per tree and calculating the percent of diseased and healthy blossom clusters in that sample. Shoot blight incidence was evaluated on 29 May and 5 June in year one and in year two on 11, 18, and 25 June. After randomly selecting enough shoots to form a sample of 100-shoots per tree, shoot blight incidence percent was calculated for each tree from the number of blighted and healthy shoots. Mean percent of blossom and shoot blight incidences for each treatment were calculated from the disease incidences on four replicate trees. For clarity, presented means consist of four replicate trees averaged across two or three time points i.e. dates listed above when fire blight incidences on flowers or shoots were rated.

We analyzed the data with MIXED procedure in SAS 9.3 (SAS Institute, 2012). The main effect of treatments on blossom and shoot blight incidence were analyzed using *F* test (α = 0.05) and if found significant, pairwise treatment comparisons were done using *t*-tests (*α* = 0.05). We presented the fire blight management results as percent of disease reduction, also known as percent disease control, calculated as: percent reduction of blossom or shoot blight incidence = [percent blossom or shoot blight incidence in water control – percent blossom or shoot blight incidence in specific treatment] 100/percent blossom or shoot blight incidence in water control.

#### *2.2.3.2 Treatments for reducing shoot blight severity*

To test the reduction of shoot blight severity with bactericide oxytetracycline hydrochloride (Arborbiotic, MFG Scientific Inc., EPA Reg. No 88482-1; Arbor-OTC® Injectable Tree Antibiotic, Arborjet Inc., Reg No. 74578-7), apple trunk injections were performed in a similar fashion described above, but by using a Quik-jet® micro-injection system instead (Arborjet Inc., Woburn, MA, USA). This device relies solely on hand-generated hydraulic pressure to inject the necessary pesticide solution volume in each port. The injection ports were created and sealed with Arborplugs (Arborjet Inc.) in the same way as described above and injected volume per tree was divided equally among the four ports. The experiments were conducted in 2 years. In year one, at petal fall growth stage (23 April) mature 12-year-old apple trees of cv. 'Jonathan' were trunk-injected with Arborbiotic using dose in **Table 3** diluted at 10% in water. The total dose per tree was calculated based

examination, i.e. slicing of interactions within main effects was performed, *F*-tests conducted and pairwise or specific time or treatment comparisons were done with *t*-tests (*α* = 0.05). We presented the fungicide management results as percent of disease reduction, also known as percent disease control, calculated as: percent reduction of disease incidence = [percent disease incidence in water control – percent disease incidence in specific treatment] 100/percent disease incidence

*Trunk-injected treatments of bactericides and SAR-activators for management of fire blight bacterium Erwinia*

To test the effect of injected bactericides and activators of plant systemic acquired resistance (SAR) for blossom and shoot blight incidence reduction, the orchard experiments were conducted over 2 years (**Table 3**). The early spring injections in year one (26 March) were conducted with Viper air/hydraulic microinjection system® at under 110 psi of air pressure and late spring injections (23 April) were done with Tree IV® air/hydraulic micro-injection system, at 60 psi air pressure (Arborjet Inc., Woburn, MA). In the year two, trunk injections on 1 and 22 May were applied using Tree IV® air/hydraulic micro-injection system at 60 psi of air pressure. The injection needles of these devices were inserted through the oneway valve silicone septum in the Arborplugs® which allowed delivery of protective solutions into he drilled injection ports. In each injection, the total injected volume per tree was divided equally among the four ports (**Table 3**). Four injection ports per each apple per tree, positioned ca. 10–15 cm above the ground level, were cardinally oriented and created by drilling 25 mm into the xylem tissue using a 9.5 mm diameter drill bit attached to a cordless drill. Each port was sealed with Arborplug® no. 4, by pushing the plug with a specialized screwdriver-like tapper hit with a hammer (Arborjet Inc., Woburn, MA, USA). The plug was positioned just

*2.2.3 Trunk injection of pesticides for Erwinia amylovora management*

*2.2.3.1 Treatments for reducing blossom and shoot blight incidence*

*amylovora on flowers and shoots of 'Gala' and 'Jonathan' apple trees.*

*Plant Diseases-Current Threats and Management Trends*

below the bark level to allow port closure with cambium callus.

In the year one, we used 14-year-old 'Gala' apple trees which were trunkinjected using the compounds and dosages listed in **Table 3**. Injections were performed at the tight cluster growth stage in apples (26 March), or 21 days before 80% bloom, and at petal fall growth stage (23 April). In the year two, experiments were conducted on a new set of 21-year-old 'Gala' apple trees, injected with the same doses in **Table 3**. Injections were applied at early tight cluster growth stage (1 May) or 13 days before 80% bloom and at petal fall (22 May). The treatment

in water control.

**146**

**Table 3.**

on the unique trunk diameters at 30 cm height using the EPA label instructions. In year two, the same apple trees injected in year one were re-injected at petal fall (22 May) using the same dose in **Table 3** delivered via a fresh set of drilled injection ports above the previous year's set of injection ports. In both years, Arborbiotic treatment as well as water control were replicated on four trees arranged in a CRD.

24 June and 1, 8, and 15, 2013 July). The mean of shoot blight severity percent in per tree basis was calculated from the 10 shoot replicates. For each time point when the disease was rated, the average shoot blight severity was calculated from the four tree replicate means. For clarity, presented means consist of four replicate trees averaged across five or six time points i.e. dates listed above when fire blight

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

We analyzed the data using MIXED procedure in SAS 9.3 (SAS Institute, 2012). If the main effect of treatment on shoot blight severity was found significant (*F* test, α = 0.05), comparison to water control was conducted using re *t*-tests (*α* = 0.05). We presented the fire blight management results as percent of disease reduction, also known as percent disease control, calculated as: percent reduction of shoot blight severity = [percent shoot blight severity in water control – percent shoot blight severity in specific treatment] 100/percent shoot blight severity in water control.

All the three fungicides trunk-injected preventively provided significant reduction of Bot canker caused by *D. corticola* for 37.2–71.1% (**Figure 1**). Phosphojet at 1.5 ml per tree gave the best disease control when averaged across both years (58.5%) but caused phytotoxicity on four out of six tree replicates in year one and these trees died before the disease was rated. In year two, Phosphojet rate was reduced to 0.75 ml and this negative effect was not detected again. Averaged across both years, Arbotect provided the second-best control of 57.5%, followed by

In the experiment 1, fungicides injected four times in total, once in fall and then three additional times in spring, during the primary scab infection period, provided significant reduction of apple scab incidence on spur and shoot leaves (**Figure 2A**).

In the experiment 2, fungicides injected 1–2 times in total, across or within two seasons of fall and spring, revealed that the injected Inspire Super treatments largely did not significantly reduce disease incidence on spur and shoot leaves when compared to the water control. In contrast, all the injected Phosphojet treatments and Agrifos sprays did. Comparisons among these treatments clearly demonstrated that on all the three rated apple organs (**Figure 2B**), Phosphojet trunk injections provided statistically better apple scab reduction i.e. control in comparison to all the Inspire Super trunk injections. On spur leaves, two Phosphojet trunk injections, fall plus spring, was the best treatment among injections by providing 46.3% control which was similar to the Inspire Super sprays (**Figure 2B**). On shoot leaves, two Phosphojet trunk injections both done in spring, provided the best scab control of 66.5% similar to nine sprays of Agrifos (**Figure 2B**). On fruit, scab control was the best in Phosphojet trunk injection done once or twice in spring, and in fall plus

**3.1 Trunk injection of pesticides for** *Diplodia corticola* **management**

**3.2 Trunk injection of pesticides for** *Venturia inaequalis* **management**

On spur leaves, the best scab reduction of 45.5% was achieved with injected Phosphojet high, but this control was not better in comparison to 78.6% in spray standard applied in spring during the primary scab season (**Figure 2A**). In contrast, control with injected Phosphojet high on shoots outperformed the spray standard with 73.6 vs. 62.9% in scab reduction (**Figure 2A**). Similarly, Alamo performed

better on shoot leaved than on spur leaves (**Figure 2A**).

severity on shoots was rated.

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

Propizol with 53.3% (**Figure 1**).

**3. Results**

**149**

A total of 10 terminal shoots per each tree were inoculated on 7 May in year one and on 30 May in year two. We used a previously reported inoculation method [114]. In brief, the upper third of leaf blade of the second or the third youngest leaf on each shoot tip was cut perpendicular to the leaf midvein with scissors dipped in *E. amylovora* suspension (year one: 4.7 <sup>10</sup><sup>7</sup> CFU/ml; year two: 5 108 CFU/ml). An additional 10 shoots per each tree were wounded with scissors dipped in distilled water and used as an in-per-tree negative control. When the disease started developing on inoculated shoots, the length of shoot blight lesion (necrosis) and the total shoot length was measured for each inoculated shoot and the shoot blight severity percent was calculated by comparing the ratio of necrotic lesion length and the total shoot length (cm). Only the total shoot length was measured for negative control shoots. The shoot necrosis lesions and total shoot lengths were measured at 7-day intervals after inoculation and were ceased when terminal bud set on shoots occurred (year one: 14, 21, and 28 May and 4, 11, and 18 June; year two: 10, 17, and

#### **Figure 1.**

*Percent reduction i.e. control of Bot canker necrosis area in trunk xylem in relation to water control on Quercus rubra trees in year one (A) and year two (B) achieved with trunk injections of fungicides Propizol (propiconazole), Arbotect (thiabendazole) and Phosphojet (potassium phosphites). Means followed by different letters are significantly different (A: p < 0.1, LSD test; B: p < 0.05, LSD test). In year one (A), the area of Bot canker necrosis in trunk xylem in water control was 5.15 cm<sup>2</sup> and in year two (B) 5.8 cm<sup>2</sup> . Each mean consists of six replicate trees.*

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

24 June and 1, 8, and 15, 2013 July). The mean of shoot blight severity percent in per tree basis was calculated from the 10 shoot replicates. For each time point when the disease was rated, the average shoot blight severity was calculated from the four tree replicate means. For clarity, presented means consist of four replicate trees averaged across five or six time points i.e. dates listed above when fire blight severity on shoots was rated.

We analyzed the data using MIXED procedure in SAS 9.3 (SAS Institute, 2012). If the main effect of treatment on shoot blight severity was found significant (*F* test, α = 0.05), comparison to water control was conducted using re *t*-tests (*α* = 0.05). We presented the fire blight management results as percent of disease reduction, also known as percent disease control, calculated as: percent reduction of shoot blight severity = [percent shoot blight severity in water control – percent shoot blight severity in specific treatment] 100/percent shoot blight severity in water control.

#### **3. Results**

on the unique trunk diameters at 30 cm height using the EPA label instructions. In year two, the same apple trees injected in year one were re-injected at petal fall (22 May) using the same dose in **Table 3** delivered via a fresh set of drilled injection ports above the previous year's set of injection ports. In both years, Arborbiotic treatment as well as water control were replicated on four trees arranged in a CRD. A total of 10 terminal shoots per each tree were inoculated on 7 May in year one

*Plant Diseases-Current Threats and Management Trends*

and on 30 May in year two. We used a previously reported inoculation method [114]. In brief, the upper third of leaf blade of the second or the third youngest leaf on each shoot tip was cut perpendicular to the leaf midvein with scissors dipped in *E. amylovora* suspension (year one: 4.7 <sup>10</sup><sup>7</sup> CFU/ml; year two: 5 108 CFU/ml). An additional 10 shoots per each tree were wounded with scissors dipped in distilled water and used as an in-per-tree negative control. When the disease started developing on inoculated shoots, the length of shoot blight lesion (necrosis) and the total shoot length was measured for each inoculated shoot and the shoot blight severity percent was calculated by comparing the ratio of necrotic lesion length and the total shoot length (cm). Only the total shoot length was measured for negative control shoots. The shoot necrosis lesions and total shoot lengths were measured at 7-day intervals after inoculation and were ceased when terminal bud set on shoots occurred (year one: 14, 21, and 28 May and 4, 11, and 18 June; year two: 10, 17, and

*Percent reduction i.e. control of Bot canker necrosis area in trunk xylem in relation to water control on Quercus*

*. Each*

*rubra trees in year one (A) and year two (B) achieved with trunk injections of fungicides Propizol (propiconazole), Arbotect (thiabendazole) and Phosphojet (potassium phosphites). Means followed by different letters are significantly different (A: p < 0.1, LSD test; B: p < 0.05, LSD test). In year one (A), the area of Bot canker necrosis in trunk xylem in water control was 5.15 cm<sup>2</sup> and in year two (B) 5.8 cm<sup>2</sup>*

**Figure 1.**

**148**

*mean consists of six replicate trees.*

#### **3.1 Trunk injection of pesticides for** *Diplodia corticola* **management**

All the three fungicides trunk-injected preventively provided significant reduction of Bot canker caused by *D. corticola* for 37.2–71.1% (**Figure 1**). Phosphojet at 1.5 ml per tree gave the best disease control when averaged across both years (58.5%) but caused phytotoxicity on four out of six tree replicates in year one and these trees died before the disease was rated. In year two, Phosphojet rate was reduced to 0.75 ml and this negative effect was not detected again. Averaged across both years, Arbotect provided the second-best control of 57.5%, followed by Propizol with 53.3% (**Figure 1**).

#### **3.2 Trunk injection of pesticides for** *Venturia inaequalis* **management**

In the experiment 1, fungicides injected four times in total, once in fall and then three additional times in spring, during the primary scab infection period, provided significant reduction of apple scab incidence on spur and shoot leaves (**Figure 2A**). On spur leaves, the best scab reduction of 45.5% was achieved with injected Phosphojet high, but this control was not better in comparison to 78.6% in spray standard applied in spring during the primary scab season (**Figure 2A**). In contrast, control with injected Phosphojet high on shoots outperformed the spray standard with 73.6 vs. 62.9% in scab reduction (**Figure 2A**). Similarly, Alamo performed better on shoot leaved than on spur leaves (**Figure 2A**).

In the experiment 2, fungicides injected 1–2 times in total, across or within two seasons of fall and spring, revealed that the injected Inspire Super treatments largely did not significantly reduce disease incidence on spur and shoot leaves when compared to the water control. In contrast, all the injected Phosphojet treatments and Agrifos sprays did. Comparisons among these treatments clearly demonstrated that on all the three rated apple organs (**Figure 2B**), Phosphojet trunk injections provided statistically better apple scab reduction i.e. control in comparison to all the Inspire Super trunk injections. On spur leaves, two Phosphojet trunk injections, fall plus spring, was the best treatment among injections by providing 46.3% control which was similar to the Inspire Super sprays (**Figure 2B**). On shoot leaves, two Phosphojet trunk injections both done in spring, provided the best scab control of 66.5% similar to nine sprays of Agrifos (**Figure 2B**). On fruit, scab control was the best in Phosphojet trunk injection done once or twice in spring, and in fall plus

#### **Figure 2.**

*Percent reduction i.e. control of apple scab in relation to water control on 'Mac Spur' trees after in experiment 1 (A) and experiment 2 (B) achieved with trunk injections and sprays of potassium phosphites (Phosphojet, Arborfos) and of difenoconazole + cyprodinil (Inspire Super). Means within each graph section i.e. apple organ followed by different letters are significantly different (t-test, p < 0.05). F - one fall injection; 3S - three spring injections; S - one spring injection. In experiment 1 (A), scab incidences in water control on spur and shoot leaves were 72.2 and 54%. In experiment 2 (B) scab incidences in water control on spur leaves, shoot leaves and fruit were 88.3, 94.4 and 95.5%, respectively. Each mean consists of six replicate trees.*

blossom blight incidence in comparison to the water control (**Figure 3**). In year one, which had low disease pressure (**Figure 3A**), there was no significant difference among all the treatments in disease reduction i.e. control (37.9–61.1%). In year two, with high infection pressure, Agrimycin was the best providing 28.9% blossom blight control (**Figure 3B**). Averaged across both years, Agrimycin and then Phosphojet were the best treatments with 45 and 40.5% achieved control, respec-

*Percent reduction i.e. control of blossom blight incidence in relation to water control on 'Gala' apple trees in year one (A) and year two (B) achieved with one to two trunk injections of 'Gala' apple trees with Agrimycin (streptomycin), Phosphojet (potassium phosphites) and Actigard (acibenzolar-S-methyl). Means within each graph followed by different letters are significantly different (t-test, p < 0.05). Blossom blight incidence in water control in year one was 47.2% (A) and in year two 72.9% (B). Each mean consists of four replicate trees*

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

In year one, none of the trunk-injected products provided significant reduction of shoot blight incidence in comparison to the water control, hence did not differ among each other (**Figure 4A**). In year two, under high disease pressure, all the injected products significantly reduced shoot blight incidence for 23.4–36.5% in comparison to the water control, but when compared they did not significantly differ between each other (**Figure 4B**). If averaged across both years, Agrimycin and then Phosphojet

achieved the best control of 53.5 and 42.8%, respectively (**Figure 4**).

tively (**Figure 3**).

**151**

*averaged across three time points when disease was rated.*

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

**Figure 3.**

spring: 62.8, 69.7 and 64.6%, significantly outperforming both the Agrifos and the Inspire Super sprays (**Figure 2B**).

#### **3.3 Trunk injection of pesticides for** *Erwinia amylovora* **management**

#### *3.3.1 Treatments for reducing blossom and shoot blight incidence*

In both year one and year two, all the trunk-injected bactericides (Agrimycin) and SAR-activators (Actigard, Phosphojet) provided significant reduction of

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

**Figure 3.**

*Percent reduction i.e. control of blossom blight incidence in relation to water control on 'Gala' apple trees in year one (A) and year two (B) achieved with one to two trunk injections of 'Gala' apple trees with Agrimycin (streptomycin), Phosphojet (potassium phosphites) and Actigard (acibenzolar-S-methyl). Means within each graph followed by different letters are significantly different (t-test, p < 0.05). Blossom blight incidence in water control in year one was 47.2% (A) and in year two 72.9% (B). Each mean consists of four replicate trees averaged across three time points when disease was rated.*

blossom blight incidence in comparison to the water control (**Figure 3**). In year one, which had low disease pressure (**Figure 3A**), there was no significant difference among all the treatments in disease reduction i.e. control (37.9–61.1%). In year two, with high infection pressure, Agrimycin was the best providing 28.9% blossom blight control (**Figure 3B**). Averaged across both years, Agrimycin and then Phosphojet were the best treatments with 45 and 40.5% achieved control, respectively (**Figure 3**).

In year one, none of the trunk-injected products provided significant reduction of shoot blight incidence in comparison to the water control, hence did not differ among each other (**Figure 4A**). In year two, under high disease pressure, all the injected products significantly reduced shoot blight incidence for 23.4–36.5% in comparison to the water control, but when compared they did not significantly differ between each other (**Figure 4B**). If averaged across both years, Agrimycin and then Phosphojet achieved the best control of 53.5 and 42.8%, respectively (**Figure 4**).

spring: 62.8, 69.7 and 64.6%, significantly outperforming both the Agrifos and the

*Percent reduction i.e. control of apple scab in relation to water control on 'Mac Spur' trees after in experiment 1 (A) and experiment 2 (B) achieved with trunk injections and sprays of potassium phosphites (Phosphojet, Arborfos) and of difenoconazole + cyprodinil (Inspire Super). Means within each graph section i.e. apple organ followed by different letters are significantly different (t-test, p < 0.05). F - one fall injection; 3S - three spring injections; S - one spring injection. In experiment 1 (A), scab incidences in water control on spur and shoot leaves were 72.2 and 54%. In experiment 2 (B) scab incidences in water control on spur leaves, shoot leaves and*

In both year one and year two, all the trunk-injected bactericides (Agrimycin)

**3.3 Trunk injection of pesticides for** *Erwinia amylovora* **management**

*fruit were 88.3, 94.4 and 95.5%, respectively. Each mean consists of six replicate trees.*

*Plant Diseases-Current Threats and Management Trends*

and SAR-activators (Actigard, Phosphojet) provided significant reduction of

*3.3.1 Treatments for reducing blossom and shoot blight incidence*

Inspire Super sprays (**Figure 2B**).

**Figure 2.**

**150**

*3.3.2 Treatments for reducing shoot blight severity*

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

(**Figure 5**).

**4. Discussion**

sulphate [20].

on larger trees.

**153**

caused by *Apiognomonia veneta*).

**4.1** *Diplodia corticola*

In both years Arborbiotic provided significant reduction i.e. control of shoot blight severity in comparison to the water control (**Figure 5**). When averaged across both years, the control of shoot blight severity reached 72.4%

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

We present the first data on management of *D. corticola* on northern red oak using fungicides thiabendazole, propiconazole and potassium phosphites delivered by trunk injection as an alternative pesticide application method which offers selective exposure of this and other wood pathogens to the injected compounds. Since this fungus invades and spreads via tree xylem on different oak species as hardwood trees and causes necrosis and vascular occlusion [7, 8], ultimately killing the tree, trunk injection of fungicides seems as the most suitable fungicide delivery method for this pathogen's biology and likely more effective for managing the resulting Bot canker disease. The achieved levels in control of Bot canker in xylem ranged from 37.2 to 71.1% with an overall average of 56.4% across all the fungicides we trunk injected. Phosphojet provided control of 58.5%, but the most reliable fungicides and across-years consistent were Arbotect (thiabendazole) and Propizol (propiconazole) which achieved control of 57.5 and 53.3%. A higher efficacy was not achieved probably because of the short time between fungicide injection and inoculation with *D. corticola* which could have reduced the uniformity in distribution of these fungicides in xylem, thus hampering the efficacy. On the debarked cork oak trees and under moist conditions, the canker length caused by *D. corticola* is reduced for 25.8–98.5% by preventive spray applications of thiophanate-methyl and/or copper-calcium sulphate, delivered immediately after the cork peeling [20]. On average, across different test locations, Bot canker control in this study was 64.7% with thiophanate-methyl and/or copper-calcium

The organic carbon-water partitioning coefficient (Ko/c) for thiabendazole is moderate to high and ranges from 1104 to 4680 ml/g, while water solubility is 50 mg/L at pH 7 and 38 mg/ml at pH 2 [118]. These parameters indicate on low to no mobility of thianbendazole in xylem as a carbon rich environment. The Ko/c of propiconazole is 1086–1817 ml/g which is moderate to high [119, 120] and water solubility is low, 100–150 mg/L [121]. This could have contributed to slow and reduced uniformity in distribution of injected fungicides in xylem. However, both Arbotect and Propizol are fungicides formulated for trunk injection on hardwood trees and if properly diluted and delivered preventively they can accumulate sufficiently to secure the internal control of specific plant diseases (e.g. Dutch elm disease caused by *O. ulmi* and *O. novo-ulmi*; sycamore/London plane anthracnose

In the future studies, we predict that the efficacy of preventive fungicide applications against *D. corticola* via trunk injection delivery can be increased: (1) with more time allowed between injection and infection with *D. corticola*, (2) with more injections per season, and (3) a larger dose per tree. These factors should allow continued and better distribution of these fungicides in the wood xylem and canopy and probably secure the higher fungicide efficacy in Bot canker control, especially

#### **Figure 4.**

*Percent reduction i.e. control of shoot blight incidence in relation to water control on 'Gala' apple trees in year one (A) and year two (B) achieved with one to two trunk injections of Agrimycin (streptomycin), Phosphojet (potassium phosphites) and Actigard (acibenzolar-S-methyl). (A) In year one, the injected treatments did not significantly reduce shoot blight incidence relative to water control. (B) Means followed by different letters are significantly different (t-test, p < 0.05). Soot blight incidence in water control in year one was 22.4% (A) and in year two 68.5% (B). Each mean consists of 4 replicate trees averaged across two time points in (A) and three time points in (B) when disease was rated.*

#### **Figure 5.**

*Percent reduction i.e. control of shoot blight severity relative to water control achieved from a single trunk injection of 'Jonathan' apple trees with Arborbiotic (oxytetracycline hydrochloride) in each year. Means with an asterisk indicate significant reduction of shoot blight severity (year one: Tukey's HSD test; year two: t-test, p < 0.05). Each mean consists of four replicate trees averaged across five time points in year 1 and six time points in year 2 when disease was rated.*

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

#### *3.3.2 Treatments for reducing shoot blight severity*

In both years Arborbiotic provided significant reduction i.e. control of shoot blight severity in comparison to the water control (**Figure 5**). When averaged across both years, the control of shoot blight severity reached 72.4% (**Figure 5**).

#### **4. Discussion**

#### **4.1** *Diplodia corticola*

We present the first data on management of *D. corticola* on northern red oak using fungicides thiabendazole, propiconazole and potassium phosphites delivered by trunk injection as an alternative pesticide application method which offers selective exposure of this and other wood pathogens to the injected compounds. Since this fungus invades and spreads via tree xylem on different oak species as hardwood trees and causes necrosis and vascular occlusion [7, 8], ultimately killing the tree, trunk injection of fungicides seems as the most suitable fungicide delivery method for this pathogen's biology and likely more effective for managing the resulting Bot canker disease. The achieved levels in control of Bot canker in xylem ranged from 37.2 to 71.1% with an overall average of 56.4% across all the fungicides we trunk injected. Phosphojet provided control of 58.5%, but the most reliable fungicides and across-years consistent were Arbotect (thiabendazole) and Propizol (propiconazole) which achieved control of 57.5 and 53.3%. A higher efficacy was not achieved probably because of the short time between fungicide injection and inoculation with *D. corticola* which could have reduced the uniformity in distribution of these fungicides in xylem, thus hampering the efficacy. On the debarked cork oak trees and under moist conditions, the canker length caused by *D. corticola* is reduced for 25.8–98.5% by preventive spray applications of thiophanate-methyl and/or copper-calcium sulphate, delivered immediately after the cork peeling [20]. On average, across different test locations, Bot canker control in this study was 64.7% with thiophanate-methyl and/or copper-calcium sulphate [20].

The organic carbon-water partitioning coefficient (Ko/c) for thiabendazole is moderate to high and ranges from 1104 to 4680 ml/g, while water solubility is 50 mg/L at pH 7 and 38 mg/ml at pH 2 [118]. These parameters indicate on low to no mobility of thianbendazole in xylem as a carbon rich environment. The Ko/c of propiconazole is 1086–1817 ml/g which is moderate to high [119, 120] and water solubility is low, 100–150 mg/L [121]. This could have contributed to slow and reduced uniformity in distribution of injected fungicides in xylem. However, both Arbotect and Propizol are fungicides formulated for trunk injection on hardwood trees and if properly diluted and delivered preventively they can accumulate sufficiently to secure the internal control of specific plant diseases (e.g. Dutch elm disease caused by *O. ulmi* and *O. novo-ulmi*; sycamore/London plane anthracnose caused by *Apiognomonia veneta*).

In the future studies, we predict that the efficacy of preventive fungicide applications against *D. corticola* via trunk injection delivery can be increased: (1) with more time allowed between injection and infection with *D. corticola*, (2) with more injections per season, and (3) a larger dose per tree. These factors should allow continued and better distribution of these fungicides in the wood xylem and canopy and probably secure the higher fungicide efficacy in Bot canker control, especially on larger trees.

**Figure 4.**

**Figure 5.**

**152**

*time points in (B) when disease was rated.*

*Plant Diseases-Current Threats and Management Trends*

*points in year 2 when disease was rated.*

*Percent reduction i.e. control of shoot blight incidence in relation to water control on 'Gala' apple trees in year one (A) and year two (B) achieved with one to two trunk injections of Agrimycin (streptomycin), Phosphojet (potassium phosphites) and Actigard (acibenzolar-S-methyl). (A) In year one, the injected treatments did not significantly reduce shoot blight incidence relative to water control. (B) Means followed by different letters are significantly different (t-test, p < 0.05). Soot blight incidence in water control in year one was 22.4% (A) and in year two 68.5% (B). Each mean consists of 4 replicate trees averaged across two time points in (A) and three*

*Percent reduction i.e. control of shoot blight severity relative to water control achieved from a single trunk injection of 'Jonathan' apple trees with Arborbiotic (oxytetracycline hydrochloride) in each year. Means with an asterisk indicate significant reduction of shoot blight severity (year one: Tukey's HSD test; year two: t-test, p < 0.05). Each mean consists of four replicate trees averaged across five time points in year 1 and six time*

#### **4.2** *Venturia inaequalis*

We evaluated the similar fungicides on apple, *M. pumila*, another hardwood tree species. When Phosphojet and Inspire Super, where the latter one contains a DMI (demethylation inhibitor) fungicide difenoconazole from the same class as Alamo or Propizol (propiconazole), were trunk-injected for management of apple scab fungus *V. inaequalis*, the best control was achieved with Phosphojet and then by Alamo.

and injectable formulation are possible for one active ingredient, a rapid and desired

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

The reduction of apple scab and our prior work on analyzing the residues of injected pesticides on apple leaves and fruit [12, 61] indicates that accumulation of trunk-injected fungicides in the wood and canopy is a time-demanding process chiefly shaped by the tree physiology and tissue resistance points [127, 128]. Trunk injection is an opposite process to the immediate deposition of fungicide solution on the tree canopy by foliar spray applications. However, even though the injected dose per tree of phosphites in Phosphojet was 1.6–2 times higher than in the Agrifos sprays, the fact that just two injections secured better control of scab on fruit and spur leaves in comparison to nine Agrifos sprays demonstrated better persistence of injected Phosphojet. This shows that trunk injection is a superior delivery method

control effect on plant pathogen or insect pest can be expected [42, 45, 126].

for phosphites as it enhances their activity for 1–2 growing seasons [12].

The fire blight bacterium *E. amylovora* is a pathogen of apple trees with a unique and complicated biology involving several lifestyles: (1) *in planta* overwintering in fire blight cankers on bark or asymptomatically in host buds or as latent infections in asymptomatic wood [101], (2) residing on different plant surfaces and colonizing flower surfaces before their infection, and (3) migration after infection to other close or far host tissues and organs through colonizing the cortical parenchyma and xylem vessels. Therefore, it seems that for the stages of pathogen overwintering in wood or bark and especially for migration via xylem, the use of trunk injection delivery of compounds active against *E. amylovora* might be the most suitable way to control this pathogen. Overall, our trunk injection experiments with antibiotic bactericides, Phospojet and Actigard, both known SAR-activators [11], demonstrated good to poor fire blight incidence reduction in years with low and high

The best control i.e. reduction of blossom blight incidence across both trial years

Vegetative flowers parts in *Malus* species and later fruit have 10- to 100-fold lower frequency of stomata on epidermis when compared to the epidermis of leaves [122]. Flowers also have a considerably smaller green tissue volume. This leads to a conclusion that due to a very low transpiration footprint of green flower parts with lower number of stomata in comparison to the leaves and fruit, accumulation of injected compounds in these parts was weaker and slow thus reducing their efficacy. Second, it is possible that the injected antibiotics could not reach the surface of stigmas where *E. amylovora* multiplies to reduce its populations as successfully as

was achieved with two trunk injections of Agrimycin (45%) and of Phosphojet (40.5%). However, under high and low infection pressures in the two trial years, the levels of control with these materials (28.9, 61.1%, 25.1, 55.9) were far from comparable to 92–99% control often achieved and expected with preventive flower spray application of Agrimycin and Kasugamycin in commercial apple orchards [129, 130]. In the case of injected Phosphojet and Actigard, the achieved blossom blight reduction probably originated from an SAR effect triggered in the nearby spur leaves by these compounds, as the SAR effect in flowers was inconsistent [11]. SAR is a defense plant response which is activated after localized plant exposure to a pathogen or after a spray applications of a synthetic or natural compound, known as an SAR-inducer or activator [131]. Our 1–2 trunk injections of Actigard reduced blossom blight incidence for only 19–42%, indicating that this delivery method cannot not improve the SAR-effect of Actigard on flowers to combat blossom blight successfully. Namely, different sources report from 3 to 91% of blossom blight control with foliar sprays of Actigard on other apple cultivars [132–134].

**4.3** *Erwinia amylovora*

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

infection pressures, respectively.

**155**

The efficacy against this subcuticular pathogen that infects just below the waxy layer on leaves and fruit, clearly depended on the apple canopy organ and the time/s of fungicide injection/s. Namely, on spurs which hold much fewer leaves in total in comparison to the shoots, the best leaf scab incidence reduction was 45.5 and 46.3%. In contrast, scab reduction on shoot leaves with Phosphojet reached 66.5 and 73.6%. On apple fruit, scab reduction reached up to 62.8, 64.6 and 69.7%. These efficacy patterns clearly demonstrate the differential influences of the tree's yearly and organ-specific physiology, the properties of injected compound, and the injection timing on the accumulation of fungicides in the canopy. Since the major water transport in xylem, occurs in spring, at least one to two injections of phosphites in early spring gave a good disease control, depending on the canopy organ. The best scab control with injected phosphites was achieved on the shoot leaves, followed by apple fruit, and then on the spur leaves. The injected phosphites probably accumulated more in the shoot leaves than in the spur leaves and they accumulate more in fruit than in spur leaves. This can be explained by the variable rates of transpiration from these organs, which influences the speed and abundance of fungicide accumulation after trunk injection. The total leaf area on shoots is larger in comparison to spurs. The fewer leaves on spurs, which are first to develop in spring and early reach their full size, have fewer total number of stomata on them in comparison to more numerous shoot leaves. Additionally, from petal fall up until terminal bud set, shoots keep growing and developing more leaves on the tips. Hence, apple shoots hold the higher number of stomata in total, thus allowing much higher transpiration intensity, abundant accumulation of injected fungicides and thus scab control. Similarly, apple scab control was lower on fruit than on shoots which could be explained by the fact that apple fruit hold 10- to 100-fold lower frequency of stomata on their epidermis in comparison to the apple leaves [122].

The chemical properties of different active ingredients impact their distribution and accumulation in the canopy. For example, potassium phosphites have higher water solubility of 500 g/L in comparison to propiconazole and difenoconazole which have low to very low water solubilities of 100–150 mg/L and 13 mg/L, respectively [121, 123]. Potassium phosphites have low organic carbon-water partitioning coefficient (Ko/c) from 228 to 587 ml/g in comparison to moderate to high of propiconazole, 1086–1817 ml/g, and of difenoconazole, 3870–11,202 ml/g, respectively [119, 120]. This difference likely allowed phosphites to move faster in xylem [124] and accumulate more in leaves and fruit than the other injected fungicides. At the same time, propiconazole and difenoconazole were probably bound to the organic phase of xylem symplast and apoplast, thus lowering their accumulation in leaves and fruit and reducing their effect on scab incidence [65]. This is often referred to as a reservoir effect and Ko/c as is an important property of a pesticide that can explains its limited or abundant accumulation in the canopy [65, 125]. Besides the Ko/c and water solubility, the inactive components of the Inspire Super pesticide formulation we injected (stickers, emulsifiers, surfactants, etc.) could reduce the abundant accumulation of difenoconazole and a better scab control. Fungicides have to be formulated for injection to secure their upward translocation in xylem and often diluted prior to trunk injection to reduce the impact of Ko/c effect. Once the high solubility, low Ko/c

#### *Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

and injectable formulation are possible for one active ingredient, a rapid and desired control effect on plant pathogen or insect pest can be expected [42, 45, 126].

The reduction of apple scab and our prior work on analyzing the residues of injected pesticides on apple leaves and fruit [12, 61] indicates that accumulation of trunk-injected fungicides in the wood and canopy is a time-demanding process chiefly shaped by the tree physiology and tissue resistance points [127, 128]. Trunk injection is an opposite process to the immediate deposition of fungicide solution on the tree canopy by foliar spray applications. However, even though the injected dose per tree of phosphites in Phosphojet was 1.6–2 times higher than in the Agrifos sprays, the fact that just two injections secured better control of scab on fruit and spur leaves in comparison to nine Agrifos sprays demonstrated better persistence of injected Phosphojet. This shows that trunk injection is a superior delivery method for phosphites as it enhances their activity for 1–2 growing seasons [12].

#### **4.3** *Erwinia amylovora*

**4.2** *Venturia inaequalis*

*Plant Diseases-Current Threats and Management Trends*

Alamo.

**154**

We evaluated the similar fungicides on apple, *M. pumila*, another hardwood tree species. When Phosphojet and Inspire Super, where the latter one contains a DMI (demethylation inhibitor) fungicide difenoconazole from the same class as Alamo or Propizol (propiconazole), were trunk-injected for management of apple scab fungus *V. inaequalis*, the best control was achieved with Phosphojet and then by

The efficacy against this subcuticular pathogen that infects just below the waxy layer on leaves and fruit, clearly depended on the apple canopy organ and the time/s of fungicide injection/s. Namely, on spurs which hold much fewer leaves in total in comparison to the shoots, the best leaf scab incidence reduction was 45.5 and 46.3%. In contrast, scab reduction on shoot leaves with Phosphojet reached 66.5 and 73.6%. On apple fruit, scab reduction reached up to 62.8, 64.6 and 69.7%. These efficacy patterns clearly demonstrate the differential influences of the tree's yearly and organ-specific physiology, the properties of injected compound, and the injection timing on the accumulation of fungicides in the canopy. Since the major water transport in xylem, occurs in spring, at least one to two injections of phosphites in early spring gave a good disease control, depending on the canopy organ. The best scab control with injected phosphites was achieved on the shoot leaves, followed by apple fruit, and then on the spur leaves. The injected phosphites probably accumulated more in the shoot leaves than in the spur leaves and they accumulate more in fruit than in spur leaves. This can be explained by the variable rates of transpiration from these organs, which influences the speed and abundance of fungicide accumulation after trunk injection. The total leaf area on shoots is larger in comparison to spurs. The fewer leaves on spurs, which are first to develop in spring and early reach their full size, have fewer total number of stomata on them in comparison to more numerous shoot leaves. Additionally, from petal fall up until terminal bud set, shoots keep growing and developing more leaves on the tips. Hence, apple shoots hold the higher number of stomata in total, thus allowing much higher transpiration intensity, abundant accumulation of injected fungicides and thus scab control. Similarly, apple scab control was lower on fruit than on shoots which could be explained by the fact that apple fruit hold 10- to 100-fold lower frequency of

stomata on their epidermis in comparison to the apple leaves [122].

The chemical properties of different active ingredients impact their distribution and accumulation in the canopy. For example, potassium phosphites have higher water solubility of 500 g/L in comparison to propiconazole and difenoconazole which have low to very low water solubilities of 100–150 mg/L and 13 mg/L, respectively [121, 123]. Potassium phosphites have low organic carbon-water partitioning coefficient (Ko/c) from 228 to 587 ml/g in comparison to moderate to high of propiconazole, 1086–1817 ml/g, and of difenoconazole, 3870–11,202 ml/g, respectively [119, 120]. This difference likely allowed phosphites to move faster in xylem [124] and accumulate more in leaves and fruit than the other injected fungicides. At the same time, propiconazole and difenoconazole were probably bound to the organic phase of xylem symplast and apoplast, thus lowering their accumulation in leaves and fruit and reducing their effect on scab incidence [65]. This is often referred to as a

reservoir effect and Ko/c as is an important property of a pesticide that can explains its limited or abundant accumulation in the canopy [65, 125]. Besides the Ko/c and water solubility, the inactive components of the Inspire Super pesticide formulation we injected (stickers, emulsifiers, surfactants, etc.) could reduce the abundant accumulation of difenoconazole and a better scab control. Fungicides have to be formulated for injection to secure their upward translocation in xylem and often diluted prior to trunk injection to reduce the impact of Ko/c effect. Once the high solubility, low Ko/c

The fire blight bacterium *E. amylovora* is a pathogen of apple trees with a unique and complicated biology involving several lifestyles: (1) *in planta* overwintering in fire blight cankers on bark or asymptomatically in host buds or as latent infections in asymptomatic wood [101], (2) residing on different plant surfaces and colonizing flower surfaces before their infection, and (3) migration after infection to other close or far host tissues and organs through colonizing the cortical parenchyma and xylem vessels. Therefore, it seems that for the stages of pathogen overwintering in wood or bark and especially for migration via xylem, the use of trunk injection delivery of compounds active against *E. amylovora* might be the most suitable way to control this pathogen. Overall, our trunk injection experiments with antibiotic bactericides, Phospojet and Actigard, both known SAR-activators [11], demonstrated good to poor fire blight incidence reduction in years with low and high infection pressures, respectively.

The best control i.e. reduction of blossom blight incidence across both trial years was achieved with two trunk injections of Agrimycin (45%) and of Phosphojet (40.5%). However, under high and low infection pressures in the two trial years, the levels of control with these materials (28.9, 61.1%, 25.1, 55.9) were far from comparable to 92–99% control often achieved and expected with preventive flower spray application of Agrimycin and Kasugamycin in commercial apple orchards [129, 130]. In the case of injected Phosphojet and Actigard, the achieved blossom blight reduction probably originated from an SAR effect triggered in the nearby spur leaves by these compounds, as the SAR effect in flowers was inconsistent [11]. SAR is a defense plant response which is activated after localized plant exposure to a pathogen or after a spray applications of a synthetic or natural compound, known as an SAR-inducer or activator [131]. Our 1–2 trunk injections of Actigard reduced blossom blight incidence for only 19–42%, indicating that this delivery method cannot not improve the SAR-effect of Actigard on flowers to combat blossom blight successfully. Namely, different sources report from 3 to 91% of blossom blight control with foliar sprays of Actigard on other apple cultivars [132–134].

Vegetative flowers parts in *Malus* species and later fruit have 10- to 100-fold lower frequency of stomata on epidermis when compared to the epidermis of leaves [122]. Flowers also have a considerably smaller green tissue volume. This leads to a conclusion that due to a very low transpiration footprint of green flower parts with lower number of stomata in comparison to the leaves and fruit, accumulation of injected compounds in these parts was weaker and slow thus reducing their efficacy. Second, it is possible that the injected antibiotics could not reach the surface of stigmas where *E. amylovora* multiplies to reduce its populations as successfully as

after the topical spray application, or that they do reach stigma surfaces but at a too low of a dose or too late for a better reduction. The reached levels of control with the injected Agrimycin probably originated from the limited accumulation i.e. presence of a suboptimal dose of this antibiotic in the green flower tissues. This only partially stopped the progress of the infection once *E. amylovora* entered the flower tissues. Therefore, the injected compounds aiming to reduce blossom blight should be formulated to translocate and accumulate faster in flower green tissues to reach a potentially higher efficacy. Otherwise, these should be injected much earlier in comparison to our injection dates, probably in fall of the previous year, to increase the time for compound accumulation and ultimately improve the disease reduction. A process of optimizing the trunk injection timing/s is a common topic research on agricultural tree crops to maximize the effect in pathogen or pest control [10, 12, 50, 56]. It appears that higher dose of injected compounds might be necessary for longer-lasting control of fire blight on both flowers and shoots.

**5. Conclusion**

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

thus increasing their efficacy.

**157**

Our results on management of three different pathogens with partially similar or different biologies, where *D. corticola* and *E. amylovora* invade and spread in xylem while *V. inaequalis* does not and infects subcuticularly, indicate that trunk injection of pesticides that are formulated for xylem translocation can be more-less similar in control of these three pathogens. However, the interaction of chemical properties of the active ingredient, the injected dose per tree, as well as the transpiration footprint of plant organs, played the key roles that determined the achieved levels of efficacy.

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

In the biology i.e. life cycle of *D. corticola*, it seems that the dominant phase is the invasion and necrosis of xylem, leading to vascular occlusion, canopy wilting and canker development on wood before it kills oak trees. Hence the logical approach to prevent this disease is trunk injection delivery of fungicides. In our two-year experiments on potted trees, the injected potassium phosphites (Phosphojet) achieved levels of Bot canker control in xylem of up to 71.1%. Averaged across both years, potassium phosphites achieved disease reduction of 58.5%, but the more consistent results were achieved with fungicides thiabendazole (Arbotect) and propiconazole (Propizol) which reduced xylem necrosis for 57.5 and 53.3% on average. The maximums in reduction in individual years for these two fungicides were of 60.5 and 69.3%, respectively. We predict that higher efficacy with these fungicides can be achieved with optimization of preventive fungicide injection which would increase the uniformity of distribution of these fungicides in xylem,

In the case of *V. inaequalis*, for which the injected fungicides would need to translocate the farthest via xylem to reach and accumulate in and on the epidermal cells of green plant surfaces in tree canopy, the most efficient apple scab reduction of 45.5– 73.6% was achieved with potassium phosphites (Phosphojet). Unlike this readily mobile compound, scab control with propiconazole that is much less xylem mobile

Finally, there is the case of a complex biology of *E. amylovora* which combines life stages of inhabiting and multiplying on plant surfaces, migrating through internal host tissues after infecting, dwelling and overwintering asymptomatically in host buds or wood, and overwintering in fire blight cankers on bark. The injected compounds active against this pathogen would need to translocate and distribute in in xylem and phloem, reach in and onto the stigma surfaces of flowers and accumulate at effective doses in these and green tissues of the apple tree canopy. Based on presented research, it seems that these multiple difficult tasks in this and our previous study [44] were best achieved with oxytetracycline hydrochloride—both on the apple flowers [44] and on shoots [11]. Overall the injected antibiotic streptomycin (Agrimycin) formulated for foliar application gave the best reduction of blossom blight ranging from 28.9 to 61.1% and of shoot blight from 36.5 to 70.4%. The shoot blight severity reduction with Arboriotic, the injectable formulation of oxytetracycline hydrochloride, reached an excellent 82%. Hence, the effect depended on the plant organ, bactericide active ingredient, injected dose and formulation. The SAR-activating potassium phosphites (Phosphojet) were the second best to antibiotics with 25.1 and 55.9% of blossom blight reduction and 23.4 and

ranged from 17.1 to 51.5%, while the least xylem mobile difenoconazole

thus securing higher accumulation in tissues exposed to infection.

underperformed with only up to 10.8% apple scab control. It is assumed that the injected potassium phosphites secured its efficacy against *V. inaequalis* through a strong plant defense response in the tissues called SAR [11, 139], as apparently it is not directly toxic to this pathogen [140]. We speculate that better efficacy with other systemic fungicides active against apple scab might be achieved if their formulations were redesigned for trunk injection i.e. to facilitate easier and faster translocation in xylem,

Even though reduction of shoot blight incidence was not statistically significant in year one, which was characterized with low infection pressure, it indicated that trunkinjected Agrimycin and Phosphojet might have potential to perform better than Actigard treatments. However, in year two, under the heavy infection pressure, this was not the case as all the injected treatments were similar. Overall, it seems that the reduction of shoot blight incidence with injected Agrimycin and Phosphojet across both years of 53.5 and 42.8%, was slightly better than the reduction of blossom blight incidence with the same materials of 45 and 40.5%, respectively. Shoots obviously have much higher green tissue area and transpiration rate in comparison to the flowers. Shoots likely accumulate higher amounts of trunk injected compounds in comparison to the green flower parts, which allowed slightly better disease reduction early after injection. Still, the shoot blight incidence reduction was far from the expected control with spray applied antibiotics in commercial apple orchards. In a trial with trunk injection of Arborfos (45.8% mono- and di-potassium salts of phosphorous acid, Mauget Inc., Arcadia, CA, USA), shoot blight was reduced for 67% on inoculated 'Paulared' apple trees [116]. The same dose per tree which we delivered in two injections of Phosphojet, achieved shoot blight incidence reduction of 23.4–62.1%. Since we have split the dose delivery temporally, this weakened shoot blight incidence reduction by Phosphojet and probably by Actigard too. In shoot inoculated trials multiple Actigard sprays achieved shoot blight reduction between 2.8 and 50.7% [135, 136] while by trunk injection we achieved only 1.7–30.9% of shoot blight reduction. Hence, the two-time trunk injection does not improve shoot blight reduction by Actigard.

The reduction of shoot blight severity with Arborbiotic (MFG Scientific Inc., USA) was excellent and reached up to 82%. Such an effect with oxytetracycline hydrochloride demonstrates that this active ingredient is readily soluble in water and that the formulation we used is designed for trunk injection. Our results indicated that the trunk injected Arborbiotic limits i.e. stops systemic spread of *E. amylovora* in xylem of apple shoots [11]. Even though oxytetracycline hydrochloride is a bacteriostatic, when we delivered it via trunk injection in apple trees only one time per year, it demonstrated prolonged effectiveness that was higher in comparison to spray applications [44, 137, 138]. Trunk injection delivery enhanced the efficacy of oxytetracycline hydrochloride in control of shoot blight severity. Finally, in our prior work we also showed that the injected Arborbiotic at a dose of 0.31 g + 2.52 ml water per each 2.5 cm of trunk diameter at 30.5 cm height, can achieve a formidable reduction of blossom and shoot blight incidence for 60.6 and 60.7%, respectively [44]. This indicates that this bactericide in this formulation and probably at a slightly higher dose is the best candidate to achieve satisfactory accumulation inside and deposition on the susceptible apple plant tissues and surfaces to secure the higher efficacy.

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

#### **5. Conclusion**

after the topical spray application, or that they do reach stigma surfaces but at a too low of a dose or too late for a better reduction. The reached levels of control with the injected Agrimycin probably originated from the limited accumulation i.e. presence of a suboptimal dose of this antibiotic in the green flower tissues. This only partially stopped the progress of the infection once *E. amylovora* entered the flower tissues. Therefore, the injected compounds aiming to reduce blossom blight should be formulated to translocate and accumulate faster in flower green tissues to reach a potentially higher efficacy. Otherwise, these should be injected much earlier in comparison to our injection dates, probably in fall of the previous year, to increase the time for compound accumulation and ultimately improve the disease reduction. A process of optimizing the trunk injection timing/s is a common topic research on agricultural tree crops to maximize the effect in pathogen or pest control [10, 12, 50, 56]. It appears that higher dose of injected compounds might be necessary for

Even though reduction of shoot blight incidence was not statistically significant in year one, which was characterized with low infection pressure, it indicated that trunkinjected Agrimycin and Phosphojet might have potential to perform better than Actigard treatments. However, in year two, under the heavy infection pressure, this was not the case as all the injected treatments were similar. Overall, it seems that the reduction of shoot blight incidence with injected Agrimycin and Phosphojet across both years of 53.5 and 42.8%, was slightly better than the reduction of blossom blight incidence with the same materials of 45 and 40.5%, respectively. Shoots obviously have much higher green tissue area and transpiration rate in comparison to the flowers. Shoots likely accumulate higher amounts of trunk injected compounds in comparison to the green flower parts, which allowed slightly better disease reduction early after injection. Still, the shoot blight incidence reduction was far from the expected control with spray applied antibiotics in commercial apple orchards. In a trial with trunk injection of Arborfos (45.8% mono- and di-potassium salts of phosphorous acid, Mauget Inc., Arcadia, CA, USA), shoot blight was reduced for 67% on inoculated 'Paulared' apple trees [116]. The same dose per tree which we delivered in two injections of Phosphojet, achieved shoot blight incidence reduction of 23.4–62.1%. Since we have split the dose delivery temporally, this weakened shoot blight incidence reduction by Phosphojet and probably by Actigard too. In shoot inoculated trials multiple Actigard sprays achieved shoot blight reduction between 2.8 and 50.7% [135, 136] while by trunk injection we achieved only 1.7–30.9% of shoot blight reduction. Hence, the two-time trunk injection does not improve shoot blight reduction by Actigard. The reduction of shoot blight severity with Arborbiotic (MFG Scientific Inc., USA) was excellent and reached up to 82%. Such an effect with oxytetracycline hydrochloride demonstrates that this active ingredient is readily soluble in water and that the formulation we used is designed for trunk injection. Our results indicated that the trunk injected Arborbiotic limits i.e. stops systemic spread of *E. amylovora* in xylem of apple shoots [11]. Even though oxytetracycline hydrochloride is a bacteriostatic, when we delivered it via trunk injection in apple trees only one time per year, it demonstrated prolonged effectiveness that was higher in comparison to spray applications [44, 137, 138]. Trunk injection delivery enhanced the efficacy of oxytetracycline hydrochloride in control of shoot blight severity. Finally, in our prior work we also showed that the injected Arborbiotic at a dose of 0.31 g + 2.52 ml water per each 2.5 cm of trunk diameter at 30.5 cm height, can achieve a formidable reduction of blossom and shoot blight incidence for 60.6 and 60.7%, respectively [44]. This indicates that this bactericide in this formulation and probably at a slightly higher dose is the best candidate to achieve satisfactory accumulation inside and deposition on the susceptible apple plant tissues and

longer-lasting control of fire blight on both flowers and shoots.

*Plant Diseases-Current Threats and Management Trends*

surfaces to secure the higher efficacy.

**156**

Our results on management of three different pathogens with partially similar or different biologies, where *D. corticola* and *E. amylovora* invade and spread in xylem while *V. inaequalis* does not and infects subcuticularly, indicate that trunk injection of pesticides that are formulated for xylem translocation can be more-less similar in control of these three pathogens. However, the interaction of chemical properties of the active ingredient, the injected dose per tree, as well as the transpiration footprint of plant organs, played the key roles that determined the achieved levels of efficacy.

In the biology i.e. life cycle of *D. corticola*, it seems that the dominant phase is the invasion and necrosis of xylem, leading to vascular occlusion, canopy wilting and canker development on wood before it kills oak trees. Hence the logical approach to prevent this disease is trunk injection delivery of fungicides. In our two-year experiments on potted trees, the injected potassium phosphites (Phosphojet) achieved levels of Bot canker control in xylem of up to 71.1%. Averaged across both years, potassium phosphites achieved disease reduction of 58.5%, but the more consistent results were achieved with fungicides thiabendazole (Arbotect) and propiconazole (Propizol) which reduced xylem necrosis for 57.5 and 53.3% on average. The maximums in reduction in individual years for these two fungicides were of 60.5 and 69.3%, respectively. We predict that higher efficacy with these fungicides can be achieved with optimization of preventive fungicide injection which would increase the uniformity of distribution of these fungicides in xylem, thus increasing their efficacy.

In the case of *V. inaequalis*, for which the injected fungicides would need to translocate the farthest via xylem to reach and accumulate in and on the epidermal cells of green plant surfaces in tree canopy, the most efficient apple scab reduction of 45.5– 73.6% was achieved with potassium phosphites (Phosphojet). Unlike this readily mobile compound, scab control with propiconazole that is much less xylem mobile ranged from 17.1 to 51.5%, while the least xylem mobile difenoconazole underperformed with only up to 10.8% apple scab control. It is assumed that the injected potassium phosphites secured its efficacy against *V. inaequalis* through a strong plant defense response in the tissues called SAR [11, 139], as apparently it is not directly toxic to this pathogen [140]. We speculate that better efficacy with other systemic fungicides active against apple scab might be achieved if their formulations were redesigned for trunk injection i.e. to facilitate easier and faster translocation in xylem, thus securing higher accumulation in tissues exposed to infection.

Finally, there is the case of a complex biology of *E. amylovora* which combines life stages of inhabiting and multiplying on plant surfaces, migrating through internal host tissues after infecting, dwelling and overwintering asymptomatically in host buds or wood, and overwintering in fire blight cankers on bark. The injected compounds active against this pathogen would need to translocate and distribute in in xylem and phloem, reach in and onto the stigma surfaces of flowers and accumulate at effective doses in these and green tissues of the apple tree canopy. Based on presented research, it seems that these multiple difficult tasks in this and our previous study [44] were best achieved with oxytetracycline hydrochloride—both on the apple flowers [44] and on shoots [11]. Overall the injected antibiotic streptomycin (Agrimycin) formulated for foliar application gave the best reduction of blossom blight ranging from 28.9 to 61.1% and of shoot blight from 36.5 to 70.4%. The shoot blight severity reduction with Arboriotic, the injectable formulation of oxytetracycline hydrochloride, reached an excellent 82%. Hence, the effect depended on the plant organ, bactericide active ingredient, injected dose and formulation. The SAR-activating potassium phosphites (Phosphojet) were the second best to antibiotics with 25.1 and 55.9% of blossom blight reduction and 23.4 and

62.1% of shoot blight reduction. Actigard underperformed with blossom blight reduction of 19 and 42% and shoot blight reduction of only 1.7 and 30.9%.

#### **Acknowledgements**

The work on *Diplodia corticola* was funded by the USDA-APHIS PPQ grant awards USDA-APHIS-10025-PPQS&T00-18-0155 to DKHM and USDA-APHIS-18PPQS&T00-C11-0SGA to SGA for the project "Evaluation of the origin, epidemiology, and fungicide use in controlling *Diplodia corticola*, a new pathogen of red oak." We thank Peter Jentsch, Director of Cornell University's Hudson Valley Research Laboratory in Highland NY for supporting this research with injection equipment.

The work on *Venturia inaequalis* was supported by the grant awards to JCW by the Michigan Apple Research Committee for the project "Trunk Injection of Fungicides and Bio-pesticides for Apple Scab Control" and the IR-4 Project "Enhancing Performance of Phosphorous Acid Salts for Apple Scab Management through Trunk Injection Delivery."

The fire blight work was funded by USDA-NIFA Pest Management Alternatives grant MICL05066 in 2012 and continuation grant MICL07748 in 2013–2014 to JCW for project "Trunk Injection: A Discriminating Delivery System for Tree Fruit IPM". The work with evaluating Arborbiotic in reduction of fire blight severity was funded by Arborjet Inc., Woburn, MA, USA.

We thank Joseph J. Doccola, director of research and development at Arborjet Inc. and John J. Aiken, regional technical manager, for donating injection equipment and chemicals used in apple scab and fire blight experiments. For assistance in conducting or facilitating experiments, we thank research staff of at Michigan State University's Trevor Nichols Research Center (TNRC) in Fennville, MI, Anthony VanWoerkom, Gail Ehret, Jerri Gillett, Jason Seward, Laura Lamb and Kyle Coffindaffer. We acknowledge Dr. Annemiek Schilder, Dr. Brad Day, Dr. Jianjun Hao, Dr. Jim Miller and Dr. Randolph Beaudry for generously sharing their laboratory resources at Michigan State University in support of this research.

**Author details**

Srđan G. Aćimović<sup>1</sup>

New Hampshire, USA

**159**

\*, Danielle K.H. Martin2

Hudson Valley Research Laboratory, Highland, New York, USA

1 Plant Pathology and Plant Microbe Biology Section, Cornell University,

2 USDA Forest Service, Forest Health Protection, Northeastern Area State and

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

*DOI: http://dx.doi.org/10.5772/intechopen.87956*

3 USDA Forest Service, Northeastern Area State and Private Forestry, Durham,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Christopher L. Meredith<sup>1</sup> and Isabel A. Munck<sup>3</sup>

Private Forestry, Morgantown, West Virginia, USA

\*Address all correspondence to: acimovic@cornell.edu

provided the original work is properly cited.

, Richard M. Turcotte<sup>2</sup>

,

#### **Conflict of interest**

All authors declare that the research was conducted without any commercial or financial relationships that could be interpreted as a potential conflict of interest.

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

#### **Author details**

62.1% of shoot blight reduction. Actigard underperformed with blossom blight reduction of 19 and 42% and shoot blight reduction of only 1.7 and 30.9%.

The work on *Diplodia corticola* was funded by the USDA-APHIS PPQ grant awards USDA-APHIS-10025-PPQS&T00-18-0155 to DKHM and USDA-APHIS-18PPQS&T00-C11-0SGA to SGA for the project "Evaluation of the origin, epidemiology, and fungicide use in controlling *Diplodia corticola*, a new pathogen of red oak." We thank Peter Jentsch, Director of Cornell University's Hudson Valley Research Laboratory in Highland NY for supporting this research with injection

The work on *Venturia inaequalis* was supported by the grant awards to JCW by the Michigan Apple Research Committee for the project "Trunk Injection of Fungicides and Bio-pesticides for Apple Scab Control" and the IR-4 Project "Enhancing Performance of Phosphorous Acid Salts for Apple Scab Management through Trunk

The fire blight work was funded by USDA-NIFA Pest Management Alternatives grant MICL05066 in 2012 and continuation grant MICL07748 in 2013–2014 to JCW for project "Trunk Injection: A Discriminating Delivery System for Tree Fruit IPM". The work with evaluating Arborbiotic in reduction of fire blight severity was

We thank Joseph J. Doccola, director of research and development at Arborjet Inc. and John J. Aiken, regional technical manager, for donating injection equipment and chemicals used in apple scab and fire blight experiments. For assistance in conducting or facilitating experiments, we thank research staff of at Michigan State University's Trevor Nichols Research Center (TNRC) in Fennville, MI, Anthony VanWoerkom, Gail Ehret, Jerri Gillett, Jason Seward, Laura Lamb and Kyle Coffindaffer. We acknowledge Dr. Annemiek Schilder, Dr. Brad Day, Dr. Jianjun Hao, Dr. Jim Miller and Dr. Randolph Beaudry for generously sharing their labora-

All authors declare that the research was conducted without any commercial or financial relationships that could be interpreted as a potential conflict of interest.

tory resources at Michigan State University in support of this research.

**Acknowledgements**

equipment.

Injection Delivery."

**Conflict of interest**

**158**

funded by Arborjet Inc., Woburn, MA, USA.

*Plant Diseases-Current Threats and Management Trends*

Srđan G. Aćimović<sup>1</sup> \*, Danielle K.H. Martin2 , Richard M. Turcotte<sup>2</sup> , Christopher L. Meredith<sup>1</sup> and Isabel A. Munck<sup>3</sup>

1 Plant Pathology and Plant Microbe Biology Section, Cornell University, Hudson Valley Research Laboratory, Highland, New York, USA

2 USDA Forest Service, Forest Health Protection, Northeastern Area State and Private Forestry, Morgantown, West Virginia, USA

3 USDA Forest Service, Northeastern Area State and Private Forestry, Durham, New Hampshire, USA

\*Address all correspondence to: acimovic@cornell.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Plant Diseases-Current Threats and Management Trends*

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[99] Bereswill S, Jock S, Bellemann P, et al. Identification of *Erwinia amylovora* by growth morphology on agar containing copper sulfate and by capsule staining with lectin. Plant Disease. 1998;**82**:158-164

[100] Schaad NW, Jones JB, Chun W. Laboratory Guide for Identification of Plant Pathogenic Bacteria. St. Paul, MN: APS Press; 2001

[101] Thomson SV. Epidemiology of fire blight. In: Vanneste JL, editor. Fire Blight: The Disease and its Causative Agent, *Erwinia amylovora*. Hamilton, NZ: CABI Publishing; 2000. pp. 9-35

[102] Santander RD, Català-Senent JF, Marco-Noales E, et al. In planta recovery of *Erwinia amylovora* viable but nonculturable cells. Trees. 2012;**26**: 75-82

[103] Santander RD, Biosca EG. *Erwinia amylovora* psychrotrophic adaptations: Evidence of pathogenic potential and survival at temperate and low environmental temperatures. PeerJ. 2017;**5**:e3931. DOI: 10.7717/peerj.3931

[104] Slack SM, Sundin GW. News on ooze, the fire blight spreader. Fruit Quarterly. 2017;**25**:9-12

[113] Bogs J, Bruchmüller I, Erbar C, et al. Colonization of host plants by the fire blight pathogen *Erwinia amylovora* marked with genes for bioluminescence and fluorescence. Phytopathology. 1998;

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*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies…*

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[124] EFSA. Conclusion on the peer review of the pesticide risk assessment of the active substance potassium phosphonates, European Food Safety Authority. EFSA Journal. 2012;**10**:1-43

[125] Tanis SR, Cregg BM, Mota-Sanchez

[126] Kondo ES. Scope and limitations of carbendazim H2PO4 injections in Dutch elm disease [*Ceratocystis ulmi*, North America]. Journal of Arboriculture.

[127] Coomes DA. Challenges to the generality of WBE theory. Trends in Ecology & Evolution. 2006;**21**:593-596

Water transport in plants obeys

[129] McGhee GC, Sundin GW. Evaluation of Kasugamycin for fire blight management, effect on nontarget

Kasugamycin resistance potential in *Erwinia amylovora*. Phytopathology.

bacteria, and assessment of

2011;**101**:192-204

[128] McCulloh KA, Sperry JS, Adler FR.

Murray's law. Nature. 2003;**421**:939-942

D, et al. Spatial and temporal distribution of trunk-injected 14Cimidacloprid in Fraxinus trees. Pest Management Science. 2012;**68**:529-536

1978;**4**:80-86

[114] Koczan JM, McGrath MJ, Zhao Y, et al. Contribution of *Erwinia amylovora* exopolysaccharides amylovoran and

[115] Momol MT, Norelli JL, Piccioni DE, et al. Internal movement of *Erwinia amylovora* through symptomless apple Scion tissues into the rootstock. Plant

[116] Spitko R. Efficacy of Microinjected Apogee and ArborFos against Fire Blight Disease Incidence and Shoot Growth of Apple. Arcadia, CA: J.J. Mauget Co.; 2008 [Accessed: 28 October 2011]

concentrations in green ash (*Fraxinus pennsylvanica*) following treatments with two trunk-injection methods. Arboriculture & Urban Forestry. 2006;

[118] National Center for Biotechnology Information. Thiabendazole. PubChem Database. Thiabendazole. 2019. https:// pubchem.ncbi.nlm.nih.gov/compound/

5430 [Accessed: 27 April 2019]

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[108] Pusey PL, Smith TJ. Relation of apple flower age to infection of hypanthium by *Erwinia amylovora*. Plant Disease. 2007;**92**:137-142

[109] Keitt GW, Ivanoff SS. Transmission of fire blight by bees and its relation to nectar concentration of apple and pear blossoms. Journal of Agricultural Research. 1941;**62**:745-753

[110] Johnson KB, Sawyer TL, Temple TN. Rates of epiphytic growth of *Erwinia amylovora* on flowers common in the landscape. Plant Disease. 2006; **90**:1331-1336

[111] Billing E. Fire blight. Why do views on host invasion by *Erwinia amylovora* differ? Plant Pathology. 2011;**60**:178-189

[112] Mohammadi M. Enhanced colonization and pathogenicity of *Erwinia amylovora* strains transformed with the near-ubiquitous pEA29 plasmid on pear and apple. Plant Pathology. 2010;**59**:252-261

*Choosing an Adequate Pesticide Delivery System for Managing Pathogens with Difficult Biologies… DOI: http://dx.doi.org/10.5772/intechopen.87956*

[113] Bogs J, Bruchmüller I, Erbar C, et al. Colonization of host plants by the fire blight pathogen *Erwinia amylovora* marked with genes for bioluminescence and fluorescence. Phytopathology. 1998; **88**:416-421

and Serology. Wallingfod: Fire Blight

*Plant Diseases-Current Threats and Management Trends*

[104] Slack SM, Sundin GW. News on ooze, the fire blight spreader. Fruit

[105] Ogawa JM, English H. Diseases of temperate zone tree fruit and nut crops. Agriculture & Natural Resources. 1991. 464 p. ISBN-13: 978-0931876974, ISBN-

[106] Ordax M, Piquer-Salcedo JE, Santander RD, et al. Medfly Ceratitis capitata as potential vector for fire blight pathogen *Erwinia amylovora*: Survival and transmission. PLoS One.

[107] Cougar Blight Model Overview,

[108] Pusey PL, Smith TJ. Relation of apple flower age to infection of hypanthium by *Erwinia amylovora*. Plant Disease. 2007;**92**:137-142

Transmission of fire blight by bees and its relation to nectar concentration of apple and pear blossoms. Journal of Agricultural Research. 1941;**62**:745-753

[110] Johnson KB, Sawyer TL, Temple TN. Rates of epiphytic growth of *Erwinia amylovora* on flowers common in the landscape. Plant Disease. 2006;

[111] Billing E. Fire blight. Why do views on host invasion by *Erwinia amylovora* differ? Plant Pathology. 2011;**60**:178-189

[112] Mohammadi M. Enhanced colonization and pathogenicity of *Erwinia amylovora* strains transformed with the near-ubiquitous pEA29 plasmid on pear and apple. Plant Pathology.

[109] Keitt GW, Ivanoff SS.

**90**:1331-1336

2010;**59**:252-261

Washington State University. Chelan & Douglas Counties. Available from: h ttps://extension.wsu.edu/chelan-dougla s/agriculture/treefruit/pestmanageme nt/cougarblightmodeloverview/ [Accessed: 23 April 2019]

Chelan & Douglas Counties,

Quarterly. 2017;**25**:9-12

10: 0931876974

2015;**10**:e0127560

[95] Slack SM, Zeng Q, Outwater CA, et al. Microbiological examination of *Erwinia amylovora* exopolysaccharide ooze. Phytopathology. 2017;**107**:403-411

CABI Publ; 2000. pp. 87-115

[96] Boucher M, Cox K, Loeb G. Establishing a Role for Flies in the Disease Cycle of Fire Blight (*Erwinia amylovora*). Fruit Quarterly. **27**:19-22

[97] Hildebrand EM. Studies on fire blight ooze. Phytopathology. 1939;**29**:

[98] Billing E, LAE B, Crosse JE, et al. Characteristics of English isolates of *Erwinia amylovora* (Burrill) Winslow

[99] Bereswill S, Jock S, Bellemann P, et al. Identification of *Erwinia amylovora*

[100] Schaad NW, Jones JB, Chun W. Laboratory Guide for Identification of Plant Pathogenic Bacteria. St. Paul, MN:

[101] Thomson SV. Epidemiology of fire blight. In: Vanneste JL, editor. Fire Blight: The Disease and its Causative Agent, *Erwinia amylovora*. Hamilton, NZ: CABI Publishing; 2000. pp. 9-35

[102] Santander RD, Català-Senent JF, Marco-Noales E, et al. In planta recovery of *Erwinia amylovora* viable but nonculturable cells. Trees. 2012;**26**:

[103] Santander RD, Biosca EG. *Erwinia amylovora* psychrotrophic adaptations: Evidence of pathogenic potential and survival at temperate and low environmental temperatures. PeerJ. 2017;**5**:e3931. DOI: 10.7717/peerj.3931

by growth morphology on agar containing copper sulfate and by capsule staining with lectin. Plant

Disease. 1998;**82**:158-164

APS Press; 2001

75-82

**166**

et al. The Journal of Applied Bacteriology. 1961;**24**:195-211

142-156

[114] Koczan JM, McGrath MJ, Zhao Y, et al. Contribution of *Erwinia amylovora* exopolysaccharides amylovoran and Levan to biofilm formation: Implications in pathogenicity. Phytopathology. 2009;**99**:1237-1244

[115] Momol MT, Norelli JL, Piccioni DE, et al. Internal movement of *Erwinia amylovora* through symptomless apple Scion tissues into the rootstock. Plant Disease. 1998;**82**:646-650

[116] Spitko R. Efficacy of Microinjected Apogee and ArborFos against Fire Blight Disease Incidence and Shoot Growth of Apple. Arcadia, CA: J.J. Mauget Co.; 2008 [Accessed: 28 October 2011]

[117] Harrell M. Imidacloprid concentrations in green ash (*Fraxinus pennsylvanica*) following treatments with two trunk-injection methods. Arboriculture & Urban Forestry. 2006; **32**:126

[118] National Center for Biotechnology Information. Thiabendazole. PubChem Database. Thiabendazole. 2019. https:// pubchem.ncbi.nlm.nih.gov/compound/ 5430 [Accessed: 27 April 2019]

[119] PPDB. Pesticide Properties DataBase—difenoconazole (Ref: CGA 169374). Pesticide Properties DataBase. 2013. Available from: http://kartofel. org/pesticides\_op/difenokonazol.PDF [Accessed: 24 October 2013]

[120] BCP Council. The Pesticide Manual: A World Compendium. Tomlin, CDS: British Crop Protection; 2011

[121] Herner AE, Acock B. USDA-ARS pesticide properties database. In:

Encyclopedia of Agrochemicals. John Wiley & Sons, Inc.; 2003 . Available from: http://onlinelibrary.wiley.com/ doi/10.1002/047126363X.agr237/abstract [Accessed: 24 October 2013]

[122] Blanke MM, Lenz F. Fruit photosynthesis. Plant, Cell & Environment. 1989;**12**:31-46

[123] Mensink B. Environmental risk limits for difenoconazole. 601716005/ 2008. Netherlands: National Institute for Public Health and the Environment; 2008 . Available from: http://www. rivm.nl/bibliotheek/rapporten/ 601716005.pdf [Accessed: 24 October 2013]

[124] EFSA. Conclusion on the peer review of the pesticide risk assessment of the active substance potassium phosphonates, European Food Safety Authority. EFSA Journal. 2012;**10**:1-43

[125] Tanis SR, Cregg BM, Mota-Sanchez D, et al. Spatial and temporal distribution of trunk-injected 14Cimidacloprid in Fraxinus trees. Pest Management Science. 2012;**68**:529-536

[126] Kondo ES. Scope and limitations of carbendazim H2PO4 injections in Dutch elm disease [*Ceratocystis ulmi*, North America]. Journal of Arboriculture. 1978;**4**:80-86

[127] Coomes DA. Challenges to the generality of WBE theory. Trends in Ecology & Evolution. 2006;**21**:593-596

[128] McCulloh KA, Sperry JS, Adler FR. Water transport in plants obeys Murray's law. Nature. 2003;**421**:939-942

[129] McGhee GC, Sundin GW. Evaluation of Kasugamycin for fire blight management, effect on nontarget bacteria, and assessment of Kasugamycin resistance potential in *Erwinia amylovora*. Phytopathology. 2011;**101**:192-204

[130] Sundin GW, Werner NA, Yoder KS, et al. Field evaluation of biological control of fire blight in the eastern United States. Plant Disease. 2009;**93**: 386-394

[131] Hammerschmidt R. Introduction: Definitions and some history. In: Walters D, Newton A, Lyon G, editors. Induced Resistance for Plant Defence. Hoboken, NJ, USA: Blackwell Publishing; 2007. pp. 1-8

[132] Thomson SV, Gouk SC, Paulin JP. Efficacy of BION®(Actigard®) to control fire blight in pear and apple orchards in USA, New Zealand and France. In: VIII International Workshop on Fire Blight 489. 1998. pp. 589-596

[133] Brisset MN, Cesbron S, Thomson SV, et al. Acibenzolar-*S*-methyl induces the accumulation of defense-related enzymes in apple and protects from fire blight. European Journal of Plant Pathology. 2000;**106**:529-536

[134] Zeller W, Zeller V. Control of fire blight with the plant activator BION®. In: VIII International Workshop on Fire Blight 489. 1998. pp. 639-646

[135] Maxson KL, Jones AL. Management of fire blight with gibberellin inhibitors and SAR inducers. In: IX International Workshop on Fire Blight 590. 2001. pp. 217-223

[136] Maxson-Stein K, He SY, Hammerschmidt R, et al. Effect of treating apple trees with acibenzolar-*S*methyl on fire blight and expression of pathogenesis-related protein genes. Plant Disease. 2002;**86**:785-790

[137] Johnson KB, Stockwell VO. Management of fire blight: A case study in microbial ecology. Annual Review of Phytopathology. 1998;**36**:227-248

[138] McManus PS, Stockwell VO, Sundin GW, et al. Antibiotic use in plant agriculture. Annual Review of Phytopathology. 2002;**40**:443-465

[139] Deliopoulos T, Kettlewell PS, Hare MC. Fungal disease suppression by inorganic salts: A review. Crop Protection. 2010;**29**:1059-1075

[140] Boneti J da S, Katsurayama Y. Viabilidade do uso de fosfitos no controle da sarna-da-macieira. Agropecuaria Catarinense. 2005;**18**: 51-54

**169**

**Chapter 10**

Future

**Abstract**

**1. Introduction**

*George A. Ameyaw*

Management of the Cacao Swollen

Shoot Virus (CSSV) Menace in

Ghana: The Past, Present and the

This chapter outlines and discusses some of the challenges associated with management of the cacao swollen shoot virus (CSSV) disease in Ghana and its impact on cocoa production. The discussion will bring to the fore some of the factors that has militated against implementation of the recommended management strategies in the past and its consequential effect on the present widespread of the disease across the various cocoa regions in West Africa. The wide variability in the different strains of the virus as manifested in recent molecular studies is highlighted as a possible contributor and explanation for the prevalence and varying virulence of the disease in new infections, especially, in the Western region of Ghana. Current research efforts and strategies aimed at minimizing of CSSV continuous spread and

devastation on Ghana's cocoa production is discussed.

**Keywords:** cocoa, swollen shoot virus disease, mealybug vector, CSSVD

The cocoa industry plays critical role in the socioeconomic development of Ghana by providing employment and source of livelihood to many farm families and other stakeholders in the cocoa value chain. Export of cocoa beans and other cocoa products is a major avenue for the generation of the much needed foreign exchange for the economies of Ghana and Cote d'Ivoire. Sustainability of the cocoa industry is therefore critical for the governments and people of these West African nations and other stakeholders in the cocoa business. Cocoa cultivation is however bedeviled with several production problems as the cocoa plant is affected by numerous diseases and pests which accounts for significant yield losses in the various cocoa producing nations across the world (**Tables 1** and **2**). Five major diseases of the cocoa plant (*Theobroma cacao* L.) namely; *Phytophthora* pod rot (black pod), witches broom, cacao swollen shoot virus, vascular streak dieback, and monilia pod rot account for over 40% annual yield loss across the different production regions [1]. The cacao swollen shoot virus disease (CSSVD) which is considered the most economically important cocoa virus disease could account for 15–50% yield loss if the severe strains are involved in infections [2, 3]. Since the discovery of this important disease in Ghana, it has been managed through the "cutting out and replanting

#### **Chapter 10**

[130] Sundin GW, Werner NA, Yoder KS, et al. Field evaluation of biological control of fire blight in the eastern United States. Plant Disease. 2009;**93**:

*Plant Diseases-Current Threats and Management Trends*

agriculture. Annual Review of Phytopathology. 2002;**40**:443-465

[139] Deliopoulos T, Kettlewell PS, Hare MC. Fungal disease suppression by inorganic salts: A review. Crop Protection. 2010;**29**:1059-1075

[140] Boneti J da S, Katsurayama Y. Viabilidade do uso de fosfitos no controle da sarna-da-macieira. Agropecuaria Catarinense. 2005;**18**:

51-54

[131] Hammerschmidt R. Introduction: Definitions and some history. In: Walters D, Newton A, Lyon G, editors. Induced Resistance for Plant Defence.

[132] Thomson SV, Gouk SC, Paulin JP. Efficacy of BION®(Actigard®) to control fire blight in pear and apple orchards in USA, New Zealand and France. In: VIII International Workshop on Fire Blight 489. 1998. pp. 589-596

[133] Brisset MN, Cesbron S, Thomson SV, et al. Acibenzolar-*S*-methyl induces the accumulation of defense-related enzymes in apple and protects from fire blight. European Journal of Plant Pathology. 2000;**106**:529-536

[134] Zeller W, Zeller V. Control of fire blight with the plant activator BION®. In: VIII International Workshop on Fire

[135] Maxson KL, Jones AL. Management of fire blight with gibberellin inhibitors and SAR inducers. In: IX International Workshop on Fire Blight 590. 2001.

Blight 489. 1998. pp. 639-646

[136] Maxson-Stein K, He SY, Hammerschmidt R, et al. Effect of treating apple trees with acibenzolar-*S*methyl on fire blight and expression of pathogenesis-related protein genes. Plant Disease. 2002;**86**:785-790

[137] Johnson KB, Stockwell VO.

[138] McManus PS, Stockwell VO, Sundin GW, et al. Antibiotic use in plant

Management of fire blight: A case study in microbial ecology. Annual Review of Phytopathology. 1998;**36**:227-248

pp. 217-223

**168**

Hoboken, NJ, USA: Blackwell Publishing; 2007. pp. 1-8

386-394

## Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present and the Future

*George A. Ameyaw*

#### **Abstract**

This chapter outlines and discusses some of the challenges associated with management of the cacao swollen shoot virus (CSSV) disease in Ghana and its impact on cocoa production. The discussion will bring to the fore some of the factors that has militated against implementation of the recommended management strategies in the past and its consequential effect on the present widespread of the disease across the various cocoa regions in West Africa. The wide variability in the different strains of the virus as manifested in recent molecular studies is highlighted as a possible contributor and explanation for the prevalence and varying virulence of the disease in new infections, especially, in the Western region of Ghana. Current research efforts and strategies aimed at minimizing of CSSV continuous spread and devastation on Ghana's cocoa production is discussed.

**Keywords:** cocoa, swollen shoot virus disease, mealybug vector, CSSVD

#### **1. Introduction**

The cocoa industry plays critical role in the socioeconomic development of Ghana by providing employment and source of livelihood to many farm families and other stakeholders in the cocoa value chain. Export of cocoa beans and other cocoa products is a major avenue for the generation of the much needed foreign exchange for the economies of Ghana and Cote d'Ivoire. Sustainability of the cocoa industry is therefore critical for the governments and people of these West African nations and other stakeholders in the cocoa business. Cocoa cultivation is however bedeviled with several production problems as the cocoa plant is affected by numerous diseases and pests which accounts for significant yield losses in the various cocoa producing nations across the world (**Tables 1** and **2**). Five major diseases of the cocoa plant (*Theobroma cacao* L.) namely; *Phytophthora* pod rot (black pod), witches broom, cacao swollen shoot virus, vascular streak dieback, and monilia pod rot account for over 40% annual yield loss across the different production regions [1].

The cacao swollen shoot virus disease (CSSVD) which is considered the most economically important cocoa virus disease could account for 15–50% yield loss if the severe strains are involved in infections [2, 3]. Since the discovery of this important disease in Ghana, it has been managed through the "cutting out and replanting


#### **Table 1.**

*Causal pathogens of common cocoa diseases in the world. Source: [48].*

system" with the aim of removing sources of inoculum from affected cocoa plantations and replanting with tolerant cocoa hybrids [3–5]. Nonetheless, reports from many reassessments and disease surveys indicate that the prevalence of the disease is still high with varying virulence across the cocoa regions. This has partly been attributed to the poor implementation of the cutting out program to manage the disease. This chapter highlights some of the past challenges that have bedeviled the "cutting out system" and discusses some of the current strategies being implemented by the various stakeholders and researchers to minimize the continuous spread and impact of the disease on cocoa production in Ghana and Cote d'Ivoire.

**171**

**Figure 1.** *Red vein banding.*

**Table 2.**

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present…*

*DOI: http://dx.doi.org/10.5772/intechopen.87009*

**2. The cacao swollen shoot virus disease (CSSVD)**

*Economic losses from some important cocoa diseases across the world. Source: [48].*

The *Cacao swollen shoot virus* disease (CSSVD) was first noted in the Eastern Region of Ghana in 1936 by a farmer in a form of cocoa stem swollen conditions [6] but its virus nature was confirmed in 1939 [7]. The disease is considered the most important cocoa viruses in West Africa due to its devastating effect on yield and possibility of causing death of cocoa plants especially when the severe strains are involved in infections [8, 9]. The virus affects all parts of the cocoa plant and the severe strains induce varying leaf symptoms and swellings of the stems and roots. Some of the leaf symptoms include; red vein banding of the immature "flush" leaves [10] (**Figure 1**); chlorotic vein flecking or banding which may occur in angular flecks (**Figure 2**). Stem swellings occur at the nodes, internodes or tips of the stem [2, 10], (**Figures 3** and **4**).

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present… DOI: http://dx.doi.org/10.5772/intechopen.87009*


**Table 2.**

*Plant Diseases-Current Threats and Management Trends*

system" with the aim of removing sources of inoculum from affected cocoa plantations and replanting with tolerant cocoa hybrids [3–5]. Nonetheless, reports from many reassessments and disease surveys indicate that the prevalence of the disease is still high with varying virulence across the cocoa regions. This has partly been attributed to the poor implementation of the cutting out program to manage the disease. This chapter highlights some of the past challenges that have bedeviled the "cutting out system" and discusses some of the current strategies being implemented by the various stakeholders and researchers to minimize the continuous spread and impact of the disease on cocoa production in Ghana and Cote d'Ivoire.

*Causal pathogens of common cocoa diseases in the world. Source: [48].*

**170**

**Table 1.**

*Economic losses from some important cocoa diseases across the world. Source: [48].*

#### **2. The cacao swollen shoot virus disease (CSSVD)**

The *Cacao swollen shoot virus* disease (CSSVD) was first noted in the Eastern Region of Ghana in 1936 by a farmer in a form of cocoa stem swollen conditions [6] but its virus nature was confirmed in 1939 [7]. The disease is considered the most important cocoa viruses in West Africa due to its devastating effect on yield and possibility of causing death of cocoa plants especially when the severe strains are involved in infections [8, 9]. The virus affects all parts of the cocoa plant and the severe strains induce varying leaf symptoms and swellings of the stems and roots. Some of the leaf symptoms include; red vein banding of the immature "flush" leaves [10] (**Figure 1**); chlorotic vein flecking or banding which may occur in angular flecks (**Figure 2**). Stem swellings occur at the nodes, internodes or tips of the stem [2, 10], (**Figures 3** and **4**).

**Figure 1.** *Red vein banding.*

**Figure 2.** *Chlorotic vein banding.*

Some strains also cause infected pods to change shape and become rounder, smaller and with smoother surfaces [11].CSSV is classified as a member of the plant-infecting pararetroviruses in the genus badnaviridae which are with non-enveloped bacilliform particles that encapsulate a circular double stranded DNA-genome [12–15]. The viral particle of CSSV is identified with length measurements in the range of 121–130 and a width of 28 nm [12]. The genome size ranges from 7.4 to 8.0 kilobase pair depending on strain [16, 17]. The CSSV genome is organized into five putative open reading frames (ORFs 1, 2, 3 X and Y) located on the plus strand of the 7.16 kbp [15].

#### **2.1 CSSVD isolates and strains**

Generally, isolates of the virus have been designated by naming them according to the nearest town or village where they are first collected and are generally

**173**

**Figure 5.**

*Molecular diversity of CSSV in West Africa [47].*

**Figure 4.** *Shoot swelling.*

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present…*

grouped according to severity of symptom expression and geographical origin [12–15]. Ghanaian CSSVD isolates are distinguished five groups of through enzyme linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) techniques and by using leaf symptoms [18]. Recent molecular studies with the use of advance sequencing methods such Next generation tools have provided the opportunity to further classify CSSV isolates across the West African sub-region into new groups based on their molecular information at the DNA level. Some of these studies have identified wide variability in the strains of the virus and virulence of the disease in new infections especially in the Western region of Ghana [3, 19, 20]. The strains of the virus have now been reclassified based on their molecular

diversity into groups A, B, C, D, E, F, G, H, G, J, K. L, M, and N [**Figure 5**].

*DOI: http://dx.doi.org/10.5772/intechopen.87009*

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present… DOI: http://dx.doi.org/10.5772/intechopen.87009*

*Plant Diseases-Current Threats and Management Trends*

Some strains also cause infected pods to change shape and become rounder, smaller and with smoother surfaces [11].CSSV is classified as a member of the plant-infecting pararetroviruses in the genus badnaviridae which are with non-enveloped bacilliform particles that encapsulate a circular double stranded DNA-genome [12–15]. The viral particle of CSSV is identified with length measurements in the range of 121–130 and a width of 28 nm [12]. The genome size ranges from 7.4 to 8.0 kilobase pair depending on strain [16, 17]. The CSSV genome is organized into five putative open reading

frames (ORFs 1, 2, 3 X and Y) located on the plus strand of the 7.16 kbp [15].

Generally, isolates of the virus have been designated by naming them according to the nearest town or village where they are first collected and are generally

**2.1 CSSVD isolates and strains**

**172**

**Figure 3.** *Tip swelling.*

**Figure 2.**

*Chlorotic vein banding.*

grouped according to severity of symptom expression and geographical origin [12–15]. Ghanaian CSSVD isolates are distinguished five groups of through enzyme linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) techniques and by using leaf symptoms [18]. Recent molecular studies with the use of advance sequencing methods such Next generation tools have provided the opportunity to further classify CSSV isolates across the West African sub-region into new groups based on their molecular information at the DNA level. Some of these studies have identified wide variability in the strains of the virus and virulence of the disease in new infections especially in the Western region of Ghana [3, 19, 20]. The strains of the virus have now been reclassified based on their molecular diversity into groups A, B, C, D, E, F, G, H, G, J, K. L, M, and N [**Figure 5**].

**Figure 5.** *Molecular diversity of CSSV in West Africa [47].*

#### **2.2 CSSVD transmission**

CSSV is semi-persistently transmitted by several species of mealybugs (*Pseudococcidae*, *Homoptera*) on cocoa [21, 22]. The vectors feed on all parts of the cocoa tree including flowers, cherelles, pods and leaves. The mealybug species differ in their ability to transmit different strains of the virus. The most efficient mealybug transmitters of the virus include the *Planococcoides njalensis* (Laing), *Planococcus citri* (Rossi) and *Ferrisia virgata* (Okll) specie which are also dominant on cocoa fields in Ghana and Cote d'Ivoire (**Figure 6**). The ages of the mealybugs are also important in the spread of the virus in that only the young adults (nymphs) are very mobile and so are more efficient transmitters than the adults which are most often sedentary [23]. The use of mechanical inoculation procedures to transmit CSSV was made possible in 1960 [24]. Considering the continual spread of the virus to new cocoa plantings, research is still focusing on other insect pests in the cocoa environment to ascertain their vector status regarding CSSV transmission in the field.

#### **2.3 CSSVD alternative hosts**

The virus is considered to have originated from wild indigenous forest trees within the cocoa environment [25–28]. This suggestion was based on studies in the Western Region of Ghana which showed that some of the CSSV isolates could be found in some forest trees such as *Cola chlamydantha* (K. Schum), trees and the prevalence of the disease was also high in areas where these trees were found. Other tree species that have subsequently been identified as wild alternative hosts of CSSV include *Erythropsis barteri* (Mast), *Sterculia tragacantha* (Lindle), *Sterculia rhinopetala* (K. Schum), *Cola gigantean* var. *glabrescens* (BronnanetKeay), *Adansonia digitata* (L.), *Bombax buonopozense*, and *Ceiba pentandra* (L.), [25]. It is, however, noteworthy to indicate that not all the wild hosts are good sources of the virus and also its availability declines to a low level in the bigger and old trees which sometimes makes the virus not readily available to the mealybug vectors. Mechanically transmission CSSV from some of the wild hosts to cocoa and vice versa was achieved in 1962 [24]. It was therefore recommended that the abovementioned forest trees known to be alternative host plants for the virus be removed as much as possible from cocoa plantations before replanting with new cocoa to prevent early

**175**

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present…*

reinfection [29]. Currently, research is focusing on other possible alternative host plants of CSSV in the forms of weeds and food crops within the cocoa ecosystem.

Generally, the spread of the virus in the field is triggered by several factors such as the size of the initial source of infection, and the age and type of the mealybug population present [8, 9, 29]. Apparently, natural spread of the virus is slow under low inoculum pressure (i.e., presence of few inoculums sources) because it is dependent on the movement of the mealybug vectors infected with the virus. It is also slow within young plantings until the trees become well established and form a continuous canopy of interlocking branches [29]. Nevertheless, virus infected mealybugs are occasionally blown by wind to uninfected trees some distance from the original site of infection resulting in jump spread of the virus to uninfected areas to initiate new outbreaks [30, 31]. New outbreaks of CSSV tended to be concentrated around the periphery of existing outbreaks (infections) or abandoned forests with alternative hosts which then spread slowly to give clearly defined expanding foci [29]. The pattern of spread within outbreaks was noted to be of a circumscribed nature and tended to be high close to source of infection. Existing outbreaks then initiates new "satellite" outbreaks through "jump" spread over wider distance by windblown mealybugs. These new "satellite" outbreaks enlarge and eventually coalesce to form large areas of mass infection. Spread of the disease within outbreaks was however noted to result from movement of mealybugs carrying the virus from infected to healthy trees. It was noted that new outbreaks get bigger close to large sources of infection and diminish further away from them [32].

Management of CSSVD in Ghana has over the years been carried out in an integrated manner involving the use of different strategies such; as the cutting out method, mealybug control, removal of alternative hosts, and the use of tolerant planting materials. These control strategies and some of their challenges are discussed below.

Cutting-out of CSSVD infected cocoa trees together with a ring of nearby apparently healthy cocoa trees have been the main method adopted to control the spread of the virus in Ghana since 1946. The aim of this strategy is to eliminate or reduce the sources of infection (inoculums) within new cocoa plantings. Once the infected cocoa trees are removed, the field is expected to be replanted with CSSVD tolerant cocoa varieties from the seed gardens. This method has gone through several challenges in its implementation thereby resulting in the continuous spread of the virus to new areas [5, 9]. Although most of these challenges are intertwined, notable among them include; late discovery of infections, lack of continuity of the program, non-co-operation of farmers due to issues of compensation payments, land tenure issues, and non-adherence to replanting recommendations after the removal of

The effectiveness of the cutting out procedure depends largely on the efficiency

of early detection of infections [9]. The disease identification system whereby

*DOI: http://dx.doi.org/10.5772/intechopen.87009*

**2.4 CSSVD spread in the field**

**3. CSSVD control strategies**

**3.1 The cutting out method**

sources of infection [5, 8, 29].

*3.1.1 Late discovery of infections*

**Figure 6.** *Mealybug infested cocoa pods.*

reinfection [29]. Currently, research is focusing on other possible alternative host plants of CSSV in the forms of weeds and food crops within the cocoa ecosystem.

#### **2.4 CSSVD spread in the field**

*Plant Diseases-Current Threats and Management Trends*

CSSV is semi-persistently transmitted by several species of mealybugs (*Pseudococcidae*, *Homoptera*) on cocoa [21, 22]. The vectors feed on all parts of the cocoa tree including flowers, cherelles, pods and leaves. The mealybug species differ in their ability to transmit different strains of the virus. The most efficient mealybug transmitters of the virus include the *Planococcoides njalensis* (Laing), *Planococcus citri* (Rossi) and *Ferrisia virgata* (Okll) specie which are also dominant on cocoa fields in Ghana and Cote d'Ivoire (**Figure 6**). The ages of the mealybugs are also important in the spread of the virus in that only the young adults (nymphs) are very mobile and so are more efficient transmitters than the adults which are most often sedentary [23]. The use of mechanical inoculation procedures to transmit CSSV was made possible in 1960 [24]. Considering the continual spread of the virus to new cocoa plantings, research is still focusing on other insect pests in the cocoa environment to

ascertain their vector status regarding CSSV transmission in the field.

The virus is considered to have originated from wild indigenous forest trees within the cocoa environment [25–28]. This suggestion was based on studies in the Western Region of Ghana which showed that some of the CSSV isolates could be found in some forest trees such as *Cola chlamydantha* (K. Schum), trees and the prevalence of the disease was also high in areas where these trees were found. Other tree species that have subsequently been identified as wild alternative hosts of CSSV include *Erythropsis barteri* (Mast), *Sterculia tragacantha* (Lindle), *Sterculia rhinopetala* (K. Schum), *Cola gigantean* var. *glabrescens* (BronnanetKeay), *Adansonia digitata* (L.), *Bombax buonopozense*, and *Ceiba pentandra* (L.), [25]. It is, however, noteworthy to indicate that not all the wild hosts are good sources of the virus and also its availability declines to a low level in the bigger and old trees which sometimes makes the virus not readily available to the mealybug vectors. Mechanically transmission CSSV from some of the wild hosts to cocoa and vice versa was achieved in 1962 [24]. It was therefore recommended that the abovementioned forest trees known to be alternative host plants for the virus be removed as much as possible from cocoa plantations before replanting with new cocoa to prevent early

**2.2 CSSVD transmission**

**2.3 CSSVD alternative hosts**

**174**

**Figure 6.**

*Mealybug infested cocoa pods.*

Generally, the spread of the virus in the field is triggered by several factors such as the size of the initial source of infection, and the age and type of the mealybug population present [8, 9, 29]. Apparently, natural spread of the virus is slow under low inoculum pressure (i.e., presence of few inoculums sources) because it is dependent on the movement of the mealybug vectors infected with the virus. It is also slow within young plantings until the trees become well established and form a continuous canopy of interlocking branches [29]. Nevertheless, virus infected mealybugs are occasionally blown by wind to uninfected trees some distance from the original site of infection resulting in jump spread of the virus to uninfected areas to initiate new outbreaks [30, 31]. New outbreaks of CSSV tended to be concentrated around the periphery of existing outbreaks (infections) or abandoned forests with alternative hosts which then spread slowly to give clearly defined expanding foci [29]. The pattern of spread within outbreaks was noted to be of a circumscribed nature and tended to be high close to source of infection. Existing outbreaks then initiates new "satellite" outbreaks through "jump" spread over wider distance by windblown mealybugs. These new "satellite" outbreaks enlarge and eventually coalesce to form large areas of mass infection. Spread of the disease within outbreaks was however noted to result from movement of mealybugs carrying the virus from infected to healthy trees. It was noted that new outbreaks get bigger close to large sources of infection and diminish further away from them [32].

#### **3. CSSVD control strategies**

Management of CSSVD in Ghana has over the years been carried out in an integrated manner involving the use of different strategies such; as the cutting out method, mealybug control, removal of alternative hosts, and the use of tolerant planting materials. These control strategies and some of their challenges are discussed below.

#### **3.1 The cutting out method**

Cutting-out of CSSVD infected cocoa trees together with a ring of nearby apparently healthy cocoa trees have been the main method adopted to control the spread of the virus in Ghana since 1946. The aim of this strategy is to eliminate or reduce the sources of infection (inoculums) within new cocoa plantings. Once the infected cocoa trees are removed, the field is expected to be replanted with CSSVD tolerant cocoa varieties from the seed gardens. This method has gone through several challenges in its implementation thereby resulting in the continuous spread of the virus to new areas [5, 9]. Although most of these challenges are intertwined, notable among them include; late discovery of infections, lack of continuity of the program, non-co-operation of farmers due to issues of compensation payments, land tenure issues, and non-adherence to replanting recommendations after the removal of sources of infection [5, 8, 29].

#### *3.1.1 Late discovery of infections*

The effectiveness of the cutting out procedure depends largely on the efficiency of early detection of infections [9]. The disease identification system whereby

trained diseased spotters carry out tree-by-tree inspection for visual symptoms of the virus inevitably means that latently infected trees which have not produced symptoms at the time of inspection are missed. Considering the pattern of CSSVD spread [29] which is mainly limited to adjacent trees around the periphery of existing outbreaks, it can be argued that, the disease spotters follow the virus and are always "one step behind". It is also generally known that CSSVD symptoms tend to be least conspicuous during the dry season when the trees deteriorate and leaves and shoots are shed or damaged by capsids [9]. Considering the common phenomenon of dehydration and less active growth of infected trees during the dry season, it is very likely that disease spotters would miss some infected trees in the field during these periods. There is thus a high possibility that these missed infections could be supporting mealybug population and also transmit the disease even though they may not show conspicuous symptoms [8, 9, 23]. The challenge of lack of efficient detection protocol for the virus at the early stages has therefore generally been considered among the reasons why the disease continues to spread at an increasing rate in Ghana. The need for efficient early detection tools has always been advocated to be one of the means to help in the effective management of the virus.

#### *3.1.2 Lack of continuity of the cutting out program*

National implementation of the cutting out program has been delayed or halted on many occasions for numerous reasons such as; farmer opposition, logistical constraints, lack of funding, and at times political interference [5]. Although, it is known that continuity in the cutting out operation would have been very essential for the control of other viral diseases in other areas it has never been achieved most especially in Ghana. There is always time lag between symptom identification in outbreaks and time to treatment and replanting.

#### *3.1.3 Non-adherence to replanting recommendations*

Replanting of treated cocoa farms according to laid down recommendations could have been successful in rehabilitating devastated cocoa areas. However, it was noted that newly planted farms in most of the cocoa areas, especially, in the Western Region, showed symptoms of re-infection with the virus [4]. This is because farms are replanted very close to the boundary of abandoned cocoa farms containing visible infections, without much attempt to remove the infected trees in the old plantations. The recommendation that replanting should only occur after the complete removal of sources of infected cocoa trees or alternative host plants, have not been applied adequately in the eradication and replanting exercise hence contributing to the prevalence of the disease across cocoa farms.

#### *3.1.4 Presence of alternative hosts in newly replanted farms*

The occurrence of wild alternative host plants of the virus in and around cocoa farms is a contributory factor to spread and early re-infection of new cocoa plantings in Ghana [25]. Even though, removal of wild alternative host trees has been recommended, it is seldom applied. The advice to farmers to leave at least a 15 m barrier with some economic crops such as citrus and oil palm around new cocoa plantings to delay reinfection from old plantations and forest trees have also not been implemented fully. Farmers always want to fully utilize all available space of land for cocoa planting and also use the alternative host trees as a source of natural shade for their cocoa during the early growth period.

**177**

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present…*

The use of synthetic chemicals for the control of the mealybug vectors has not been effective over the years. This is attributed to the morphology of the mealybugs having a protective wax covering and also the building of mud tents over them by black ants [33–36]. Attempts by scientists with the use of biological means have also

Even though it has long be envisaged that planting of cocoa varieties tolerant to CSSVD would be the most effective means to manage the disease [38], most of the available cocoa planting materials has however shown low levels of resistance under field conditions. It is notable that many of the inter-Amazon hybrids developed and recommended by the British Research Team and currently available to farmers [38] have only partial resistance to CSSVD [38–40]. Although partial resistance is beneficial for being able to tolerate the virus to give appreciable yield in the short to medium term, the need for varieties that could offer greater resistance cannot be overemphasized. The continuous search for varieties that could offer long-term resistance that has been going on over the years using modern breeding approaches such as mutation and tissue culture techniques in Ghana and elsewhere is therefore

Long term field assessment of the mild strain cross protection experiments carried out at the Cocoa Research Institute of Ghana (CRIG) has shown that, the immunity conferred on the healthy cocoa plants from the available mild strains N1 and SS365B eventually breaks down after 20 years [42, 43]. These reports support past works and suggestions that further investigations on the effectiveness of the mild strain phenomenon need to be carried out before its adoption as a manage-

The cutting out and rehabilitation program was re-launched in June 2018 to concurrently cut out CSSVD infected cocoa trees along the borders of Ghana and Cote d'Ivoire. The expectation is to progressively remove about 100,000 ha of infected CSSVD outbreaks across the cocoa regions in Ghana by 2023. The current cutting out activities involves the total removal of CSSVD infected cocoa trees in blocks of outbreaks and replanting with tolerant cocoa varieties. Payment of compensation to farmers and land owners has been incorporated into the program to sustain farmers' livelihood during the periods of cocoa tree removal and replanting. Additionally, farmers would be supported in their cocoa farm establishment and maintenance activities such as provision of temporary and permanent shade plants, farm weeding and fertilizer application. Cutting out activities are to be intensified

in high prevalent areas such as the Western and Eastern regions of Ghana.

Scientific research activities to support the program have been strengthened to include development of early detection tools, mealybug vectors control, identification of other alternative hosts and vectors as well as development of resistant cocoa varieties. Characterization of the diversity and virulence of the disease across the West African Sub-region is also to progress to understand the nature of the virus

*DOI: http://dx.doi.org/10.5772/intechopen.87009*

not been successful [37].

very appropriate [41].

*3.1.7 Mild strains cross protection*

ment strategy for the virus [44–46].

**4. Current strategies on CSSVD management**

*3.1.6 Use of resistant cocoa varieties*

*3.1.5 Lack of effective control methods for mealybugs*

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present… DOI: http://dx.doi.org/10.5772/intechopen.87009*

#### *3.1.5 Lack of effective control methods for mealybugs*

The use of synthetic chemicals for the control of the mealybug vectors has not been effective over the years. This is attributed to the morphology of the mealybugs having a protective wax covering and also the building of mud tents over them by black ants [33–36]. Attempts by scientists with the use of biological means have also not been successful [37].

#### *3.1.6 Use of resistant cocoa varieties*

*Plant Diseases-Current Threats and Management Trends*

*3.1.2 Lack of continuity of the cutting out program*

outbreaks and time to treatment and replanting.

*3.1.3 Non-adherence to replanting recommendations*

contributing to the prevalence of the disease across cocoa farms.

*3.1.4 Presence of alternative hosts in newly replanted farms*

shade for their cocoa during the early growth period.

trained diseased spotters carry out tree-by-tree inspection for visual symptoms of the virus inevitably means that latently infected trees which have not produced symptoms at the time of inspection are missed. Considering the pattern of CSSVD spread [29] which is mainly limited to adjacent trees around the periphery of existing outbreaks, it can be argued that, the disease spotters follow the virus and are always "one step behind". It is also generally known that CSSVD symptoms tend to be least conspicuous during the dry season when the trees deteriorate and leaves and shoots are shed or damaged by capsids [9]. Considering the common phenomenon of dehydration and less active growth of infected trees during the dry season, it is very likely that disease spotters would miss some infected trees in the field during these periods. There is thus a high possibility that these missed infections could be supporting mealybug population and also transmit the disease even though they may not show conspicuous symptoms [8, 9, 23]. The challenge of lack of efficient detection protocol for the virus at the early stages has therefore generally been considered among the reasons why the disease continues to spread at an increasing rate in Ghana. The need for efficient early detection tools has always been advocated to be one of the means to help in the effective management

National implementation of the cutting out program has been delayed or halted

on many occasions for numerous reasons such as; farmer opposition, logistical constraints, lack of funding, and at times political interference [5]. Although, it is known that continuity in the cutting out operation would have been very essential for the control of other viral diseases in other areas it has never been achieved most especially in Ghana. There is always time lag between symptom identification in

Replanting of treated cocoa farms according to laid down recommendations could have been successful in rehabilitating devastated cocoa areas. However, it was noted that newly planted farms in most of the cocoa areas, especially, in the Western Region, showed symptoms of re-infection with the virus [4]. This is because farms are replanted very close to the boundary of abandoned cocoa farms containing visible infections, without much attempt to remove the infected trees in the old plantations. The recommendation that replanting should only occur after the complete removal of sources of infected cocoa trees or alternative host plants, have not been applied adequately in the eradication and replanting exercise hence

The occurrence of wild alternative host plants of the virus in and around cocoa farms is a contributory factor to spread and early re-infection of new cocoa plantings in Ghana [25]. Even though, removal of wild alternative host trees has been recommended, it is seldom applied. The advice to farmers to leave at least a 15 m barrier with some economic crops such as citrus and oil palm around new cocoa plantings to delay reinfection from old plantations and forest trees have also not been implemented fully. Farmers always want to fully utilize all available space of land for cocoa planting and also use the alternative host trees as a source of natural

**176**

of the virus.

Even though it has long be envisaged that planting of cocoa varieties tolerant to CSSVD would be the most effective means to manage the disease [38], most of the available cocoa planting materials has however shown low levels of resistance under field conditions. It is notable that many of the inter-Amazon hybrids developed and recommended by the British Research Team and currently available to farmers [38] have only partial resistance to CSSVD [38–40]. Although partial resistance is beneficial for being able to tolerate the virus to give appreciable yield in the short to medium term, the need for varieties that could offer greater resistance cannot be overemphasized. The continuous search for varieties that could offer long-term resistance that has been going on over the years using modern breeding approaches such as mutation and tissue culture techniques in Ghana and elsewhere is therefore very appropriate [41].

#### *3.1.7 Mild strains cross protection*

Long term field assessment of the mild strain cross protection experiments carried out at the Cocoa Research Institute of Ghana (CRIG) has shown that, the immunity conferred on the healthy cocoa plants from the available mild strains N1 and SS365B eventually breaks down after 20 years [42, 43]. These reports support past works and suggestions that further investigations on the effectiveness of the mild strain phenomenon need to be carried out before its adoption as a management strategy for the virus [44–46].

#### **4. Current strategies on CSSVD management**

The cutting out and rehabilitation program was re-launched in June 2018 to concurrently cut out CSSVD infected cocoa trees along the borders of Ghana and Cote d'Ivoire. The expectation is to progressively remove about 100,000 ha of infected CSSVD outbreaks across the cocoa regions in Ghana by 2023. The current cutting out activities involves the total removal of CSSVD infected cocoa trees in blocks of outbreaks and replanting with tolerant cocoa varieties. Payment of compensation to farmers and land owners has been incorporated into the program to sustain farmers' livelihood during the periods of cocoa tree removal and replanting. Additionally, farmers would be supported in their cocoa farm establishment and maintenance activities such as provision of temporary and permanent shade plants, farm weeding and fertilizer application. Cutting out activities are to be intensified in high prevalent areas such as the Western and Eastern regions of Ghana.

Scientific research activities to support the program have been strengthened to include development of early detection tools, mealybug vectors control, identification of other alternative hosts and vectors as well as development of resistant cocoa varieties. Characterization of the diversity and virulence of the disease across the West African Sub-region is also to progress to understand the nature of the virus

at the molecular level. Diversity studies to classify the virus into distinct groups of strains and isolates as identified in the Western region and adjoining areas of the Brong Ahafo Region is also being aggressively pursued to explain the difference in virulence and symptoms of the disease in different outbreaks.

#### **5. Conclusion**

The cutting out approach to remove sources of infection from farms still remains the most feasible method to manage the spread of CSSVD in an integrated system with other agronomic practices. Emphasis should therefore be placed on finding the most logical and efficient means of carrying out the program with the support of farmers and other relevant stakeholders. Accordingly, extensive farmer education on the effects of the disease and the rationale behind removal of sources of infection from outbreak areas is very imperative. Additionally, policies and rate of treatment and replanting needs careful coordination both at the District and Regional levels and this should take into consideration the severity of the disease and the availability of adequate manpower and resources to implement the program according to laid down recommendations.

The ineffectiveness of the cutting out strategy as noted in the past should be placed in the appropriate context of the many problems or challenges in its implementation together with the logistical and manpower constraints that have characterized the cutting out scheme from the outset. This chapter highlighted some of the challenges of the cutting out system and still recommends its application in an integrated approach involving the use of different measures in a coordinated manner. The focus for current and future scientific research and topics for consideration include; the use of modern breeding techniques to develop resistant cocoa varieties for CSSV, studies on chemical and biological control of the mealybug vectors, development of early detection tools, and identification of other vectors and hosts of the virus.

#### **Acknowledgements**

The author thanks fellow scientists for reading through the manuscript and for their useful contributions and suggestions. This paper is published with the kind permission of the Executive Director of the Cocoa Research Institute of Ghana (CRIG).

#### **Author details**

George A. Ameyaw Cocoa Research Institute of Ghana (CRIG), Ghana

\*Address all correspondence to: gaakumfi@yahoo.co.uk

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**179**

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present…*

[9] Thresh JM. Control of cacao swollen shoots disease. A review of the present situation. Technical Bulletin; West African Cocoa Research Institute; No. 4,

[10] Posnette AF. Swollen-shoot virus disease of cacao. Tropical Agricultura.

[11] Posnette AF. The diagnosis of swollen shoot disease of cacao. Tropical

[12] Brunt AA, Kenten RH, Nixon HL. Some properties of cocoa swollen hoot virus. Journal of General Microbiology.

[13] Kenten RH, Legg JT. Varietal resistance of cocoa to swollen shoot disease in West Africa. FAO Plant Protection Bulletin. 1971;**19**:2-12

[14] Adomako D, Lesemann DE, Paul HL, Owusu GK. Improved methods for the purification and detection of cacao swollen shoot virus. Annals of Applied

[15] Muller E, Sackey S. Molecular variability analysis of five new complete cacao swollen shoot virus genomic sequences. Archives of Virology.

[16] Lot H, Djiekpor E, Jacquuemond M. Characterization of the genome of cacao swollen shoot virus. Journal of General

[17] Hagen LS, Jacquemod M, Lepingle A, Lot H, Tepfer M. Nucleotide sequence and genome organisation of cacao swollen shoot virus. Virology. 1993;**196**:619-628

[18] Sagerman W, Lesemann D-E, Paul

HL, Adomako D, Owusu GK. Detection and comparison of some Ghanaian isolates of cocoa swollen shoot virus (CSSV) by enzyme-linked

Biology. 1983;**103**:109-116

Virology. 1991;**72**:1735-1739

2005;**150**:53-66

Agriculture. 1943;**21**:156-158

WACRI, Tafo, Ghana; 1958

1941;**18**:87-90

1964;**36**:303-309

*DOI: http://dx.doi.org/10.5772/intechopen.87009*

[1] Flood J, Guest D, Holmes KA, Keane P, Padi B, Sulistyowati E. Cocoa under attack. In: Flood J, Murphy R, editors. Cocoa Futures: A Source Book of Some Important Issues Facing the Cocoa Industry. Chinchina, Colombia: CABI-FEDERACAFE, USDA. The Commodities Press; 2004. pp. 33-53.

[2] Posnette AF. Viruses of cocoa in West Africa: 1. Cocoa viruses 1A, 1B, 1C, and 1D. Annals of Applied Biology.

[3] Muller E, Ravel S, Agret C, Abrokwah F, Dzahini-Obiatey H, Galyuon I, et al. Next generation sequencing elucidates cacao badnavirus diversity and reveals the existence of more than ten viral species. Virus

Research. 2018;**244**:235-251

[4] Dzahini-Obiatey H, Ameyaw GA, Ollennu LA. Control of cocoa swollen shoot disease by eradicating infected trees in Ghana: A survey of treated and replanted areas. Crop Protection.

[5] Ameyaw GA, Dzahini-Obiatey HK, Domfeh O. Perspectives on cocoa swollen shoot virus disease (CSSVD) management in Ghana. Crop Protection.

[6] Steven WF. Swollen shoot and die-back—A new disease of cocoa. Gold

[7] Posnette AF. Transmission of "swollen shoot" disease of cacao. Tropical Agriculture, Trinidad and

[8] Owusu GK. The cocoa swollen shoot virus problem in Ghana. In: Plumb RT, Thresh JM, editors. Plant Virus Epidemiology. Oxford: Blackwell Scientific Publications;

Coast Farmer. 1936;**5**:144

Tobago. 1940;**17**:98

1983. pp. 73-83

ISBN 958-97441-1-7

**References**

1947;**34**:388-402

2006;**25**:647-652

2014;**65**:64-70

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present… DOI: http://dx.doi.org/10.5772/intechopen.87009*

#### **References**

*Plant Diseases-Current Threats and Management Trends*

according to laid down recommendations.

**Acknowledgements**

**Author details**

George A. Ameyaw

Cocoa Research Institute of Ghana (CRIG), Ghana

provided the original work is properly cited.

\*Address all correspondence to: gaakumfi@yahoo.co.uk

**5. Conclusion**

virulence and symptoms of the disease in different outbreaks.

at the molecular level. Diversity studies to classify the virus into distinct groups of strains and isolates as identified in the Western region and adjoining areas of the Brong Ahafo Region is also being aggressively pursued to explain the difference in

The cutting out approach to remove sources of infection from farms still remains the most feasible method to manage the spread of CSSVD in an integrated system with other agronomic practices. Emphasis should therefore be placed on finding the most logical and efficient means of carrying out the program with the support of farmers and other relevant stakeholders. Accordingly, extensive farmer education on the effects of the disease and the rationale behind removal of sources of infection from outbreak areas is very imperative. Additionally, policies and rate of treatment and replanting needs careful coordination both at the District and Regional levels and this should take into consideration the severity of the disease and the availability of adequate manpower and resources to implement the program

The ineffectiveness of the cutting out strategy as noted in the past should be placed in the appropriate context of the many problems or challenges in its implementation together with the logistical and manpower constraints that have characterized the cutting out scheme from the outset. This chapter highlighted some of the challenges of the cutting out system and still recommends its application in an integrated approach involving the use of different measures in a coordinated manner. The focus for current and future scientific research and topics for consideration include; the use of modern breeding techniques to develop resistant cocoa varieties for CSSV, studies on chemical and biological control of the mealybug vectors, development of early detection tools, and identification of other vectors and hosts of the virus.

The author thanks fellow scientists for reading through the manuscript and for their useful contributions and suggestions. This paper is published with the kind permission of the Executive Director of the Cocoa Research Institute of Ghana (CRIG).

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

**178**

[1] Flood J, Guest D, Holmes KA, Keane P, Padi B, Sulistyowati E. Cocoa under attack. In: Flood J, Murphy R, editors. Cocoa Futures: A Source Book of Some Important Issues Facing the Cocoa Industry. Chinchina, Colombia: CABI-FEDERACAFE, USDA. The Commodities Press; 2004. pp. 33-53. ISBN 958-97441-1-7

[2] Posnette AF. Viruses of cocoa in West Africa: 1. Cocoa viruses 1A, 1B, 1C, and 1D. Annals of Applied Biology. 1947;**34**:388-402

[3] Muller E, Ravel S, Agret C, Abrokwah F, Dzahini-Obiatey H, Galyuon I, et al. Next generation sequencing elucidates cacao badnavirus diversity and reveals the existence of more than ten viral species. Virus Research. 2018;**244**:235-251

[4] Dzahini-Obiatey H, Ameyaw GA, Ollennu LA. Control of cocoa swollen shoot disease by eradicating infected trees in Ghana: A survey of treated and replanted areas. Crop Protection. 2006;**25**:647-652

[5] Ameyaw GA, Dzahini-Obiatey HK, Domfeh O. Perspectives on cocoa swollen shoot virus disease (CSSVD) management in Ghana. Crop Protection. 2014;**65**:64-70

[6] Steven WF. Swollen shoot and die-back—A new disease of cocoa. Gold Coast Farmer. 1936;**5**:144

[7] Posnette AF. Transmission of "swollen shoot" disease of cacao. Tropical Agriculture, Trinidad and Tobago. 1940;**17**:98

[8] Owusu GK. The cocoa swollen shoot virus problem in Ghana. In: Plumb RT, Thresh JM, editors. Plant Virus Epidemiology. Oxford: Blackwell Scientific Publications; 1983. pp. 73-83

[9] Thresh JM. Control of cacao swollen shoots disease. A review of the present situation. Technical Bulletin; West African Cocoa Research Institute; No. 4, WACRI, Tafo, Ghana; 1958

[10] Posnette AF. Swollen-shoot virus disease of cacao. Tropical Agricultura. 1941;**18**:87-90

[11] Posnette AF. The diagnosis of swollen shoot disease of cacao. Tropical Agriculture. 1943;**21**:156-158

[12] Brunt AA, Kenten RH, Nixon HL. Some properties of cocoa swollen hoot virus. Journal of General Microbiology. 1964;**36**:303-309

[13] Kenten RH, Legg JT. Varietal resistance of cocoa to swollen shoot disease in West Africa. FAO Plant Protection Bulletin. 1971;**19**:2-12

[14] Adomako D, Lesemann DE, Paul HL, Owusu GK. Improved methods for the purification and detection of cacao swollen shoot virus. Annals of Applied Biology. 1983;**103**:109-116

[15] Muller E, Sackey S. Molecular variability analysis of five new complete cacao swollen shoot virus genomic sequences. Archives of Virology. 2005;**150**:53-66

[16] Lot H, Djiekpor E, Jacquuemond M. Characterization of the genome of cacao swollen shoot virus. Journal of General Virology. 1991;**72**:1735-1739

[17] Hagen LS, Jacquemod M, Lepingle A, Lot H, Tepfer M. Nucleotide sequence and genome organisation of cacao swollen shoot virus. Virology. 1993;**196**:619-628

[18] Sagerman W, Lesemann D-E, Paul HL, Adomako D, Owusu GK. Detection and comparison of some Ghanaian isolates of cocoa swollen shoot virus (CSSV) by enzyme-linked

immunosorbent assay (ELISA) and immunoelectron microscopy (EM) using an antiserum to CSSV strain 1A. Journal of Phytopathology. 1985;**114**:78-89

[19] Nomatter C, Kouakou K, Aka R, Ameyaw G, Gutierrez OA, Herrmann H-W, et al. The proposed new species, cacao red vein virus, and three previously recognized badnavirus species are associated with cacao swollen shoot disease. Virology Journal. 2017;**14**:199-213

[20] Ameyaw GA, Chigandu N, Dzahini-Obiatey HK, Domfeh O, Gutierrez O, Judith K. Brown Variable detection potential of some newly designed primers on Ghanaian CSSV isolates. Lima, Peru: International Symposium on Cocoa Research (ISCR); 13-17 November 2017

[21] Posnette AF, Strickland AH. Virus diseases of cocoa in West Africa. Technique of insect transmission. Annals of Applied Biology. 1948;**35**:53-63

[22] Campbell CAM. The assessment of mealybugs (*Pseudococcidae*) and other Homoptera on mature cocoa trees in Ghana. Bulletin of Entomological Research. 1983;**73**:137-151

[23] Cornwell PB. Effect of wind currents on vector dispersal. Annual Report; West African Cocoa Research Institute; 1956. p. 46

[24] Brunt AA, Kenten RH. The mechanical transmission of cocoa swollen shoot virus to and from cocoa and other hosts. Annals of Applied Biology. 1962;**50**:749-754

[25] Posnette AF, Robertson NF, Todd JM. Virus diseases of cocoa in West Africa. V. Alternative host plants. Annals of Applied Biology. 1950;**37**:229-240

[26] Posnette AF. The role of wild hosts. In: Thresh JM, editor. Pests, Pathogens

and Vegetation. London, U.K.: Pitman; 1981. pp. 71-78

[27] Dale WT, Attafuah A. The host range of cocoa viruses. Annual Report of the West African Cocoa Research Institute; 1955/1956. pp. 28-30

[28] Attafuah A, Glendenning DR. Studies on resistance and tolerance to cocoa viruses in Ghana. I. A survey of T17 progeny. The Annals of Applied Biology. 1965;**56**:219

[29] Thresh JM, Owusu GK, Ollennu LAA. Cocoa swollen shoot virus: An archetypal crowd disease. Journal of Plant Diseases and Protection. 1988;**95**:428-446

[30] Cornwell PB. Movements of the vectors of virus diseases of cacao in Ghana. II. Wind movement and aerial dispersal. Bulletin of Entomological Research. 1960;**51**:175-201

[31] Strickland AH. The dispersal of *Pseudococcidae* (*Hemiptera*-*Homoptera*) by air currents in the Gold Coast. Proceedings of the Royal Entomological society, London. 1950;**25**:1-9

[32] Thresh JM, Lister RM. Coppicing experiments on the spread and control of cacao swollen shoot disease in Nigeria. Annals of Applied Biology. 1960;**48**:65-74

[33] Donald RG. Mealybug studies. Annual Report of West African Cocoa Research Institute; 1953/1954. p. 101

[34] Decker FE. Mealybug studies; biological control of mealybugs. Annual Report of West African Cocoa Research Institute. 1953. p. 23

[35] Hanna AD, Judenko EB, Heatherington W. Systemic insecticides for the control of insects transmitting the swollen shoot virus of cocoa in the Gold Coast. Bulletin of Entomological Research. 1955;**46**:669-710

**181**

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present…*

SS365B on growth and yield of cacao. Journal of General Plant Pathology. 2018;**84**:369-375. DOI: 10.1007/

[44] Owusu GK, Ollennu LAA, Dzahini-Obiatey HK. The prospects for mild strain cross-protection to control cocoa swollen shoot disease in Ghana. In: Proceedings of 12th International Cocoa Research Conference; Salvador, Bahia, Brazil; Lagos, Nigeria: Cocoa Producers' Alliance (COPAL); 1996. pp. 121-127

[45] Hughes JA, LAA O. Mild strain protection of cocoa in Ghana against cocoa swollen shoot virus—A review. Plant Pathology. 1994;**43**:442-457

[46] Ollennu LAA, Owusu GK, Dzahini-Obiatey HK. Recent studies of mild strain cross protection with cocoa swollen shoot virus. Journal of the Ghana Science Association. 1999;**2**:5-11

[47] Muller E. Cacao Swollen Shoot Virus (CSSV) History, Biology, and Genome. In: Bailey BA, Meinhard LW, editors. Cacao Diseases—A History of Old Enemies and New Encounters. Switzerland: Springer International

[48] INCOPED. Proceedings of 5th International Seminar on Cocoa Pests and Diseases (INCOPED); 15-17 October 2006; San Jose, Costa Rica

Publishing AG; 2016

s10327-018-0794-3

*DOI: http://dx.doi.org/10.5772/intechopen.87009*

[36] Hanna AD. Research in the use of systemic insecticides to control the mealybug vectors of swollen shoot disease of cocoa. Phytopharmacology.

[37] Acknonor JB. Preliminary studies on breeding and predation on *Scymnus* (*pullus*) sp. and *Hyperaspis egregia* Mader on *Plannococcus njalensis* (Laing) In: Ollennu LAA, Owusu GK, Padi B, editors. Proceedings of the First International Cocoa Pests and Diseases Seminar; 6-10 November 1995; Accra,

[38] Legg JT, Lockwood G. Resistance of cocoa to swollen shoot virus in Ghana. I. Field trials. The Annals of Applied

[39] Adu-Ampomah Y. The cocoa breeding program in Ghana:

Achievements and prospects for the future. Cocoa Growers' Bulletin.

[40] Legg J, Owusu GK, Ollennu LAA, Lovi NK. The Problems of Controlling Cocoa Swollen Shoot Disease in Ghana. In: Proceedings of 6th International Cocoa Research Conference; Caracas, Venezuela; Lagos, Nigeria: Cocoa Producers' Alliance (COPAL); 1981.

[41] Adomako B, Adu-Ampomah Y, Ollennu LAA, Dzahini-Obiatey H, Takrama JF. Breeding for cocoa varieties resistant/tolerant to cocoa swollen shoot virus. Annual Report; Cocoa Research Institute of Ghana; 2003. pp. 201-203

[42] Ameyaw GA, Dzahini-Obiatey HK, Domfeh O, Ollennu LAA, Owusu G. Appraisal of cocoa swollen shoot virus mild isolates for cross protection of cocoa against severe strains in Ghana. Plant Disease. 2016;**100**(4):810-815

[43] Domfeh O, Ameyaw GA, Dzahini-Obiatey HK. The effects of mild cacao swollen shoot virus strains N1 and

1954:425-432

Ghana; pp. 238-241

Biology. 1981;**97**:75-89

1996;**50**:17-21

pp. 267-278

*Management of the Cacao Swollen Shoot Virus (CSSV) Menace in Ghana: The Past, Present… DOI: http://dx.doi.org/10.5772/intechopen.87009*

[36] Hanna AD. Research in the use of systemic insecticides to control the mealybug vectors of swollen shoot disease of cocoa. Phytopharmacology. 1954:425-432

*Plant Diseases-Current Threats and Management Trends*

and Vegetation. London, U.K.: Pitman;

[27] Dale WT, Attafuah A. The host range of cocoa viruses. Annual Report of the West African Cocoa Research Institute; 1955/1956. pp. 28-30

[28] Attafuah A, Glendenning DR. Studies on resistance and tolerance to cocoa viruses in Ghana. I. A survey of T17 progeny. The Annals of Applied

[29] Thresh JM, Owusu GK, Ollennu LAA. Cocoa swollen shoot virus: An archetypal crowd disease. Journal of Plant Diseases and Protection.

[30] Cornwell PB. Movements of the vectors of virus diseases of cacao in Ghana. II. Wind movement and aerial dispersal. Bulletin of Entomological

[31] Strickland AH. The dispersal of *Pseudococcidae* (*Hemiptera*-*Homoptera*) by air currents in the Gold Coast. Proceedings of the Royal Entomological

[32] Thresh JM, Lister RM. Coppicing experiments on the spread and control of cacao swollen shoot disease in Nigeria. Annals of Applied Biology.

[33] Donald RG. Mealybug studies. Annual Report of West African Cocoa Research Institute; 1953/1954. p. 101

[34] Decker FE. Mealybug studies; biological control of mealybugs. Annual Report of West African Cocoa Research

Heatherington W. Systemic insecticides for the control of insects transmitting the swollen shoot virus of cocoa in the Gold Coast. Bulletin of Entomological

Research. 1960;**51**:175-201

society, London. 1950;**25**:1-9

1960;**48**:65-74

Institute. 1953. p. 23

[35] Hanna AD, Judenko EB,

Research. 1955;**46**:669-710

1981. pp. 71-78

Biology. 1965;**56**:219

1988;**95**:428-446

immunosorbent assay (ELISA) and immunoelectron microscopy (EM) using an antiserum to CSSV strain 1A. Journal of Phytopathology.

[19] Nomatter C, Kouakou K, Aka R, Ameyaw G, Gutierrez OA, Herrmann H-W, et al. The proposed new species, cacao red vein virus, and three previously recognized badnavirus species are associated with cacao swollen shoot disease. Virology Journal. 2017;**14**:199-213

[20] Ameyaw GA, Chigandu N, Dzahini-Obiatey HK, Domfeh O, Gutierrez O, Judith K. Brown Variable detection potential of some newly designed primers on Ghanaian CSSV isolates. Lima, Peru: International Symposium on Cocoa Research (ISCR); 13-17

[21] Posnette AF, Strickland AH. Virus diseases of cocoa in West Africa. Technique of insect transmission. Annals of Applied Biology.

[22] Campbell CAM. The assessment of mealybugs (*Pseudococcidae*) and other Homoptera on mature cocoa trees in Ghana. Bulletin of Entomological

Research. 1983;**73**:137-151

Institute; 1956. p. 46

Biology. 1962;**50**:749-754

[23] Cornwell PB. Effect of wind currents on vector dispersal. Annual Report; West African Cocoa Research

[24] Brunt AA, Kenten RH. The mechanical transmission of cocoa swollen shoot virus to and from cocoa and other hosts. Annals of Applied

[25] Posnette AF, Robertson NF, Todd JM. Virus diseases of cocoa in West Africa. V. Alternative host plants. Annals of Applied Biology. 1950;**37**:229-240

[26] Posnette AF. The role of wild hosts. In: Thresh JM, editor. Pests, Pathogens

1985;**114**:78-89

November 2017

1948;**35**:53-63

**180**

[37] Acknonor JB. Preliminary studies on breeding and predation on *Scymnus* (*pullus*) sp. and *Hyperaspis egregia* Mader on *Plannococcus njalensis* (Laing) In: Ollennu LAA, Owusu GK, Padi B, editors. Proceedings of the First International Cocoa Pests and Diseases Seminar; 6-10 November 1995; Accra, Ghana; pp. 238-241

[38] Legg JT, Lockwood G. Resistance of cocoa to swollen shoot virus in Ghana. I. Field trials. The Annals of Applied Biology. 1981;**97**:75-89

[39] Adu-Ampomah Y. The cocoa breeding program in Ghana: Achievements and prospects for the future. Cocoa Growers' Bulletin. 1996;**50**:17-21

[40] Legg J, Owusu GK, Ollennu LAA, Lovi NK. The Problems of Controlling Cocoa Swollen Shoot Disease in Ghana. In: Proceedings of 6th International Cocoa Research Conference; Caracas, Venezuela; Lagos, Nigeria: Cocoa Producers' Alliance (COPAL); 1981. pp. 267-278

[41] Adomako B, Adu-Ampomah Y, Ollennu LAA, Dzahini-Obiatey H, Takrama JF. Breeding for cocoa varieties resistant/tolerant to cocoa swollen shoot virus. Annual Report; Cocoa Research Institute of Ghana; 2003. pp. 201-203

[42] Ameyaw GA, Dzahini-Obiatey HK, Domfeh O, Ollennu LAA, Owusu G. Appraisal of cocoa swollen shoot virus mild isolates for cross protection of cocoa against severe strains in Ghana. Plant Disease. 2016;**100**(4):810-815

[43] Domfeh O, Ameyaw GA, Dzahini-Obiatey HK. The effects of mild cacao swollen shoot virus strains N1 and

SS365B on growth and yield of cacao. Journal of General Plant Pathology. 2018;**84**:369-375. DOI: 10.1007/ s10327-018-0794-3

[44] Owusu GK, Ollennu LAA, Dzahini-Obiatey HK. The prospects for mild strain cross-protection to control cocoa swollen shoot disease in Ghana. In: Proceedings of 12th International Cocoa Research Conference; Salvador, Bahia, Brazil; Lagos, Nigeria: Cocoa Producers' Alliance (COPAL); 1996. pp. 121-127

[45] Hughes JA, LAA O. Mild strain protection of cocoa in Ghana against cocoa swollen shoot virus—A review. Plant Pathology. 1994;**43**:442-457

[46] Ollennu LAA, Owusu GK, Dzahini-Obiatey HK. Recent studies of mild strain cross protection with cocoa swollen shoot virus. Journal of the Ghana Science Association. 1999;**2**:5-11

[47] Muller E. Cacao Swollen Shoot Virus (CSSV) History, Biology, and Genome. In: Bailey BA, Meinhard LW, editors. Cacao Diseases—A History of Old Enemies and New Encounters. Switzerland: Springer International Publishing AG; 2016

[48] INCOPED. Proceedings of 5th International Seminar on Cocoa Pests and Diseases (INCOPED); 15-17 October 2006; San Jose, Costa Rica

**Chapter 11**

**Abstract**

Emergence of Benzimidazole- and

Strobilurin-Quinone Outside

Inhibitor-Resistant Strains of

*Colletotrichum gloeosporioides*

sensu lato, the Causal Fungus

of Japanese Pear Anthracnose,

Japanese pear anthracnose (JPA) can cause severe tree defoliation during the growing season. Infected trees become weak and produce fewer flower buds the following spring. This economically serious fungal plant disease has affected cultivated pears in Japan since 1910. Initially, JPA was controlled by benzimidazole fungicides. However, benzimidazole-resistant pathogen strains emerged in the late 1990s, and the range of JPA has expanded in Japan. Since then strobilurin-quinone outside inhibitors (ST-QoIs) such as azoxystrobin and kresoxim-methyl became popular, but ST-QoI-resistant pathogen strains appeared. By 2005, JPA control became difficult once again. In this chapter, we outline the history of JPA fungicide resistance problems, assess advantages and disadvantages of available fungicide options, and develop JPA management strategies based on evidences we obtained

**Keywords:** anthracnose, benzimidazole, deciduous disease, Japanese pear,

A sudden and severe outbreak of Japanese pear anthracnose (JPA) occurred in July 1999 on the Japanese pear cultivars "Housui" and "Niitaka" (*Pyrus pyrifolia* Nakai var. culta Nakai) in Saga prefecture on Kyushu Island, which is one of the major Japanese pear-producing regions located in southwestern Japan [1, 2].

and Alternative Fungicides

*Nobuya Tashiro, Youichi Ide, Mayumi Noguchi,*

to Resistant Strains

from a series of field and lab studies.

*Pyrus pyrifolia*, ST-QoI

**1. Introduction**

**183**

*Hisayoshi Watanabe and Mizuho Nita*

### **Chapter 11**

Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains of *Colletotrichum gloeosporioides* sensu lato, the Causal Fungus of Japanese Pear Anthracnose, and Alternative Fungicides to Resistant Strains

*Nobuya Tashiro, Youichi Ide, Mayumi Noguchi, Hisayoshi Watanabe and Mizuho Nita*

### **Abstract**

Japanese pear anthracnose (JPA) can cause severe tree defoliation during the growing season. Infected trees become weak and produce fewer flower buds the following spring. This economically serious fungal plant disease has affected cultivated pears in Japan since 1910. Initially, JPA was controlled by benzimidazole fungicides. However, benzimidazole-resistant pathogen strains emerged in the late 1990s, and the range of JPA has expanded in Japan. Since then strobilurin-quinone outside inhibitors (ST-QoIs) such as azoxystrobin and kresoxim-methyl became popular, but ST-QoI-resistant pathogen strains appeared. By 2005, JPA control became difficult once again. In this chapter, we outline the history of JPA fungicide resistance problems, assess advantages and disadvantages of available fungicide options, and develop JPA management strategies based on evidences we obtained from a series of field and lab studies.

**Keywords:** anthracnose, benzimidazole, deciduous disease, Japanese pear, *Pyrus pyrifolia*, ST-QoI

#### **1. Introduction**

A sudden and severe outbreak of Japanese pear anthracnose (JPA) occurred in July 1999 on the Japanese pear cultivars "Housui" and "Niitaka" (*Pyrus pyrifolia* Nakai var. culta Nakai) in Saga prefecture on Kyushu Island, which is one of the major Japanese pear-producing regions located in southwestern Japan [1, 2].

At first, phytotoxicity was suspected owing to extensive and rapid symptom development throughout the orchard. Subsequent investigation revealed that it was JPA caused by *Colletotrichum gloeosporioides* sensu lato (Cgsl) [1, 2].

JPA was first reported in Japan by Kurosawa in 1910 [3]. He observed JPA in Fukuoka prefecture which is adjacent to Saga prefecture in June 1910. The infection caused black spots on the leaves and severe defoliation. Disease incidence and severity differed among varieties. It was severe on "Doitsu," moderate on "Nijusseiki," and mild on "Chojuro." Morphological analyses indicated that the causal organism was *C. gloeosporioides*. Kurosawa stated that Bordeaux mixture could be an effective treatment and damaged leaves should be incinerated to prevent the spread of the disease. Based on Kurosawa's research, Hara introduced JPA in his textbook entitled *Fruit Tree Disease Theory* [4]. Ikata reported that JPA was uncommon and caused no severe damage except for an outbreak in Fukuoka prefecture in 1910 [5]. There were no further reports on JPA until 1974.

In 1974, severe JPA infestations on the "Yakumo" cultivar were reported in Fukushima prefecture of the Tohoku region, which is located in northeastern Japan. The outbreak caused severe defoliation. Ochiai et al. monitored the progress of the outbreak and isolated the causal organism [6]. Ochiai and Hayashi discussed the pathogenicity of isolated *Colletotrichum* sp. and indicated that disease severity differed among host cultivars [7]. They also mentioned the effect of the infection timing (the number of days elapsed after leaf expansion) [8], temperature and leaf wetness on infection and disease incidence [9], and growth medium and temperature on pathogen growth [10]. However, there were no published reports on pathogen control methods.

In 1987 and 1998, severe incidences of JPA were reported in Kochi prefecture in Shikoku Island, located in southwestern Japan. Morita et al. reported the symptoms and transition of the outbreak. They documented the efficacy of thiophanatemethyl/maneb wettable powder (WP), maneb WP, and benomyl WP at controlling this disease [11]. There was also a report of an outbreak of moderately benzimidazole-resistant strains in 1998 [12].

which leads to a fewer number of flowers in the next spring, which caused serious

*Black spots on anthracnose-infected petioles and leaves caused by* Colletotrichum gloeosporioides *sensu lato observed in Japanese pear cv. Housui (*Pyrus pyrifolia *Nakai var.* culta *Nakai) in the field. (A) Very minute black spots symptoms on the petioles, (B) leaf symptoms showing numerous black spots of various sizes, (C) large blackish brown spots with a diameter of about 1–2 cm, (D) yellowing leaves with large blackish brown*

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

Fungal cultures were isolated from the large dark brown lesions on leaves and smaller lesions on petioles of the 'Housui' and "Niitaka" Japanese pear cultivars. Morphologically, these isolates were identical. The isolates formed light salmon flesh-colored conidial masses on spore-inducing media (K2HPO4 1 g, MgSO4 0.5 g, peptone 5 g, lactose 10 g, agar 30 g, and distilled water 1000 mL) (**Figure 3**). Foliar

disease symptoms similar to those observed in the orchards (**Figure 3**). The inocu-

The conidia are cylindrical with an average size of 15.8 μm 5.0 μm (**Figure 3**). The mycelia from these isolates grow at 10–35°C with an optimum at 28°C. PCR using primer CgInt [13] to detect Cgsl disclosed a band at 450 bp similar to that

Based on its morphological characteristics, a similar foliar disease observed on "Kousui" in Akita prefecture in the Tohoku region of Japan was thought to be anthracnose caused by *Colletotrichum acutatum* sensu lato [14]. However, DNAbased identification failed to establish *C. acutatum* sensu lato as the cause of JPA in the pear orchards of other regions of Japan [12, 15]. Therefore, most of the JPA

In China, *C. fructicola* was reported as an anthracnose pathogen causing leaf black spot in sandy pear (*Pyrus pyrifolia* Nakai) in 2015 [16]. In 2019, 12 species of *Colletotrichum* spp. including *C. fructicola* and *C. gloeosporioides* were reported as

) on 'Housui' reproduced

yield loss in the following year (**Figure 2**).

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

obtained by using Cgsl as a control.

spray inoculation of a conidial suspension (105 mL<sup>1</sup>

pathogens in Japan are probably caused by Cgsl.

lated fungi were re-isolated to confirm Koch's postulates [1, 2].

**2.2 Causal organism**

**Figure 1.**

*spots.*

**185**

Probably because JPA happened sporadically over a long time period and in small and isolated geographical areas, there was a very limited effort to identify fungicides that are effective against JPA. Therefore, no registered fungicides were available for JPA when the major JPA outbreak occurred in Saga prefecture in 1999.

#### **2. JPA symptoms and causal organism**

#### **2.1 Symptoms**

In JPA-affected orchards in Saga prefecture, Japanese pear cultivars "Housui" and "Niitaka" developed minute black spots formed on the leaf laminae and petioles starting in mid-June. The leaves appear as though they have been stabbed with a fine needle. The perforations are visible when the leaves are held up to the sunlight. Since the lesions are very small, it is difficult for the grower to notice the initial disease symptoms unless the leaves are inspected very closely. The initially tiny black dots then expand into small curved black spots 0.5–1 mm in diameter. Certain lesions may develop into large blackish-brown spots 2 cm in diameter. By that time, the leaves rapidly turn yellow and abscise (**Figure 1**).

When the JPA outbreaks were observed in 1999, JPA caused a severe defoliation by mid-July and markedly reduced tree vigor. The intense defoliation caused new leaves to emerge soon after the event; however, these new leaves were quickly and fatally infested with JPA. In addition, defoliation triggered flowering in autumn

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

#### **Figure 1.**

At first, phytotoxicity was suspected owing to extensive and rapid symptom development throughout the orchard. Subsequent investigation revealed that it was JPA

JPA was first reported in Japan by Kurosawa in 1910 [3]. He observed JPA in Fukuoka prefecture which is adjacent to Saga prefecture in June 1910. The infection caused black spots on the leaves and severe defoliation. Disease incidence and severity differed among varieties. It was severe on "Doitsu," moderate on "Nijusseiki," and mild on "Chojuro." Morphological analyses indicated that the causal organism was *C. gloeosporioides*. Kurosawa stated that Bordeaux mixture could be an effective treatment and damaged leaves should be incinerated to prevent the spread of the disease. Based on Kurosawa's research, Hara introduced JPA in his textbook entitled *Fruit Tree Disease Theory* [4]. Ikata reported that JPA was uncommon and caused no severe damage except for an outbreak in Fukuoka pre-

In 1974, severe JPA infestations on the "Yakumo" cultivar were reported in Fukushima prefecture of the Tohoku region, which is located in northeastern Japan. The outbreak caused severe defoliation. Ochiai et al. monitored the progress of the outbreak and isolated the causal organism [6]. Ochiai and Hayashi discussed the pathogenicity of isolated *Colletotrichum* sp. and indicated that disease severity differed among host cultivars [7]. They also mentioned the effect of the infection timing (the number of days elapsed after leaf expansion) [8], temperature and leaf wetness on infection and disease incidence [9], and growth medium and temperature on pathogen growth [10]. However, there were no published reports

In 1987 and 1998, severe incidences of JPA were reported in Kochi prefecture in Shikoku Island, located in southwestern Japan. Morita et al. reported the symptoms and transition of the outbreak. They documented the efficacy of thiophanatemethyl/maneb wettable powder (WP), maneb WP, and benomyl WP at controlling

Probably because JPA happened sporadically over a long time period and in small and isolated geographical areas, there was a very limited effort to identify fungicides that are effective against JPA. Therefore, no registered fungicides were available for JPA when the major JPA outbreak occurred in Saga prefecture in 1999.

In JPA-affected orchards in Saga prefecture, Japanese pear cultivars "Housui" and "Niitaka" developed minute black spots formed on the leaf laminae and petioles starting in mid-June. The leaves appear as though they have been stabbed with a fine needle. The perforations are visible when the leaves are held up to the sunlight. Since the lesions are very small, it is difficult for the grower to notice the initial disease symptoms unless the leaves are inspected very closely. The initially tiny black dots then expand into small curved black spots 0.5–1 mm in diameter. Certain lesions may develop into large blackish-brown spots 2 cm in diameter. By that

When the JPA outbreaks were observed in 1999, JPA caused a severe defoliation by mid-July and markedly reduced tree vigor. The intense defoliation caused new leaves to emerge soon after the event; however, these new leaves were quickly and fatally infested with JPA. In addition, defoliation triggered flowering in autumn

caused by *Colletotrichum gloeosporioides* sensu lato (Cgsl) [1, 2].

*Plant Diseases-Current Threats and Management Trends*

fecture in 1910 [5]. There were no further reports on JPA until 1974.

this disease [11]. There was also a report of an outbreak of moderately

on pathogen control methods.

benzimidazole-resistant strains in 1998 [12].

**2. JPA symptoms and causal organism**

time, the leaves rapidly turn yellow and abscise (**Figure 1**).

**2.1 Symptoms**

**184**

*Black spots on anthracnose-infected petioles and leaves caused by* Colletotrichum gloeosporioides *sensu lato observed in Japanese pear cv. Housui (*Pyrus pyrifolia *Nakai var.* culta *Nakai) in the field. (A) Very minute black spots symptoms on the petioles, (B) leaf symptoms showing numerous black spots of various sizes, (C) large blackish brown spots with a diameter of about 1–2 cm, (D) yellowing leaves with large blackish brown spots.*

which leads to a fewer number of flowers in the next spring, which caused serious yield loss in the following year (**Figure 2**).

#### **2.2 Causal organism**

Fungal cultures were isolated from the large dark brown lesions on leaves and smaller lesions on petioles of the 'Housui' and "Niitaka" Japanese pear cultivars. Morphologically, these isolates were identical. The isolates formed light salmon flesh-colored conidial masses on spore-inducing media (K2HPO4 1 g, MgSO4 0.5 g, peptone 5 g, lactose 10 g, agar 30 g, and distilled water 1000 mL) (**Figure 3**). Foliar spray inoculation of a conidial suspension (105 mL<sup>1</sup> ) on 'Housui' reproduced disease symptoms similar to those observed in the orchards (**Figure 3**). The inoculated fungi were re-isolated to confirm Koch's postulates [1, 2].

The conidia are cylindrical with an average size of 15.8 μm 5.0 μm (**Figure 3**). The mycelia from these isolates grow at 10–35°C with an optimum at 28°C. PCR using primer CgInt [13] to detect Cgsl disclosed a band at 450 bp similar to that obtained by using Cgsl as a control.

Based on its morphological characteristics, a similar foliar disease observed on "Kousui" in Akita prefecture in the Tohoku region of Japan was thought to be anthracnose caused by *Colletotrichum acutatum* sensu lato [14]. However, DNAbased identification failed to establish *C. acutatum* sensu lato as the cause of JPA in the pear orchards of other regions of Japan [12, 15]. Therefore, most of the JPA pathogens in Japan are probably caused by Cgsl.

In China, *C. fructicola* was reported as an anthracnose pathogen causing leaf black spot in sandy pear (*Pyrus pyrifolia* Nakai) in 2015 [16]. In 2019, 12 species of *Colletotrichum* spp. including *C. fructicola* and *C. gloeosporioides* were reported as

#### **Figure 2.**

*Defoliation in summer and flowering in autumn caused by* Colletotrichum gloeosporioides *sensu lato observed in Japanese pear cv. Housui (*Pyrus pyrifolia *Nakai var.* culta *Nakai) in the fields. (A) Severe defoliation in the field of Japanese pear cv. Housui (*Pyrus pyrifolia *Nakai var.* culta *Nakai) (courtesy M. Suzuki); (B) yellowish discolored fallen leaves with a lot of black spots; (C) twigs with no leaves in summer; (D) emerging of new leaves and flowers soon after intense defoliation, the number of flowers in the next spring was dramatically decreased to cause great yield loss in the following year (courtesy M. Suzuki); (E) black spots symptoms on newly emerged leaves after defoliation, these new leaves quickly withered; (F) defoliation of almost all of the leaves in the summer, leading to the reduced vigor of the trees.*

pathogens causing anthracnose on pear leaves and fruit [17]. In Japan, we did not confirm anthracnose symptoms on Japanese pear fruit caused by Cgsl. A report of JPA outbreak from Akita prefecture, where they suspected *C. acutatum* sensu lato to be the causal agent, did not include anthracnose on fruits. However, *C. fioriniae* destroyed "Niitaka" fruit in Oita prefecture in 2013 [18]. In Korea, two species of *C. gloeosporioides* sensu lato [19] and *C. acutatum* sensu lato [20] were reported as the causal organisms of Asian pear fruit rot.

*Colletotrichum karstii* has been reported as a new GLS pathogen [26]. Apple GLS

*Causal pathogen of Japanese pear (*Pyrus pyrifolia *Nakai var.* culta *Nakai) anthracnose and representative symptoms induced by inoculation of conidial suspension under unwounded condition. (A) A 7-day-old colony of strain C-17 grown on potato dextrose agar medium at 25°C, (B) salmon pink spore mass produced on sporeinducing medium (K2HPO4 1 g, MgSO4 0.5 g, peptone 5 g, lactose 10 g, agar 30 g, distilled water 1000 mL), (C) conidia of strain C-17 produced on spore-inducing medium (scale bar = 20 μm), (D) shoots of Japanese pear cv. Housui that showed severe symptoms by inoculation of conidial suspensions of strain C-15 under unwounded condition, (E) black spot lesions of different sizes reproduced by unwounded inoculation of spore*

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

**3. Development of fungicide control technology for JPA (until 2004)**

Our aim was to select efficacious fungicides at the Fruit Tree Experiment Station in Saga prefecture [1, 28]. The JPA fungicide spray timing was the same as that for Japanese pear ring rot caused by *Botryosphaeria berengeriana* De Notaris f. sp. piricola (Nose) in Koganezawa and Sakuma. Thus, these diseases had to be

addressed simultaneously, and fungicide efficacy on ring rot was also evaluated [2].

On the other hand, the compendium makes no reference to foliar anthracnose in pear or Asian pear [21], which occurs on leaves and causes severe defoliation. Since this disease may be unique to Asian and Japanese pear, further investigations of its

caused by *Glomerella cingulata* was reported in China in 2012 [27].

**Figure 3.**

*suspension.*

**187**

causal pathogens using molecular diagnostic tools are required.

The *Compendium of Apple and Pear Diseases and Pests* describes apple and pear bitter rot as a common disease and mentions that apple anthracnose causes speckle spots followed by defoliation [21]. In 1988, Leite et al. [22] described a new apple leaf spot disease on the Gala and Golden Delicious cultivars in Brazil and demonstrated that it was caused by *G. cingulata* which is the sexual stage of *C. gloeosporioides*. This disease was named *Glomerella* leaf spot (GLS). This report was the first to cite any *Colletotrichum* sp. as the causative agent of leaf spot in the apple orchard. Under favorable conditions, a GLS infestation may result in 75% defoliation by harvest time. It can weaken trees and reducing yield [23, 24]. GLS was first reported in the United States in 1998 as a severe leaf spot on Gala apples [25].

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

#### **Figure 3.**

pathogens causing anthracnose on pear leaves and fruit [17]. In Japan, we did not confirm anthracnose symptoms on Japanese pear fruit caused by Cgsl. A report of JPA outbreak from Akita prefecture, where they suspected *C. acutatum* sensu lato to be the causal agent, did not include anthracnose on fruits. However, *C. fioriniae* destroyed "Niitaka" fruit in Oita prefecture in 2013 [18]. In Korea, two species of *C. gloeosporioides* sensu lato [19] and *C. acutatum* sensu lato [20] were reported as

*Defoliation in summer and flowering in autumn caused by* Colletotrichum gloeosporioides *sensu lato observed in Japanese pear cv. Housui (*Pyrus pyrifolia *Nakai var.* culta *Nakai) in the fields. (A) Severe defoliation in the field of Japanese pear cv. Housui (*Pyrus pyrifolia *Nakai var.* culta *Nakai) (courtesy M. Suzuki); (B) yellowish discolored fallen leaves with a lot of black spots; (C) twigs with no leaves in summer; (D) emerging of new leaves and flowers soon after intense defoliation, the number of flowers in the next spring was dramatically decreased to cause great yield loss in the following year (courtesy M. Suzuki); (E) black spots symptoms on newly emerged leaves after defoliation, these new leaves quickly withered; (F) defoliation of almost*

The *Compendium of Apple and Pear Diseases and Pests* describes apple and pear bitter rot as a common disease and mentions that apple anthracnose causes speckle spots followed by defoliation [21]. In 1988, Leite et al. [22] described a new apple leaf spot disease on the Gala and Golden Delicious cultivars in Brazil and demon-

*gloeosporioides*. This disease was named *Glomerella* leaf spot (GLS). This report was the first to cite any *Colletotrichum* sp. as the causative agent of leaf spot in the apple orchard. Under favorable conditions, a GLS infestation may result in 75% defoliation by harvest time. It can weaken trees and reducing yield [23, 24]. GLS was first reported in the United States in 1998 as a severe leaf spot on Gala apples [25].

strated that it was caused by *G. cingulata* which is the sexual stage of *C.*

the causal organisms of Asian pear fruit rot.

*all of the leaves in the summer, leading to the reduced vigor of the trees.*

*Plant Diseases-Current Threats and Management Trends*

**Figure 2.**

**186**

*Causal pathogen of Japanese pear (*Pyrus pyrifolia *Nakai var.* culta *Nakai) anthracnose and representative symptoms induced by inoculation of conidial suspension under unwounded condition. (A) A 7-day-old colony of strain C-17 grown on potato dextrose agar medium at 25°C, (B) salmon pink spore mass produced on sporeinducing medium (K2HPO4 1 g, MgSO4 0.5 g, peptone 5 g, lactose 10 g, agar 30 g, distilled water 1000 mL), (C) conidia of strain C-17 produced on spore-inducing medium (scale bar = 20 μm), (D) shoots of Japanese pear cv. Housui that showed severe symptoms by inoculation of conidial suspensions of strain C-15 under unwounded condition, (E) black spot lesions of different sizes reproduced by unwounded inoculation of spore suspension.*

*Colletotrichum karstii* has been reported as a new GLS pathogen [26]. Apple GLS caused by *Glomerella cingulata* was reported in China in 2012 [27].

On the other hand, the compendium makes no reference to foliar anthracnose in pear or Asian pear [21], which occurs on leaves and causes severe defoliation. Since this disease may be unique to Asian and Japanese pear, further investigations of its causal pathogens using molecular diagnostic tools are required.

#### **3. Development of fungicide control technology for JPA (until 2004)**

Our aim was to select efficacious fungicides at the Fruit Tree Experiment Station in Saga prefecture [1, 28]. The JPA fungicide spray timing was the same as that for Japanese pear ring rot caused by *Botryosphaeria berengeriana* De Notaris f. sp. piricola (Nose) in Koganezawa and Sakuma. Thus, these diseases had to be addressed simultaneously, and fungicide efficacy on ring rot was also evaluated [2].

#### **3.1 Selection of effective fungicides**

#### *3.1.1 Benzimidazoles, benomyl, and thiophanate-methyl*

Benzimidazole fungicides, which inhibit β-tubulin assembly during mitosis, were introduced ca. 1970. This group includes thiophanate-methyl, carbendazim, and benomyl. Benomyl (methyl [1-(butylcarbamoyl)benzimidazole-2-yl]carbamate) was registered under the brand name Benlate (50% wettable powder) by DuPont in Japan in 1971. Sumitomo Chemical Co., Ltd. (Tokyo, Japan) acquired the business in 2002. Thiophanate-methyl, dimethyl 4,40 -(o-phenylene) bis(3 thioallophanate), was registered in Japan in 1971 under the brand name Topsin-M (70% wettable powder; Nippon Soda Co., Ltd., Tokyo, Japan).

Initially, benomyl and thiophanate-methyl were considered as broadspectrum fungicides with low phytotoxicity, and these materials controlled the diseases caused by *Ascomycetes*, *Deuteromycetes*, and *Basidiomycetes*. Thus, benzimidazoles were frequently used on a wide range of crop groups. However, the pathogens rapidly developed field resistance; then, the usage of these fungicides decreased over time. They are still widely used on certain crops as they are broad-spectrum antifungal agents. In Japan, they are often applied to fruit trees.

Benomyl WP and thiophanate-methyl WP have been used since 1975 to prevent Asian pear scab (APS) caused by *Venturia nashicola*. These benzimidazoles were initially highly efficacious [29]. Therefore, their usage increased in frequency. APS fungus resistant to benzimidazoles were first detected in 1980 [30–33]; then the efficacy of benzimidazoles at suppressing APS diminished. In 1985, a demethylation inhibitor (DMI) with significant efficacy against the APS pathogen was introduced [34–37]; then, the use of benzimidazoles against APS was discontinued.

Benzimidazoles are very effective at suppressing ring rot [38] and powdery mildew [39] caused by *Phyllactinia mali* (Duby) U. Braun. Instead of targeting scab disease from April to June, growers applied benzimidazoles three to four times from mid-June until harvest to prevent ring rot and powdery mildew. This time window is also the main JPA infection period. Since benzimidazoles were effective against anthracnose caused by *C. gloeosporioides* sensu lato [11, 40–43], these materials were used often to prevent JPA.

#### *3.1.2 Fungicide screening against the JPA pathogen*

We conducted preventive application screening using "Housui" leaves and using fungicides registered for Japanese pears in Japan. Fungicide suspensions were diluted to predetermine concentrations and sprayed onto the leaves on branches excised from the "Housui" tree. The leaves were air-dried and sprayed with a Cgsl spore suspension (�105 mL�<sup>1</sup> ). The inoculated leaves were maintained in humid conditions at 25°C for 2 days. The lesions on the leaves were counted 7 days after inoculation.

Propyneb WP, dithianon FL, fluazinam FL, organic copper FL, azoxystrobin FL, kresoxim-methyl DF, captan WP, and mancozeb WP had excellent preventive efficacies (**Table 1**). In contrast, the benzimidazoles, benomyl, and thiophanate-methyl which were previously considered effective against anthracnose caused by Cgsl [11] were significantly less efficacious against both strains than the best treatment (**Table 1**), indicating the presence of benzimidazoleresistant strains.

**Generic name**

**189**

Benomyl Thiophanate-methyl

Fluazinam Dithianon

Propineb Kresoxim-methyl

Azoxystrobin Oxyquinoline

Captan Captan/oxyquinoline

Captan/benomyl

Iminoctadine

Mancozeb Hexaconazole Difenoconazole

Fosetyl Mepanipyrim *1Standards on the use of pesticide in agricultural*

*2Control (%) = (1 – average lesion number per leaf with fungicide* 

**Table 1.** *Preventive*

 *effect of various fungicides for anthracnose*

 *on Japanese pear.*

tris(albesilate)

 copper

 copper

Benlate WP Topsin-M WP

Frowncide SC

Delan FL

Antracol WG

Storoby DF

Amistar 10 FL Quinondo FL

Orthocide WP 80

Oxyrane WP

Caplate WP

Bellkute WP

Zimandithane

Anvil FL Score WG Aliette WP Frupica FL

 *chemical regulation law of Japan.* *application/average*

 *lesion number per control leaf) 100.*

 WP

1 1 29 M9 M3

11 11 —

M4 M4/ M4/1

M7 M3

3 3 P7

9

50.0 70.0 39.5 42.0 70.0 50.0 10.0 35.0 80.0 20.0/30.0 60.0/10.0

40.0 80.0

2.0 10.0 80.0 40.0

250 700 198 420 1400

250 100 350 1000 400/600 1000/167

400 2000

20 25 1000

200

**Trade name in Japan**

 **FRAC code**

 **Active ingredient (%)**

 **Rate applied (mg L1**

**)1**

**Control (%)2**

**Strain C-17**

0 6.8 100 98.9 100 100 99.6 98.5 93.8 74.1 86.8 53.1 95.1 33.8 36.3 7.4

0

0

8.1

40.0

28.6

96.1

46.4

91.6

70.1

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

94.3

96.8

99.8

98.6

100

99.1

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

100

93.6

90.2

 **Strain C-25**


*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

> **Table 1.**

*Preventive effect of various fungicides for anthracnose on Japanese*

 *pear.*

**3.1 Selection of effective fungicides**

fruit trees.

was discontinued.

used often to prevent JPA.

spore suspension (�105 mL�<sup>1</sup>

inoculation.

resistant strains.

**188**

*3.1.2 Fungicide screening against the JPA pathogen*

*3.1.1 Benzimidazoles, benomyl, and thiophanate-methyl*

*Plant Diseases-Current Threats and Management Trends*

business in 2002. Thiophanate-methyl, dimethyl 4,40

(70% wettable powder; Nippon Soda Co., Ltd., Tokyo, Japan).

Benzimidazole fungicides, which inhibit β-tubulin assembly during mitosis, were introduced ca. 1970. This group includes thiophanate-methyl, carbendazim, and benomyl. Benomyl (methyl [1-(butylcarbamoyl)benzimidazole-2-yl]carbamate) was registered under the brand name Benlate (50% wettable powder) by DuPont in Japan in 1971. Sumitomo Chemical Co., Ltd. (Tokyo, Japan) acquired the

thioallophanate), was registered in Japan in 1971 under the brand name Topsin-M

Initially, benomyl and thiophanate-methyl were considered as broadspectrum fungicides with low phytotoxicity, and these materials controlled the diseases caused by *Ascomycetes*, *Deuteromycetes*, and *Basidiomycetes*. Thus, benzimidazoles were frequently used on a wide range of crop groups. However, the pathogens rapidly developed field resistance; then, the usage of these fungicides decreased over time. They are still widely used on certain crops as they are broad-spectrum antifungal agents. In Japan, they are often applied to

Benomyl WP and thiophanate-methyl WP have been used since 1975 to

Benzimidazoles are very effective at suppressing ring rot [38] and powdery mildew [39] caused by *Phyllactinia mali* (Duby) U. Braun. Instead of targeting scab disease from April to June, growers applied benzimidazoles three to four times from mid-June until harvest to prevent ring rot and powdery mildew. This time window is also the main JPA infection period. Since benzimidazoles were effective against anthracnose caused by *C. gloeosporioides* sensu lato [11, 40–43], these materials were

We conducted preventive application screening using "Housui" leaves and using

). The inoculated leaves were maintained in humid

fungicides registered for Japanese pears in Japan. Fungicide suspensions were diluted to predetermine concentrations and sprayed onto the leaves on branches excised from the "Housui" tree. The leaves were air-dried and sprayed with a Cgsl

conditions at 25°C for 2 days. The lesions on the leaves were counted 7 days after

FL, kresoxim-methyl DF, captan WP, and mancozeb WP had excellent preventive efficacies (**Table 1**). In contrast, the benzimidazoles, benomyl, and thiophanate-methyl which were previously considered effective against anthracnose caused by Cgsl [11] were significantly less efficacious against both strains than the best treatment (**Table 1**), indicating the presence of benzimidazole-

Propyneb WP, dithianon FL, fluazinam FL, organic copper FL, azoxystrobin

benzimidazoles were initially highly efficacious [29]. Therefore, their usage increased in frequency. APS fungus resistant to benzimidazoles were first detected in 1980 [30–33]; then the efficacy of benzimidazoles at suppressing APS diminished. In 1985, a demethylation inhibitor (DMI) with significant efficacy against the APS pathogen was introduced [34–37]; then, the use of benzimidazoles against APS

prevent Asian pear scab (APS) caused by *Venturia nashicola*. These


#### *3.1.3 Confirming the lack of susceptibility to benzimidazoles among the JPA pathogen strains*

Based on the results of the previous study (**Table 1**), we investigated the susceptibility of 122 Cgsl strains to benomyl. The strains were isolated from infected leaves collected in 1999 from nine orchards known to have frequent outbreaks of this disease. Before the experiment, the pathogenicity of these strains was confirmed by inoculation tests. The strains were divided into those with minimum inhibitory concentration (MIC) ≤ 0.39 mg L<sup>1</sup> and those with MIC ≥ 1600 mg L<sup>1</sup> (**Table 2**). The former were deemed susceptible. The latter were considered highly resistant and were prevalent at all nine orchards investigated (**Table 2**). These highly resistant strains were also highly resistant to thiophanate-methyl, which are very similar to benomyl in the mode of action (**Table 3**). When "Housui" leaves were sprayed with benomyl (250 mg L<sup>1</sup> ) and then inoculated with the highly


resistant Cgsl strains, the treated leaves became severely diseased, i.e., benomyl did

*Control efficacy of benomyl against benzimidazole-sensitive (SCG-25 and SCG-30) strains and highly benzimidazole-resistant (SCG-17 and SCG-72) strains of* C. gloeosporioides *sensu lato on the leaves of the*

**Tested leaves Lesions/leaf Tested leaves Lesions/leaf** SCG-25 27 14.6<sup>a</sup> 26 126.8a 88.5<sup>a</sup> SCG-30 28 6.3<sup>a</sup> 24 98.6<sup>a</sup> 93.6a SCG-17 26 152.5<sup>b</sup> 21 142.3a 0<sup>b</sup> SCG-72 22 96.5<sup>b</sup> 24 106.8<sup>a</sup> 9.6<sup>b</sup>

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

*The Japanese pear variety "Housui" (2-year-old trees) was sprayed with wettable powder of benomyl and thoroughly*

*30; highly benzimidazole-resistant strains, SCG-17, SCG-72) were then inoculated. Seven days after inoculation, the development of symptoms was assessed. Values followed by different letter differ significantly in a multiple comparison*

*Control (%) = (1 – average lesion number per leaf on the trees with benomyl application/average lesion number per*

**) sprayed trees Control trees Control (%)<sup>2</sup>**

*) of each strain (benzimidazole-sensitive strains, SCG-25 and SCG-*

Benzimidazole-resistant Cgsl that occurred at a high frequency over a wide range in the Japanese pear-growing areas of Saga prefecture caused benzimidazoles to be no longer effective against JPA. In addition, benzimidazole-resistant Cgsl was also confirmed in Chiba, Oita, and Kochi prefectures. Only highly resistant strains were observed in Chiba prefecture [44], a mixture of highly and moderately resistant strains was detected in Oita prefecture [45], and only moderately resistant

**3.2 Change in detection frequency of highly benzimidazole-resistant strains**

To determine the changes in detection frequency of benzimidazole-resistant strains, Cgsl strains from orchards where benzimidazoles were discontinued were challenged with benomyl in 1999, 2000, 2001, and 2004. The discontinuation of benzimidazole fungicides in each orchard was confirmed from fungicide spray records. The frequency of benzimidazole-resistant strain ranged from 81 to 88% during the study, and there was no indication of a reduction over time (**Figure 4**). Therefore, reintroduction of benzimidazoles to the pear-producing areas of this

The proportion of benzimidazole-resistant Cgsl strains causing JPA did not decrease even 4 years after discontinuation. Pathogen populations in abscised leaves may be carried over to the following year, and pathogen latently infected with twigs may remain viable for several years [46]. Also, both the resistant and sensitive

Impacts on the detection frequency of benzimidazole-resistant isolates after the discontinuation were highly variable for other crops and pathogens. The discontinuation of benzimidazole immediately reduced the ratios of highly resistant *Botrytis cinerea* strains causing grape gray mold [47] and *Gloeosporium theae-sinensis* causing tea anthracnose [48]. The ratio of highly resistant *Venturia nashicola* strains causing Japanese pear scab was immediately reduced upon benzimidazole discontinuation; however, the overall ratio of resistant strains did not decline as moderately and weakly resistant strains emerged [49]. As with JPA, the frequency of highly

strains may have similar levels of competitiveness or fitness.

not suppress JPA (**Table 4**).

*leaf on the control trees) 100.*

*Japanese pear variety "Housui".*

**Strain Benomyl (250 mg L<sup>1</sup>**

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

*dried. Conidial suspensions (approx. 10<sup>5</sup> mL<sup>1</sup>*

*based on the Tukey–Kramer HSD test (P <sup>&</sup>lt; 0.05). <sup>2</sup>*

*1*

**Table 4.**

region was not recommended.

**191**

strains were confirmed for Kochi prefecture [12].

*1*

**after discontinuing benzimidazoles**

*1 Values in parentheses are the percentage of the total for each category.*

#### **Table 2.**

*Benomyl sensitivity of* C. gloeosporioides *sensu lato, the causal organism of anthracnose in Japanese pear varieties "Housui" and "Niitaka" at Imari district in Saga Prefecture in 1999.*


**Table 3.**

*Effect of benomyl and thiophanate-methyl on the mycelial growth of benzimidazole-sensitive (SCG-25, SCG-30, and SCG-64) strains and highly benzimidazole-resistant (SCG-08, SCG-17, and SCG-72) strains of C. gloeosporioides obtained from lesions of Japanese pear anthracnose.*

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*


*1 The Japanese pear variety "Housui" (2-year-old trees) was sprayed with wettable powder of benomyl and thoroughly dried. Conidial suspensions (approx. 10<sup>5</sup> mL<sup>1</sup> ) of each strain (benzimidazole-sensitive strains, SCG-25 and SCG-30; highly benzimidazole-resistant strains, SCG-17, SCG-72) were then inoculated. Seven days after inoculation, the development of symptoms was assessed. Values followed by different letter differ significantly in a multiple comparison based on the Tukey–Kramer HSD test (P <sup>&</sup>lt; 0.05). <sup>2</sup>*

*Control (%) = (1 – average lesion number per leaf on the trees with benomyl application/average lesion number per leaf on the control trees) 100.*

#### **Table 4.**

*3.1.3 Confirming the lack of susceptibility to benzimidazoles among the JPA pathogen*

**Source orchard Variety Number of strains Number of strains for each MIC (mg L<sup>1</sup>**

Minamihata-1 Housui 13 0 0 13 Minamihata-2 Housui 15 4 0 11 Minamihata-3 Housui 14 0 0 14 Minamihata-4 Niitaka 13 0 0 13 Okawa-1 Housui 10 0 0 10 Okawa-2 Housui 10 1 0 9 Okawa-3 Housui 12 0 0 12 Okawa-4 Housui 22 4 0 18 Okawa-5 Niitaka 13 3 0 10 Total 122 12 (9.8)1 0 (0.0) 110 (90.2)

*Benomyl sensitivity of* C. gloeosporioides *sensu lato, the causal organism of anthracnose in Japanese pear*

**Strain Location of isolation<sup>1</sup> Year of isolation EC50 (mg L<sup>1</sup>**

SCG-25 Minamihata town 1999 0.151 0.151 SCG-30 Ohkawa town 1999 0.166 0.206 SCG-64 Ohkawa town 1999 0.155 0.186 SCG-08 Minamihata town 1999 485 2856 SCG-17 Minamihata town 1999 481 2386 SCG-72 Ohkawa town 1999 491 3211

*Effect of benomyl and thiophanate-methyl on the mycelial growth of benzimidazole-sensitive (SCG-25, SCG-30, and SCG-64) strains and highly benzimidazole-resistant (SCG-08, SCG-17, and SCG-72) strains*

Based on the results of the previous study (**Table 1**), we investigated the susceptibility of 122 Cgsl strains to benomyl. The strains were isolated from infected leaves collected in 1999 from nine orchards known to have frequent outbreaks of this disease. Before the experiment, the pathogenicity of these strains was confirmed by inoculation tests. The strains were divided into those with minimum inhibitory concentration (MIC) ≤ 0.39 mg L<sup>1</sup> and those with MIC ≥ 1600 mg L<sup>1</sup> (**Table 2**). The former were deemed susceptible. The latter were considered highly resistant and were prevalent at all nine orchards investigated (**Table 2**). These highly resistant strains were also highly resistant to thiophanate-methyl, which are very similar to benomyl in the mode of action (**Table 3**). When "Housui" leaves

) and then inoculated with the highly

**0.78 25–100 >1600**

**) range**

**) values of**

**Benomyl Thiophanate-methyl**

*strains*

*1*

*1*

**190**

**Table 3.**

**Table 2.**

were sprayed with benomyl (250 mg L<sup>1</sup>

*Plant Diseases-Current Threats and Management Trends*

*Values in parentheses are the percentage of the total for each category.*

*varieties "Housui" and "Niitaka" at Imari district in Saga Prefecture in 1999.*

*Minamihata town and Ohkawa town are both in the Imari area of Saga prefecture.*

*of C. gloeosporioides obtained from lesions of Japanese pear anthracnose.*

*Control efficacy of benomyl against benzimidazole-sensitive (SCG-25 and SCG-30) strains and highly benzimidazole-resistant (SCG-17 and SCG-72) strains of* C. gloeosporioides *sensu lato on the leaves of the Japanese pear variety "Housui". 1*

resistant Cgsl strains, the treated leaves became severely diseased, i.e., benomyl did not suppress JPA (**Table 4**).

Benzimidazole-resistant Cgsl that occurred at a high frequency over a wide range in the Japanese pear-growing areas of Saga prefecture caused benzimidazoles to be no longer effective against JPA. In addition, benzimidazole-resistant Cgsl was also confirmed in Chiba, Oita, and Kochi prefectures. Only highly resistant strains were observed in Chiba prefecture [44], a mixture of highly and moderately resistant strains was detected in Oita prefecture [45], and only moderately resistant strains were confirmed for Kochi prefecture [12].

#### **3.2 Change in detection frequency of highly benzimidazole-resistant strains after discontinuing benzimidazoles**

To determine the changes in detection frequency of benzimidazole-resistant strains, Cgsl strains from orchards where benzimidazoles were discontinued were challenged with benomyl in 1999, 2000, 2001, and 2004. The discontinuation of benzimidazole fungicides in each orchard was confirmed from fungicide spray records. The frequency of benzimidazole-resistant strain ranged from 81 to 88% during the study, and there was no indication of a reduction over time (**Figure 4**). Therefore, reintroduction of benzimidazoles to the pear-producing areas of this region was not recommended.

The proportion of benzimidazole-resistant Cgsl strains causing JPA did not decrease even 4 years after discontinuation. Pathogen populations in abscised leaves may be carried over to the following year, and pathogen latently infected with twigs may remain viable for several years [46]. Also, both the resistant and sensitive strains may have similar levels of competitiveness or fitness.

Impacts on the detection frequency of benzimidazole-resistant isolates after the discontinuation were highly variable for other crops and pathogens. The discontinuation of benzimidazole immediately reduced the ratios of highly resistant *Botrytis cinerea* strains causing grape gray mold [47] and *Gloeosporium theae-sinensis* causing tea anthracnose [48]. The ratio of highly resistant *Venturia nashicola* strains causing Japanese pear scab was immediately reduced upon benzimidazole discontinuation; however, the overall ratio of resistant strains did not decline as moderately and weakly resistant strains emerged [49]. As with JPA, the frequency of highly

#### **Figure 4.**

*Change in detection frequency of highly benzimidazole-resistant strains after discontinuing benzimidazoles.*

resistant strains did not change for *V. nashicola* [50–52] and *V. inaequalis* which cause pear and apple scab, respectively [53].

#### **3.3 Residual efficacy and rainfastness of fungicides effective against benzimidazole-resistant strains of** *Colletotrichum gloeosporioides* **sensu lato**

#### *3.3.1 Residual efficacy of the sprayed fungicides*

To ensure effective pathogen control, it is important to know the length of time fungicidal efficacy persists after product application. Experiments were conducted to determine the period of residual fungicidal activity against JPA. Each fungicide was sprayed onto "Housui" trees in Japanese pear orchards where JPA had never been previously detected. Branches with their leaves intact were excised and brought to the laboratory. A conidial suspension (105 mL<sup>1</sup> ) was sprayed onto the leaves. Relative product efficacy was scored based on the number of foliar lesions. Duration of efficacy after product application was also evaluated. Two experiments, where each had different sets of treatments, were conducted in late July and mid-September 2002. In each treatment, 100 leaves from new branches were examined.

*3.3.2 Rainfastness of the sprayed fungicides*

4.0 mL leaf<sup>1</sup>

**Generic name**

Kresoximmethyl

Captan/ benomyl

*fungicide application) 100.*

**Generic name Trade name in**

Copper (II) sulfate IC Bordeaux

**Japan**

48Q

*1*

**Table 5.**

Captan/ oxyquinoline copper

*1 See Table 5.*

**Table 6.**

**Trade name in Japan**

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

**FRAC code**

**Active ingredient (%)**

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

Azoxystrobin Amistar 10 FL 11 10.0 100 84 78 45

Dithianon Delan FL M9 42.0 420 83 90 50 Fluazinam Frowncide SC 29 39.5 198 77 69 39

*Control (%) = (1 – mean ratio of diseased leaves of trees with fungicide application/mean ratio of diseased leaves of trees without*

**Active ingredient (%)**

Dithianon Delan FL M9 42.0 420 92 83 41 Fluazinam Frowncide SC 29 39.5 198 59 0 0

Propineb Antracol WG M3 70.0 1400 0 0 —

*Residence period of sprayed fungicides against anthracnose on the Japanese pear "Housui" (1).*

**FRAC code**

Storobi DF 11 50.0 250 75 15 20

Caplate WP M4/1 60.0/10.0 1000/167 98 68 46

**Rate applied (mg L<sup>1</sup> )**

M1 31.2 10,400 69 70 0

Oxyrane WP M4/ 20.0/30.0 400/600 67 57 0

**Rate applied (mg L<sup>1</sup> )**

**Changes of control (%)1**

**14 days after**

**Changes of control (%)1**

**14 days after**

**21 days after**

**7 days after**

**21 days after**

**7 days after**

control.

**193**

efficient and successful disease control program.

The JPA pathogen propagates and infects during rainfall. The amount of rain determines the degree of attenuation of the fungicide spray on the pear leaves. Thus, the establishment of the rainfastness of various fungicides helps develop an

Several fungicide treatments were tested on pot-grown "Housui" trees in 2002.

lative rain. The efficacy of the fungicide was visually assessed to estimate % disease

The level of JPA suppression was high when the leaves received no rainfall, resulting in 100% disease control (=no disease development). As expected, disease control efficacy decreased with increasing cumulative rainfall. For azoxystrobin FL and dithianon FL, the disease control was ≥70% at 200 mm cumulative rainfall after fungicide application (**Figure 5**). Fluazinam FL and kresoxim-methyl DF achieved ≥70% disease control at 100 mm cumulative rainfall, but the disease control

) before treatment application, at 100, 200, 300, and 400 mm cumu-

One day after fungicide application, a rainfall treatment of 17 mm h<sup>1</sup> and 50 mm d<sup>1</sup> was conducted using an artificial rainfall machine (DIK-6000; Daiki Rika Kogyo Co., Ltd., Tokyo, Japan). The leaves were excised from each tree and inoculated with a pathogen conidial suspension (2 105 conidia mL<sup>1</sup> and

*Residence period of sprayed fungicides against anthracnose on the Japanese pear "Housui" (2).*

For the late July experiment, a mean % disease control (=% suppression of the mean disease incidence relative to the mean disease incidence of the positive control) of >70% was taken as the threshold of satisfactory disease control. The disease control sustainability was measured as days post-application. Dithianon FL and azoxystrobin FL continued to suppress disease onset for 14 days after application (**Table 5**). Satisfactory disease control was observed for fluazinam FL, kresoximmethyl DF, and captan/benomyl WP until 7 days after application. However, at 14 days after the application, the disease control effect (%) dropped to 69 and 68% for fluazinam FL and captan/benomyl WP, respectively, and 15% for kresoximmethyl DF. Thus, these fungicides, especially kresoxim-methyl DF, had comparatively shorter disease control durations.

In the mid-September experiment, dithianon FL presented with satisfactory disease control efficacy until 14 days after application as in the previous experiment (**Table 6**). The efficacies of the other fungicides were inferior to that of dithianon FL, and none of the treatment achieved the mean % disease control of >70%. Propineb WG showed no disease control efficacy whatsoever.

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*


*1 Control (%) = (1 – mean ratio of diseased leaves of trees with fungicide application/mean ratio of diseased leaves of trees without fungicide application) 100.*

#### **Table 5.**

resistant strains did not change for *V. nashicola* [50–52] and *V. inaequalis* which

*Change in detection frequency of highly benzimidazole-resistant strains after discontinuing benzimidazoles.*

**benzimidazole-resistant strains of** *Colletotrichum gloeosporioides* **sensu lato**

To ensure effective pathogen control, it is important to know the length of time fungicidal efficacy persists after product application. Experiments were conducted to determine the period of residual fungicidal activity against JPA. Each fungicide was sprayed onto "Housui" trees in Japanese pear orchards where JPA had never been previously detected. Branches with their leaves intact were excised and

leaves. Relative product efficacy was scored based on the number of foliar lesions. Duration of efficacy after product application was also evaluated. Two experiments, where each had different sets of treatments, were conducted in late July and mid-September 2002. In each treatment, 100 leaves from new branches were examined. For the late July experiment, a mean % disease control (=% suppression of the mean disease incidence relative to the mean disease incidence of the positive control) of >70% was taken as the threshold of satisfactory disease control. The disease control sustainability was measured as days post-application. Dithianon FL and azoxystrobin FL continued to suppress disease onset for 14 days after application (**Table 5**). Satisfactory disease control was observed for fluazinam FL, kresoximmethyl DF, and captan/benomyl WP until 7 days after application. However, at 14 days after the application, the disease control effect (%) dropped to 69 and 68% for fluazinam FL and captan/benomyl WP, respectively, and 15% for kresoximmethyl DF. Thus, these fungicides, especially kresoxim-methyl DF, had compara-

In the mid-September experiment, dithianon FL presented with satisfactory disease control efficacy until 14 days after application as in the previous experiment (**Table 6**). The efficacies of the other fungicides were inferior to that of dithianon FL, and none of the treatment achieved the mean % disease control of >70%.

) was sprayed onto the

**3.3 Residual efficacy and rainfastness of fungicides effective against**

brought to the laboratory. A conidial suspension (105 mL<sup>1</sup>

Propineb WG showed no disease control efficacy whatsoever.

cause pear and apple scab, respectively [53].

*Plant Diseases-Current Threats and Management Trends*

**Figure 4.**

*3.3.1 Residual efficacy of the sprayed fungicides*

tively shorter disease control durations.

**192**

*Residence period of sprayed fungicides against anthracnose on the Japanese pear "Housui" (1).*


#### **Table 6.**

*Residence period of sprayed fungicides against anthracnose on the Japanese pear "Housui" (2).*

#### *3.3.2 Rainfastness of the sprayed fungicides*

The JPA pathogen propagates and infects during rainfall. The amount of rain determines the degree of attenuation of the fungicide spray on the pear leaves. Thus, the establishment of the rainfastness of various fungicides helps develop an efficient and successful disease control program.

Several fungicide treatments were tested on pot-grown "Housui" trees in 2002. One day after fungicide application, a rainfall treatment of 17 mm h<sup>1</sup> and 50 mm d<sup>1</sup> was conducted using an artificial rainfall machine (DIK-6000; Daiki Rika Kogyo Co., Ltd., Tokyo, Japan). The leaves were excised from each tree and inoculated with a pathogen conidial suspension (2 105 conidia mL<sup>1</sup> and 4.0 mL leaf<sup>1</sup> ) before treatment application, at 100, 200, 300, and 400 mm cumulative rain. The efficacy of the fungicide was visually assessed to estimate % disease control.

The level of JPA suppression was high when the leaves received no rainfall, resulting in 100% disease control (=no disease development). As expected, disease control efficacy decreased with increasing cumulative rainfall. For azoxystrobin FL and dithianon FL, the disease control was ≥70% at 200 mm cumulative rainfall after fungicide application (**Figure 5**). Fluazinam FL and kresoxim-methyl DF achieved ≥70% disease control at 100 mm cumulative rainfall, but the disease control

efficacy fell to <70% at 200 mm cumulative rainfall. For captan/oxyquinolinecopper WP and captan/benomyl WP, the mean disease control efficacy was 90% and >60% at 100 mm cumulative rainfall but sharply declined to 0 and 23%, respectively, at 200 mm cumulative rainfall (**Figure 5**).

>200 mm, the trees were immediately resprayed to compensate for the product washed off by the rain. Experiments were conducted in mid-June 2001 and mid-May 2002 using a slightly modified spray guideline. The treatments were applied either 20 days after the previous treatment or when the post-application cumulative rainfall was 200 mm. Several heavy rain events increased the cumulative rainfall to >200 mm, but all fungicide treatments were applied before the cumulative rainfall

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

As with the previous experiments, 70% control was set as the efficacy threshold. For all 3 years, preventive azoxystrobin FL and dithianon FL application provided >70% disease control (**Table 7**). Both treatments resulted in consistently high disease control efficacy as they did in the residual efficacy and rainfastness tests

Kresoxim-methyl DF demonstrated >80% disease control efficacy in one of the

residual activity experimental runs in 2000 and 2002, but the results were not consistent among three trials (**Table 7**). In the other trials, the mean % disease control of kresoxim-methyl DF varied from 15 to 75% in the residual efficacy test (**Table 5**), and the mean % disease control efficacy dropped very sharply to below 70% in the rainfastness test at 200 mm cumulative rain fall (**Figure 5**). Thus, the environmental conditions, especially the amount of precipitations, may negatively

Fluazinam showed good levels of disease control (75%) in this experiment (**Table 7**), but it did not perform well with the residual tests (**Tables 5** and **6**), and the mean % disease control dropped at 200 mm cumulative rainfall (**Figure 5**). A trend with propineb was similar where 80% mean disease control was observed in this experiment, but it did not provide any level of control in the residual efficacy test (**Table 6**). We need to investigate more to determine what created these

The lack of disease prevention efficacy for benomyl WP was expected as benzimidazole-resistant strains were detected in this orchard (**Table 7**). The disease prevention efficacy of captan/benomyl WP was 70% in all 3 years, possibly because of benzimidazole-resistant strains and low rainfastness of captan, which is also shown in the rainfastness test (**Figure 5**). Thus, captan probably needs to be applied with a non-benzimidazole material, and if sprayed with captan alone, it should be applied using a 100 mm cumulative rainfall

Fungicide application on a 10- to 14-day schedule from the first cover until harvest is the main disease control method that growers use. JPA is very difficult to control after the leaves have been infected with it. Dithianon, fluazinam, strobilurin-quinone outside inhibitor (ST-QoI) fungicides, and captan/benomyl

WP provide good disease control when they are applied preventively.

*3.3.4 Use of fungicides effective to JPA against ring rot of Japanese pear*

We evaluated fungicide efficacy against Japanese pear ring rot because spray application timing was the same as that for JPA [2]. The ST-QoIs azoxystrobin and kresoxim-methyl were highly efficacious against ring rot (**Table 8**). Captan/benomyl also showed high efficacy. In contrast, the efficacy of dithianon against ring rot was highly variable (from 100 to 0% control) during the years it was tested. Fluazinam provided unsatisfactory disease control efficacy

reached 300 mm.

(**Tables 5** and **6**).

differences.

threshold.

against ring rot.

**195**

impact kresoxim-methyl DF to be effective.

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

#### *3.3.3 Preventive efficacy of fungicide treatments against JPA in Japanese pear orchards*

In the "Housui" orchard, an experiment was conducted over three seasons to determine the efficacy of preventive fungicide application against JPA. Two experiments were conducted in late June 2000. Trees were sprayed at 10- to 14-day intervals. When the cumulative rainfall after the previous application was

#### **Figure 5.**

*Reduction of the control effect of various fungicides on Japanese pear anthracnose associated with artificial rainfall after spraying; error bar, 95% confidence interval.*

#### *Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

>200 mm, the trees were immediately resprayed to compensate for the product washed off by the rain. Experiments were conducted in mid-June 2001 and mid-May 2002 using a slightly modified spray guideline. The treatments were applied either 20 days after the previous treatment or when the post-application cumulative rainfall was 200 mm. Several heavy rain events increased the cumulative rainfall to >200 mm, but all fungicide treatments were applied before the cumulative rainfall reached 300 mm.

As with the previous experiments, 70% control was set as the efficacy threshold. For all 3 years, preventive azoxystrobin FL and dithianon FL application provided >70% disease control (**Table 7**). Both treatments resulted in consistently high disease control efficacy as they did in the residual efficacy and rainfastness tests (**Tables 5** and **6**).

Kresoxim-methyl DF demonstrated >80% disease control efficacy in one of the residual activity experimental runs in 2000 and 2002, but the results were not consistent among three trials (**Table 7**). In the other trials, the mean % disease control of kresoxim-methyl DF varied from 15 to 75% in the residual efficacy test (**Table 5**), and the mean % disease control efficacy dropped very sharply to below 70% in the rainfastness test at 200 mm cumulative rain fall (**Figure 5**). Thus, the environmental conditions, especially the amount of precipitations, may negatively impact kresoxim-methyl DF to be effective.

Fluazinam showed good levels of disease control (75%) in this experiment (**Table 7**), but it did not perform well with the residual tests (**Tables 5** and **6**), and the mean % disease control dropped at 200 mm cumulative rainfall (**Figure 5**). A trend with propineb was similar where 80% mean disease control was observed in this experiment, but it did not provide any level of control in the residual efficacy test (**Table 6**). We need to investigate more to determine what created these differences.

The lack of disease prevention efficacy for benomyl WP was expected as benzimidazole-resistant strains were detected in this orchard (**Table 7**). The disease prevention efficacy of captan/benomyl WP was 70% in all 3 years, possibly because of benzimidazole-resistant strains and low rainfastness of captan, which is also shown in the rainfastness test (**Figure 5**). Thus, captan probably needs to be applied with a non-benzimidazole material, and if sprayed with captan alone, it should be applied using a 100 mm cumulative rainfall threshold.

Fungicide application on a 10- to 14-day schedule from the first cover until harvest is the main disease control method that growers use. JPA is very difficult to control after the leaves have been infected with it. Dithianon, fluazinam, strobilurin-quinone outside inhibitor (ST-QoI) fungicides, and captan/benomyl WP provide good disease control when they are applied preventively.

#### *3.3.4 Use of fungicides effective to JPA against ring rot of Japanese pear*

We evaluated fungicide efficacy against Japanese pear ring rot because spray application timing was the same as that for JPA [2]. The ST-QoIs azoxystrobin and kresoxim-methyl were highly efficacious against ring rot (**Table 8**). Captan/benomyl also showed high efficacy. In contrast, the efficacy of dithianon against ring rot was highly variable (from 100 to 0% control) during the years it was tested. Fluazinam provided unsatisfactory disease control efficacy against ring rot.

efficacy fell to <70% at 200 mm cumulative rainfall. For captan/oxyquinolinecopper WP and captan/benomyl WP, the mean disease control efficacy was 90% and >60% at 100 mm cumulative rainfall but sharply declined to 0 and 23%,

*3.3.3 Preventive efficacy of fungicide treatments against JPA in Japanese pear orchards*

*Reduction of the control effect of various fungicides on Japanese pear anthracnose associated with artificial*

*rainfall after spraying; error bar, 95% confidence interval.*

In the "Housui" orchard, an experiment was conducted over three seasons to determine the efficacy of preventive fungicide application against JPA. Two experiments were conducted in late June 2000. Trees were sprayed at 10- to 14-day intervals. When the cumulative rainfall after the previous application was

respectively, at 200 mm cumulative rainfall (**Figure 5**).

*Plant Diseases-Current Threats and Management Trends*

**Figure 5.**

**194**


#### **Table 7.**

**Generic name**

**197**

Benomyl Fluazinam Dithianon Kresoxim-methyl

Azoxystrobin Oxyquinoline

Captan/benomyl

*1Sprays have been done in May to August each year.*

*2See Table 5.*

**Table 8.** *Control effect of several fungicides*

 *against ring rot on the Japanese pear*

*"Housui".*

*1*

 copper

Benlate WP Frowncide SC

Delan FL Storobi DF Amistar 10 FL

Quinondo FL

Caplate WP

1 29 M9

11 11 —

M4/1

50.0 39.5 42.0 50.0 10.0 35.0 60.0/10.0 Ratio of diseased leaves in control

250 198 420 250 100 350 1000/167

32 30 19 39 47

0 17 45.0%

 40.6%

 10.9%

69

78

—

 —

73

100

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

70

54

0

100

37

—

—

 —

**Trade name in Japan**

 **FRAC code**

 **Active ingredient (%)**

 **Rate applied (mg L1**

**)** **In 2000**

 **In 2001**

 **In 2002**

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

**Control (%)2**

*Control effect of several fungicides against anthracnose on the Japanese pear "Housui".*

*1*


**Table 8.** *ControleffectofseveralfungicidesagainstringrotontheJapanesepear"Housui".*

*1*

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

**Generic name**

**196**

Benomyl Fluazinam Dithianon

Propineb Kresoxim-methyl

Azoxystrobin Oxyquinoline

Captan/benomyl

Hexaconazole *1Sprays have been done in May to August each year.*

*2See Table 5.*

**Table 7.** *Control effect of several fungicides*

 *against anthracnose*

 *on the Japanese pear*

*"Housui".*

*1*

Caplate WP

Anvil FL

 copper

 Quinondo FL

Storobi DF Amistar 10 FL

Benlate WP Frowncide SC

Delan FL Antracol WG

1 29 M9 M3

11 11 —

M4/1

3

2.0

Ratio of diseased leaves in control

60.0/10.0

10.0 35.0

50.0

70.0

42.0

39.5

50.0

250 198 420 1400

250 100 350 1000/167

20

65

29 67.0%

70.5%

 38.3%

 63.0%

—

——

—

72

 67

 **Trade name in Japan**

 **FRAC code**

 **Active ingredient (%)**

 **Rate applied (mg L1**

**)**

**Control (%)2**

> **In 2000**

> > **Experiment**

3 75

82 80 61 81 26

87

91

—

 —

 88

79

69

 87

—

——

99

93

 95

—

85

—

——

*Plant Diseases-Current Threats and Management Trends*

 **1 Experiment**

 **2**

**In 2001**

 **In 2002**

#### **3.4 Temporary suspension of the 1999 JPA outbreak**

Before the 1999 JPA outbreak, the main disease to control in Japanese pear cultivation was Asian pear scab (APS). Sterol demethylation inhibitor (DMI), belonging to sterol biosynthesis inhibitors (SBIs), was the product most frequently used to control this disease. Iminoctadine tris(albesilate), captan/oxyquinoline copper, and captan were applied for APS a few times. Benzimidazoles were applied three to four times to control ring rot and powdery mildew. However, by 2000, benzimidazoles were no longer recommended in Japanese pear production due to its resistance issue. In their place, local systemic fungicides such as strobilurins (azoxystrobin and kresoxim-methyl) and protective fungicides such as dithianon and fluazinam were applied.

eggplant leaf mold (*Mycovellosiella nattrassii*) [61], *Corynespora* cucumber leaf spot (*Corynespora cassicola*) [62], citrus gray mold (*Botrytis cinerea*) [63], European pear black spot (*Alternaria alternata*) [64], *Alternaria* apple blotch (*Alternaria alternata* apple pathotype) [65], grapevine leaf blight (*Pseudocercospora vitis*) [66], strawberry anthracnose (*Colletotrichum gloeosporioides*) [67, 68], tea gray blight

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

(*Pestalotiopsis longiseta*) [69], apple bitter rot (*Colletotrichum gloeosporioides*) [70],

*gloeosporioides*) [72], apple scab (*Venturia inaequalis*) [73], grapevine downy mildew (*Plasmopara viticola*) [74], cucurbits gummy stem blight (*Didymella bryoniae*) [75],

**4.2 Emergence of strains of** *Colletotrichum gloeosporioides* **sensu lato resistant to**

Over nearly a decade in the Saga and Oita prefectures, ST-QoIs were sprayed three to four times annually between June and early August as countermeasures against JPA and APS. That is, many growers heavily depended on ST-QoIs, especially late in the season because ST-QoIs are phytotoxic to Japanese pear leaves at their early growth stage. In addition, ST-QoIs were also highly efficacious against

The alternative material, thiuram FL, has a 30-day PHI; therefore, it cannot be used after mid-July. The other options, such as iminoctadine tris(albesilate)/captan WP, have a relatively shorter PHI (14 days), and captan WP has a 3-day PHI. Captan/oxyquinoline copper WP (3-day PHI), captan WP (3-day PHI), and iminoctadine tris(albesilate)/captan WP (14-day PHI) showed adequate efficacy against JPA [28, 44, 54, 80]. However, the ST-QoIs were preferred over these choices by growers as they were more effective than these; in addition, the common

As JPA became very prevalent in 2010–2011 in the Oita and Saga prefectures where above-mentioned spraying system. We assessed ST-QoI sensitivity in Cgsl isolates by placing mycelial discs on potato dextrose agar (PDA) containing 100 μg mL<sup>1</sup> azoxystrobin and 1000 μg mL<sup>1</sup> salicylhydroxamic acid (SHAM). Mycelial elongation was measured 4 days post-inoculation [81]. Isolates from Saga [80] and Oita [45] prefecture grew on the PDA containing azoxystrobin (**Table 9**,

To determine the effect of ST-QoI pretreatment on JPA development, conidial suspensions were sprayed on "Housui" leaves previously exposed to azoxystrobin FL. The appearance of JPA lesions caused by the sensitive strain was nearly zero

**Source orchard Number of tested strains Number of resistant strains<sup>1</sup>**

*Number of strains that grew on PDA with 1000 μg mL<sup>1</sup> SHAM and 100 μg mL<sup>1</sup> azoxystrobin cultured 4 days*

*Azoxystrobin sensitivity of* C. gloeosporioides *sensu lato, the causal organism of anthracnose in Japanese pear varieties "Housui" and "Niitaka" at Imari district in Saga prefecture and Hita district in Oita prefecture both*

Imari district in Saga prefecture 61 20 (32.8%)<sup>2</sup> Hita district in Oita prefecture 254 49 (16.2%)

*Values in parentheses are the percentage of the resistant strains.*

component of these materials, captan, tends to cause stains on the fruit.

rice blast (*Magnaporthe oryzae*) [71], mango anthracnose (*Colletotrichum*

(*Sphaerotheca aphanis* var. aphanis) [77].

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

**ST-QoIs**

APS [78, 79].

**Figure 6**).

*1*

*at 25°C. 2*

**Table 9.**

**199**

*Kyushyu island in 2011.*

chrysanthemum white rust (*Puccinia horiana*) [76], wheat powdery mildew (*Erysiphe* (Blumeria) *graminis f.sp. tritici*), and strawberry powdery mildew

Dithianon FL, fluazinam FL, ST-QoI fungicides (azoxystrobin FL, kresoximmethyl DF), captan/oxyquinoline copper WP, and captan/benomyl WP were effective against JPA, APS, and ring spot, and all except dithianon were efficacious against powdery mildew. Therefore, these materials were incorporated into the spray calendar with heavy reliance on DMIs, which were popular at that time. As a result, JPA incidence was drastically reduced.

Although Dithianon FL has high JPA control efficacy, it has a 60-day pre-harvest interval (PHI) in Japan. Thus, it cannot be used after mid-June which is a critical JPA control period. The PHI of fluazinam SC was 30 days, so it could be applied until mid-July. Captan/oxyquinoline copper WP has a very short PHI of only 3 days. On the other hand, it leaves visible residues on the fruit and may not be sprayed too soon before harvest.

In contrast, the ST-QoIs (azoxystrobin FL, kresoxim-methyl DF, and pyraclostrobin with boscalid WP in a pre-mix) showed excellent anti-JPA efficacy [28, 44, 54]. These fungicides have a 1-day PHI and can, therefore, be applied up until the day before harvest. Moreover, they leave no visible residues on the fruit. Consequently, the application frequency of ST-QoIs against JPA increased.

#### **4. Emergence of strobilurin (ST)-QoI fungicide-resistant strains and new treatment recommendations after 2011**

#### **4.1 ST-QoIs**

ST-QoIs or strobilurins were first used in the 1990s and became one of the most important fungicides of the past 25 years. They inhibit ubiquinol oxidation at the quinone outside (Qo) binding site on the cytochrome bc1 complex in the inner mitochondrial membranes of fungi [55]. At the time of introduction, ST-QoIs showed very high efficacy against many different pathogen-crop combinations; however, ST-QoI fungicides are highly prone to inducing resistance in target pathogens that can lead to reduced field efficacy. The ST-QoI resistance risk has been rated high by the Fungicide Resistance Action Committee (FRAC) [56]. ST-QoIresistant strains have been detected in 60 fungal and oomycete pathogen species worldwide including powdery and downy mildews, gray mold, *Alternaria* disease, scab, and anthracnose [57]. Currently, disease control strategies that are overly reliant on ST-QoIs are considered undesirable [57]. A major source of ST-QoI resistance is a point mutation in the cytochrome b gene that substitutes alanine for glycine at amino acid position 143. This site may be associated with the pathogen binding affinity of the fungicide [58].

In Japan, ST-QoI resistance has emerged in cucumber powdery mildew (*Podosphaera xanthii*), downy mildew (*Pseudoperonospora cubensis*) [59, 60], *Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

eggplant leaf mold (*Mycovellosiella nattrassii*) [61], *Corynespora* cucumber leaf spot (*Corynespora cassicola*) [62], citrus gray mold (*Botrytis cinerea*) [63], European pear black spot (*Alternaria alternata*) [64], *Alternaria* apple blotch (*Alternaria alternata* apple pathotype) [65], grapevine leaf blight (*Pseudocercospora vitis*) [66], strawberry anthracnose (*Colletotrichum gloeosporioides*) [67, 68], tea gray blight (*Pestalotiopsis longiseta*) [69], apple bitter rot (*Colletotrichum gloeosporioides*) [70], rice blast (*Magnaporthe oryzae*) [71], mango anthracnose (*Colletotrichum gloeosporioides*) [72], apple scab (*Venturia inaequalis*) [73], grapevine downy mildew (*Plasmopara viticola*) [74], cucurbits gummy stem blight (*Didymella bryoniae*) [75], chrysanthemum white rust (*Puccinia horiana*) [76], wheat powdery mildew (*Erysiphe* (Blumeria) *graminis f.sp. tritici*), and strawberry powdery mildew (*Sphaerotheca aphanis* var. aphanis) [77].

#### **4.2 Emergence of strains of** *Colletotrichum gloeosporioides* **sensu lato resistant to ST-QoIs**

Over nearly a decade in the Saga and Oita prefectures, ST-QoIs were sprayed three to four times annually between June and early August as countermeasures against JPA and APS. That is, many growers heavily depended on ST-QoIs, especially late in the season because ST-QoIs are phytotoxic to Japanese pear leaves at their early growth stage. In addition, ST-QoIs were also highly efficacious against APS [78, 79].

The alternative material, thiuram FL, has a 30-day PHI; therefore, it cannot be used after mid-July. The other options, such as iminoctadine tris(albesilate)/captan WP, have a relatively shorter PHI (14 days), and captan WP has a 3-day PHI. Captan/oxyquinoline copper WP (3-day PHI), captan WP (3-day PHI), and iminoctadine tris(albesilate)/captan WP (14-day PHI) showed adequate efficacy against JPA [28, 44, 54, 80]. However, the ST-QoIs were preferred over these choices by growers as they were more effective than these; in addition, the common component of these materials, captan, tends to cause stains on the fruit.

As JPA became very prevalent in 2010–2011 in the Oita and Saga prefectures where above-mentioned spraying system. We assessed ST-QoI sensitivity in Cgsl isolates by placing mycelial discs on potato dextrose agar (PDA) containing 100 μg mL<sup>1</sup> azoxystrobin and 1000 μg mL<sup>1</sup> salicylhydroxamic acid (SHAM). Mycelial elongation was measured 4 days post-inoculation [81]. Isolates from Saga [80] and Oita [45] prefecture grew on the PDA containing azoxystrobin (**Table 9**, **Figure 6**).

To determine the effect of ST-QoI pretreatment on JPA development, conidial suspensions were sprayed on "Housui" leaves previously exposed to azoxystrobin FL. The appearance of JPA lesions caused by the sensitive strain was nearly zero


*1 Number of strains that grew on PDA with 1000 μg mL<sup>1</sup> SHAM and 100 μg mL<sup>1</sup> azoxystrobin cultured 4 days at 25°C.*

*2 Values in parentheses are the percentage of the resistant strains.*

#### **Table 9.**

**3.4 Temporary suspension of the 1999 JPA outbreak**

*Plant Diseases-Current Threats and Management Trends*

and fluazinam were applied.

soon before harvest.

**4.1 ST-QoIs**

**198**

result, JPA incidence was drastically reduced.

Before the 1999 JPA outbreak, the main disease to control in Japanese pear cultivation was Asian pear scab (APS). Sterol demethylation inhibitor (DMI), belonging to sterol biosynthesis inhibitors (SBIs), was the product most frequently used to control this disease. Iminoctadine tris(albesilate), captan/oxyquinoline copper, and captan were applied for APS a few times. Benzimidazoles were applied three to four times to control ring rot and powdery mildew. However, by 2000, benzimidazoles were no longer recommended in Japanese pear production due to its

resistance issue. In their place, local systemic fungicides such as strobilurins (azoxystrobin and kresoxim-methyl) and protective fungicides such as dithianon

In contrast, the ST-QoIs (azoxystrobin FL, kresoxim-methyl DF, and pyraclostrobin with boscalid WP in a pre-mix) showed excellent anti-JPA efficacy [28, 44, 54]. These fungicides have a 1-day PHI and can, therefore, be applied up until the day before harvest. Moreover, they leave no visible residues on the fruit. Consequently, the application frequency of ST-QoIs against JPA increased.

**4. Emergence of strobilurin (ST)-QoI fungicide-resistant strains and**

In Japan, ST-QoI resistance has emerged in cucumber powdery mildew (*Podosphaera xanthii*), downy mildew (*Pseudoperonospora cubensis*) [59, 60],

ST-QoIs or strobilurins were first used in the 1990s and became one of the most important fungicides of the past 25 years. They inhibit ubiquinol oxidation at the quinone outside (Qo) binding site on the cytochrome bc1 complex in the inner mitochondrial membranes of fungi [55]. At the time of introduction, ST-QoIs showed very high efficacy against many different pathogen-crop combinations; however, ST-QoI fungicides are highly prone to inducing resistance in target pathogens that can lead to reduced field efficacy. The ST-QoI resistance risk has been rated high by the Fungicide Resistance Action Committee (FRAC) [56]. ST-QoIresistant strains have been detected in 60 fungal and oomycete pathogen species worldwide including powdery and downy mildews, gray mold, *Alternaria* disease, scab, and anthracnose [57]. Currently, disease control strategies that are overly reliant on ST-QoIs are considered undesirable [57]. A major source of ST-QoI resistance is a point mutation in the cytochrome b gene that substitutes alanine for glycine at amino acid position 143. This site may be associated with the pathogen

**new treatment recommendations after 2011**

binding affinity of the fungicide [58].

Dithianon FL, fluazinam FL, ST-QoI fungicides (azoxystrobin FL, kresoximmethyl DF), captan/oxyquinoline copper WP, and captan/benomyl WP were effective against JPA, APS, and ring spot, and all except dithianon were efficacious against powdery mildew. Therefore, these materials were incorporated into the spray calendar with heavy reliance on DMIs, which were popular at that time. As a

Although Dithianon FL has high JPA control efficacy, it has a 60-day pre-harvest interval (PHI) in Japan. Thus, it cannot be used after mid-June which is a critical JPA control period. The PHI of fluazinam SC was 30 days, so it could be applied until mid-July. Captan/oxyquinoline copper WP has a very short PHI of only 3 days. On the other hand, it leaves visible residues on the fruit and may not be sprayed too

> *Azoxystrobin sensitivity of* C. gloeosporioides *sensu lato, the causal organism of anthracnose in Japanese pear varieties "Housui" and "Niitaka" at Imari district in Saga prefecture and Hita district in Oita prefecture both Kyushyu island in 2011.*

squash (sorbitan fatty acid ester 70.0% and polyoxyethylene resin acid ester 5.5%; Maruwa Biochemical Co., Ltd., Tokyo, Japan). These agents render the spray spots inconspicuous by lowering droplet surface tension. All the three agents reduced the visibility of the captan residues on the plant surfaces. There is a concern that the addition of the spreader can decrease the amount of fungicide that attached to the host plant [82, 83]. However, the mixture had nearly the same efficacy levels as

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

By 2014, pear producers had fully recognized the presence of benzimidazoleand ST-QoI-resistant pathogen strains and stopped relying on ST-QoI to manage JPA. The current recommended JPA management protocol for Japanese pear is dithianon FL in early June; thiuram FL, captan/oxyquinoline copper, and

iminoctadine tris(albesilate)/captan WP from mid-June to early July; and captan WP with a spreader several times after mid-July. The occurrence of JPA has abated as growers are now comparatively less dependent on ST-QoI fungicides [80]. We also advocate proper spray coverage. For example, we recommend everyrow spray over alternate-row spray with an air-blast sprayer (**Figure 7**), because of better fungicide coverage achieved by the former. It has been shown in one of our studies that JPA is more effectively controlled when fungicides are sprayed onto all rows [84]. Moreover, infected and abscised leaves should be promptly removed

Our test results of 1999 and the data obtained at the experiment stations in other prefectures promoted the registration of additional fungicides to control this disease. In 2019, 11 products were registered for use against JPA in Japan (**Table 11**). This step provides a wider selection of fungicides to control or manage this disease.

Pyribencarb (methyl{2-chloro-5-[(1E)-1-(6-methyl-2-pyridylmethoxyimino) ethyl]benzyl} carbamate) was formulated by Kumiai Chemical Industry Co., Ltd. and Ihara Chemical Industry Co., Ltd. in Japan. It is a novel benzylcarbamate-type QoI fungicide (BC-QoI) and is active against a wide range of fungal plant pathogens [86]. Pyribencarb is both preventive and curative [87], and its chemical structure

captan alone in the field trial [80].

**Figure 7.**

**201**

*4.3.2 Current recommendation against JPA*

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

*Fungicide application by air-blast sprayer in the Japanese pear orchard.*

from orchards to reduce the inoculum pool [85].

**5.1 Benzylcarbamate (BC)-QoI and pyribencarb**

**5. Potential options for JPA management in the future**

#### **Figure 6.**

*Effect of azoxystrobin on the mycelial growth of azoxystrobin-resistant and azoxystrobin-sensitive strains of* Colletotrichum gloeosporioides *sensu lato, the causal fungus of Japanese pear anthracnose, 4 days after inoculation of mycelial disks (4 mm) at 25°C; medium, potato dextrose agar medium with 25 μg mL<sup>1</sup> azoxystrobin and 1000 μg mL<sup>1</sup> SHAM; strains above the line, azoxystrobin-resistant strains; strains under the line, azoxystrobin-sensitive strains.*

(99.6% control). In contrast, the two resistant strains induced many lesions, and there was a very low rate of disease control (**Table 10**).

#### **4.3 Effective spraying program in the presence of benzimidazole- and ST-QoI-resistant strains**

#### *4.3.1 Use of the adjuvant to reduce the risk of phytotoxicity caused by captan*

Products containing captan provide a sufficient level of disease control, but they blemish the fruit to reduce its quality. We investigated the application of spreaders such as Makupika (polyoxyethylene methylpolysiloxane 93.0%; Ishihara Bio-Science Co., Ltd., Tokyo, Japan) and Santokuten 80 (polyoxyethylene dodecyl ether 80.0%; Sumitomo Chemical Co., Ltd., Tokyo, Japan). We also tested the adjuvant


*1 The Japanese pear variety "Housui" (2-year-old trees) were sprayed with wettable powder of azoxystrobin and thoroughly dried. Conidial suspensions (approx. 105 mL<sup>1</sup> ) of each strain (azoxystrobin-sensitive strains, 1–7; azoxystrobin-resistant strains, 3–1, 3–2) were then inoculated. Seven days after inoculation, the development of symptoms was assessed.*

*2 All strains was isolataed at Hita city of Oita prefecture in 2011.*

*3 Control (%) = (1 – average lesion number per leaf on the trees with azoxystrobin application/average lesion number per leaf on the control trees) 100.*

#### **Table 10.**

*Control efficacy of azoxystorobin against azoxystrobin-sensitive (1–7) strains and azoxystrobin-resistant (3–1 and 3–2) strains of* C. gloeosporioides *sensu lato on the leaves of the Japanese pear variety "Housui". 1*

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

**Figure 7.** *Fungicide application by air-blast sprayer in the Japanese pear orchard.*

squash (sorbitan fatty acid ester 70.0% and polyoxyethylene resin acid ester 5.5%; Maruwa Biochemical Co., Ltd., Tokyo, Japan). These agents render the spray spots inconspicuous by lowering droplet surface tension. All the three agents reduced the visibility of the captan residues on the plant surfaces. There is a concern that the addition of the spreader can decrease the amount of fungicide that attached to the host plant [82, 83]. However, the mixture had nearly the same efficacy levels as captan alone in the field trial [80].

#### *4.3.2 Current recommendation against JPA*

By 2014, pear producers had fully recognized the presence of benzimidazoleand ST-QoI-resistant pathogen strains and stopped relying on ST-QoI to manage JPA. The current recommended JPA management protocol for Japanese pear is dithianon FL in early June; thiuram FL, captan/oxyquinoline copper, and iminoctadine tris(albesilate)/captan WP from mid-June to early July; and captan WP with a spreader several times after mid-July. The occurrence of JPA has abated as growers are now comparatively less dependent on ST-QoI fungicides [80].

We also advocate proper spray coverage. For example, we recommend everyrow spray over alternate-row spray with an air-blast sprayer (**Figure 7**), because of better fungicide coverage achieved by the former. It has been shown in one of our studies that JPA is more effectively controlled when fungicides are sprayed onto all rows [84]. Moreover, infected and abscised leaves should be promptly removed from orchards to reduce the inoculum pool [85].

#### **5. Potential options for JPA management in the future**

Our test results of 1999 and the data obtained at the experiment stations in other prefectures promoted the registration of additional fungicides to control this disease. In 2019, 11 products were registered for use against JPA in Japan (**Table 11**). This step provides a wider selection of fungicides to control or manage this disease.

#### **5.1 Benzylcarbamate (BC)-QoI and pyribencarb**

Pyribencarb (methyl{2-chloro-5-[(1E)-1-(6-methyl-2-pyridylmethoxyimino) ethyl]benzyl} carbamate) was formulated by Kumiai Chemical Industry Co., Ltd. and Ihara Chemical Industry Co., Ltd. in Japan. It is a novel benzylcarbamate-type QoI fungicide (BC-QoI) and is active against a wide range of fungal plant pathogens [86]. Pyribencarb is both preventive and curative [87], and its chemical structure

(99.6% control). In contrast, the two resistant strains induced many lesions, and

*Effect of azoxystrobin on the mycelial growth of azoxystrobin-resistant and azoxystrobin-sensitive strains of* Colletotrichum gloeosporioides *sensu lato, the causal fungus of Japanese pear anthracnose, 4 days after inoculation of mycelial disks (4 mm) at 25°C; medium, potato dextrose agar medium with 25 μg mL<sup>1</sup> azoxystrobin and 1000 μg mL<sup>1</sup> SHAM; strains above the line, azoxystrobin-resistant strains; strains under*

Products containing captan provide a sufficient level of disease control, but they blemish the fruit to reduce its quality. We investigated the application of spreaders such as Makupika (polyoxyethylene methylpolysiloxane 93.0%; Ishihara Bio-Science Co., Ltd., Tokyo, Japan) and Santokuten 80 (polyoxyethylene dodecyl ether 80.0%; Sumitomo Chemical Co., Ltd., Tokyo, Japan). We also tested the adjuvant

**Tested leaves Lesions/leaf Tested leaves Lesions/leaf** –7 5 0.2 5 56.8 99.6 –1 5 24.8 5 26.8 7.6 –2 4 5.6 4 16.3 65.5

*The Japanese pear variety "Housui" (2-year-old trees) were sprayed with wettable powder of azoxystrobin and*

*azoxystrobin-resistant strains, 3–1, 3–2) were then inoculated. Seven days after inoculation, the development of*

*Control (%) = (1 – average lesion number per leaf on the trees with azoxystrobin application/average lesion number*

*Control efficacy of azoxystorobin against azoxystrobin-sensitive (1–7) strains and azoxystrobin-resistant (3–1 and 3–2) strains of* C. gloeosporioides *sensu lato on the leaves of the Japanese pear variety "Housui".*

**) sprayed trees Control trees Control (%)3**

*) of each strain (azoxystrobin-sensitive strains, 1–7;*

*1*

**4.3 Effective spraying program in the presence of benzimidazole- and**

*4.3.1 Use of the adjuvant to reduce the risk of phytotoxicity caused by captan*

there was a very low rate of disease control (**Table 10**).

*Plant Diseases-Current Threats and Management Trends*

**ST-QoI-resistant strains**

**Strain<sup>2</sup> Azoxystrobin (100 mg L<sup>1</sup>**

*thoroughly dried. Conidial suspensions (approx. 105 mL<sup>1</sup>*

*All strains was isolataed at Hita city of Oita prefecture in 2011.*

*the line, azoxystrobin-sensitive strains.*

**Figure 6.**

*1*

*2*

*3*

**200**

**Table 10.**

*symptoms was assessed.*

*per leaf on the control trees) 100.*



 *maximum of application per season.*

*4Standards on the use of pesticide in agricultural chemical regulation law of Japan.*

#### **Table 11.**

*Registered fungicides for Japanese pear anthracnose in Japan.1* resembles that of ST-QoIs such as kresoxim-methyl and azoxystrobin. However, it has a substitution of the carbonyl moiety on the benzene ring [88]. The binding site of pyribencarb on cytochrome b may be slightly different from that of the

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

Pyribencarb more effectively controlled ST-QoI-resistant gray mold isolates than other ST-QoI fungicides [90]. It also had relatively higher efficacy against ST-QoI-resistant *Pestalotiopsis longiseta* which causes tea gray blight [69]. Pyribencarb

Since pyribencarb has an excellent effect on JPA [44], it has been recommended to use it in orchards where ST-QoI-resistant strains are present or ST-QoI effects are reduced. However, there have been no reports of the effects of pyribencarb in an orchard where ST-QoI-resistant strains exist. Moreover, the risk of fungal pathogen resistance development of pyribencarb is high [91]. Therefore, it is necessary to take careful approaches to prevent the similar mistake we made with ST-QoIs. The number of pyribencarb application must be limited, and the application should be mixed with another broad-spectrum protective fungicide with a different mode of

Pyribencarb may be used less than three times per season on Japanese pear (**Table 11**). The Japan Fungicide Resistance Action Committee (Japan FRAC) guidelines recommend that QoIs be used up to twice annually on Japanese pear [92]. But we believe that it should be used only once between mid-June and early July which is the most critical disease control period of JPA and JPS for proper fungicide resistance management. In addition, pyribencarb must always be co-applied with the protective (multisite) fungicide such as captan, thiuram, iminoctadine tris (albesilate), and iminoctadine tris(albesilate)/captan to reduce the resistant risk. This treatment protocol may enhance disease control efficacy, lower pathogen density, and delay resistant strain development. In the future, comparative field trials would help validate the efficacy of the current treatment recommendations.

Fludioxonil is a benzodioxole that affects the signal transduction in the target fungal pathogen. These agents are also known as phenylpyrroles or PP-fungicides. According to the FRAC, the risk of pathogen resistance to this chemical class is low to medium [91]. Fludioxonil had extremely strong efficacy against JPA [93]. As of 2019, however, it has not yet been registered for use on Japanese pear in Japan. Data from field trials are being compiled for fludioxonil registration, and it is hoped that products containing fludioxonil will soon be available so that they may be inte-

Highly efficacious fungicides tend to be used the most. At the same time, the risks of fungicide-resistant fungal pathogen strains against the heavily used fungicide increase with the usage in the field. Fungicides that are prone to inducing pathogen resistance must be used properly by targeting the correct pathogens, applying the agents only at the appropriate times during the season, reducing application frequency, and mixing with other fungicides that are at low risk of inducing pathogen resistance. A mathematical model-based study suggested that the efficacy of high-risk fungicides may be substantially extended if they are mixed with low-risk fungicides [94]. This hypothesis should be validated by field trials, which are costly, time-consuming, and labor-intensive. On the other hand, these

shows differential cross-resistance patterns to ST-QoI [89].

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

ST-QoIs [89].

action.

**5.2 Benzodioxoles and fludioxonil**

grated into our JPA management strategies.

**6. Conclusions**

**203**

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

resembles that of ST-QoIs such as kresoxim-methyl and azoxystrobin. However, it has a substitution of the carbonyl moiety on the benzene ring [88]. The binding site of pyribencarb on cytochrome b may be slightly different from that of the ST-QoIs [89].

Pyribencarb more effectively controlled ST-QoI-resistant gray mold isolates than other ST-QoI fungicides [90]. It also had relatively higher efficacy against ST-QoI-resistant *Pestalotiopsis longiseta* which causes tea gray blight [69]. Pyribencarb shows differential cross-resistance patterns to ST-QoI [89].

Since pyribencarb has an excellent effect on JPA [44], it has been recommended to use it in orchards where ST-QoI-resistant strains are present or ST-QoI effects are reduced. However, there have been no reports of the effects of pyribencarb in an orchard where ST-QoI-resistant strains exist. Moreover, the risk of fungal pathogen resistance development of pyribencarb is high [91]. Therefore, it is necessary to take careful approaches to prevent the similar mistake we made with ST-QoIs. The number of pyribencarb application must be limited, and the application should be mixed with another broad-spectrum protective fungicide with a different mode of action.

Pyribencarb may be used less than three times per season on Japanese pear (**Table 11**). The Japan Fungicide Resistance Action Committee (Japan FRAC) guidelines recommend that QoIs be used up to twice annually on Japanese pear [92]. But we believe that it should be used only once between mid-June and early July which is the most critical disease control period of JPA and JPS for proper fungicide resistance management. In addition, pyribencarb must always be co-applied with the protective (multisite) fungicide such as captan, thiuram, iminoctadine tris (albesilate), and iminoctadine tris(albesilate)/captan to reduce the resistant risk. This treatment protocol may enhance disease control efficacy, lower pathogen density, and delay resistant strain development. In the future, comparative field trials would help validate the efficacy of the current treatment recommendations.

#### **5.2 Benzodioxoles and fludioxonil**

Fludioxonil is a benzodioxole that affects the signal transduction in the target fungal pathogen. These agents are also known as phenylpyrroles or PP-fungicides. According to the FRAC, the risk of pathogen resistance to this chemical class is low to medium [91]. Fludioxonil had extremely strong efficacy against JPA [93]. As of 2019, however, it has not yet been registered for use on Japanese pear in Japan. Data from field trials are being compiled for fludioxonil registration, and it is hoped that products containing fludioxonil will soon be available so that they may be integrated into our JPA management strategies.

#### **6. Conclusions**

Highly efficacious fungicides tend to be used the most. At the same time, the risks of fungicide-resistant fungal pathogen strains against the heavily used fungicide increase with the usage in the field. Fungicides that are prone to inducing pathogen resistance must be used properly by targeting the correct pathogens, applying the agents only at the appropriate times during the season, reducing application frequency, and mixing with other fungicides that are at low risk of inducing pathogen resistance. A mathematical model-based study suggested that the efficacy of high-risk fungicides may be substantially extended if they are mixed with low-risk fungicides [94]. This hypothesis should be validated by field trials, which are costly, time-consuming, and labor-intensive. On the other hand, these

**Generic name**

**202**

Dithianon Kresoxim-methyl

Azoxystrobin

Thiuram Thiuram Pyraclostrobin/boscalid

Captan/oxyquinoline

Captan Iminoctadine

captan

Pyribencarb Captan/penthiopyrad

*12019 confirmed on September 1, 2019.*

*2Legal pre-harvest*

*3The maximum number of application*

*4Standards on the use of pesticide in agricultural*

**Table 11.** *Registered fungicides for Japanese pear anthracnose*

 *in Japan.1*

 *interval.*

 *per season.*

 *chemical regulation law of Japan.*

tris(albesilate)/

Dyepower WP

Fantasista WDG Fruitguard WDG

 M4/7

 11

40.0 70.0/7.5

3

 3

1

 3

133.3 700/75

2013 2019

—

[44]

 M7/M4

 20.0/45.0

 14

 5

 copper

Oxyrane WP

Orthocide WP 80

 M4

 M4/M1

 20.0/30.0

80.0

3

 9

 3

 9

Storoby DF

Amistar 10 FL

Thionoc FL

Trenox FL Naria WDG

 M3

 11/7

 M3

 11

 11

50.0 10.0 40.0 40.0 6.8/13.6

1

 3

30

 5

30

 5

1

 5

1

 3

250 100 800 800 34/68 400/600

1000 200/450

2003 2006 2008 2008 2008 2009 2011 2012

[44]

[44, 80]

[44, 54, 80]

[44]

[44, 80]

[44, 80]

[28, 54]

*Plant Diseases-Current Threats and Management Trends*

[28, 54]

**Trade name in**

**FRAC**

**Active ingredient**

**LPHI2,4**

**MNAPS3,4**

**Rate applied**

**Resistered**

 **year in**

**References**

**Japan**

2003

[28, 44,

54, 80]

**(mg L1**

**)4**

**code**

**(%)**

**Japan**

Delan FL

M9

42.0

60

 4

420 field-based data are invaluable in the development of effective measures against fungicide-resistant plant pathogens.

We conceptualized a series of efforts to develop the best plant disease control practice at agricultural sites as an evidence-based control (EBC) [95–103]. The management of plant diseases needs to be developed based on the accumulated evidences, but not anecdotal observations. To gather useful evidence, the data need to be collected from the combination of field, controlled environment, and lab experiments, and then these data must be statistically validated to come up with repeatable and reliable information.

In this chapter, we demonstrated the use of EBC using the development of JPA management strategies against recent outbreaks as an example. JPA outbreak in 1999 and a detection of benzimidazole-resistant Cgsl strains [1, 28] triggered us to investigate alternatives such as fungicides ST-QoI, dithianon, and fluazinam, which were registered for use on Japanese pear [1, 2, 28, 54]. We also established the residual efficacy and rainfastness of these alternative fungicides [54]. We also obtained the evidence of long-term retention of benzimidazole-resistant strains in the field. Based on these results, an effective fungicide spray program without the use of benzimidazoles was established, and JPA was effectively controlled 2 years after the outbreak [1, 28].

However, JPA became conspicuous in 2006 and 2007 in two geographically distant regions, Kyushu (southeast) and Kanto (central). Outbreaks were reported in Oita prefecture in the Kyushu region in 2006 [45] and in Chiba and Kanagawa prefectures in the Kanto region in 2007 [44, 104]. Also a resurgence of JPA was reported around 2011 in Saga prefecture where the 1999 outbreak occurred [80]. Excessive dependence on ST-QoI fungicides induced ST-QoI-resistant Cgsl strains in Oita and Saga prefecture, which contributed to these new outbreaks [45, 80]. In Chiba and Kanagawa prefecture, the occurrence of QoI-resistant strains has not been investigated, but we suspect that the situation is very similar to Oita and Saga prefectures.

**Author details**

Nobuya Tashiro<sup>1</sup>

and Mizuho Nita<sup>5</sup>

Japan

Japan

**205**

\*, Youichi Ide2

2 Saga Prefectural Agricultural Research Center, Saga, Japan

, Mayumi Noguchi<sup>3</sup>

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

1 Saga Prefectural Upland Farming Research and Extension Center, Karatsu, Saga,

3 Saga Prefectural Nishimatsuura Agricultural Extension Center, Imari, Saga, Japan

4 Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Usa, Oita,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

5 Alson H. Smith Jr. Agricultural Research and Extension Center, Virginia

Polytechnic Institute and State University, Winchester, VA, USA

\*Address all correspondence to: tashirongreen12@gmail.com

provided the original work is properly cited.

, Hisayoshi Watanabe<sup>4</sup>

In order to increase the number of options to be used in late-season JPA management, we tested the efficacy of adjuvants to reducing visible chemical residues on fruits. Information from these experiments enabled us to determine appropriate and effective combinations of fungicides against JPA without relying on either the benzimidazole or ST-QoI. We intend to keep conducting similar holistic evidencebased approaches to develop effective management strategies for other pathosystems.

#### **Acknowledgements**

We appreciate the collaboration and information exchange with Dr. Yohei Kaneko of the CAFRC. In addition, we sincerely thank Dr. Kayo Manabe of Nippon Steel Eco-tech Corporation for assisting in the collection of references and Ms. Noriko Orihara and Mr. Makoto Suzuki of the Kanagawa Agricultural Technology Center for providing photos of JPA outbreak. Moreover, for implementation of the study, we sincerely thank the staff members of the Plant Protection Laboratory of Saga Prefectural Fruit Tree Experiment Station including Ms. Hisako Fukumoto, Setsumi Morinaga, and Hatsumi Nakayama and students of Saga Prefectural Agricultural College Fruit Tree Branch School.

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

#### **Author details**

field-based data are invaluable in the development of effective measures against

We conceptualized a series of efforts to develop the best plant disease control practice at agricultural sites as an evidence-based control (EBC) [95–103]. The management of plant diseases needs to be developed based on the accumulated evidences, but not anecdotal observations. To gather useful evidence, the data need to be collected from the combination of field, controlled environment, and lab experiments, and then these data must be statistically validated to come up with

In this chapter, we demonstrated the use of EBC using the development of JPA management strategies against recent outbreaks as an example. JPA outbreak in 1999 and a detection of benzimidazole-resistant Cgsl strains [1, 28] triggered us to investigate alternatives such as fungicides ST-QoI, dithianon, and fluazinam, which were registered for use on Japanese pear [1, 2, 28, 54]. We also established the residual efficacy and rainfastness of these alternative fungicides [54]. We also obtained the evidence of long-term retention of benzimidazole-resistant strains in the field. Based on these results, an effective fungicide spray program without the use of benzimidazoles was established, and JPA was effectively controlled 2 years

However, JPA became conspicuous in 2006 and 2007 in two geographically distant regions, Kyushu (southeast) and Kanto (central). Outbreaks were reported in Oita prefecture in the Kyushu region in 2006 [45] and in Chiba and Kanagawa prefectures in the Kanto region in 2007 [44, 104]. Also a resurgence of JPA was reported around 2011 in Saga prefecture where the 1999 outbreak occurred [80]. Excessive dependence on ST-QoI fungicides induced ST-QoI-resistant Cgsl strains in Oita and Saga prefecture, which contributed to these new outbreaks [45, 80]. In Chiba and Kanagawa prefecture, the occurrence of QoI-resistant strains has not been investigated, but we suspect that the situation is very similar to Oita and Saga

In order to increase the number of options to be used in late-season JPA management, we tested the efficacy of adjuvants to reducing visible chemical residues on fruits. Information from these experiments enabled us to determine appropriate and effective combinations of fungicides against JPA without relying on either the benzimidazole or ST-QoI. We intend to keep conducting similar holistic evidence-

We appreciate the collaboration and information exchange with Dr. Yohei Kaneko of the CAFRC. In addition, we sincerely thank Dr. Kayo Manabe of Nippon Steel Eco-tech Corporation for assisting in the collection of references and Ms. Noriko Orihara and Mr. Makoto Suzuki of the Kanagawa Agricultural Technology Center for providing photos of JPA outbreak. Moreover, for implementation of the study, we sincerely thank the staff members of the Plant Protection Laboratory of Saga Prefectural Fruit Tree Experiment Station including Ms. Hisako Fukumoto, Setsumi Morinaga, and Hatsumi Nakayama and students of Saga Prefectural

based approaches to develop effective management strategies for other

fungicide-resistant plant pathogens.

*Plant Diseases-Current Threats and Management Trends*

repeatable and reliable information.

after the outbreak [1, 28].

prefectures.

pathosystems.

**204**

**Acknowledgements**

Agricultural College Fruit Tree Branch School.

Nobuya Tashiro<sup>1</sup> \*, Youichi Ide2 , Mayumi Noguchi<sup>3</sup> , Hisayoshi Watanabe<sup>4</sup> and Mizuho Nita<sup>5</sup>

1 Saga Prefectural Upland Farming Research and Extension Center, Karatsu, Saga, Japan

2 Saga Prefectural Agricultural Research Center, Saga, Japan

3 Saga Prefectural Nishimatsuura Agricultural Extension Center, Imari, Saga, Japan

4 Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Usa, Oita, Japan

5 Alson H. Smith Jr. Agricultural Research and Extension Center, Virginia Polytechnic Institute and State University, Winchester, VA, USA

\*Address all correspondence to: tashirongreen12@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(in Japanese)

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**206**

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[62] Fuji M, Yamaguchi J, Furuta A, So K. Sensitivity of *Corynespora* leaf spot on cucumber fungus isolated from Saga Prefecture to strobilurin fungicides and methods for testing the sensitivity. Japanese Journal of Phytopathology. 2003;**69**:299-300 (Japanese abstract)

[63] Kansako M, Yoneda Y, Shimadu K, Ishii H. Occurrence of strobilurinresistant *Botrytis cinerea*, pathogen of citrus gray mold. Japanese Journal of Phytopathology. 2005;**71**:249 (Japanese abstract)

[64] Tanahashi M, Nakano T, Ishii H, Kodama M, Otani H. Possible occurrence of strobilurin resistant strains in *Alternaria alternata* causing black spot of European pear. Japanese Journal of Phytopathology. 2006;**72**:275 (Japanese abstract)

[65] Tsushima Y, Yukita K, Fukushi Y, Akahira T. Resistance to strobilurin fungicides of *Alternaria alternata* apple pathotype and simplified test method. Japanese Journal of Phytopathology. 2007;**73**:51-52 (Japanese abstract)

[66] Koya N, Inoue K, Kawaguchi A. Occurrence of strobilurin resistant isolates of *Pseudocercospora vitis*, grapevine leaf blight fungus. Japanese Journal of Phytopathology. 2008;**74**: 73-74 (Japanese abstract)

[67] Inada M, Ishii H, Chung W-H, Chung WH, Yamada T, Yamaguchi J, et al. Occurrence of strobilurin-resistant strains of *Colletotrichum gloeosporioides* (*Glomerella cingulata*), the causal fungus of strawberry anthracnose. Japanese Journal of Phytopathology. 2008;**74**: 114-117 (in Japanese with English summary)

*inaequalis* causing apple scab in Aomori Prefecture, Japan. Annual Report of the Society of Plant Protection of North Japan. 2017;**68**:115-119 (in Japanese with

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

[81] Watanebe H. Methods for detecting

*Colletotrichum gloeosporioides* (Japanese pear anthracnose). Plant Protection. 2017;**71**:327-330 (in Japanese)

[82] Tashiro N, Sadamatsu M. The effect of additional wetting spreaders on the effect of fungicides for control of Japanese pear black spot caused by *Alternaria alternata* Japanese pear pathotype. Bulletin of Saga Prefectural Fruit Tree Experiment Station. 1996;**13**:104-113 (in Japanese with English summary)

[83] Tashiro N. The effective use of the spreading agent in the disease control of fruit trees. Plant Protection. 2009;**63**:

[84] Ide Y, Tashiro N. Effect of rowpassage styles by speed sprayer on the efficacy against the scab by *Venturia nashicola* and anthracnose by *Colletotrichum gloeosporioides* and chemical adhesion on Japanese pear

212-217 (in Japanese)

leaves. Japanese Journal of Phytopathology. 2007;**73**:289-294 (in Japanese with English summary)

(in Japanese)

2001. WO 01/10825 A1

(Japanese abstract)

[87] Takagaki M, Kawata M, Fukumoto S, Miura I. Efficacy and in vitro activity of new fungicide KUF-1204 on *Botrytis cinerea*. Japanese Journal of Phytopathology. 2005;**71**:256

[85] Kaneko Y, Fukuta H. Use of 'Makupika'spreader for the purpose of reducing fruit stains with captan wettable powder just before the pear harvest period in Chiba Prefecture. CAFRC. Research Bulletin. 2010;**9**:49-55

[86] Ozaki M, Fukumoto S, Tamai R, Yonekura N, Ikegaya K, Kawashima T, et al. Kumiai Chemical Industry Co, Ltd. and Ihara Chemical Industry Co. Ltd.;

[88] Kataoka S, Takagaki M, Kaku K, Shimizu T. Mechanism of action and

QoI fungicide resistance in

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[74] Furuya S, Mochizuki M, Saito S, Kobayashi H, Takayanagi T, Suzuki S. Monitoring of QoI fungicide resistance in *Plasmopara viticola* populations in Japan. Pest Management Science. 2010;

[75] Orihara N, Uekusa H, Miyakawa K, Okamoto M, Kobatashi N. Occurrence of QoI-resistant strains of *Didymella bryoniae*, causal fungus of gummy stem

prefecture. Annual Report of the Kanto-Tosan Plant Protection Society. 2013;**60**:

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[77] Ishii H. Current status of QoI fungicide resistance. Plant Protecton. 2015;**69**:469-474 (in Japanese)

[78] Ogata T, Kanno E. Application period and selection of effective chemicals for the Japanese pear scab control in autumn. Annual Report of the Society of Plant Protection of North Japan. 2000;**51**:141-143 (in

[79] Tomita Y, Ogawara T, Miyamoto T. Fungicidal control of Japanese pear scab caused by *Venturia nashicola* in Ibaraki Prefecture. Plant Protection. 2011;**65**:

[80] Noguchi M. Occurrence of QoIresistance to *Glomerella cingulate* on Japanese pear and countermeasures in Saga Prefecture. Plant Protection. 2015;

blight of cucurbits in Kanagawa

31-33 (in Japanese)

Japanese)

Japanese)

**211**

131-133 (in Japanese)

**69**:494-497 (in Japanese)

English summary)

**66**:1268-1272

[68] Hirayama Y, Kawamoto Y, Matsutani S, Nishizaki M, Okayama K. Occurrence of fungicides resistant isolates of *Glomerella cingulata* causing the strawberry anthracnose in Nara Prefecture. Annual Report Kansai Plant Protection. 2008;**50**:93-94 (in Japanese)

[69] Tomihama T, Nonaka T, Omatsu N, Nishi Y. Occurrence of QoI resistance in *Pestalotiopsis longiseta*, the pathogen causing gray blight disease in tea plants. Kyushu Plant Protection Research. 2009;**55**:83-88 (in Japanese with English summary)

[70] Akahira T, Hanaoka T. Occurrence of trifloxystrobin resistant strains of *Colletotrichum gloeosporioides* (*Glomerella cingulata*), the causal fungus of apple bitter rot in Aomori Prefecture. Japanese Journal of Phytopathology. 2013;**79**:197-198 (Japanese abstract)

[71] Miyagawa N, Fuji M. Occuurrence of QoI-fungicide-resistant strains of *Magnaporthe oryzae* on rice and fungicidal effective. In: Abstracts of the 23rd Symposium of PSJ Research Committee on Fungicide Resistance, Gifu, Japan. 2013. pp. 25-35 (in Japanese with English summary)

[72] Takushi T, Kadekaru K, Arasaki C, Taba S. Occurrence of strobilurinresistant strains of *Colletotrichum gloeosporioides*, the causal fungus of mango anthracnose. Japanese Journal of Phytopathology. 2014;**80**:119-123 (in Japanese with English summary)

[73] Hirayama K, Akahira T, Hanaoka T. QoI-resistant strains of *Venturia*

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

*inaequalis* causing apple scab in Aomori Prefecture, Japan. Annual Report of the Society of Plant Protection of North Japan. 2017;**68**:115-119 (in Japanese with English summary)

[60] Ishii H, Fraaije BA, Sugiyama T, Noguchi K, Nishimura K, Takeda T, et al. Occurrence and molecular

**91**:1166-1171

summary)

abstract)

characterization of strobilurin resistance in cucumber powdery mildew and downy mildew. Phytopathology. 2001;

*Plant Diseases-Current Threats and Management Trends*

[67] Inada M, Ishii H, Chung W-H, Chung WH, Yamada T, Yamaguchi J, et al. Occurrence of strobilurin-resistant strains of *Colletotrichum gloeosporioides* (*Glomerella cingulata*), the causal fungus of strawberry anthracnose. Japanese Journal of Phytopathology. 2008;**74**: 114-117 (in Japanese with English

[68] Hirayama Y, Kawamoto Y,

Matsutani S, Nishizaki M, Okayama K. Occurrence of fungicides resistant isolates of *Glomerella cingulata* causing the strawberry anthracnose in Nara Prefecture. Annual Report Kansai Plant Protection. 2008;**50**:93-94 (in Japanese)

[69] Tomihama T, Nonaka T, Omatsu N, Nishi Y. Occurrence of QoI resistance in *Pestalotiopsis longiseta*, the pathogen causing gray blight disease in tea plants. Kyushu Plant Protection Research. 2009;**55**:83-88 (in Japanese with English

[70] Akahira T, Hanaoka T. Occurrence of trifloxystrobin resistant strains of

(*Glomerella cingulata*), the causal fungus of apple bitter rot in Aomori Prefecture. Japanese Journal of Phytopathology. 2013;**79**:197-198 (Japanese abstract)

[71] Miyagawa N, Fuji M. Occuurrence of QoI-fungicide-resistant strains of *Magnaporthe oryzae* on rice and

fungicidal effective. In: Abstracts of the 23rd Symposium of PSJ Research Committee on Fungicide Resistance, Gifu, Japan. 2013. pp. 25-35 (in Japanese

[72] Takushi T, Kadekaru K, Arasaki C, Taba S. Occurrence of strobilurinresistant strains of *Colletotrichum gloeosporioides*, the causal fungus of mango anthracnose. Japanese Journal of Phytopathology. 2014;**80**:119-123 (in Japanese with English summary)

[73] Hirayama K, Akahira T, Hanaoka T.

QoI-resistant strains of *Venturia*

*Colletotrichum gloeosporioides*

with English summary)

summary)

summary)

[61] Yano K, Kawada Y. Occurrence of

*Mycovellosiella nattrassii*, causal fungus of leaf mold of eggplants. Japanese Journal of Phytopathology. 2003;**69**: 220-223 (in Japanese with English

[62] Fuji M, Yamaguchi J, Furuta A, So K. Sensitivity of *Corynespora* leaf spot on cucumber fungus isolated from Saga Prefecture to strobilurin fungicides and methods for testing the sensitivity. Japanese Journal of Phytopathology. 2003;**69**:299-300 (Japanese abstract)

[63] Kansako M, Yoneda Y, Shimadu K, Ishii H. Occurrence of strobilurinresistant *Botrytis cinerea*, pathogen of citrus gray mold. Japanese Journal of Phytopathology. 2005;**71**:249 (Japanese

[64] Tanahashi M, Nakano T, Ishii H,

[65] Tsushima Y, Yukita K, Fukushi Y, Akahira T. Resistance to strobilurin fungicides of *Alternaria alternata* apple pathotype and simplified test method. Japanese Journal of Phytopathology. 2007;**73**:51-52 (Japanese abstract)

[66] Koya N, Inoue K, Kawaguchi A. Occurrence of strobilurin resistant isolates of *Pseudocercospora vitis*, grapevine leaf blight fungus. Japanese Journal of Phytopathology. 2008;**74**:

73-74 (Japanese abstract)

**210**

Kodama M, Otani H. Possible occurrence of strobilurin resistant strains in *Alternaria alternata* causing black spot of European pear. Japanese Journal of Phytopathology. 2006;**72**:275

(Japanese abstract)

strobilurin-resistant strains of

[74] Furuya S, Mochizuki M, Saito S, Kobayashi H, Takayanagi T, Suzuki S. Monitoring of QoI fungicide resistance in *Plasmopara viticola* populations in Japan. Pest Management Science. 2010; **66**:1268-1272

[75] Orihara N, Uekusa H, Miyakawa K, Okamoto M, Kobatashi N. Occurrence of QoI-resistant strains of *Didymella bryoniae*, causal fungus of gummy stem blight of cucurbits in Kanagawa prefecture. Annual Report of the Kanto-Tosan Plant Protection Society. 2013;**60**: 31-33 (in Japanese)

[76] Matsuura S. Current status of QoI (strobilurin) fungicides sensitivity in isolates of *Puccinia horiana*, the causal agent of chrysanthemum white rust, occur in western Japan. Plant Protection. 2019;**73**:370-373 (in Japanese)

[77] Ishii H. Current status of QoI fungicide resistance. Plant Protecton. 2015;**69**:469-474 (in Japanese)

[78] Ogata T, Kanno E. Application period and selection of effective chemicals for the Japanese pear scab control in autumn. Annual Report of the Society of Plant Protection of North Japan. 2000;**51**:141-143 (in Japanese)

[79] Tomita Y, Ogawara T, Miyamoto T. Fungicidal control of Japanese pear scab caused by *Venturia nashicola* in Ibaraki Prefecture. Plant Protection. 2011;**65**: 131-133 (in Japanese)

[80] Noguchi M. Occurrence of QoIresistance to *Glomerella cingulate* on Japanese pear and countermeasures in Saga Prefecture. Plant Protection. 2015; **69**:494-497 (in Japanese)

[81] Watanebe H. Methods for detecting QoI fungicide resistance in *Colletotrichum gloeosporioides* (Japanese pear anthracnose). Plant Protection. 2017;**71**:327-330 (in Japanese)

[82] Tashiro N, Sadamatsu M. The effect of additional wetting spreaders on the effect of fungicides for control of Japanese pear black spot caused by *Alternaria alternata* Japanese pear pathotype. Bulletin of Saga Prefectural Fruit Tree Experiment Station. 1996;**13**:104-113 (in Japanese with English summary)

[83] Tashiro N. The effective use of the spreading agent in the disease control of fruit trees. Plant Protection. 2009;**63**: 212-217 (in Japanese)

[84] Ide Y, Tashiro N. Effect of rowpassage styles by speed sprayer on the efficacy against the scab by *Venturia nashicola* and anthracnose by *Colletotrichum gloeosporioides* and chemical adhesion on Japanese pear leaves. Japanese Journal of Phytopathology. 2007;**73**:289-294 (in Japanese with English summary)

[85] Kaneko Y, Fukuta H. Use of 'Makupika'spreader for the purpose of reducing fruit stains with captan wettable powder just before the pear harvest period in Chiba Prefecture. CAFRC. Research Bulletin. 2010;**9**:49-55 (in Japanese)

[86] Ozaki M, Fukumoto S, Tamai R, Yonekura N, Ikegaya K, Kawashima T, et al. Kumiai Chemical Industry Co, Ltd. and Ihara Chemical Industry Co. Ltd.; 2001. WO 01/10825 A1

[87] Takagaki M, Kawata M, Fukumoto S, Miura I. Efficacy and in vitro activity of new fungicide KUF-1204 on *Botrytis cinerea*. Japanese Journal of Phytopathology. 2005;**71**:256 (Japanese abstract)

[88] Kataoka S, Takagaki M, Kaku K, Shimizu T. Mechanism of action and selectivity of a novel fungicide, pyribencarb. Journal of Pesticide Science. 2010;**35**:99-106

[89] Ishii H. Fungicide research in Japan —An overview. In: Dehne HW, Deising HB, Gisi U, Kuck KH, Russell PE, Lyr H, editors. Modern Fungicides and Antifungal Compounds V. 15th International Reinhardsbrunn Symposium May 06–10, 2007, Friedrichroda, Germany. DPG Selbstverlag, Braunschweig. 2008. pp. 11-17

[90] Takagaki M, Kataoka S, Fukumoto S, Ishii H, Yamaguchi J, Inada M, et al. The efficacy of the novel fungicide pyribencarb to the several QoI resistant fungal strains. Japanese Journal of Phytopathology. 2006;**72**:274-275 (Japanese abstract)

[91] Fungicide Resistance Action Committee. FRAC Code List 2019: Fungal control agents sorted by cross resistance pattern and mode of action (including FRAC Code numbering). Available at: https://www.frac.info/ docs/default-source/publications/ frac-code-list/frac-code-list-2019.pdf? sfvrsn=98ff4b9a\_2 [Accessed: 14 October 2019]

[92] Japan Fungicide Resistance Action Committee. Guide line of QoIs in Japan. Available at: https://www.jcpa.or.jp/ labo/jfrac/guidelines.html [Accessed: 14 October 2019]

[93] Kaneko Y. Screening of substitutes for quinone outside inhibitor fungicides for control of Japanese pear anthracnose caused by *Glomerella cingulata* and their effects on several Japanese pear diseases. CAFRC Research Bulletin. 2016;**8**:1-7 (in Japanese with English summary)

[94] Hobbelen PHF, Paveley ND, van den Bosch F. Delaying selection for fungicide insensitivity by mixing fungicides at a low and high risk of

resistance development: S modeling analysis. Phytopathology. 2011;**101**: 1224-1233

[103] Tashiro N, Yamaguchi S, Nakashima K, Syouji K, Matsuo Y, Yamaguchi M. Evidence-based control (EBC) to greenhouse mandarin sooty

mold caused by *Cladosporium cladosporioides*. Japanese Journal of Phytopathology. 2016;**82**:79 (Japanese

[104] Kanagawa Prefecture. Anthracnose of Japanese pear (in Japanese). In: Temporary Pest Outbreak Forecast Information. Vol. 3. 2007. pp. 1-2

*DOI: http://dx.doi.org/10.5772/intechopen.90018*

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains…*

abstract)

(in Japanese)

**213**

[95] Tashiro N. New concept of disease and pest management: Construction of a control system with less frequent spray by EBC (evidence-based control). In: Proceedings of the 9th Forum of MAFF on Disease and Pest Management for Agricultural Product. Tokyo, Japan. 2003. pp. 18-27 (in Japanese)

[96] Tashiro N. Evidence-based control: The new concept of pest management. Journal of Evidence-Based Control. 2011;**7**:26-29 (in Japanese)

[97] Tashiro N. Evidence-based control: The new concept of pest management. Plant Protection. 2005;**59**:69-73 (in Japanese)

[98] Tashiro N. EBC (evidence-based control) is necessary for development of plant protection in the future. Annual Report of the Society of Plant Protection of North Japan. 2007;**58**:1-15 (in Japanese)

[99] Kawaguchi A. Concept for study of plant disease control at research committee for the EBC (evidence-based control), on the 7th workshop. Journal of Evidence-based Control. 2011;**7**:26-29 (in Japanese with English summary)

[100] Kawaguchi A. Concept and practice of EBC (evidence-based control) for on-farm research. Plant Protection. 2012;**66**:450-455 (in Japanese)

[101] Ide Y. Evidence of the chemical control to the disease on fruit trees. Plant Protection. 2012;**66**:456-459 (in Japanese)

[102] Tashiro N. Study on the improvement of fruit tree disease management using evidence-based control. Journal of General Plant Pathology. 2015;**81**:470-475

*Emergence of Benzimidazole- and Strobilurin-Quinone Outside Inhibitor-Resistant Strains… DOI: http://dx.doi.org/10.5772/intechopen.90018*

[103] Tashiro N, Yamaguchi S, Nakashima K, Syouji K, Matsuo Y, Yamaguchi M. Evidence-based control (EBC) to greenhouse mandarin sooty mold caused by *Cladosporium cladosporioides*. Japanese Journal of Phytopathology. 2016;**82**:79 (Japanese abstract)

selectivity of a novel fungicide, pyribencarb. Journal of Pesticide

—An overview. In: Dehne HW, Deising HB, Gisi U, Kuck KH, Russell PE, Lyr H, editors. Modern

Fungicides and Antifungal Compounds V. 15th International Reinhardsbrunn Symposium May 06–10, 2007, Friedrichroda, Germany. DPG Selbstverlag, Braunschweig. 2008.

[90] Takagaki M, Kataoka S, Fukumoto S, Ishii H, Yamaguchi J, Inada M, et al. The efficacy of the novel fungicide pyribencarb to the several QoI resistant fungal strains. Japanese Journal of Phytopathology. 2006;**72**:274-275

[91] Fungicide Resistance Action Committee. FRAC Code List 2019: Fungal control agents sorted by cross resistance pattern and mode of action (including FRAC Code numbering). Available at: https://www.frac.info/ docs/default-source/publications/ frac-code-list/frac-code-list-2019.pdf? sfvrsn=98ff4b9a\_2 [Accessed: 14

[92] Japan Fungicide Resistance Action Committee. Guide line of QoIs in Japan. Available at: https://www.jcpa.or.jp/ labo/jfrac/guidelines.html [Accessed:

[93] Kaneko Y. Screening of substitutes for quinone outside inhibitor fungicides for control of Japanese pear anthracnose caused by *Glomerella cingulata* and their effects on several Japanese pear diseases. CAFRC Research Bulletin. 2016;**8**:1-7 (in Japanese with English summary)

[94] Hobbelen PHF, Paveley ND, van den Bosch F. Delaying selection for fungicide insensitivity by mixing fungicides at a low and high risk of

(Japanese abstract)

October 2019]

14 October 2019]

**212**

pp. 11-17

[89] Ishii H. Fungicide research in Japan

*Plant Diseases-Current Threats and Management Trends*

resistance development: S modeling analysis. Phytopathology. 2011;**101**:

[95] Tashiro N. New concept of disease and pest management: Construction of a control system with less frequent spray by EBC (evidence-based control). In: Proceedings of the 9th Forum of MAFF on Disease and Pest Management for Agricultural Product. Tokyo, Japan. 2003. pp. 18-27 (in Japanese)

[96] Tashiro N. Evidence-based control: The new concept of pest management. Journal of Evidence-Based Control.

[97] Tashiro N. Evidence-based control: The new concept of pest management. Plant Protection. 2005;**59**:69-73

[98] Tashiro N. EBC (evidence-based control) is necessary for development of plant protection in the future. Annual Report of the Society of Plant Protection

[99] Kawaguchi A. Concept for study of plant disease control at research

committee for the EBC (evidence-based control), on the 7th workshop. Journal of Evidence-based Control. 2011;**7**:26-29 (in Japanese with English summary)

[100] Kawaguchi A. Concept and practice of EBC (evidence-based control) for on-farm research. Plant

[101] Ide Y. Evidence of the chemical control to the disease on fruit trees. Plant Protection. 2012;**66**:456-459

Protection. 2012;**66**:450-455

[102] Tashiro N. Study on the improvement of fruit tree disease management using evidence-based control. Journal of General Plant Pathology. 2015;**81**:470-475

of North Japan. 2007;**58**:1-15

2011;**7**:26-29 (in Japanese)

(in Japanese)

(in Japanese)

(in Japanese)

(in Japanese)

1224-1233

Science. 2010;**35**:99-106

[104] Kanagawa Prefecture. Anthracnose of Japanese pear (in Japanese). In: Temporary Pest Outbreak Forecast Information. Vol. 3. 2007. pp. 1-2 (in Japanese)

**215**

**Chapter 12**

Control

implications worldwide.

*Bacillus*

**1. Introduction**

**Abstract**

Biological Control of Citrus

*Sonia Villamizar and Juan Carlos Caicedo*

Canker: New Approach for Disease

Citrus canker is a disease that affects the major types of commercial citrus crops.

*Xanthomonas citri* subsp. *citri*, the etiological agent, reaches to mesophyll tissue through the stomata and afterward induces cell hyperplasia. Disease management has been based on both tree eradication and copper spray treatment. Overuse of copper for control of bacterial citrus canker has led to the development and prevalence of copper-resistant strains of Xcc. Several genera of both soil- and plant-associated bacteria became powerful tools in sustainable agriculture for control of Xcc and reduction of citrus canker disease severity. In this chapter we present bacteria able to interfere with quorum sensing as well to display antibacterial activity against *Xcc* by production of secondary metabolite. These bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used for the treatment of citrus canker disease, with significant environmental, economic, and health

**Keywords:** quorum quenching, *Pseudomonas*, biofilm, secondary metabolites,

The steady increase in global overpopulation has forced the agricultural producer to introduce environmentally aggressive practices (e.g., undiscriminating use of pesticides and chemical fertilizers), in order to respond to the rising request of cultivated crops for food. The growing breach between supply and request and the negative impact on the environment have stimulated researchers to develop alterna-

The interactions between plants and their associated microorganisms have generated a huge interest. A deep understanding of these processes allows the implementation of innovative agricultural applications. Plants produce an extensive collection of organic compounds comprising sugars, organic acids, and vitamins, which can be served as nutrients or signals for microbial communities. On the other hand, microorganisms release phytohormones, small molecules, or volatile compounds, which may act directly or indirectly reducing disease severity caused by phytopathogenic agents. Some of these actions are nutrient competition, antibiotic activity, plant immunity activation or plant growth, and morphogenesis activation [1].

tive strategies, pursuing to promote a sustainable agriculture.

#### **Chapter 12**

## Biological Control of Citrus Canker: New Approach for Disease Control

*Sonia Villamizar and Juan Carlos Caicedo*

#### **Abstract**

Citrus canker is a disease that affects the major types of commercial citrus crops. *Xanthomonas citri* subsp. *citri*, the etiological agent, reaches to mesophyll tissue through the stomata and afterward induces cell hyperplasia. Disease management has been based on both tree eradication and copper spray treatment. Overuse of copper for control of bacterial citrus canker has led to the development and prevalence of copper-resistant strains of Xcc. Several genera of both soil- and plant-associated bacteria became powerful tools in sustainable agriculture for control of Xcc and reduction of citrus canker disease severity. In this chapter we present bacteria able to interfere with quorum sensing as well to display antibacterial activity against *Xcc* by production of secondary metabolite. These bacteria may represent a highly valuable tool in the process of biological control and offer an alternative to the traditional copper treatment currently used for the treatment of citrus canker disease, with significant environmental, economic, and health implications worldwide.

**Keywords:** quorum quenching, *Pseudomonas*, biofilm, secondary metabolites, *Bacillus*

#### **1. Introduction**

The steady increase in global overpopulation has forced the agricultural producer to introduce environmentally aggressive practices (e.g., undiscriminating use of pesticides and chemical fertilizers), in order to respond to the rising request of cultivated crops for food. The growing breach between supply and request and the negative impact on the environment have stimulated researchers to develop alternative strategies, pursuing to promote a sustainable agriculture.

The interactions between plants and their associated microorganisms have generated a huge interest. A deep understanding of these processes allows the implementation of innovative agricultural applications. Plants produce an extensive collection of organic compounds comprising sugars, organic acids, and vitamins, which can be served as nutrients or signals for microbial communities. On the other hand, microorganisms release phytohormones, small molecules, or volatile compounds, which may act directly or indirectly reducing disease severity caused by phytopathogenic agents. Some of these actions are nutrient competition, antibiotic activity, plant immunity activation or plant growth, and morphogenesis activation [1].

Prokaryotes and mainly the bacterial domain are the numerically dominant component of most microbial communities in plants. Numerous genera of both soil- and plant-associated bacteria turn out to be powerful tools in sustainable agriculture, because these bacteria display extremely versatile secondary metabolisms with valuable biological activities, including quorum quenching and antibiotic activity. The aim of this chapter is to present two different approaches for biological control of bacterial citrus canker. This antagonism specifically focus in a quorum quenching of DSF pathway and antibacterial activity by *Pseudomonas* bacteria against *Xanthomonas citri* subsp. *citri* ethological agent of citrus canker disease.

#### **2. Citrus canker disease**

One of the most important diseases of citrus is citrus canker, affecting almost all commercial varieties. Bacterium *Xanthomonas citri* subsp. *citri* (*Xcc*) is the etiological agent of citrus canker. In the last decade, citrus canker disease rise as one of the main threats to citrus industry, because of the rise of copper-resistant Xcc strains. Factors such as bacterial species and weather conditions determine the disease severity. The geographical origin of the disease is not clear; some researchers report that the first disease cases appeared at Southern China [2]. However, according to Fawcett and Jenkins in 1933, the disease originated in regions of India and Java [3]. These reports suggest, therefore, that the origin of the disease occurred in tropical areas of Asia, where it is assumed that citrus species originated and has been distributed to other areas through grafting [4]. In America, the first report of the disease occurred in the United States in 1915 [5]. Currently, citrus canker is present in more than 30 countries in Asia, the Indian Ocean and Pacific Islands, South America, and the Southeastern United States [4].

Traditional control of citrus canker disease centered on the application of copper-based products seeks the reduction of bacterial population in leaf surfaces. However, multiple applications are needed in order to obtain a significant reduction in bacterial burden on phyllosphere. Weather conditions, i.e., wind and rain, decrease drastically the effectiveness of copper applications. The drawbacks of the long-term use of copper compounds to control plant pathogens in the field include selection of copper resistance and horizontal transfer in bacterial populations [6].

#### **2.1 Disease cycle and transmission mechanisms**

Invasion and colonization of the citrus host by *Xcc* occur by stomata and wounds in plant tissues, infecting leaves, fruits, and stems. The bacterium *Xcc* multiplies within the intercellular spaces in the mesophilic tissue, inducing cellular hyperplasia, leading to rupture of the leaf epidermis and resulting in high and spongy lesions surrounded by a margin soaked in water. Upon leaf epidermis eruption, a great number of bacteria are released to the environment to reach other leaves and plants. Rain, wind, and agriculture tools are the main agents of natural dispersion of disease; the insect larvae of the citrus tree cause extensive wounds in the foliar tissues and greatly increase the spread of the disease. Rainwater collected from foliage with lesions contains between 105 and 108 cfu/ml [4].

#### **2.2 Types of disease**

There are three different types of citrus canker caused by two species of *Xanthomonas*, citrus canker type: A, B, and C. The differentiation of these types is mainly based on the geographical distribution and pathogen host range [7].

**217**

**Figure 1.**

*Biological Control of Citrus Canker: New Approach for Disease Control*

*aurantifolii* type C (XauC) and only infects *C. aurantifolii* [9].

visualized with transmitted light (**Figure 1**).

**2.4 Management and treatment**

The Asian type of canker (canker A) is caused by *Xanthomonas citri* subsp. *citri*. Canker A is the most common and widespread disease, and its geographical distribution continues increasing. The disease is endemic in more than 30 countries: Asia, in the Pacific of India, Pakistan, the Indian Ocean islands, Southeast Asia, South America, Southeast China, and Japan. The bacterium Xcc has a wide range of host and produces the disease in the great majority of the citrus species as *C. paradisi*, *C. aurantifoli*, *C. sinensis*, and *C. reticulata* [8]. Type B canker is caused by the bacterium *Xanthomonas fuscans* subsp. *aurantifolii* type B (*XauB*) [9]. Type B canker has similar symptoms to type A canker; however, the symptoms take longer to appear as a consequence of the slower growth rate of XauB, and the host range is restricted to *C. limon* but has also been sporadically isolated from *C. sinensis and C. paradisi* [10]. C-type canker has only been identified in the state of São Paulo, Brazil [11], and has the same symptoms as type A citrus canker, caused by *Xanthomonas fuscans* subsp*.* 

The diseased plants are characterized by the occurrence of conspicuous raised necrotic lesions that develop on leaves, branches, and fruits. In the leaves, the first appearance is circular patches of 2–10 mm in diameter; their appearance is oily and usually appears on the abaxial surface reflecting stomatal entrance*.* The lesions are often similar in shape and size. Subsequently, both epidermal surfaces may become ruptured by pathogen-induced tissue hyperplasia. In the leaves, stems, thorns, and fruits, circular lesions became like a raised boil, growing in spongy white or yellow pustules. These pustules then darken and thicken brown cork type, which is rough to the touch. Often, a watery swell develops around the necrotic tissue and is easily

Bacterial citrus canker management involves different approaches ranging from strict quarantine measures to chemical control. Quarantining is a practical usually used in Brazil and United States of America. Extinction of infected and adjacent

*Symptoms of citrus canker. Left, early stage of the disease. Right, hyperplasia and rupture of the foliar tissue.*

*DOI: http://dx.doi.org/10.5772/intechopen.88000*

**2.3 Symptomatology**

*Biological Control of Citrus Canker: New Approach for Disease Control DOI: http://dx.doi.org/10.5772/intechopen.88000*

The Asian type of canker (canker A) is caused by *Xanthomonas citri* subsp. *citri*. Canker A is the most common and widespread disease, and its geographical distribution continues increasing. The disease is endemic in more than 30 countries: Asia, in the Pacific of India, Pakistan, the Indian Ocean islands, Southeast Asia, South America, Southeast China, and Japan. The bacterium Xcc has a wide range of host and produces the disease in the great majority of the citrus species as *C. paradisi*, *C. aurantifoli*, *C. sinensis*, and *C. reticulata* [8]. Type B canker is caused by the bacterium *Xanthomonas fuscans* subsp. *aurantifolii* type B (*XauB*) [9]. Type B canker has similar symptoms to type A canker; however, the symptoms take longer to appear as a consequence of the slower growth rate of XauB, and the host range is restricted to *C. limon* but has also been sporadically isolated from *C. sinensis and C. paradisi* [10]. C-type canker has only been identified in the state of São Paulo, Brazil [11], and has the same symptoms as type A citrus canker, caused by *Xanthomonas fuscans* subsp*. aurantifolii* type C (XauC) and only infects *C. aurantifolii* [9].

#### **2.3 Symptomatology**

*Plant Diseases-Current Threats and Management Trends*

America, and the Southeastern United States [4].

**2.1 Disease cycle and transmission mechanisms**

foliage with lesions contains between 105

**2.2 Types of disease**

**2. Citrus canker disease**

Prokaryotes and mainly the bacterial domain are the numerically dominant component of most microbial communities in plants. Numerous genera of both soil- and plant-associated bacteria turn out to be powerful tools in sustainable agriculture, because these bacteria display extremely versatile secondary metabolisms with valuable biological activities, including quorum quenching and antibiotic activity. The aim of this chapter is to present two different approaches for biological control of bacterial citrus canker. This antagonism specifically focus in a quorum quenching of DSF pathway and antibacterial activity by *Pseudomonas* bacteria against *Xanthomonas citri* subsp. *citri* ethological agent of citrus canker disease.

One of the most important diseases of citrus is citrus canker, affecting almost all commercial varieties. Bacterium *Xanthomonas citri* subsp. *citri* (*Xcc*) is the etiological agent of citrus canker. In the last decade, citrus canker disease rise as one of the main threats to citrus industry, because of the rise of copper-resistant Xcc strains. Factors such as bacterial species and weather conditions determine the disease severity. The geographical origin of the disease is not clear; some researchers report that the first disease cases appeared at Southern China [2]. However, according to Fawcett and Jenkins in 1933, the disease originated in regions of India and Java [3]. These reports suggest, therefore, that the origin of the disease occurred in tropical areas of Asia, where it is assumed that citrus species originated and has been distributed to other areas through grafting [4]. In America, the first report of the disease occurred in the United States in 1915 [5]. Currently, citrus canker is present in more than 30 countries in Asia, the Indian Ocean and Pacific Islands, South

Traditional control of citrus canker disease centered on the application of copper-based products seeks the reduction of bacterial population in leaf surfaces. However, multiple applications are needed in order to obtain a significant reduction in bacterial burden on phyllosphere. Weather conditions, i.e., wind and rain, decrease drastically the effectiveness of copper applications. The drawbacks of the long-term use of copper compounds to control plant pathogens in the field include selection of copper resistance and horizontal transfer in bacterial populations [6].

Invasion and colonization of the citrus host by *Xcc* occur by stomata and wounds in plant tissues, infecting leaves, fruits, and stems. The bacterium *Xcc* multiplies within the intercellular spaces in the mesophilic tissue, inducing cellular hyperplasia, leading to rupture of the leaf epidermis and resulting in high and spongy lesions surrounded by a margin soaked in water. Upon leaf epidermis eruption, a great number of bacteria are released to the environment to reach other leaves and plants. Rain, wind, and agriculture tools are the main agents of natural dispersion of disease; the insect larvae of the citrus tree cause extensive wounds in the foliar tissues and greatly increase the spread of the disease. Rainwater collected from

and 108

There are three different types of citrus canker caused by two species of *Xanthomonas*, citrus canker type: A, B, and C. The differentiation of these types is mainly based on the geographical distribution and pathogen host range [7].

cfu/ml [4].

**216**

The diseased plants are characterized by the occurrence of conspicuous raised necrotic lesions that develop on leaves, branches, and fruits. In the leaves, the first appearance is circular patches of 2–10 mm in diameter; their appearance is oily and usually appears on the abaxial surface reflecting stomatal entrance*.* The lesions are often similar in shape and size. Subsequently, both epidermal surfaces may become ruptured by pathogen-induced tissue hyperplasia. In the leaves, stems, thorns, and fruits, circular lesions became like a raised boil, growing in spongy white or yellow pustules. These pustules then darken and thicken brown cork type, which is rough to the touch. Often, a watery swell develops around the necrotic tissue and is easily visualized with transmitted light (**Figure 1**).

#### **2.4 Management and treatment**

Bacterial citrus canker management involves different approaches ranging from strict quarantine measures to chemical control. Quarantining is a practical usually used in Brazil and United States of America. Extinction of infected and adjacent

trees is one of the major prophylactic measures against citrus canker in commercial citrus crops. Once a symptomatic tree is identified, it is uprooted, stacked, and burned, as prophylactic measure surrounding trees is destroyed as mentioned before [12].

Prevention of primary infection in the new sprouts perhaps is the major effective approach to reduce citrus canker spread. The eradication methodology comprises conducting periodic surveys of the orchard, identifying and eliminating the outbreaks of the disease before its proliferation. Brazilian regulation stipulates that any field that has a number of diseased trees greater than 0.5% of the total orchard must be eliminated. After eradication, the contaminated field should be sprayed with copper fungicide based on 1.5 kg of metallic copper per 1 mL of water (0.15% of metallic copper). The contaminated plantations are prohibited and are forbidden from marketing the production until eradication works are completed.

The use of bactericidal products based on copper by spray application is a practice widely used for more than two decades for the bacterial citrus canker control. The prolonged exposure of bacterial strains to copper has led to the rise of resistant strains in endemic areas. Behlau et al. reported that the genes *copAB* and *cohAB* may encode copper-binding proteins responsible for the copper resistance in *Xanthomonas citri* subsp*. citri* [13].

#### **3.** *Xanthomonas citri* **subsp.** *citri*

The genus *Xanthomonas* includes a vast group of phytopathogenic bacteria belonging to the group of γ proteobacteria. *Xanthomonas* infects 124 species of monocotyledonous and 268 dicotyledonous plants [14]. *Xanthomonas* are Gramnegative bacillus endowed with a sole polar flagellum. After 24-hour incubation at 29°C, yellow and shiny colonies appear in a culture media. Xanthomonadin is an unique pigment, and it is responsible for the yellow color of bacterial colonies; the biological role is explained in detail below. The exopolysaccharide known as xanthan gum gives the shiny appearance to colonies [15]. Although the genus itself has a very broad host range, individual members are often specialized to cause disease in a limited number of taxonomically related hosts as mentioned above.

#### **3.1 Isolation and identification**

The bacterium *Xcc* can be isolated from symptomatic plants and its diverse infected tissues. Xcc grows easily in regular microbiological culture media. In order to isolate Xcc, infected tissues must be excised and washed, and subsequently the surface must be sterilized for 3 minutes in a 10% NaClO solution. The water-soaked tissue at the lesion margin is streaked across agar medium containing 50 ppm kasugamycin. *X. citri* strains grow easily on regular nutrient agar media containing 0.5% tryptone, 0.3% yeast extract, 0.09% CaCl2, 0.05% K2HPO4, and 1.5% agar in tap water, pH 7.2 [16]. After 48 hours of incubation at 29°C, mucoid yellow colonies begin to appear in microbiological medium.

#### **3.2 Determinants of virulence in** *Xanthomonas citri* **subsp.** *citri*

#### *3.2.1 Adhesins*

An essential stage in bacterial host colonization is its attachment ability. Adhesins are bacterial surface structures that facilitate the attachment to host tissues. The

**219**

*Biological Control of Citrus Canker: New Approach for Disease Control*

(type IV pili, chaperone/usher pili, two-partner secretion) [17].

nature of these structures is mainly polysaccharidic, e.g., lipopolysaccharides and exopolysaccharides. However, some of these structures share a proteinaceous nature

Bacteria inside in *Xanthomonas* genus exhibit at least six different types of protein secretion system (i.e., T1SS to T6SS), which vary in their arrangement, function, and in a recognition of secretion substrates [18]. Like many other Gramnegative phytopathogenic bacteria, *Xcc* employs mainly secretion systems T3SS, T4SS, and T5SS and their effectors as effective tools in an attempt to invade and to

Protein transport from bacterial periplasm to the extracellular environment occurs mainly by T2SS secretion system. Extracellular enzymes as lipases, proteases, and cell wall-degrading enzymes are translocated using this secretion system. Possibly the major enzymes responsible for the degradation of the plant cell wall are secreted by T2SS. T2SS translocator apparatus is composed of up to 12–15 constituents, most of which are linked to the bacterial inner membrane [19]. The T3SS secretion system also known as "needle" delivers effectors directly into host cells. These act as virulence factors influencing cell host activities [20]. In the Xcc genome, 24 effectors have been identified [21]. One of the main effectors delivered by the T3SS in *Xcc* belongs to the AvrBs3/PthA family. Xcc contains four PthA genes that encode transcription activator-like effector (TALE); of these four genes, pthA4 is responsible for the formation of citrus canker lesions. In citrus host the gene known as CsLOB is targeted by the TALE encoded by the Xcc gene pthA4; this gene was assessed in two susceptible host to Xcc infection, i.e., grape fruit and sweet orange [22]. CsLOB1-specific function still remains unclear;

some previous studies suggest that CsLOB1 is involved in the regulation of development of lateral organ and metabolism of nitrogen and anthocyanin. Some plant hormones such as auxin, gibberellin, and cytokines also have proven to exert

tus. Nowadays, the structural disposition is well established:

(iv).An extracellular pili formed by VirB2 and VirB5.

(v).VirB1 which is a periplasmic transglycosylase [26].

T4SS secretion system is an important virulence factor in a wide range of bacterial pathogens. This secretion system involves the secretion of protein or DNA into the host cells [24]. Xcc harbors two gene arrays encoding for T4SS components [25]; one of them has chromosomal location, and the other one is located at the plasmid pXAC64. Proteins VirB1–VirB11 and VirD4 make up the T4SS translocator appara-

(i).Three ATPases (VirB4, VirB11, and VirD4) located at the cytoplasm. These enzymes have been involved in the process of providing the necessary energy

(ii).Fourteen repetitions of VirB7-VirB9-virB10 trimer. These repetitions form the periplasmic core. It is noteworthy that VirB10 is anchored on both inner and outer membranes; on the other hand, VirB7 is a lipoprotein located at

(iii).An inner membrane complex formed by VirB3, VirB6, and VirB8.

*DOI: http://dx.doi.org/10.5772/intechopen.88000*

*3.2.2 Protein secretion systems and their effectors*

multiply in a susceptible host.

an effect on CsLOB1 gene [23].

for the secretion process.

the outer membrane.

nature of these structures is mainly polysaccharidic, e.g., lipopolysaccharides and exopolysaccharides. However, some of these structures share a proteinaceous nature (type IV pili, chaperone/usher pili, two-partner secretion) [17].

#### *3.2.2 Protein secretion systems and their effectors*

*Plant Diseases-Current Threats and Management Trends*

*Xanthomonas citri* subsp*. citri* [13].

**3.** *Xanthomonas citri* **subsp.** *citri*

**3.1 Isolation and identification**

begin to appear in microbiological medium.

before [12].

trees is one of the major prophylactic measures against citrus canker in commercial citrus crops. Once a symptomatic tree is identified, it is uprooted, stacked, and burned, as prophylactic measure surrounding trees is destroyed as mentioned

Prevention of primary infection in the new sprouts perhaps is the major effective approach to reduce citrus canker spread. The eradication methodology comprises conducting periodic surveys of the orchard, identifying and eliminating the outbreaks of the disease before its proliferation. Brazilian regulation stipulates that any field that has a number of diseased trees greater than 0.5% of the total orchard must be eliminated. After eradication, the contaminated field should be sprayed with copper fungicide based on 1.5 kg of metallic copper per 1 mL of water (0.15% of metallic copper). The contaminated plantations are prohibited and are forbidden

from marketing the production until eradication works are completed.

The use of bactericidal products based on copper by spray application is a practice widely used for more than two decades for the bacterial citrus canker control. The prolonged exposure of bacterial strains to copper has led to the rise of resistant strains in endemic areas. Behlau et al. reported that the genes *copAB* and *cohAB* may encode copper-binding proteins responsible for the copper resistance in

The genus *Xanthomonas* includes a vast group of phytopathogenic bacteria belonging to the group of γ proteobacteria. *Xanthomonas* infects 124 species of monocotyledonous and 268 dicotyledonous plants [14]. *Xanthomonas* are Gramnegative bacillus endowed with a sole polar flagellum. After 24-hour incubation at 29°C, yellow and shiny colonies appear in a culture media. Xanthomonadin is an unique pigment, and it is responsible for the yellow color of bacterial colonies; the biological role is explained in detail below. The exopolysaccharide known as xanthan gum gives the shiny appearance to colonies [15]. Although the genus itself has a very broad host range, individual members are often specialized to cause disease

in a limited number of taxonomically related hosts as mentioned above.

**3.2 Determinants of virulence in** *Xanthomonas citri* **subsp.** *citri*

The bacterium *Xcc* can be isolated from symptomatic plants and its diverse infected tissues. Xcc grows easily in regular microbiological culture media. In order to isolate Xcc, infected tissues must be excised and washed, and subsequently the surface must be sterilized for 3 minutes in a 10% NaClO solution. The water-soaked tissue at the lesion margin is streaked across agar medium containing 50 ppm kasugamycin. *X. citri* strains grow easily on regular nutrient agar media containing 0.5% tryptone, 0.3% yeast extract, 0.09% CaCl2, 0.05% K2HPO4, and 1.5% agar in tap water, pH 7.2 [16]. After 48 hours of incubation at 29°C, mucoid yellow colonies

An essential stage in bacterial host colonization is its attachment ability. Adhesins

are bacterial surface structures that facilitate the attachment to host tissues. The

**218**

*3.2.1 Adhesins*

Bacteria inside in *Xanthomonas* genus exhibit at least six different types of protein secretion system (i.e., T1SS to T6SS), which vary in their arrangement, function, and in a recognition of secretion substrates [18]. Like many other Gramnegative phytopathogenic bacteria, *Xcc* employs mainly secretion systems T3SS, T4SS, and T5SS and their effectors as effective tools in an attempt to invade and to multiply in a susceptible host.

Protein transport from bacterial periplasm to the extracellular environment occurs mainly by T2SS secretion system. Extracellular enzymes as lipases, proteases, and cell wall-degrading enzymes are translocated using this secretion system. Possibly the major enzymes responsible for the degradation of the plant cell wall are secreted by T2SS. T2SS translocator apparatus is composed of up to 12–15 constituents, most of which are linked to the bacterial inner membrane [19].

The T3SS secretion system also known as "needle" delivers effectors directly into host cells. These act as virulence factors influencing cell host activities [20]. In the Xcc genome, 24 effectors have been identified [21]. One of the main effectors delivered by the T3SS in *Xcc* belongs to the AvrBs3/PthA family. Xcc contains four PthA genes that encode transcription activator-like effector (TALE); of these four genes, pthA4 is responsible for the formation of citrus canker lesions. In citrus host the gene known as CsLOB is targeted by the TALE encoded by the Xcc gene pthA4; this gene was assessed in two susceptible host to Xcc infection, i.e., grape fruit and sweet orange [22]. CsLOB1-specific function still remains unclear; some previous studies suggest that CsLOB1 is involved in the regulation of development of lateral organ and metabolism of nitrogen and anthocyanin. Some plant hormones such as auxin, gibberellin, and cytokines also have proven to exert an effect on CsLOB1 gene [23].

T4SS secretion system is an important virulence factor in a wide range of bacterial pathogens. This secretion system involves the secretion of protein or DNA into the host cells [24]. Xcc harbors two gene arrays encoding for T4SS components [25]; one of them has chromosomal location, and the other one is located at the plasmid pXAC64. Proteins VirB1–VirB11 and VirD4 make up the T4SS translocator apparatus. Nowadays, the structural disposition is well established:


A recent study has shown that T4SS in Xcc displays the ability to secrete toxins; these toxins are known as VirD4-interacting proteins (XVIPs), and they are recruited by VirD4. The biological role of XVIPs is targeting and destabilizing the peptidoglycan layer in the cell wall of bacterial contenders in the ecological niche. This feature is distinctive in *Xcc*, and the protein VirD4XAC2623 endows the bacterium with an extra ability to compete in the phyllosphere [27].

#### **4. Biological control of Xcc approaches**

#### **4.1 Biological control based on DSF quorum quencher pathway**

A wide majority of bacterial genera have developed a cell-to-cell communication system known as quorum sensing (QS). This communication system is based on a signal translation mechanism whose objective is to coordinate the expression of genes at the population level in order to respond and fit to environmental changes. The cell-to-cell communication system is based on the production, secretion, and perception of small molecules known as autoinducers. A basal quantity of autoinducers are produced by every single cell, subsequently, which is secreted to extracellular milieu reflecting the bacterial population density. At high population density, the autoinducers reach a critical concentration and enable to cognate receptor to sense them. Consequently, this biological event results in triggering a cascade of diverse cell functions [28]. In the *Xanthomonas* genus, the bacteria display a quorum sensing system in which the autoinducer molecule is a short acid fat called diffusible signal factor (DSF). The DSF autoinducer family is cis-2-unsaturated fatty acids. In Xcc this DSF was characterized as cis-11-methyl-2-dodecenoic acid. The gene cluster that encodes element of quorum sensing system in *Xanthomonas* genus is the *rpf* cluster [29].

Since quorum sensing helps to coordinate community-based bacterial behavior, it is not essential for bacterial survival; therefore, the inhibition of QS interrupts only the desired phenotype, i.e., virulence, biofilm formation, and bacterial resistance to different antibiotics. Interference with QS can provide a route for disease control. This interference may involve signal degradation (quorum quenching) or excess signal production (pathogen confusion) [30]. Quorum quenching is a mechanism adopted by a number of bacteria to break the QS signaling of competitors, giving these organisms an advantage within a particular habitat [31]. It is rational that microorganisms can develop mechanisms to disarm the QS systems of competing organisms in order to increase their competitive strength in an ecosystem [32].

We have conducted a recent study that allows the isolation and identification of bacteria isolated from citrus leaves belonging to plant of field crops with and without citrus canker symptoms. From a total of 114 isolates recovered, 7 bacteria able to disrupt DSF quorum sensing pathway in *Xac* (quorum quencher bacteria) were identified. These bacteria were identified by API kits (bioMérieux's API®) and sequencing of PCR-mediated amplification products of the 16S rRNA genes as *Bacillus amyloliquefaciens*, *Bacillus vallismortis*, *Pseudomonas oryzihabitans, Pseudomonas aeruginosa, Raoultella planticola*, *Kosakonia cowanii*, and *Citrobacter freundii* [33].

Virulence assays were conducted under controlled growth conditions, and canker lesions were quantified at 21 days post inoculation. These assays demonstrated that, when citrus leaves were inoculated with mixtures of Xcc and quorum quencher bacteria, the number of cancer lesions decreased significantly reducing the severity disease (**Figure 2**).

**221**

**Figure 2.**

*Biological Control of Citrus Canker: New Approach for Disease Control*

*Virulence assay. Leaves infected by spray method at the same concentration 1 × 106*

*type. Right, Xcc plus Pseudomonas oryzihabitans. Picture was taken after 21 days of infection.*

Quorum quencher bacteria impaired the attachment and biofilm formation of Xcc to leave the surface. These are essential steps in the maintenance, survival, and initial establishment of tissue pathogenicity in citrus canker. In fact, it is completely accepted that QS plays an important, if not an essential, role in the formation of bacterial biofilm [34]. Studies of scanning electron microscopy SEM confirmed the substantial reduction in the adherence ability of Xcc after 10 hours when it was coinfected with quorum quencher bacteria relative to the control used, i.e., the leaves infected with Xcc alone. After 7 days post-infection with Xcc and the inhibitory bacteria of DSF, SEM has shown the absence of biofilm formation on the surface of leaves co-inoculated with *P. oryzihabitans* and *B. amyloliquefaciens*, relative to the

 *UFC/mL. Left, Xcc wild* 

A possible mechanism for explaining the modification or degradation of DSF molecule produced by Xcc could be the quorum quencher bacteria using the DSF molecule as a possible substrate for the UDP-sugar transferase enzyme. The addition of one unit of sugar (from UDP-sugars, i.e., UDP-glucose or UDP-galactose to the short chain of fatty acid impossible the recognition of this version of modified DSF molecule by sensor RpfC. These UDP-sugar pools are produced by the activity of carbamoyl phosphate synthetase enzyme, which is encoded by *carA* and *carB* genes. The nucleotide sequence of the *carAB* locus in the DSF inhibitory bacteria *Pseudomonas oryzihabitans* and *Bacillus amyloliquefaciens* has a strong similarity to the sequences of *carAB* genes present in the *Pseudomonas* G strain isolated and identified as efficient quorum quencher bacteria in *Xanthomonas campestris* [35].

**4.2 Biological control based on antibacterial activity of** *Pseudomonas* **strains**

*Pseudomonas* species show traits that allow them to act as effective biological control agents (BCAs) against several phytopathogens. Among these traits the most common shared by a broad range of *Pseudomonas* strains are (a) pronounced colonizing ability of plant surfaces, internal plant tissues, and phytopathogen structures [36]; (b) the ability for production of numerous kinds of antibiotic providing additional advantage in antagonism with local microbiota and phytopathogens [37]; and (c) the ability to trigger resistance responses in host plants [38]. Thus, mechanisms of direct antagonism as antibiosis or indirect mechanisms such

*DOI: http://dx.doi.org/10.5772/intechopen.88000*

control used, i.e., the infected leaves just with *Xcc*.

#### **Figure 2.**

*Plant Diseases-Current Threats and Management Trends*

**4. Biological control of Xcc approaches**

rium with an extra ability to compete in the phyllosphere [27].

**4.1 Biological control based on DSF quorum quencher pathway**

A recent study has shown that T4SS in Xcc displays the ability to secrete toxins; these toxins are known as VirD4-interacting proteins (XVIPs), and they are recruited by VirD4. The biological role of XVIPs is targeting and destabilizing the peptidoglycan layer in the cell wall of bacterial contenders in the ecological niche. This feature is distinctive in *Xcc*, and the protein VirD4XAC2623 endows the bacte-

A wide majority of bacterial genera have developed a cell-to-cell communication system known as quorum sensing (QS). This communication system is based on a signal translation mechanism whose objective is to coordinate the expression of genes at the population level in order to respond and fit to environmental changes. The cell-to-cell communication system is based on the production, secretion, and perception of small molecules known as autoinducers. A basal quantity of autoinducers are produced by every single cell, subsequently, which is secreted to extracellular milieu reflecting the bacterial population density. At high population density, the autoinducers reach a critical concentration and enable to cognate receptor to sense them. Consequently, this biological event results in triggering a cascade of diverse cell functions [28]. In the *Xanthomonas* genus, the bacteria display a quorum sensing system in which the autoinducer molecule is a short acid fat called diffusible signal factor (DSF). The DSF autoinducer family is cis-2-unsaturated fatty acids. In Xcc this DSF was characterized as cis-11-methyl-2-dodecenoic acid. The gene cluster that encodes element of quorum sensing system in *Xanthomonas* genus is the

Since quorum sensing helps to coordinate community-based bacterial behavior, it is not essential for bacterial survival; therefore, the inhibition of QS interrupts only the desired phenotype, i.e., virulence, biofilm formation, and bacterial resistance to different antibiotics. Interference with QS can provide a route for disease control. This interference may involve signal degradation (quorum quenching) or excess signal production (pathogen confusion) [30]. Quorum quenching is a mechanism adopted by a number of bacteria to break the QS signaling of competitors, giving these organisms an advantage within a particular habitat [31]. It is rational that microorganisms can develop mechanisms to disarm the QS systems of competing organisms in order to increase their competitive

We have conducted a recent study that allows the isolation and identification of bacteria isolated from citrus leaves belonging to plant of field crops with and without citrus canker symptoms. From a total of 114 isolates recovered, 7 bacteria able to disrupt DSF quorum sensing pathway in *Xac* (quorum quencher bacteria) were identified. These bacteria were identified by API kits (bioMérieux's API®) and sequencing of PCR-mediated amplification products of the 16S rRNA genes as *Bacillus amyloliquefaciens*, *Bacillus vallismortis*, *Pseudomonas oryzihabitans, Pseudomonas aeruginosa, Raoultella planticola*, *Kosakonia cowanii*,

Virulence assays were conducted under controlled growth conditions, and canker lesions were quantified at 21 days post inoculation. These assays demonstrated that, when citrus leaves were inoculated with mixtures of Xcc and quorum quencher bacteria, the number of cancer lesions decreased significantly reducing

**220**

*rpf* cluster [29].

strength in an ecosystem [32].

and *Citrobacter freundii* [33].

the severity disease (**Figure 2**).

*Virulence assay. Leaves infected by spray method at the same concentration 1 × 106 UFC/mL. Left, Xcc wild type. Right, Xcc plus Pseudomonas oryzihabitans. Picture was taken after 21 days of infection.*

Quorum quencher bacteria impaired the attachment and biofilm formation of Xcc to leave the surface. These are essential steps in the maintenance, survival, and initial establishment of tissue pathogenicity in citrus canker. In fact, it is completely accepted that QS plays an important, if not an essential, role in the formation of bacterial biofilm [34]. Studies of scanning electron microscopy SEM confirmed the substantial reduction in the adherence ability of Xcc after 10 hours when it was coinfected with quorum quencher bacteria relative to the control used, i.e., the leaves infected with Xcc alone. After 7 days post-infection with Xcc and the inhibitory bacteria of DSF, SEM has shown the absence of biofilm formation on the surface of leaves co-inoculated with *P. oryzihabitans* and *B. amyloliquefaciens*, relative to the control used, i.e., the infected leaves just with *Xcc*.

A possible mechanism for explaining the modification or degradation of DSF molecule produced by Xcc could be the quorum quencher bacteria using the DSF molecule as a possible substrate for the UDP-sugar transferase enzyme. The addition of one unit of sugar (from UDP-sugars, i.e., UDP-glucose or UDP-galactose to the short chain of fatty acid impossible the recognition of this version of modified DSF molecule by sensor RpfC. These UDP-sugar pools are produced by the activity of carbamoyl phosphate synthetase enzyme, which is encoded by *carA* and *carB* genes. The nucleotide sequence of the *carAB* locus in the DSF inhibitory bacteria *Pseudomonas oryzihabitans* and *Bacillus amyloliquefaciens* has a strong similarity to the sequences of *carAB* genes present in the *Pseudomonas* G strain isolated and identified as efficient quorum quencher bacteria in *Xanthomonas campestris* [35].

#### **4.2 Biological control based on antibacterial activity of** *Pseudomonas* **strains**

*Pseudomonas* species show traits that allow them to act as effective biological control agents (BCAs) against several phytopathogens. Among these traits the most common shared by a broad range of *Pseudomonas* strains are (a) pronounced colonizing ability of plant surfaces, internal plant tissues, and phytopathogen structures [36]; (b) the ability for production of numerous kinds of antibiotic providing additional advantage in antagonism with local microbiota and phytopathogens [37]; and (c) the ability to trigger resistance responses in host plants [38]. Thus, mechanisms of direct antagonism as antibiosis or indirect mechanisms such as competition for nutrients (e.g., siderophore production), besides induction of systemic resistance responses, actively participate in phytopathogenic disease suppression by the pseudomonads [39]. The *Pseudomonas* strains most usually recognized for their biocontrol activity against both eukaryotic and prokaryotic phytopathogenic microorganisms are *P. fluorescens*, *P. protegens*, *P. chlororaphis*, and *P. putida* [40].

In recent study (in press), we have isolated and identified from soil samples added with a compost five *Pseudomonas* strains which displayed a strong activity against Xcc. Virulence assays in very susceptible citrus host using these strains result in a deep decrease of canker lesions, which suggest a great reduction in citrus canker severity. This effect could be attributed to the great production of secondary metabolites by the *Pseudomonas* strains isolated.

#### **5. Conclusions**

Quorum sensing is an important target for prophylactic and therapeutic interventions. Identification of new bacteria species as ABC could be a new alternative for the treatment of copper traditionally used for the treatment of citrus canker disease, thus reducing selection pressure for copper resistance. We believe that the search for microorganisms that act as inhibitors of quorum sensing in phytopathogenic bacteria also as antagonist agent could be an effective strategy in a broader context. Since the organisms characterized here were originally isolated from the citrus phylloplane, the present study also contributes to an understanding of the potential interactions of bacteria on leaf surfaces**.**

#### **Acknowledgements**

The authors thank Professor Jesus A Ferro from the Technology Department, Faculdade de Ciencias Agrarias e Veterinarias, Universidade estadual Paulista, UNESP, Jaboticabal, SP, Brasil, and to CREBIO Centro de Recursos Biologicos e Biologia Genomica Univ. Estadual Paulista, Jaboticabal SP, Brazil, for the DNA sequencing.

**223**

**Author details**

Sonia Villamizar1

(UNESP), Jaboticabal, Brazil

and Juan Carlos Caicedo2

Universidad de Santander, Bucaramanga, Colombia

provided the original work is properly cited.

\*Address all correspondence to: caicedocepeda@gmail.com

\*

1 School of Agricultural and Veterinarian Sciences, São Paulo State University

2 Faculty of Exact, Natural and Agricultural Sciences, Research Group CIBAS,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Biological Control of Citrus Canker: New Approach for Disease Control*

*DOI: http://dx.doi.org/10.5772/intechopen.88000*

#### **Conflict of interest**

The authors declare no conflict of interest.

*Biological Control of Citrus Canker: New Approach for Disease Control DOI: http://dx.doi.org/10.5772/intechopen.88000*

#### **Author details**

*Plant Diseases-Current Threats and Management Trends*

metabolites by the *Pseudomonas* strains isolated.

potential interactions of bacteria on leaf surfaces**.**

The authors declare no conflict of interest.

and *P. putida* [40].

**5. Conclusions**

**Acknowledgements**

**Conflict of interest**

sequencing.

as competition for nutrients (e.g., siderophore production), besides induction of systemic resistance responses, actively participate in phytopathogenic disease suppression by the pseudomonads [39]. The *Pseudomonas* strains most usually recognized for their biocontrol activity against both eukaryotic and prokaryotic phytopathogenic microorganisms are *P. fluorescens*, *P. protegens*, *P. chlororaphis*,

In recent study (in press), we have isolated and identified from soil samples added with a compost five *Pseudomonas* strains which displayed a strong activity against Xcc. Virulence assays in very susceptible citrus host using these strains result in a deep decrease of canker lesions, which suggest a great reduction in citrus canker severity. This effect could be attributed to the great production of secondary

Quorum sensing is an important target for prophylactic and therapeutic interventions. Identification of new bacteria species as ABC could be a new alternative for the treatment of copper traditionally used for the treatment of citrus canker disease, thus reducing selection pressure for copper resistance. We believe that the search for microorganisms that act as inhibitors of quorum sensing in phytopathogenic bacteria also as antagonist agent could be an effective strategy in a broader context. Since the organisms characterized here were originally isolated from the citrus phylloplane, the present study also contributes to an understanding of the

The authors thank Professor Jesus A Ferro from the Technology Department, Faculdade de Ciencias Agrarias e Veterinarias, Universidade estadual Paulista, UNESP, Jaboticabal, SP, Brasil, and to CREBIO Centro de Recursos Biologicos e Biologia Genomica Univ. Estadual Paulista, Jaboticabal SP, Brazil, for the DNA

**222**

Sonia Villamizar1 and Juan Carlos Caicedo2 \*

1 School of Agricultural and Veterinarian Sciences, São Paulo State University (UNESP), Jaboticabal, Brazil

2 Faculty of Exact, Natural and Agricultural Sciences, Research Group CIBAS, Universidad de Santander, Bucaramanga, Colombia

\*Address all correspondence to: caicedocepeda@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[15] Swings J, Vauterin L, Kersters K. The bacterium *Xanthomonas*. In: Swings JG, Civerolo EL, editors. Xanthomonas. 1st ed. Dordrecht: Springer; 1993. pp. 121-146

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[17] Soto GE, Hultgren SJ. Bacterial adhesins: Common themes and variations in architecture and assembly. Journal of Bacteriology. 1999;**181**:1059-1071

[18] Gerlach RG, Hensel M. Protein secretion systems and adhesins: The molecular armory of Gramnegative pathogens. International Journal of Medical Microbiology. 2007;**297**:401-415

[19] Sandkvist M. Biology of type II secretion. Molecular Microbiology. 2001;**40**:271-283

[20] Büttner D, Bonas U. Regulation and secretion of *Xanthomonas* virulence factors. FEMS Microbiology Reviews. 2010;**34**:107-133

[21] Moreira LM, de Souza RF, Almeida NF Jr, Setubal JC, Oliveira JC, Furlan LR, et al. Comparative genomics analyses of citrus-associated bacteria. Annual Review of Phytopathology. 2004;**42**:163-184

[22] Yang H, Junli Z, Hongge J, Davide S, Ting L, Wolf BF, et al. Lateral organ boundaries 1 is a disease susceptibility gene for citrus bacterial canker disease. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**:E521-E529

[23] Majer C, Hochholdinger F. Defining the boundaries: Structure and function of LOB domain proteins. Trends in

Plant Science. 2011;**16**(1):47-52. DOI: 10.1016/j.tplants.2010.09.009

[24] Cascales E, Christie PJ. The versatile bacterial type IV secretion systems. Nature Reviews. Microbiology. 2003;**1**:137-150

[25] da Silva AC, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB, et al. Comparison of the genomes of two *Xanthomonas* pathogens with differing host specificities. Nature. 2002;**417**:459-463

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[32] Frederix M, Downey AJ. Quorum sensing: Regulating the regulators.

**224**

*Plant Diseases-Current Threats and Management Trends*

pv. *alfalfae* (ex Riker and Jones, 1935) dye 1978 as *X. alfalfae* subsp. *alfalfae* (ex Riker et al., 1935) sp. nov. nom. rev.; and "var. fuscans" of *X. campestris* pv. *phaseoli* (ex Smith, 1987) dye 1978 as *X. fuscans* subsp. *fuscans* sp. nov. Systematic and Applied Microbiology.

[9] Moreira LM, Almeida NF Jr., Potnis N, Digiampietri LA, Adi SS, Bortolossi JC, et al. Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of *Xanthomonas fuscans* subsp. *aurantifolii*.

[10] Namekata T, Balmer E, University of São Paulo. Estudos comparativos entre *Xanthomonas citri* (Hasse) Dow., agente causal do cancro cítrico e *Xanthomonas citri* (Hasse) Dow., n.f.sp. aurantifolia, agente causal da cancrose do limoeiro

[11] Malavolta Júnior VA, Yamashiro T, Nogueira EMC, Feichtenberger E. Distribuição do tipo C de *Xanthomonas campestris* pv. citri no Estado de São Paulo. Summa Phytopathologica. 1984;**10**:11

[12] Sharma SK, Sharma RR. Citrus canker approaching century: A review. Tree and Forestry Science and Biotechnology. 2009;**2**(Special

[13] Behlau F, Canteros BI, Minsavage GV, Jones JB, Graham JH. Molecular characterization of copper resistance genes from *Xanthomonas citri* subsp. *citri* and *Xanthomonas alfalfae* subsp. *citrumelonis*. Applied and Environmental Microbiology.

[14] Chan JWYF, Goodwin PH. The molecular genetics of virulence of *Xanthomonas campestris*. Biotechnology

Advances. 1999;**17**:489-508

BMC Genomics. 2010;**11**:238

Galego. Piracicaba; 1971

Issue):54-56

2011;**77**:4089-4096

2005;**28**:494-518

[1] Ortiz-Castro R, Contreras-Cornejo HA, Macías-Rodríguez L, López-Bucio J. The role of microbial signals in plant growth and development. Plant

Signaling and Behavior. 2009;**4**:701-712

[3] Fawcett HS, Jenkins AE. Records of citrus canker from herbarium specimens of the genus Citrus in England and the United States. Phytopathology.

[4] Das AK. Pathogenic variability in *Xanthomonas axonopodis* pv. citri, causal agent of citrus canker. Journal of Mycology and Plant Pathology.

[5] Dopson RN. The eradication of citrus canker. The Plant Disease Reporter.

[6] Civerolo EL. Bacterial canker disease of citrus. Journal of the Rio Grande Valley Horticultural Society.

[7] Stall RE, Seymour CP. Canker: A threat to citrus in the Gulf Coast states.

[8] Schaad NW, Postnikova E, Lacy GH, Sechler A, Agarkova I, Stromberg PE, et al. Reclassification of *Xanthomonas campestris* pv. citri (ex Hasse 1915) dye 1978 forms A, B/C/D, and E as *X. smithii* subsp. *citri* (ex Hasse) sp. nov. nom. rev. comb. nov., *X. fuscans* subsp. *aurantifolii* (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and *X. alfalfae* subsp. *citrumelo* (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; *X. campestris* pv. *malvacearum* (ex smith 1901) dye 1978 as *X. smithii* subsp. *smithii* nov. comb. nov. nom. nov.; *X. campestris*

Plant Disease. 1983;**67**:581-585

[2] Lee HA. Further data on the susceptibility of rutaceous plants to citrus-canker. Journal of Agricultural

Research. 1918;**15**:661-665

1933;**23**:820-824

2002;**54**:274-279

1964;**48**:30-31

1984;**37**:127-145

**References**

Advances in Microbial Physiology. 2011;**58**:23-80

[33] Caicedo JC, Villamizar S, Ferro MIT, Kupper KC, Ferro JA. Bacteria from the citrus phylloplane can disrupt cell–cell signalling in *Xanthomonas citri* and reduce citrus canker disease severity. Plant Pathology. 2016;**65**:782-791

[34] Steven B, shild Vik A, Friedman L, Kolter R. Biofilms: The matrix revisited. Trends in Microbiology. 2005;**13**(1):20-26

[35] Newman KL, Chatterjee S, Ho KA, Lindow SE. Virulence of plant pathogenic bacteria attenuated by degradation of fatty acid cell-tocell signaling factors. Molecular Plant-Microbe Interaction. 2008;**21**(3):326-334

[36] Mercado-Blanco J, Bakker PA. Interactions between plants and beneficial *Pseudomonas* spp.: Exploiting bacterial traits for crop protection. Antonie Van Leeuwenhoek. 2007;**92**:367- 389. DOI: 10.1007/s10482-007-9167-1

[37] Gross H, Loper JE. Genomics of secondary metabolite production by *Pseudomonas* spp. Natural Product Reports. 2009;**26**:1408-1446

[38] Djavaheri M, Mercado-Blanco J, Versluis C. Iron-regulated metabolites produced by *Pseudomonas fluorescens* WCS374r are not required for eliciting induced systemic resistance (ISR) against *Pseudomonas syringae* pv. tomato in Arabidopsis. Microbiology Open. 2012;**1**:311-325

[39] Zabiha HR, Savaghebi GR, Khavazi K. Pseudomonas bacteria and phosphorus fertilization, affecting wheat (*Triticum aestivum L*.) yield and P uptake under green house and field conditions. Acta Physiologiae Plantarum. 2011;**33**:145-152

[40] Ramette A, Frapolli M, Fischer-Le Saux M, Gruffaz C, Meyer JM, Défago G, et al. *Pseudomonas* protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin. Systematic and Applied Microbiology. 2011;**34**:180-188

*Plant Diseases-Current Threats and Management Trends*

et al. *Pseudomonas* protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin. Systematic and Applied Microbiology. 2011;**34**:180-188

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[33] Caicedo JC, Villamizar S, Ferro MIT, Kupper KC, Ferro JA. Bacteria from the citrus phylloplane can disrupt cell–cell signalling in *Xanthomonas citri* and reduce citrus canker disease severity. Plant Pathology. 2016;**65**:782-791

[34] Steven B, shild Vik A, Friedman L, Kolter R. Biofilms: The matrix revisited. Trends in Microbiology.

[35] Newman KL, Chatterjee S, Ho KA, Lindow SE. Virulence of plant pathogenic bacteria attenuated by degradation of fatty acid cell-tocell signaling factors. Molecular Plant-Microbe Interaction.

[36] Mercado-Blanco J, Bakker PA. Interactions between plants and beneficial *Pseudomonas* spp.: Exploiting bacterial traits for crop protection. Antonie Van Leeuwenhoek. 2007;**92**:367- 389. DOI: 10.1007/s10482-007-9167-1

[37] Gross H, Loper JE. Genomics of secondary metabolite production by *Pseudomonas* spp. Natural Product Reports. 2009;**26**:1408-1446

[38] Djavaheri M, Mercado-Blanco J, Versluis C. Iron-regulated metabolites produced by *Pseudomonas fluorescens* WCS374r are not required for eliciting induced systemic resistance (ISR) against *Pseudomonas syringae* pv. tomato in Arabidopsis. Microbiology Open.

[39] Zabiha HR, Savaghebi GR, Khavazi K. Pseudomonas bacteria and phosphorus fertilization, affecting wheat (*Triticum aestivum L*.) yield and P uptake under green house and field conditions. Acta Physiologiae Plantarum. 2011;**33**:145-152

[40] Ramette A, Frapolli M, Fischer-Le Saux M, Gruffaz C, Meyer JM, Défago G,

2011;**58**:23-80

2005;**13**(1):20-26

2008;**21**(3):326-334

**226**

2012;**1**:311-325

### *Edited by Snježana Topolovec-Pintarić*

Plant pathogens, the causal agent of infectious plant diseases, influence our lives more than just as an economic impact through yield lost. The study of plant pathogens has given rise to the development of new sciences, new technologies for plant breeding, and the agrochemical industry for pesticide developments. Yet, all our actions and efforts to suppress or eradicate them constantly pressures these various organisms to evolve and adapt for survival. Therefore today, when facing climate changes, accelerated transport of plants and plant products, and world population growth, we have to ask quo vadis phytopathology. Like Alice in Wonderland "If we wish to go anywhere we must run twice as fast as that" so we need to constantly broaden our knowledge. However, today's literature abounds with knowledge about plant pathogens. Hence, this book intends to present to the reader all the latest material and knowledge about plant pathogens, changes or refinements in plant disease epidemiology, and new approaches and materials used for plant pathogen control. Hopefully, this book will be of interest to those working within the field and looking for an up-to-date introduction. We hope it also interests students and thereby, will influence the future development of phytopathology and our better coexistence with plant pathogens.

Published in London, UK © 2020 IntechOpen © Lunasix / iStock

Plant Diseases-Current Threats and Management Trends

Plant Diseases

Current Threats and Management Trends

*Edited by Snježana Topolovec-Pintarić*