**Meet the editor**

Dr. N. Thajuddin is working as an Associate Professor & Head of the Department of Microbiology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, India. He has more than 20 years of teaching and research experience in the field of Microbiology. His work on survey of marine cyanobacteria from India resulted in the establishment of marine cyanobacterial

germplasm of 350 strains at National Facility for Marine Cyanobacteria, from which three technologies (blue colourants, β lactamase enzyme and aqua feed) were developed. He had a one-year postdoctoral training on molecular taxonomy and phylogeny of cyanobacteria at Department of Biology, Rensselaer Polytechnic Institute, Troy, New York, USA through the Department of Biotechnology Overseas Fellowship. He has published 120 articles in microbiology and related fields and has received project grants from government agencies such as DBT, MoES, and UGC. He is a life member of various academic bodies and member of editorial boards in several journals. Dr. Thajuddin has successfully guided 17 Ph.D and 22 M.Phil. scholars in diverse field of Microbiology and organized a national level symposia, workshops, refresher courses and training programmes related to Microbiology and Biotechnology. He has deposited around 250 nucleotide sequences in the GenBank (NCBI) and has visited USA, Hong Kong, Malaysia and Singapore to disseminate his expertise and to keep himself abreast of the advanced techniques in the field of Microbiology.

### Contents

#### **Preface XI**



### Preface

Fungicides are a class of pesticides used for killing or inhibiting the growth of fungus. They are extensively used in pharmaceutical industry, agriculture, in protection of seed during storage and in preventing the growth of fungi that produce toxins. Hence, fungicides production is constantly increasing as a result of their great importance to agriculture. Some fungicides affect humans and beneficial microorganisms including insects, birds and fish thus public concern about their effects is increasing day by day. In order to enrich the knowledge on beneficial and adverse effects of fungicides this book encompasses various aspects of the fungicides including fungicide resistance, mode of action, management fungal pathogens and defense mechanisms, ill effects of fungicides interfering the endocrine system, combined application of various fungicides and the need of GRAS (generally recognized as safe) fungicides. This volume will be useful source of information on fungicides for post graduate students, researchers, agriculturists, environmentalists and decision makers.

 This volume includes 13 chapters. The first chapter elaborates the problems associated with fungicide application, disease epidemiology, decision-making process, fungicide's physical mode of action, resistance and its management with two case studies on strawberry and grape. The second chapter describes the problem of *Botrytis* resistance, monitoring methods, anti-resistant strategies, *Botrytis cinerea* MDR types, their mechanisms of resistance etc. in detail. The third chapter presents overall information on the multiple fungicide resistant *Botrytis* affecting German soft fruits such as raspberry and strawberry fruits. This article gave sufficient background information on fungicide resistance in *Botrytis cinerea*, resistance assay developed, reproducibility of the assay, temporal and special distribution of fungicide resistance, the major issues of multiple fungicide resistance, factors conducive for the spread of *B. cinerea,* fungicide application*,* etc*.* It will be useful not only to the researchers but also to the regional soft fruit growers. Fourth chapter is a comprehensive account of reviewing the Fenhexamid resistance in the *Botrytis* species responsible for grey mould disease. The authors have included their research outputs and incorporated recent publications in this chapter which helps to understand basic and applied aspects of this research field.

The effects of fungicide application on the FHB disease complex with a combined approach of *in vitro* and *in vivo* field trials are discussed in the chapter 5. Sixth chapter explaining the utilization of beneficial bacteria such as *Pseudomonas fluorescens*, *Bacillus subtilis* and *Bacillus megaterium* against pathogenic fungi *Rhizoctonia solani* and *Pythium debarianum.* Seventh chapter presents sufficient background information on origin, distribution, fungal pathogens and diseases of apple, fungal toxins, defence mechanisms, alternative measures to control post harvest blue mould of apple etc. and it will be useful to the researchers as well as people involved in cultivation of apple. The author of the eighth chapter has sufficiently discussed the effect of old and new fungicides on various fruits, combined application of various fungicides and thermotherapy on various horticultural commodities, and the need of GRAS (generally recognized as safe) fungicides as an alternative to traditional fungicides which are ineffective against resistant strains. Ninth chapter describes the role of Mitogen Activated Protein Kinase (MAPK) signaling in secondary metabolism and adaptation to abiotic/fungicide stress in *Fusarium* using ∆*Fvhog1* and ∆*Fvmk2* CWI MAPK mutants of *F. verticillioides* by comparing their sensitivity to different oxidative stressors. The authors of this chapter elaborate the role of HOG1 MAPK signaling in stress and fungicide tolerance, the role of MAPK pathways in secondary metabolism of *Fusarium* species, complexity of oxidative stress signaling in fungi and sensitivity of different *Fusarium* species to hydrogen peroxide. Tenth chapter presents the Endocrine Disruptor Compounds (EDCs) such as tributyltin (androgen) and vinclozolin (anti-androgen) and their effects on vertebrate and invertebrate taxa including non target organisms. The ill effects of these fungicides interfering the endocrine system in the synthesis, secretion, transport, action or elimination of natural hormones, including in the induction of cell tumors, reduction of ejaculated sperm numbers and prostate weight and delayed puberty are clearly described in this article.

As many pathogens develop resistance under field conditions due to frequent application of various fungicides, the eleventh chapter paper presents detailed information on the laboratory mutant of *Bipolarize oryzae* resistant to Carbendazim and follows intensive studies on molecular mechanisms of the fungicide resistance to benzimidazole compound. Twelfth chapter describes the epidemiological context of *Mycosphaerella graminicola,* the effect of other factors such as external contamination by ascospores, cultivars resistance, leaf colonization stages, fungicide efficiency using qPCR and correlation between qPCR analysis and visual symptoms. Thirteenth chapter explains the efficacy of fungicide applications, the importance of nozzle tips, pressure, nozzle spacing and angle, optimum spray particle size in getting the greatest coverage at lower levels of the soybean canopy. A list of important bibiliography is included at the end of the each chapter to assist the readers in enriching their knowledge on fungicides.

This volume will be useful source of information on fungicides for post graduate students, researchers, agriculturists, environmentalists and decision makers. I am very much thankful to the contributors for their excellent articles. I am also grateful to InTech Publisher for their concern, efforts and encouragement in the task of publishing this volume.

X Preface

this article.

knowledge on fungicides.

The effects of fungicide application on the FHB disease complex with a combined approach of *in vitro* and *in vivo* field trials are discussed in the chapter 5. Sixth chapter explaining the utilization of beneficial bacteria such as *Pseudomonas fluorescens*, *Bacillus subtilis* and *Bacillus megaterium* against pathogenic fungi *Rhizoctonia solani* and *Pythium debarianum.* Seventh chapter presents sufficient background information on origin, distribution, fungal pathogens and diseases of apple, fungal toxins, defence mechanisms, alternative measures to control post harvest blue mould of apple etc. and it will be useful to the researchers as well as people involved in cultivation of apple. The author of the eighth chapter has sufficiently discussed the effect of old and new fungicides on various fruits, combined application of various fungicides and thermotherapy on various horticultural commodities, and the need of GRAS (generally recognized as safe) fungicides as an alternative to traditional fungicides which are ineffective against resistant strains. Ninth chapter describes the role of Mitogen Activated Protein Kinase (MAPK) signaling in secondary metabolism and adaptation to abiotic/fungicide stress in *Fusarium* using ∆*Fvhog1* and ∆*Fvmk2* CWI MAPK mutants of *F. verticillioides* by comparing their sensitivity to different oxidative stressors. The authors of this chapter elaborate the role of HOG1 MAPK signaling in stress and fungicide tolerance, the role of MAPK pathways in secondary metabolism of *Fusarium* species, complexity of oxidative stress signaling in fungi and sensitivity of different *Fusarium* species to hydrogen peroxide. Tenth chapter presents the Endocrine Disruptor Compounds (EDCs) such as tributyltin (androgen) and vinclozolin (anti-androgen) and their effects on vertebrate and invertebrate taxa including non target organisms. The ill effects of these fungicides interfering the endocrine system in the synthesis, secretion, transport, action or elimination of natural hormones, including in the induction of cell tumors, reduction of ejaculated sperm numbers and prostate weight and delayed puberty are clearly described in

As many pathogens develop resistance under field conditions due to frequent application of various fungicides, the eleventh chapter paper presents detailed information on the laboratory mutant of *Bipolarize oryzae* resistant to Carbendazim and follows intensive studies on molecular mechanisms of the fungicide resistance to benzimidazole compound. Twelfth chapter describes the epidemiological context of *Mycosphaerella graminicola,* the effect of other factors such as external contamination by ascospores, cultivars resistance, leaf colonization stages, fungicide efficiency using qPCR and correlation between qPCR analysis and visual symptoms. Thirteenth chapter explains the efficacy of fungicide applications, the importance of nozzle tips, pressure, nozzle spacing and angle, optimum spray particle size in getting the greatest coverage at lower levels of the soybean canopy. A list of important bibiliography is included at the end of the each chapter to assist the readers in enriching their

**Dr. Nooruddin Thajuddin**  Associate Professor and Head, Department of Microbiology, Bharathidasan University, India

### **Optimizing Fungicide Applications for Plant Disease Management: Case Studies on Strawberry and Grape**

Angel Rebollar-Alviter1 and Mizuho Nita2

*1Centro Regional Morelia, Universidad Autonoma Chapingo, 2Virginia Polytechnic Institute and State University, Alson H. Smith Jr. Agricutural Research and Extension Center, Winchester, VA, 1Mexico 2USA* 

#### **1. Introduction**

Fungicides are important tools for management of plant diseases caused by fungal and oomycete pathogens. Without use of fungicides, major crop losses are inevitable, and food supply networks as we know today are most likely not able to sustain itself. As a result, fungicides are applied in regular basis in many parts of the world; however, their applications need to be optimized in order to obtain the best result in disease management due to multiple factors such as fungicide efficacy, the risk of resistance development, environmental concerns, pesticide residue in harvest, impact on beneficial organisms, etc. The ultimate goal is to keep the losses from diseases to a level that does not represent a threat to the crop production and to the economy of the grower while reducing the number of applications as much as possible. In order to achieve this goal, growers commonly employ integrated pest management (IPM) approaches where multiple management options are used together to achieve best efficacy with minimum chemical usage. Especially in environmentally challenging growing areas, use of fungicides is an important component of the IPM approach. Abusive uses of fungicides can cost not only growers' budget, but also cost society and environment. Therefore, fungicide usages need to be carefully planned with a good understanding of plant disease epidemics, their components (host, environment and pathogen), fungicide mode of action (biochemical, biological, physical), risk of resistance development, and host physiology, among other aspects. In this chapter, we will review these components that are involved in decision-making process to optimize fungicide application. The main focus of discussion is on management of diseases of strawberry and grape, because both are high value, intensively managed crops where application of fungicides are conducted on a regular basis.

In both strawberry and grape productions, it is not uncommon to observe an excessive number of fungicide applications, which happens sometimes as a result of the lack of knowledge of the pathogen biology and epidemiology, fungicide mode of action and fungicide residues. Or simply growers do not want take risks because of high costs and values of these crops. Moreover, the availability of several groups and mixtures of fungicides in the market is creating confusion among growers who are constantly in need of learning how to integrate a new chemistry in their plant disease management program. It is further confusing not only to growers but also to educators and researchers as well. Some of new formulations or molecules are simply a mixture of known active ingredients, or a different brand name yet the same active ingredient, or a different chemical name with the same mode of action, or a mixture of known active ingredients with a different percentage, etc.

In some agricultural settings such as the wet areas of the Midwest and Eastern US, tropical and subtropical areas of Central Mexico, the need of fungicide use is continuous during the course of the crop development; therefore it is a challenge for growers to keep their fungicide program season after season. Although it is not always considered, there are many factors that influence the decision making process of a grower to apply fungicides. If you put in a simple sense, what a grower wants is to manage a population of pathogens at the end of the day; however at the same time, he/she needs to be aware of the existence of the right tools that provide her/him an economical, effective, and sustainable (in both economic and environmental sense) solution. In addition, because of social pressure against the use of chemical in agriculture, fungicides applications for plant disease management need to be carefully selected. Since development of any plant disease is a result of a complex interaction among host, pathogen, environment, and sometimes a vector of the pathogen, the optimization of a fungicide application program should be based on the knowledge of disease dynamics, fungicide and mode of action in relation to development of epidemics (Madden 2006; Madden et al. 2007). In order to establish season-long programs to manage key diseases, growers need to learn and understand knowledge of information related to the factors that affect the efficacy of a fungicide, the biology and epidemiology of the disease, and crop physiology and the environment.

In this chapter, we explore the factors that growers, consultants, and researchers need to consider in order to establish optimized season-long programs with ecologically and economically sound approaches. We describe different components that influence the development of epidemics and their impact on crop disease management and the whole season approach to manage diseases, disease epidemics, fungicide resistant and its management, integrated pest management, and uses of disease risk assessment tools. In addition, we present two case studies managing diseases using fungicides based on information considering different tactics and strategies to reduce the number of fungicide application, and risk resistance development on grape and strawberry.

#### **2. Components of epidemics and fungicides**

Plant diseases are the result of the interaction among the host, the pathogen and the environment. Plant pathologists often describe this relationship, or model, as a plant disease triangle (Francl 2001; Agrios 2005). Each component of the disease triangle plays an important role on the development of diseases. When there is a compatible interaction between a host and a pathogen (i.e., a pathogen can cause disease on a host), the environment is the element that triggers development of a plant disease. Thus, a basic idea of plant disease management is to break the disease triangle from forming by understanding

values of these crops. Moreover, the availability of several groups and mixtures of fungicides in the market is creating confusion among growers who are constantly in need of learning how to integrate a new chemistry in their plant disease management program. It is further confusing not only to growers but also to educators and researchers as well. Some of new formulations or molecules are simply a mixture of known active ingredients, or a different brand name yet the same active ingredient, or a different chemical name with the same mode of action, or a mixture of known active ingredients with a different

In some agricultural settings such as the wet areas of the Midwest and Eastern US, tropical and subtropical areas of Central Mexico, the need of fungicide use is continuous during the course of the crop development; therefore it is a challenge for growers to keep their fungicide program season after season. Although it is not always considered, there are many factors that influence the decision making process of a grower to apply fungicides. If you put in a simple sense, what a grower wants is to manage a population of pathogens at the end of the day; however at the same time, he/she needs to be aware of the existence of the right tools that provide her/him an economical, effective, and sustainable (in both economic and environmental sense) solution. In addition, because of social pressure against the use of chemical in agriculture, fungicides applications for plant disease management need to be carefully selected. Since development of any plant disease is a result of a complex interaction among host, pathogen, environment, and sometimes a vector of the pathogen, the optimization of a fungicide application program should be based on the knowledge of disease dynamics, fungicide and mode of action in relation to development of epidemics (Madden 2006; Madden et al. 2007). In order to establish season-long programs to manage key diseases, growers need to learn and understand knowledge of information related to the factors that affect the efficacy of a fungicide, the biology and epidemiology of the disease,

In this chapter, we explore the factors that growers, consultants, and researchers need to consider in order to establish optimized season-long programs with ecologically and economically sound approaches. We describe different components that influence the development of epidemics and their impact on crop disease management and the whole season approach to manage diseases, disease epidemics, fungicide resistant and its management, integrated pest management, and uses of disease risk assessment tools. In addition, we present two case studies managing diseases using fungicides based on information considering different tactics and strategies to reduce the number of fungicide

Plant diseases are the result of the interaction among the host, the pathogen and the environment. Plant pathologists often describe this relationship, or model, as a plant disease triangle (Francl 2001; Agrios 2005). Each component of the disease triangle plays an important role on the development of diseases. When there is a compatible interaction between a host and a pathogen (i.e., a pathogen can cause disease on a host), the environment is the element that triggers development of a plant disease. Thus, a basic idea of plant disease management is to break the disease triangle from forming by understanding

application, and risk resistance development on grape and strawberry.

**2. Components of epidemics and fungicides** 

percentage, etc.

and crop physiology and the environment.

the role of each element. For instance, planting a disease resistant variety is a way to disturb the disease triangle by eliminating the host so that the triangle cannot be formed.

When we consider the change of plant diseases over time and space, we are dealing with plant disease epidemics (Madden 2006; Madden et al. 2007). Since time is another factor added to the triangle, some use a modified disease triangle, which becomes a tetrahedron (Francl 2001). Sometimes it is a challenging task for agricultural educators (such as crop specialists) to describe the concept because it deals with another dimension (time). However, it is important to inform growers that the disease you see today is a consequence of an infection that happened a certain time ago, or even a consequence of multiple infections that happened over the course of time.

Since we are dealing with the progress of disease(s) over time, we need to understand the life cycle (often referred as a disease cycle) of pathogens, which are divided into two groups, monocyclic (one disease cycle per season) and polycyclic (multiple disease cycles per season). Based on the disease cycle, management strategies can differ. For example, one of strategies of plant disease management can involve elimination or reduction of the amount of primary inoculum, which reduces the rate of infection by reducing the probability of pathogens to find healthy host tissues and/or by limiting the time the pathogen and host populations interact (Nutter 2007). In some monocyclic disease cases, only one application of fungicide might be needed. For example *Fusarium graminearum*, a causal agent of Fusarium head blight of wheat causes infection on kernels during anthesis, therefore, protection of wheat during this stage of development is the key for the management of this disease (Nita et al. 2005).

On the other hand, when a continuous release of pathogen inoculum is occurring and host tissues are susceptible over time, multiple applications might be needed. In order to deal with polycyclic diseases, often several applications are needed to delay the onset of the epidemic. In this case, the impact of fungicide applications will be on the rate of the epidemic by reducing the probability of successful infection and/or successful completion of life cycle (= production of spores) (Fry 1982). Early studies by J. E. Van der Plank (1963) introduced many of these concepts, and it was followed by many plant disease epidemiologists who utilized these concepts to develop plant disease models and management strategies such as a use of disease risk assessment (forecasting or warning) tools (Zadoks and Schein 1979; Zadoks 1984; Madden et al. 2007). Some of disease risk assessment tools aim to determine the critical time when the disease become a threat and/or have an economic impact. Disease risk assessment tools can be very useful to reduce the costs of disease control and increase safety of the produce by helping growers to use fungicides in a timely and more efficient manner (Zadoks 1984; Hardwick 2006; Madden et al. 2007).

#### **3. Fungicide resistance and plant disease management**

When we discuss about fungal disease management, discussions on the issue of fungicide resistance cannot be avoided. Development of fungicide resistance fungal isolates was documented as early as 1960's when *Penicillium* spp*.* on citrus (citrus storage rot) was found to be resistant against Aromatic hydrocarbons (Eckert 1981). The other examples from that decade are resistance to organomercurials by cereal leaf spot and strip caused by *Pyrenophora* spp., dodine resistant apple scab (*Venturia inequalis*), and QoI (**Q**uinone-**O**utside **I**nhibitor) resistance against grape powdery mildew (caused by *Erysiphe necator*) in the field in Europe, and North America in 1990's to 2000's (Staub 1991; Baudoin et al. 2008).

Fungicide resistance develops when a working mode of action loses its efficacy against target fungal pathogen. When fungicide resistance appears in the field, it is often the case that a particular fungicide (or a mode of action), has been used for a several years or seasons, and growers find that the efficacy of that fungicide has been noticeably reduced or even lost. This type of resistance is often called 'field resistance' or 'practical resistance' in contrast to the cases when the resistance isolate is found only in the laboratory conditions (= laboratory resistance) (Staub 1991). Some of laboratory resistance isolates can only survive under protected conditions because they are not adequately fit to compete and survive in the field thus, the presence of these laboratory resistant isolate may or may not be a threat to the real world. Attempts were made to predict the development of field resistance based on populations of laboratory resistance isolate; however, it has been difficult. For example, although the presence of resistant isolates of *Botrytis* against dicarboximides was found in laboratory, the development of field resistance was slower than expected (Leroux et al. 1988).

The resistant mechanisms, whether a single gene or multi-locus function, maybe present naturally among wild population in a small quantity and a repeated application of a particular mode of action select these rare populations to thrive. In some cases, the development of fungicide resistance appears to be a sudden event. This type of resistance is also called 'qualitative', 'single-step', or 'discrete' resistance (Brent and Hollomon 2007). This qualitative resistance tends to appear relatively soon after the introduction of the compromised mode of action and stay once appeared. One of examples would be benzimidazole resistance of apple scab pathogen (*Venturia inequalis*) where resistant isolates appeared only after two seasons of benzimidazole fungicide application (Shabi et al. 1983; Staub 1991). In some cases, a gradual recovery of sensitivity can happen; however, as soon as an application of the compromised mode of action resumes, the resistance tends to come back quickly as in the example of potato late blight pathogen (*Phytophthora infestans*) to phenylamide fungicides (Gisi and Cohen 1996).

In the other cases, development of fungicide resistance is gradual. This type of resistance is called 'quantitative', 'multi-step' or 'continuous' (Brent and Hollomon 2007). Examples of quantitative resistance are the cases of many fungal pathogens to the DMI (sterol **D**e**M**ethylation **I**nhbitors) where the reduction of efficacy can be observed over several years or seasons (Staub 1991). For quantitative resistance, reduced use of fungicides of the same mode of action tends to revert populations back to more sensitive state. This decline of the resistance could be due to incomplete resistance, or lack of fitness, or both (Staub 1991).

Another concern on the resistance is the phenomenon called 'cross-resistance' where resistance to one fungicide translates into resistant to other fungicides, which are affected by the same gene mutation(s). Often time it happens with fungicides that are different in chemical composition, while share the same mode of action. One example is the case of benzimidazole fungicide resistance where pathogen strains that resist benomyl were resistance to carbendazim, thiophanate-methyl, or thiabendazole (Brent and Hollomon 2007). Moreover, in some cases, a fungal strain can be resistant to two or more different mode of action or acquire 'multiple resistance'. For example, *Botrytis cinerea* a causal agent of

*Pyrenophora* spp., dodine resistant apple scab (*Venturia inequalis*), and QoI (**Q**uinone-**O**utside **I**nhibitor) resistance against grape powdery mildew (caused by *Erysiphe necator*) in the field

Fungicide resistance develops when a working mode of action loses its efficacy against target fungal pathogen. When fungicide resistance appears in the field, it is often the case that a particular fungicide (or a mode of action), has been used for a several years or seasons, and growers find that the efficacy of that fungicide has been noticeably reduced or even lost. This type of resistance is often called 'field resistance' or 'practical resistance' in contrast to the cases when the resistance isolate is found only in the laboratory conditions (= laboratory resistance) (Staub 1991). Some of laboratory resistance isolates can only survive under protected conditions because they are not adequately fit to compete and survive in the field thus, the presence of these laboratory resistant isolate may or may not be a threat to the real world. Attempts were made to predict the development of field resistance based on populations of laboratory resistance isolate; however, it has been difficult. For example, although the presence of resistant isolates of *Botrytis* against dicarboximides was found in laboratory, the

The resistant mechanisms, whether a single gene or multi-locus function, maybe present naturally among wild population in a small quantity and a repeated application of a particular mode of action select these rare populations to thrive. In some cases, the development of fungicide resistance appears to be a sudden event. This type of resistance is also called 'qualitative', 'single-step', or 'discrete' resistance (Brent and Hollomon 2007). This qualitative resistance tends to appear relatively soon after the introduction of the compromised mode of action and stay once appeared. One of examples would be benzimidazole resistance of apple scab pathogen (*Venturia inequalis*) where resistant isolates appeared only after two seasons of benzimidazole fungicide application (Shabi et al. 1983; Staub 1991). In some cases, a gradual recovery of sensitivity can happen; however, as soon as an application of the compromised mode of action resumes, the resistance tends to come back quickly as in the example of potato late blight pathogen (*Phytophthora infestans*) to

In the other cases, development of fungicide resistance is gradual. This type of resistance is called 'quantitative', 'multi-step' or 'continuous' (Brent and Hollomon 2007). Examples of quantitative resistance are the cases of many fungal pathogens to the DMI (sterol **D**e**M**ethylation **I**nhbitors) where the reduction of efficacy can be observed over several years or seasons (Staub 1991). For quantitative resistance, reduced use of fungicides of the same mode of action tends to revert populations back to more sensitive state. This decline of the resistance could be due to incomplete resistance, or lack of fitness, or both (Staub 1991). Another concern on the resistance is the phenomenon called 'cross-resistance' where resistance to one fungicide translates into resistant to other fungicides, which are affected by the same gene mutation(s). Often time it happens with fungicides that are different in chemical composition, while share the same mode of action. One example is the case of benzimidazole fungicide resistance where pathogen strains that resist benomyl were resistance to carbendazim, thiophanate-methyl, or thiabendazole (Brent and Hollomon 2007). Moreover, in some cases, a fungal strain can be resistant to two or more different mode of action or acquire 'multiple resistance'. For example, *Botrytis cinerea* a causal agent of

in Europe, and North America in 1990's to 2000's (Staub 1991; Baudoin et al. 2008).

development of field resistance was slower than expected (Leroux et al. 1988).

phenylamide fungicides (Gisi and Cohen 1996).

bunch rot of grape and many other plant diseases, is commonly resistant to both benzimidazole and dicarboximide fungicides (Elad et al. 1992).

As noted previously, repeated application of the same mode of action often increase the risk of development of fungicide resistant population. Because of that, intensively managed agricultural crops, such as wine grapes and strawberries, the risk of fungicide resistance development is higher due to frequent application of fungicides throughout a season. For example, in the eastern US grape growing regions, wine grape growers apply more than 10 applications of fungicide year after year (Wolf 2008), but in other regions such as the Central part of Mexico, more than 20 fungicide applications can be done on strawberries in a growing season. Once fungicide resistance is developed against a certain mode of action, it is not only a loss for growers, but also a huge loss to chemical companies that invested a considerable amount of money and time to develop the product. Currently, there are more than 150 different fungicidal compounds used worldwide (Brent and Hollomon 2007). The total sales of fungicide are estimated to be \$7.4 billion in US dollars, and grapes are one of the largest consumers of fungicides.

#### **4. Management of fungicide resistance**

There are several tactics to reduce the risk of fungicide resistance development. Common approaches implemented from fungicide manufacturers and regulatory agencies are 1) set a limit on the number of application per year, and 2) production of a pre-mixed material. The aim of setting a limitation or a cap on the number of applications per season is to reduce the rate of shifting from sensitive to resistance population by providing a gap between usages. If fungicide sensitive population is less fit than sensitive population, then the interval will provide a time for sensitive population to take over the resistant population. However, in some cases, the cap on the usage did not help the development of fungicide resistance. For example, Baudoin et al. (2008) found that although growers were following a '3 times per season' cap set by an international organization, Fungicide Resistance Action Committee (FRAC), QoI (e.g. strobirulins) resistant grape powdery mildew appeared in the field after 10-15 applications over several years of use. It seems that the 'cost' of having QoI fungicide resistance (i.e., *G143A* mitochondorial cytochrome b gene) does not affect the fitness of the fungus. On the other hand, the cap approach could help reducing the risk of DMI resistant isolates since fungal population seems to revert back to be sensitive when DMI is not used in the field (Staub 1991; Brent and Hollomon 2007).

The aim for pre-mixed material is to create a mixture of fungicides with multiple modes of action. There is evidence of reduced rate of fungicide resistance by mixing two (or more) different mode of action. For instance, Stott et al. (1990) compared the population shift of barley powdery mildew (caused by *Erysiphe graminis*) and showed that DMI and ethirimol sensitive populations did not shift to resistant population when both materials were used together. This approach seems to be favored by fungicide manufacturing companies; however, as we noted earlier, these pre-mixed materials can cause confusion among growers especially when seemingly new materials were combinations of previously introduced modes of action in reality.

When a host crop requires intensive disease management, the aforementioned two approaches may not be enough to effectively manage the development of fungicide resistance. For instance, in order to manage grape powdery mildew under eastern US growing conditions, wine grape growers typically use a fungicide (or multiple fungicides) for powdery mildew practically every time they spray (10-15 times per year). If they use a DMI early in the season, they may not have much choice later. Thus, careful planning and execution of plant disease management becomes very important. In order to achieve the goal, many successful growers extensively practice the Integrated Pest Management (IPM) or Integrated Plant Disease Management (IPDM) approach.

#### **5. IPM approaches revisited**

A basic concept of IPM is to combine multiple approaches of disease management in order to achieve the best result (Agrios 2005). These approaches are 1) cultural control, 2) use of genetic resistance, 3) biological control, and 4) chemical control. In the case of grape disease management, cultural practice can include (but not limited to) site selection, proper nutrition management, selective pruning of dormant canes, canopy management (shoot training, leaf removal, etc), etc. Genetic resistance can be introduced by selecting disease resistant varieties such as some of French hybrids. Often time the challenge is to select resistant varieties with high market demands. One of success stories of such a case is variety called 'Norton'. This highly disease resistant variety for wine making has gained popularity in the Eastern US grape growing regions since 1990's (Ambers and Ambers 2004). There are several biological agents available for use in grape production; however, none of them seems to produce reproducible results. It is partly due to the fact that growers want to use them as if they were using chemical options. Chemical management approaches should be considered only after these non-chemical approaches are considered. Integration of these approaches not only increases the efficacy of overall management strategies, but also, can reduce the monetary cost associated with chemical management approach (e.g., costs for purchasing chemicals, labor and fuel to apply chemicals, etc).

Even after other IPM approaches are considered, growers often need to resort to chemical management options because of environmental conditions that highly favor disease development. There are a few more items to be considered before application of fungicide in order to increase the efficacy. First of all, growers need to identify target pathogen(s) correctly. Then, growers need to select the best tool for management of the target pathogen(s) based on the situation at hand. In order to guide this complicated decision making process, it is necessary to have a better understanding of pathogen and host biology, as well as awareness on legal requirements.

As with any other pest management, identification of the target organism is a very critical component of plant disease management. For instance, symptoms of downy mildew of grape (caused by *Plasmopara viticola*) and powdery mildew of grape (caused by *Erisyphae necator*) may look similar to untrained eyes; however, materials for downy mildew are most likely not effective against powdery mildew, and *vise versa*. Thus, misidentification of disease symptoms can result in unnecessary application of fungicides.

After correct identification of the target disease(s), growers need to determine the best tool(s) for the situation at hand. Both host crop physiology and pathogen population changes throughout the course of the season, and these changes can influence disease triangle of the target pathogen. As we covered in the previous section, in order for a

resistance. For instance, in order to manage grape powdery mildew under eastern US growing conditions, wine grape growers typically use a fungicide (or multiple fungicides) for powdery mildew practically every time they spray (10-15 times per year). If they use a DMI early in the season, they may not have much choice later. Thus, careful planning and execution of plant disease management becomes very important. In order to achieve the goal, many successful growers extensively practice the Integrated Pest Management (IPM)

A basic concept of IPM is to combine multiple approaches of disease management in order to achieve the best result (Agrios 2005). These approaches are 1) cultural control, 2) use of genetic resistance, 3) biological control, and 4) chemical control. In the case of grape disease management, cultural practice can include (but not limited to) site selection, proper nutrition management, selective pruning of dormant canes, canopy management (shoot training, leaf removal, etc), etc. Genetic resistance can be introduced by selecting disease resistant varieties such as some of French hybrids. Often time the challenge is to select resistant varieties with high market demands. One of success stories of such a case is variety called 'Norton'. This highly disease resistant variety for wine making has gained popularity in the Eastern US grape growing regions since 1990's (Ambers and Ambers 2004). There are several biological agents available for use in grape production; however, none of them seems to produce reproducible results. It is partly due to the fact that growers want to use them as if they were using chemical options. Chemical management approaches should be considered only after these non-chemical approaches are considered. Integration of these approaches not only increases the efficacy of overall management strategies, but also, can reduce the monetary cost associated with chemical management approach (e.g., costs for

Even after other IPM approaches are considered, growers often need to resort to chemical management options because of environmental conditions that highly favor disease development. There are a few more items to be considered before application of fungicide in order to increase the efficacy. First of all, growers need to identify target pathogen(s) correctly. Then, growers need to select the best tool for management of the target pathogen(s) based on the situation at hand. In order to guide this complicated decision making process, it is necessary to have a better understanding of pathogen and host biology,

As with any other pest management, identification of the target organism is a very critical component of plant disease management. For instance, symptoms of downy mildew of grape (caused by *Plasmopara viticola*) and powdery mildew of grape (caused by *Erisyphae necator*) may look similar to untrained eyes; however, materials for downy mildew are most likely not effective against powdery mildew, and *vise versa*. Thus, misidentification of

After correct identification of the target disease(s), growers need to determine the best tool(s) for the situation at hand. Both host crop physiology and pathogen population changes throughout the course of the season, and these changes can influence disease triangle of the target pathogen. As we covered in the previous section, in order for a

or Integrated Plant Disease Management (IPDM) approach.

purchasing chemicals, labor and fuel to apply chemicals, etc).

disease symptoms can result in unnecessary application of fungicides.

as well as awareness on legal requirements.

**5. IPM approaches revisited** 

pathogen to successfully infect a host crop, a susceptible host, a pathogenic pathogen, and a disease-conducive environment have to be present at the same time. However, a pathogen may not produce spores at a right timing or a host may not be susceptible at a certain time of its lifecycle. Even if there are spores and hosts are susceptible, if the environmental conditions are not conducive for infection, disease cannot be developed. Thus, it is very important to understand both pathogen's and host's lifecycles, as well as environmental conditions for infection, so that growers can place fungicide application to efficiently disrupt the formation of the disease triangle without wasting their effort.

Changes in host physiology throughout the season, especially fruit development, can be a key factor to determine when and how fungicide should be applied. For example, it is important to protect flowers of strawberry from Botrytis infection because flower infection result in latent infection on berries later in the season (Mertely et al. 2002). Results from Merteley et al., (2002) indicate that Botrytis fruit rot can be controlled with an application of fenhexamid when applied at anthesis. They were also able to relate a linear regression equation between time of application and Botrytis fruit rot incidence, which can guide growers to adjust their spray timings. Legard et al. (2005) integrated information of the crop physiology, epidemiological information and fungicide efficacy to develop reduced fungicides programs to control Botrytis fruit rot in Florida. Their results indicate that in the early stage of the season low rates of captan were as effective as high rates for disease control, and later in the season the control was significantly improved by applications of fenhexamid at the second bloom peak period. In the case of grape production, ontogenic resistance has been well documented against many of major pathogens such as black rot (Hoffman al. 2004), powdery mildew (Ficke et al. 2002; Gadoury et al. 2003), and downy mildew (Kennelly et al. 2005). Grape berries become resistance against downy mildew, powdery mildew, and black rot approximately 4-5 weeks and 3-4 weeks after bloom for French and for American varieties, respectively. By knowing this information, growers can concentrate their effort to protect berries during this critical period.

In addition to biological factors, legal or legislative factors can influence fungicide application decision-making process. For instance, a product containing mancozeb has a 66 day PHI (pre-harvest interval) set by the EPA (Environmental Protection Agency) for an application on grape in the US. Thus, grape growers need to adjust their spray program against downy mildew or black rot when they are expecting to harvest within 2 months. Also, REI (re-entry interval) can be a limiting factor. A product Topsin-M (thiophanatemethyl) has a REI of 2-days for grapes, and a product Pristine (boscalid + pyraclostrobin) has a warning on the label that growers cannot work on grape canes within 5 days after application. Thus, it is difficult to use either Topsin-M or Pristine when constant canopy management is required. The other factors can influence fungicide application is an incompatibility issue. For example, several fungicides, including chlorothalonil can cause phytotoxicity on 'Concord' and related American grape varieties (Goffinet and Pearson 1991).

#### **6. Physical mode of action of fungicides**

There is yet another factor to be considered prior to an application of fungicide, that is, physical mode of action of fungicide. Physical mode of action (PMoA) describes the effect a fungicide with respect to the time of placement of a fungicide in relation to the hostpathogen interaction, that is on pre-infection, post-infection, pre- and post-symptom, and vapor activity (Szkolnik 1981; Wong and Wilcox 2001) and the duration and degree of the fungicides activity (Pfender 2006).

PMoA of protectant fungicides is pre-infection effect. It can reduce the infection efficiency as a result of the placement of a fungicidal material on plant tissues. McKenzie et al. (2009) found that applying captan 2 days before inoculation on strawberry crown rot (caused by *Colletotrichum gloeosporioides*), disease intensity was consistently reduced at the end of the season. Azoxystrobin, pyraclostrobin and thiophanate-methyl performed better if applied 1 day after inoculation, but their effect reducing the disease was variable. Based on these results the recommendation was to spray captan throughout the season in a protectant strategy, and azoxystrobin, pyraclostrobin and thiophanate-methyl if an infection event was present in order to keep the disease at low levels.

On the other hand, systemic fungicides with more curative (eradicating) activity can impact the processes of infection and establishment by pathogen, thus these are post-infection and can be pre- or post-symptom effect. Vapor activity can facilitate pre- and post-symptom effects. A single fungicide can provide both protectant and curative activities. For example, fungicides such as strobilurins (QoI) will mainly impact on spore germination, as they interfere with mitochondrial respiration (Bartlett et al. 2002), giving an excellent protectant activity. At the same time, the QoI can provide good curative activity against rusts such as *Puccinia hemerocallidis* and *Puccinia graminis* subsp *graminicola* (Godwin et al. 1992; Pfender 2006). In some cases such as *Cercospora beticola*, that causes Cercospora diseases on sugarbeet, good post symptom activity (eradicant) an antisporulant activity of this group of fungicides has been reported (Ypema and Gold 1999; Anesiadis et al. 2003). In other cases such as downy mildew of grape, caused by *Plasmopara viticola* (Wong and Wilcox 2001) and *Phytophthora cactorum* on strawberries (Rebollar-Alviter et al 2007) these fungicides provide good protectant activities, but do not perform well in post-infection treatments. Other groups inhibiting the sterol biosynthesis (SBI/DMI) do not have direct effect on spore germination, but impact more directly on mycelial growth. Hoffman et al. (2004) found that a DMI, myclobutanil, provides a better post-infection activity against black rot of grape, compared with azoxystrobin, which provided a slight evidence of a post-infection activity.

#### **7. Fungicide use based on disease risk assessment tools**

Now we have covered basics of plant disease development, management approaches, fungicide resistant issues, and physical model of action, the next step is to combine these together. As we briefly touched earlier, one of approaches taken by many researchers and growers are the use of disease risk assessment (or forecasting or warning) tools to minimize the use of fungicides by determining the best timing for application. There are several examples of risk assessment tools used together with the knowledge of the physical mode of action of fungicides. For example, Madden et al. (2000) evaluated an electronic warning system for downy mildew based on infection of leaves of American grapes, *Vitis labrusca*, productions of sporangia and sporangial survival over a period of 7 years. Sprayings were done when the system indicated that environmental conditions were favorable for sporangia production. Their results indicated that during this time the use of the warning system reduced the number of applications of metalaxyl plus mancozeb from one to six applications compared to the standard calendar based program. Wong and Wilcox (2001)

pathogen interaction, that is on pre-infection, post-infection, pre- and post-symptom, and vapor activity (Szkolnik 1981; Wong and Wilcox 2001) and the duration and degree of the

PMoA of protectant fungicides is pre-infection effect. It can reduce the infection efficiency as a result of the placement of a fungicidal material on plant tissues. McKenzie et al. (2009) found that applying captan 2 days before inoculation on strawberry crown rot (caused by *Colletotrichum gloeosporioides*), disease intensity was consistently reduced at the end of the season. Azoxystrobin, pyraclostrobin and thiophanate-methyl performed better if applied 1 day after inoculation, but their effect reducing the disease was variable. Based on these results the recommendation was to spray captan throughout the season in a protectant strategy, and azoxystrobin, pyraclostrobin and thiophanate-methyl if an infection event was

On the other hand, systemic fungicides with more curative (eradicating) activity can impact the processes of infection and establishment by pathogen, thus these are post-infection and can be pre- or post-symptom effect. Vapor activity can facilitate pre- and post-symptom effects. A single fungicide can provide both protectant and curative activities. For example, fungicides such as strobilurins (QoI) will mainly impact on spore germination, as they interfere with mitochondrial respiration (Bartlett et al. 2002), giving an excellent protectant activity. At the same time, the QoI can provide good curative activity against rusts such as *Puccinia hemerocallidis* and *Puccinia graminis* subsp *graminicola* (Godwin et al. 1992; Pfender 2006). In some cases such as *Cercospora beticola*, that causes Cercospora diseases on sugarbeet, good post symptom activity (eradicant) an antisporulant activity of this group of fungicides has been reported (Ypema and Gold 1999; Anesiadis et al. 2003). In other cases such as downy mildew of grape, caused by *Plasmopara viticola* (Wong and Wilcox 2001) and *Phytophthora cactorum* on strawberries (Rebollar-Alviter et al 2007) these fungicides provide good protectant activities, but do not perform well in post-infection treatments. Other groups inhibiting the sterol biosynthesis (SBI/DMI) do not have direct effect on spore germination, but impact more directly on mycelial growth. Hoffman et al. (2004) found that a DMI, myclobutanil, provides a better post-infection activity against black rot of grape, compared with azoxystrobin, which provided a slight evidence of a post-infection activity.

Now we have covered basics of plant disease development, management approaches, fungicide resistant issues, and physical model of action, the next step is to combine these together. As we briefly touched earlier, one of approaches taken by many researchers and growers are the use of disease risk assessment (or forecasting or warning) tools to minimize the use of fungicides by determining the best timing for application. There are several examples of risk assessment tools used together with the knowledge of the physical mode of action of fungicides. For example, Madden et al. (2000) evaluated an electronic warning system for downy mildew based on infection of leaves of American grapes, *Vitis labrusca*, productions of sporangia and sporangial survival over a period of 7 years. Sprayings were done when the system indicated that environmental conditions were favorable for sporangia production. Their results indicated that during this time the use of the warning system reduced the number of applications of metalaxyl plus mancozeb from one to six applications compared to the standard calendar based program. Wong and Wilcox (2001)

fungicides activity (Pfender 2006).

present in order to keep the disease at low levels.

**7. Fungicide use based on disease risk assessment tools** 

evaluated the physical mode of action of azoxystrobin, mancozeb and metalaxyl against *Plasmopara viticola*, the causal agent of grape downy mildew. Azoxystrobin was effective in pre-infection treatments, but was ineffective when applied as a post-infection treatment. However, good effect was observed on reduction of sporulation, and reduction lesion size in post-symptom applications. Mancozeb was also excellent protectant but did not have any effect on post infection applications. Metalaxyl provided good pre-infection, post-infection and eradicant activity. Kennely et al. (2005) indicated that mefenoxam has strong vapor activity against *Plasmopara viticola*, grapevine downy mildew and 48 h of systemic activity in post-infection applications. Caffi et al. (2010) evaluated a warning system to control primary infections of downy mildew on grapevine, and results indicated that the number of applications can be reduced by more than 50% with significant savings in cost per ha without compromising downy mildew control.

Working with anthracnose fruit rot of strawberry, Turecheck et al. (2006) evaluated the preand post-infection activity of pyraclostrobin on the incidence of anthracnose fruit rot at different times of wetness periods and temperatures. Results indicated that pyraclostrobin was less effective when applied in post-infection with the longest wetness duration (12 and 24) and high temperature (22 and 30 C). The post-infection application had a significant effect when applications were made within 3 and 8 h after the wetness period. Under field conditions, applications made after 24 h after an infection event were able to successfully control the disease, indicating the possibility to incorporate pyraclostrobin in a disease management program in strawberry in a curative form if infection events occurred in the previous 24 h. In a similar study, Peres et al. (2010) indicated that anthracnose fruit rot was effectively controlled with captan on pre-infection under short wetting period and fludioxonil + ciprodinil was effective when applied in pre-inoculation, but also when applied at 4, 8, and 12 h after inoculation, but the efficacy was higher under short wetting periods (6 o 8 h). These studies indicate that performance of fungicide is strongly influenced by wetness duration regardless of the ability of the fungicide move in plant tissues.

Thus, growers face multiple layers of factors such as host-pathogen dynamics, fungicide resistance, physical and biochemical mode of action, IPM strategies, etc. in order to make decisions on fungicide application. Also, note that we were focus only on biological considerations, but not covering many of social and environmental factors such as society's concerns on fungicide use, issues on waste water management, and so on. In addition, there is a whole art and science of fungicide application technologies that is beyond our scope of this chapter. Instead of widening our topics, we would like to focus on the factors we discussed in this chapter by presenting two case studies that are compilations' of multiple studies to establish an optimal use of fungicide(s).

#### **8. Case study 1: Phomopsis cane blight and leaf spot of grape**

A series of studies by Nita et al. (2006a; 2006b; 2007a; 2007b; 2008) showed a multi-prong approach to develop a sound management strategy against Phomopsis cane and leaf spot of grape. Phomopsis cane and leaf spot is a common disease of grape in the U.S. and other grape growing regions around the world (Pearson and Goheen 1988; Pscheidt and Pearson 1989). The fungus, *Phomopsis viticola* (Sacc.), is the causal agent of the disease (Pearson and Goheen 1988). Typical symptoms on leaves are yellow spots, which varies in size (less than 1 mm to a few mm). On canes and rachis, it causes necrotic lesions that can be expanded to cause canker. The infected tissues become weak and prone to be damaged by wind. With heavy infection on rachis, fruit drop can be observed. Infections on fruits cause a fruit rot and thus directly decrease yield and fruit quality. Up to 30% loss of the crop has been reported in the Southern Ohio grape growing regions (Erincik, et al. 2001).

The source of inoculum in a given season consists of canes or trunks that were infected during previous growing seasons (Pscheidt and Pearson 1989). The fungus survives in the infected tissues over the winter, and in the spring, numerous pycnidia arise on infected canes. Conidia from these pycnidia are splashed by rain onto new growth (i.e., canes, leaves, and clusters) to cause infection. According to previous studies, *P. viticola* can be active in relatively cool weather conditions (7-8 C) (Erincik et al. 2003). Since they do not produce new spores during the season, it is considered a monocyclic disease.

In order to evaluate efficacy of currently available fungicides, Nita et al. (2007a) examined several fungicides for their protectant and potential curative activity against Phomopsis cane and leaf spot of grape. Fungicides with variety of mode of action, strobilurin, thiophanate-methyl (benzomidazole), myclobutanil (DMI), EBDC (mancozeb), calcium polysulfide (lime sulfur), were tested as protectant as well as curative application in a controlled environment study. Protectant application was applied a few hours prior to an artificial inoculation of leaves and shoots using spore suspension water that contained 5 x 106 spores per ml. Various patterns of post-inoculation (curative) application were tested. The shortest period between inoculation and application of a fungicide was 4 hours and the longest was 48 hours. In addition, a treatment with or without an adjuvant (product name Regulaid or JMS Stylet Oil) was also tested. These adjuvants were added in a hope that it might help facilitate movement of chemical into tissues. In addition, up to 150% of labeled rate of fungicide was examined to see a potential dose effect. Results indicated that all materials tested, regardless of a higher rate and/or a presence of adjuvant, did not show evidence of curative activity. On the other hand, strobilurin, calcium polysulfide, and EBDC showed a good protectant activity, up to >85% disease control [(treatment disease severitynegative (=untreated) control disease intensity)/negative control disease intensity], indicating that the management strategy for Phomopsis cane and leaf spot has to focus on protection of vines.

Then the same group evaluated the effect of dormant season fungicide applications of copper and calcium polysulfide against Phomopsis cane and leaf spot of grape disease intensity and inoculum production (Nita et al. 2006a). These dormant season fungicide applications aimed to reduce the source of inoculum by disturbing fungal colonies surviving on grape trunk tissues. Results indicated that fall and spring and spring applications of calcium polysulfide (10% in volume) provided 12 to 88% reduction in disease intensity and inoculum production. Thus, the reduction of disease intensity was not sufficient. Although inoculum production (the number of pycnidium per square cm) was significantly reduced, none of tested canes had zero pycnidium, indicating that there will be a plenty of inoculum available even with the best treatment. In the same study, the authors examined calendarbased applications of mancozeb or calcium polysulfide (0.5% in volume), which reduced 47 to 100% disease incidence and severity. The result indicated that although sanitation approach against this disease did not provide reasonable reduction in disease development, early season applications of a protectant fungicide (mancozeb or calcium polysulfide) provided a better efficacy. These results confirmed previously discussed management recommendations (Pearson and Goheen 1988; Pscheidt and Pearson 1989).

cause canker. The infected tissues become weak and prone to be damaged by wind. With heavy infection on rachis, fruit drop can be observed. Infections on fruits cause a fruit rot and thus directly decrease yield and fruit quality. Up to 30% loss of the crop has been

The source of inoculum in a given season consists of canes or trunks that were infected during previous growing seasons (Pscheidt and Pearson 1989). The fungus survives in the infected tissues over the winter, and in the spring, numerous pycnidia arise on infected canes. Conidia from these pycnidia are splashed by rain onto new growth (i.e., canes, leaves, and clusters) to cause infection. According to previous studies, *P. viticola* can be active in relatively cool weather conditions (7-8 C) (Erincik et al. 2003). Since they do not produce

In order to evaluate efficacy of currently available fungicides, Nita et al. (2007a) examined several fungicides for their protectant and potential curative activity against Phomopsis cane and leaf spot of grape. Fungicides with variety of mode of action, strobilurin, thiophanate-methyl (benzomidazole), myclobutanil (DMI), EBDC (mancozeb), calcium polysulfide (lime sulfur), were tested as protectant as well as curative application in a controlled environment study. Protectant application was applied a few hours prior to an artificial inoculation of leaves and shoots using spore suspension water that contained 5 x 106 spores per ml. Various patterns of post-inoculation (curative) application were tested. The shortest period between inoculation and application of a fungicide was 4 hours and the longest was 48 hours. In addition, a treatment with or without an adjuvant (product name Regulaid or JMS Stylet Oil) was also tested. These adjuvants were added in a hope that it might help facilitate movement of chemical into tissues. In addition, up to 150% of labeled rate of fungicide was examined to see a potential dose effect. Results indicated that all materials tested, regardless of a higher rate and/or a presence of adjuvant, did not show evidence of curative activity. On the other hand, strobilurin, calcium polysulfide, and EBDC showed a good protectant activity, up to >85% disease control [(treatment disease severitynegative (=untreated) control disease intensity)/negative control disease intensity], indicating that the management strategy for Phomopsis cane and leaf spot has to focus on

Then the same group evaluated the effect of dormant season fungicide applications of copper and calcium polysulfide against Phomopsis cane and leaf spot of grape disease intensity and inoculum production (Nita et al. 2006a). These dormant season fungicide applications aimed to reduce the source of inoculum by disturbing fungal colonies surviving on grape trunk tissues. Results indicated that fall and spring and spring applications of calcium polysulfide (10% in volume) provided 12 to 88% reduction in disease intensity and inoculum production. Thus, the reduction of disease intensity was not sufficient. Although inoculum production (the number of pycnidium per square cm) was significantly reduced, none of tested canes had zero pycnidium, indicating that there will be a plenty of inoculum available even with the best treatment. In the same study, the authors examined calendarbased applications of mancozeb or calcium polysulfide (0.5% in volume), which reduced 47 to 100% disease incidence and severity. The result indicated that although sanitation approach against this disease did not provide reasonable reduction in disease development, early season applications of a protectant fungicide (mancozeb or calcium polysulfide) provided a better efficacy. These results confirmed previously discussed management

recommendations (Pearson and Goheen 1988; Pscheidt and Pearson 1989).

reported in the Southern Ohio grape growing regions (Erincik, et al. 2001).

new spores during the season, it is considered a monocyclic disease.

protection of vines.

Nita et al. (2006b) also evaluated a warning system (based on temperature and wetness duration following rain) for Phomopsis cane and leaf spot of grape by applying fungicides based on prediction of infection events considering three criteria for risk: light, moderate and high. The infection condition was determined previously by Erincik et al. 2003. This study was conducted to determine if the warning system would provide a reasonable disease control compared with a calendar-based, 7-day interval protectant fungicide application. The warning-system based approach resulted in two to three times less number of applications while the percentage of control was often not significantly lower than the 7 day protectant schedule based on mancozeb, which constantly provided 70-80% and over 95% disease incidence and severity control, respectively.

The same group expanded this study by examining Phomopsis cane and leaf spot disease survey data using various statistical tools and modeling approach (Nita et al. 2007b; Nita et al. 2008). They found out that the variation of disease incidence observed in 20 different commercial vineyard locations over three consecutive years could be explained by a combination of local weather conditions and fungicide application trends. They further found that growers who had a better early season fungicide program (i.e., a use of dormant application of lime sulfur and/or mancozeb application soon after bud break) tended to have lower disease incidence than others who did not protect their vines during that time.

These series of studies showed that pre-season dormant application does not provide satisfactory reduction of this disease, and there are no potential curative materials; however, a dormant season application can be used in a conjunction with early season protectant fungicide applications, a warning system approach can be a good tool to be used, and more importantly, protection of grape tissues during early part of the season was found to be critical for the management of Phomopsis cane and leaf spot of grape. The Eastern and Midwestern US grape growing regions often receive a series of rains in April to May when new grape shoots are emerging, and pathogen can infect tissues under relatively low temperatures conditions, 7-8 C (Erincik et al. 2003; Nita et al. 2003). Therefore, good protection of newly emerging shoots (when new shoots are about 2.5-7.5 cm in length) using a protectant fungicide is a standard recommendation for this disease (Pscheidt and Pearson 1989; Nita et al. 2007b).

#### **9. Case study 2: Leather rot of strawberry**

Crown and root rots, such as those caused by *Colletotrichum* spp, *Phytophthora* spp. and *Verticillium* spp., and fruit rots, such as *Botrytis cinerea*, *Colletotrichum acutatum*, and *Phytophthora cactorum* are among the most important pathogens causing disease on strawberry that cause more losses around the world.

Leather rot caused by *P. cactorum* is one of most common disease on strawberry, especially in systems such as matted row and annual systems. The disease is less severe and not very frequent in high tunnel system, mainly because plastic tunnels prevent rain to reach plants and induce splash dispersal of the pathogen. On strawberry all stages of fruit development may be infected by this pathogen, including flowers. On green fruits dark areas covering the entire fruit may develop which later appear leathery and eventually mummify. Mature fruits do not always show the typical symptoms, except they appear discolored and whitish in some areas. However, diseased fruits are in general easy to distinguish because the bad off-odor and taste, which is caused by phenolic compounds (Jelen et al. 2005). In Ohio, losses over 50% have reported (Ellis and Grove 1983) and in areas with medium to low technology levels in open field strawberry plantings under annual production systems in countries such as Mexico, the disease can be a problem during the rainy season of the year (June to October) where losses can reach up to 30% of production.

Development of leather rot is favored by excessive wet weather, especially on saturated soils with poor drainage. In this pathosystem, oospores represent the primary inoculum, which is a survival structure. With moisture, oospores germinate to produce sporangia. Sporangia can germinate and produce a germ tube for infection, or can give a rise to zoospores that can swim in water. With a rain event, both sporangia and zoospore are splash dispersed to fruits to cause infection. Once established, new sporangia can form on the infected fruit to cause another infection. Thus, it is considered a polycyclic disease. Extensive studies conducted on the epidemiology of the disease in the past decade have shown that wetness duration and temperature (17 to 25 C) are important factors for disease development. Splashing of zoospores and sporangia is caused by rainfall and wetness periods can be as short as 2 h are sufficient for the oomycete to cause infection (Grove et al 1985a; Grove et al. 1985b; Madden et al. 1991). Typically there is a latent period of 5 days for full development of symptoms.

Management of leather rot is based on the use of fungicides and cultural practices such as avoiding saturated soils by proper site selection, improving soil drainage and applying straw mulches between rows. Applying straw mulch between row spaces prevents fruits from touching the soil and standing water, and reduces the splashing of water droplets containing sporangia and zoospores (Madden et al. 1991). Protective fungicide program using captan and thiram are widely used; however, both fungicides are not able to control the disease when weather conditions favor leather rot development. Therefore fungicide with a different biochemical, and physical mode of action with the ability to penetrate plant tissues need to be used.

In order to select the proper fungicide, the efficacy of fungicides was defined in the field (Rebollar-Alviter et al. 2005). During 2003 and 2004, the efficacy of pyraclostrobin, azoxystrobin, potassium phosphite and mefenoxam was evaluated in Wooster Ohio, USA against leather rot of strawberry grown in a matted row system. Treatments were applied as a preventive application at the initiations of bloom. In order to create conditions that favor leather rot development, straw between the rows was removed and then plots were flooded until water puddle between the rows at different times using an overhead irrigation system. Results from these experiments indicated that during the two years of testing, disease incidence on fruits varied from 58 to 67% in the controls. No significant differences were detected among the fungicides treatments. Disease incidences ranged from 0.3 to 0.5% with the QoI fungicides (azoxystrobin and pyraclostrobin), 0.8 to 5.4% with potassium phosphite, and 0.3 to 11% with mefenoxam (Rebollar-Alviter et al. 2005). Interestingly, these experiments showed that both QoI fungicides tested were highly effective for control of leather rot of strawberry. Thus, these QoI fungicides can be used in a disease management program alternating with potassium phosphite and/or mefenoxam, which are known to be efficacious to control the disease (Ellis et al. 1998).

In order to understand some aspects of the physical mode of action of the QoI, potassium phosphite, and mefenoxam fungicides that were tested in the previous work, a greenhouse

off-odor and taste, which is caused by phenolic compounds (Jelen et al. 2005). In Ohio, losses over 50% have reported (Ellis and Grove 1983) and in areas with medium to low technology levels in open field strawberry plantings under annual production systems in countries such as Mexico, the disease can be a problem during the rainy season of the year

Development of leather rot is favored by excessive wet weather, especially on saturated soils with poor drainage. In this pathosystem, oospores represent the primary inoculum, which is a survival structure. With moisture, oospores germinate to produce sporangia. Sporangia can germinate and produce a germ tube for infection, or can give a rise to zoospores that can swim in water. With a rain event, both sporangia and zoospore are splash dispersed to fruits to cause infection. Once established, new sporangia can form on the infected fruit to cause another infection. Thus, it is considered a polycyclic disease. Extensive studies conducted on the epidemiology of the disease in the past decade have shown that wetness duration and temperature (17 to 25 C) are important factors for disease development. Splashing of zoospores and sporangia is caused by rainfall and wetness periods can be as short as 2 h are sufficient for the oomycete to cause infection (Grove et al 1985a; Grove et al. 1985b; Madden et al. 1991). Typically there is a latent period of 5 days for full development of symptoms.

Management of leather rot is based on the use of fungicides and cultural practices such as avoiding saturated soils by proper site selection, improving soil drainage and applying straw mulches between rows. Applying straw mulch between row spaces prevents fruits from touching the soil and standing water, and reduces the splashing of water droplets containing sporangia and zoospores (Madden et al. 1991). Protective fungicide program using captan and thiram are widely used; however, both fungicides are not able to control the disease when weather conditions favor leather rot development. Therefore fungicide with a different biochemical, and physical mode of action with the ability to penetrate plant

In order to select the proper fungicide, the efficacy of fungicides was defined in the field (Rebollar-Alviter et al. 2005). During 2003 and 2004, the efficacy of pyraclostrobin, azoxystrobin, potassium phosphite and mefenoxam was evaluated in Wooster Ohio, USA against leather rot of strawberry grown in a matted row system. Treatments were applied as a preventive application at the initiations of bloom. In order to create conditions that favor leather rot development, straw between the rows was removed and then plots were flooded until water puddle between the rows at different times using an overhead irrigation system. Results from these experiments indicated that during the two years of testing, disease incidence on fruits varied from 58 to 67% in the controls. No significant differences were detected among the fungicides treatments. Disease incidences ranged from 0.3 to 0.5% with the QoI fungicides (azoxystrobin and pyraclostrobin), 0.8 to 5.4% with potassium phosphite, and 0.3 to 11% with mefenoxam (Rebollar-Alviter et al. 2005). Interestingly, these experiments showed that both QoI fungicides tested were highly effective for control of leather rot of strawberry. Thus, these QoI fungicides can be used in a disease management program alternating with potassium phosphite and/or mefenoxam, which are known to be

In order to understand some aspects of the physical mode of action of the QoI, potassium phosphite, and mefenoxam fungicides that were tested in the previous work, a greenhouse

(June to October) where losses can reach up to 30% of production.

tissues need to be used.

efficacious to control the disease (Ellis et al. 1998).

study was conducted. Fungicides were applied on pre-infection, 2, 4 and 7 days before inoculation with a zoospore suspension (105 zoospores/ml) and 13, 24, 36 and 48 h after inoculation. A wetness period of 12 h was applied to plants and fruits either before or after inoculation, and disease incidence was recorded 6 days after inoculation. Results indicated that all fungicides applied in pre-infection provided excellent protection activity against the disease when applied up to 7 days before inoculation. These studies confirmed the protectant activity of all fungicides in previous experiments in strawberry. However, the results when the fungicides were applied in post-inoculation (curative application), both QoI fungicides had some effect 13 h after inoculation reducing disease incidence by 60%. Nevertheless when both fungicides were sprayed 24, 36 and 48 h after inoculation there was no disease control. In contrast, the systemic fungicides potassium phosphite and mefenoxam successfully controlled the disease up to 36 h after inoculation with no significant differences between these two fungicides. At 48 h both fungicides still had some moderate control, but not enough to be considered in a curative strategy for disease management (Rebollar-Alviter et al. 2007a).

These results were then used in conjunction with the previous knowledge on the disease epidemiology in order to evaluate disease management programs and to optimize fungicide application. A 3-year study was conducted in a field to examine efficacy of several modes of action (mefenoxam, phenilamides; azoxystrobin and pyraclostrobin, QoI, and potassium phosphite, phosphonate) against leather rot. In previous studies on a forecasting system for leather rot; occurrence of rain was considered a better indicator of risk of disease development than temperature condition or length of wetness duration (Reynolds et al. 1988; Madden et al. 1991). This is probably because this pathogen requires very short wetness periods (2 h) to infect (Grove et al. 1985a), and it can also infect under a wide range of temperatures. Therefore, specific infection conditions (i.e., temperature or length of wetness duration) would not clearly define the risk conditions. Rather, a detection of individual rainstorm and the amount of rainfall during critical periods is a better indicator for post-infection application of a fungicide. The amount of rainfall is critical because it will be a predictor for the dissemination of the spores to susceptible fruits (Ntahimpera et al. 1998).

Based on previous experiments where post infection activities of mefenoxam and potassium phosphite indicated that this fungicides were able to control the disease up to 36 h after inoculation, and considering that epidemic is basically driven by moderate to heavy rain events (Reynolds et al. 1987; Reynolds et al. 1988), scheduling fungicides after the occurrence of rain events taking in to account fungicide persistence in plant (at least 7 days) and other factors that affect the efficacy of fungicides, as well as weather predictions, it would be possible to reduce the number of applications during the critical time for disease development. These experiments indicated that post infection treatments applied after flooding events were as effective as those applied on a calendar basis, but with 1 to 3 fewer sprayings. One spraying of mefenoxam was sufficient to keep the disease under control when applied within 36 h after a rain event. Similarly, 2 sprayings of potassium phosphite were enough to control the disease when sprayings were done within the same time after the occurrence of a rain event. Whereas in calendar based applications (7 days schedule) four sprayings were necessary to control the disease using programs based on azoxystrobin and potassium phosphite, 1 spraying of mefenoxam and 2 of potassium phosphite were enough to control the disease under high disease pressure (Rebollar-Alviter et al. 2010).

The disease control programs evaluated either as protectant strategy or curative responding to rain events were able to control the disease under weather conditions favoring disease development. Calendar based fungicide applications as well as those responding to rain events take in to consideration the risk of disease development and agree with current recommendation to manage fungicide resistance. Growers have a choice to use the protectant (calendar-based program) or curative strategy under a matted row production system in Ohio and similar strawberry production areas to extend the life of the fungicide by a proper use of fungicide resistance techniques.

As additional factor that contributes to optimize fungicide application for the management of leather rot of strawberry is the distribution of the sensitivity to the tested fungicides. A study was conducted in order to determine the sensitivity of *P. cactorum* to azoxystrobin and pyraclostrobin fungicides among isolates from different parts of the state of Ohio, and other states of the USA, so the risk of resistance development by using these fungicides on strawberry could be determined. The sensitivity of 89 isolates of *P. cactorum* was determined to both fungicides on mycelia and zoospore germination. The results showed that there was a wide distribution of sensitivity to azoxystrobin, indicating a great diversity among the isolates evaluated. Thus, the sensitivity distributions can be used as a baseline sensitivity to monitor shifts in fungicide resistance in *P. cactorum* (Rebollar-Alviter et al. 2007b).

These series of studies showed that both calendar-based and disease risk-based fungicide application can result in a satisfactory disease management. Also a proper combination of protectant and curative approach can extend the life of the fungicide. The results obtained from these experiments are based on growing conditions in the Midwestern US with matted row perennial production; however, it can be also applicable to other type of production systems. For example, in subtropical areas of the central part of Mexico (Michoacan and Guanajuato States), strawberries are grown as an annual crop and season is drastically different from the Midwest; however, rain season coincides with fruit set and first harvest as it is in the Midwestern US. Thus, the same principals for leather rot management can be applied.

#### **10. Concluding remarks**

In this chapter, we reviewed major components that are associated with fungicide application decision-making process: basic understanding of disease epidemiology; fungicide resistance and its management; fungicide physical mode of action; and use of plant disease risk assessment tools that can integrate these components. We also discussed two case studies where multiple studies are conducted to develop optimal management recommendations. We believe that this chapter demonstrated the complication involved in an optimization of fungicide uses which growers face every day, and presented some of approaches that can be used to investigate this intriguing study subject.

#### **11. Acknowledgement**

Authors are very thankful to our mentors Drs. Laurence V. Madden and Michael A. Ellis of the Department of Plant Pathology at The Ohio State University for their invaluable advice and support during the time experiments from cases 1 and 2 were conducted.

#### **12. References**

14 Fungicides – Beneficial and Harmful Aspects

The disease control programs evaluated either as protectant strategy or curative responding to rain events were able to control the disease under weather conditions favoring disease development. Calendar based fungicide applications as well as those responding to rain events take in to consideration the risk of disease development and agree with current recommendation to manage fungicide resistance. Growers have a choice to use the protectant (calendar-based program) or curative strategy under a matted row production system in Ohio and similar strawberry production areas to extend the life of the fungicide

As additional factor that contributes to optimize fungicide application for the management of leather rot of strawberry is the distribution of the sensitivity to the tested fungicides. A study was conducted in order to determine the sensitivity of *P. cactorum* to azoxystrobin and pyraclostrobin fungicides among isolates from different parts of the state of Ohio, and other states of the USA, so the risk of resistance development by using these fungicides on strawberry could be determined. The sensitivity of 89 isolates of *P. cactorum* was determined to both fungicides on mycelia and zoospore germination. The results showed that there was a wide distribution of sensitivity to azoxystrobin, indicating a great diversity among the isolates evaluated. Thus, the sensitivity distributions can be used as a baseline sensitivity to

These series of studies showed that both calendar-based and disease risk-based fungicide application can result in a satisfactory disease management. Also a proper combination of protectant and curative approach can extend the life of the fungicide. The results obtained from these experiments are based on growing conditions in the Midwestern US with matted row perennial production; however, it can be also applicable to other type of production systems. For example, in subtropical areas of the central part of Mexico (Michoacan and Guanajuato States), strawberries are grown as an annual crop and season is drastically different from the Midwest; however, rain season coincides with fruit set and first harvest as it is in the Midwestern US. Thus, the same principals for leather rot management can be

In this chapter, we reviewed major components that are associated with fungicide application decision-making process: basic understanding of disease epidemiology; fungicide resistance and its management; fungicide physical mode of action; and use of plant disease risk assessment tools that can integrate these components. We also discussed two case studies where multiple studies are conducted to develop optimal management recommendations. We believe that this chapter demonstrated the complication involved in an optimization of fungicide uses which growers face every day, and presented some of

Authors are very thankful to our mentors Drs. Laurence V. Madden and Michael A. Ellis of the Department of Plant Pathology at The Ohio State University for their invaluable advice

approaches that can be used to investigate this intriguing study subject.

and support during the time experiments from cases 1 and 2 were conducted.

monitor shifts in fungicide resistance in *P. cactorum* (Rebollar-Alviter et al. 2007b).

by a proper use of fungicide resistance techniques.

applied.

**10. Concluding remarks** 

**11. Acknowledgement** 

Agrios, G. N. (2005). Plant Pathology. San Diego, Academic Press.


Goffinet, M. C. and R. C. Pearson (1991). Anatomy of Russeting Induced in Concord Grape Berries by the Fungicide Chlorothalonil. Am. J. Enol. Vitic. 42(4): 281-289. Grove, G. G., L. V. Madden and M. A. Ellis (1985a). Influence of temperature and wetness

Grove, G. G., L. V. Madden and M. A. Ellis (1985b). Splash dispersal of Phytophthora cactorum from infected strawberry fruit. Phytopathology 75(5): 611-615. Hardwick, N. V. (2006). Disease Forecasting. The Epidemiology of Plant Diseases. B. M. Cooke, G. J. Jones and B. Kaye. Dordrecht, The Netherlands, Springer. Hoffman, L. E., W. F. Wilcox, D. M. Gadoury, R. C. Seem and D. G. Riegel (2004). Integrated

Jeleń, H. H., J. Krawczyk, T. O. Larsen, A. Jarosz and B. Gołębniak (2005). Main compounds

Kennelly, M. M., D. M. Gadoury, W. F. Wilcox, P. A. Magarey and R. C. Seem (2005).

Legard, D. E., S. J. MacKenzie, J. C. Mertely, C. K. Chandler and N. A. Peres (2005).

Leroux, P., M. Gredt and P. Boeda (1988). Resistance to inhibitors of sterol biosynthesis in

MacKenzie, S. J., J. C. Mertely and N. A. Peres (2009). Curative and Protectant Activity of

Madden, L. V. (2006). Botanical epidemiology: some key advances and its continuing role in disease management." European Journal of Plant Pathology 115: 2-23. Madden, L. V., M. A. Ellis, G. G. Grove, K. M. Reynolds and L. L. Wilson (1991).

Madden, L. V., M. A. Ellis, N. Lalancette, G. Hughes and L. L. Wilson (2000). Evaluation of a

Madden, L. V., G. Hughes and F. van den Bosch (2007). The study of plant disease

Mertely, J. C., S. J. MacKenzie and D. E. Legard (2002). Timing of Fungicide Applications for

Nita, M., M. A. Ellis and L. V. Madden (2007a). Evaluation of the curative and protectant

sanitation, and spray timing. Phytopathology 94: 641-650.

in Applied Microbiology 40(4): 255-259.

and Rachises. Phytopathology 95(12): 1445-1452.

herpotrichoides. Pesticide Science 23(2): 119-129.

*gloeosporioides*. Plant Disease 93(8): 815-820.

epidemics. St. Paul, MN, APS press.

Plant Disease 86(9): 1019-1024.

92(7): 1053-1061.

445.

554.

annual winter strawberry. Plant Disease 89: 1353-1358.

Phytopathology 75: 700-703.

duration on sporulation of *Phytophthora cactorum* on infected strawberry fruit.

control of grape black rot: Influence of host phenology, inoculum availability,

responsible for off-odour of strawberries infected by *Phytophthora cactorum*. Letters

Seasonal Development of Ontogenic Resistance to Downy Mildew in Grape Berries

Development of a reduced use fungicide program for control of Botrytis fruit rot on

field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella

Fungicides for Control of Crown Rot of Strawberry Caused by *Colletotrichum* 

"Epidemiology and control of leather rot of strawberries. Plant Disease 75(5): 439-

Disease Warning System for Downy Mildew of Grapes. Plant Disease 84(5): 549-

Botrytis cinerea Based on Development Stage of Strawberry Flowers and Fruit.

activity of fungicides and fungicide-adjuvant mixtures on Phomopsis cane and leaf spot of grape: a controlled environment study. Crop Protection 26(9): 1377-1384. Nita, M., M. A. Ellis and L. V. Madden (2008). Variation in Disease Incidence of Phomopsis

Cane and Leaf Spot of Grape in Commercial Vineyards in Ohio. Plant Disease


protectant efficacy of azoxystrobin and potassium phosphite for controlling leather rot of strawberry caused by *Phytophthora cactorum*. Crop Protection 29(4): 349-353.


### **Resistance to Botryticides**

Snježana Topolovec-Pintarić

*Department for Plant Pathology, Faculty of Agriculture, University of Zagreb Croatia* 

#### **1. Introduction**

18 Fungicides – Beneficial and Harmful Aspects

Shabi, E., T. Katan and K. Marion (1983). Inheritance of resistance to benomyl in isolates of

Staub, T. (1991). Fungicide Resistance: Practical Experience with Antiresistance Strategies and the Role of Integrated Use. Annual Review of Phytopathology 29(1): 421-442. Stott, I. P. H., Noon, R. A., Heaney, S. P. (1990). Flutriafol, ethirimol, and thiabendazole seed

Turechek, W. W., N. l. A. Peres and N. A. Werner (2006). Pre- and Post-Infection Activity of

Van der Plank, J. E. (1963). Plant Diseases: Epidemics and Control. New York, Academic

Wolf, T. K., Ed. (2008). Wine grape production guide for eastern North America. Ithaca,

Wong, F. P. and W. F. Wilcox (2001). Comparative Physical Modes of Action of

Ypema, H. L. and R. E. Gold (1999). Kresoxim-methyl: modification of a naturally occurring

Zadoks, J. C. (1984). A quarter century of disease warning, 1958-1983. Plant Disease 68(4):

Zadoks, J. C. and R. D. Schein (1979). Epidemiology and plant disease management. New

compound to produce a new fungicide. Plant Disease 83(1): 4-19.

treatment - an update on field performance and resistance monitoring. Brighton Crop Protection Conference, Pests and Diseases - 1990. Vol. 3. pp. 1169-1174. Szkolnik, M. (1981). Physical mode of action of sterol-inhibiting fungicide against apple

Pyraclostrobin for Control of Anthracnose Fruit Rot of Strawberry Caused by

N.Y., Natural Resource, Agriculture, and Engineering Service (NRAES)

Azoxystrobin, Mancozeb, and Metalaxyl Against Plasmopara viticola (Grapevine

strawberry leather rot epidemics. Phytopathology 78: 822-827.

Venturia inaequalis from Israel. Plant Pathology 32(2): 207-211.

diseases. Plant Disease 65: 981-985.

Cooperative Extension.

Press.

352-355.

*Colletotrichum acutatum*. Plant Disease 90(7): 862-868.

Downy Mildew). Plant Disease 85(6): 649-656.

York, Oxford University Press Inc.

protectant efficacy of azoxystrobin and potassium phosphite for controlling leather rot of strawberry caused by *Phytophthora cactorum*. Crop Protection 29(4): 349-353. Reynolds, K. M., M. A. Ellis and L. V. Madden (1987). Progress in development of a strawberry leather rot forecasting system. Avd. Strawberry Prod. 6: 18-22. Reynolds, K. M., M. A. Ellis and L. V. Madden (1988). Effect of weather variables on

> Plant pathogenic fungi and oomycetes as causal of plant diseases are responsible for economical looses in agricultural production worldwide. Therefore, their chemical control by products named fungicides is needed and justified especially when disease tend to became an epidemic. Without fungicides both yield and quality would be severely reduced by the ravages of fungi. The improved performance, specificity and environmental safety of the modern fungicides led to become their ever more widely used. But, as great Renaissance man Leonardo da Vinci said: "*The nature never breaks her own laws*", the fungi constantly found the new ways to adapt to conditions that human creates and keep existing and living. Fungus develops insensibility to chemical compound aimed to their suppression under constant pressure of often and continuous use of fungicide with specific mechanisms of action. This ability is nothing else than natural phenomenon or evolution. Today this phenomenon is less mysterious than three decades ago when first arise although some new challenges have spring up. Phenomenon of insensibility of fungus to the chemical compound used for controlling it is named resistance. With the increased use and specificity of the product comes a greater risk that resistance will developed because certain members of the target fungal population will not be affected by the product and therefore fungus cannot be controlled adequately any more. That is, they are genetically resistant to it. Although some plant diseases may be managed through the alteration of cultural practices, many diseases are only managed acceptably with the application of fungicides. One of them is grey mould of wine grape caused by ascomycete fungus *Botrytis cinerea* Pers.:Fr. (teleomorph *Botryotinia fuckeliana* (de Bary) Whetzel). Even today the only effective control of *B. cinerea* remains application of fungicides specifically named botryticides. In the past *B. cinerea* has proved to be very prone to resistance development which makes it difficult to control. Those drown attention of scientists and catalyse studies of resistance phenomenon in *B. cinerea*. Furthermore, resistance phenomenon intensified the genetic studies of this fungus because it was assumed that limited understanding of the genetic structure of *B. cinerea* populations is reflecting in difficulties in managing the disease. Despite of gained knowledge about *B. cinerea* resistance and managing solutions the resistance is still an ever present threat with new cases arising and some old problems still continuing. A new segment of the topic becomes issue of multiple drug resistance (MDR). MDR phenomenon is common in human pathogens but it has been rarely described before in field strains of plant pathogenic fungi. Gaining knowledge about MDR revealed existence and involvement of some different mechanisms for resistance development. Fungicide resistance mechanisms

can relate to qualitative factors such as absence or presence of a sensitive target site. Beside this, qualitative factors like uptake, transport, storage and metabolism also need to be considered. The MDR phenomenon of *B. cinerea* was firstly recorded in 1998. Since than, more data of MDR monitoring were obtained indicating that *B. cinerea* MDR types in combination with other *B. cinerea* resistant types could represent a significant threat for future chemical control of *B. cinerea*.

#### **2. Bunch rot of grapes: High standard disease of grapevine**

Bunch rot of grapes is one of the grapevine diseases of great economical importance because it leads to substantial losses in yield and lowering in quality. Vineyard ecosystem is often difficult to manipulate both the crop and its environment. Also, it is a stage where *B. cinerea* can express its dual nature in causing the destructive bunch rot and, under certain conditions, the non-destructive noble rot, which is not paralleled in plant pathology. Noble rot yields vines of a special quality that are high economical. In the continental climate the bunch rot disease can inflict damages up to 50 or 60 percent and under the Mediterranean climate 3 to 5 percent. The damages are continuing in vine making process. Rotting of grape berries caused by fungus is probably old as winegrowing and some descriptions date from time of Roman Plinius the Older (1. century). Even the genus name *Botrytis* is derived from Latin for "grapes like ashes" by Micheli who erected the genus in 1729. Name of disease, grey mould, actually describes the grey coating spread over the bunch especially beacon before vintage when the most damage is already done. The coating is somatic filamentous body or sporulating mycelia of fungus *B. cinerea*. In grapevine *B. cinerea* causes massive losses of yield and quality of grape berries for vine production especially during cool and wet climatic conditions. This fungus is able to act as saprophyte, necrotroph as well as pathogen. In vineyards *B. cinerea* is present as part of the environmental micro-bionta and predominantly being saprophytic it colonize wounds or senescing tissue. From an economic point of view, only while acting as true pathogen infecting flowers and grape berries are of importance in terms of lowering quantity and quality of yield. Although there are numerous scientific contributions that continue to be published, there are still gaps in our knowledge about the etiology and epidemiology of bunch rot disease in vineyards. Disease starts with infections in flowering and even earlier. Establishment of *B. cinerea* on moribund and injured tissues normally allows pathogen to infect health tissues. Source of inoculum which will initiate further cycle of the disease are sclerotia and mycelium formed in the outer layers of the dead bark of shoots, cane or on plant debris of various origin. The sclerotia may be directly infective as sources of conidia yet some sclerotia are not conidia-bearing but form reproductive body apothecia. The ascospores produced from apothecia can also initiate primary infections although sexual stage is not considered as significant for epidemiology of grey mould yet Anotnin de Bary described easily found apothecia on dead vine leaves in late 1866. Sclerotia are rare in the regions with warm dry summers and therefore it is unlikely apothecia will be found either. Sporulation on sclerotia is repeated and this extend period of conidial production and infection. Rain and splashing water under natural conditions dislodge conidia from germinating sclerotia and conidia are dispersing in air currents, in splashing water droplets and by insects. The "fruit fly" *Drosopilla melanogaster* is considered as plurimodal vector of *B. cinerea*. The concentration of conidia in the air is increasing as the grapevines maturing. The mycelium spread through outer layers of the dead bark of shoots and the bark of invade cane is bleached to almost white colour. *Botrytis*

can relate to qualitative factors such as absence or presence of a sensitive target site. Beside this, qualitative factors like uptake, transport, storage and metabolism also need to be considered. The MDR phenomenon of *B. cinerea* was firstly recorded in 1998. Since than, more data of MDR monitoring were obtained indicating that *B. cinerea* MDR types in combination with other *B. cinerea* resistant types could represent a significant threat for

Bunch rot of grapes is one of the grapevine diseases of great economical importance because it leads to substantial losses in yield and lowering in quality. Vineyard ecosystem is often difficult to manipulate both the crop and its environment. Also, it is a stage where *B. cinerea* can express its dual nature in causing the destructive bunch rot and, under certain conditions, the non-destructive noble rot, which is not paralleled in plant pathology. Noble rot yields vines of a special quality that are high economical. In the continental climate the bunch rot disease can inflict damages up to 50 or 60 percent and under the Mediterranean climate 3 to 5 percent. The damages are continuing in vine making process. Rotting of grape berries caused by fungus is probably old as winegrowing and some descriptions date from time of Roman Plinius the Older (1. century). Even the genus name *Botrytis* is derived from Latin for "grapes like ashes" by Micheli who erected the genus in 1729. Name of disease, grey mould, actually describes the grey coating spread over the bunch especially beacon before vintage when the most damage is already done. The coating is somatic filamentous body or sporulating mycelia of fungus *B. cinerea*. In grapevine *B. cinerea* causes massive losses of yield and quality of grape berries for vine production especially during cool and wet climatic conditions. This fungus is able to act as saprophyte, necrotroph as well as pathogen. In vineyards *B. cinerea* is present as part of the environmental micro-bionta and predominantly being saprophytic it colonize wounds or senescing tissue. From an economic point of view, only while acting as true pathogen infecting flowers and grape berries are of importance in terms of lowering quantity and quality of yield. Although there are numerous scientific contributions that continue to be published, there are still gaps in our knowledge about the etiology and epidemiology of bunch rot disease in vineyards. Disease starts with infections in flowering and even earlier. Establishment of *B. cinerea* on moribund and injured tissues normally allows pathogen to infect health tissues. Source of inoculum which will initiate further cycle of the disease are sclerotia and mycelium formed in the outer layers of the dead bark of shoots, cane or on plant debris of various origin. The sclerotia may be directly infective as sources of conidia yet some sclerotia are not conidia-bearing but form reproductive body apothecia. The ascospores produced from apothecia can also initiate primary infections although sexual stage is not considered as significant for epidemiology of grey mould yet Anotnin de Bary described easily found apothecia on dead vine leaves in late 1866. Sclerotia are rare in the regions with warm dry summers and therefore it is unlikely apothecia will be found either. Sporulation on sclerotia is repeated and this extend period of conidial production and infection. Rain and splashing water under natural conditions dislodge conidia from germinating sclerotia and conidia are dispersing in air currents, in splashing water droplets and by insects. The "fruit fly" *Drosopilla melanogaster* is considered as plurimodal vector of *B. cinerea*. The concentration of conidia in the air is increasing as the grapevines maturing. The mycelium spread through outer layers of the dead bark of shoots and the bark of invade cane is bleached to almost white colour. *Botrytis*

**2. Bunch rot of grapes: High standard disease of grapevine** 

future chemical control of *B. cinerea*.

mycelium sometimes invades the nodes and buds on lower parts of the shoots especially if they had bad wood maturation in the autumn. Buds with dormant mycelia will be finally killed and this will reduce bud burst on the basal parts of the fruit canes in the next spring. Sclerotia and mycelium can also exist on various plants surrounding vineyard and from there conidia disperse in air currents are imported to vineyard. Sclerotia and conidia can be developed on pruned cans left *in situ* or on mummified berries. Abundance of described carry over inoculum in the beginning of new vegetation season at pre-flowering stage is quantitatively related and therefore important for flower infections. Infections are favoured by wet period, at least 12 hours duration, and temperature between 15 and 20 C. Primary infections of grapes occurs at bloom time or at the end of it when *B. cinerea* starts it's life cycle as biotroph infecting flowers through the stigma and style and then into the stylar end to the ovary. Infected flowers are symptomless and only microscopic examination will reveal necrosis of stamens and growth of the pathogen on the style and stigma. These flower infections are invariably followed by a period of latency when fungus remains in a quiescent phase in receptacle area. Flower infection is believed to be an important stage in the epidemiology of *B. cinerea* in grapes. Furthermore, early infections of the generative organs can destroy flower bunches. Infected flowers, also could become potent inocula within developing bunches for berry rot. Because of the abundance of necrotic floral debris in the vineyards, the end of flowering represents an important epidemiological stage for *B. cinerea.* The floral debris provides an excellent nutrient source for the conidia. Floral debris bearing mycelium are dispersed in wind and rain (Jarvis, 1980) onto leaves and berries. After infection at bloom time following symptomless latent phase, generally until berries begin to ripen. Latency could be explained by the ability of the young berries to synthesize stilbenes until veraison (Pezet & Pont, 1992), maintaining the fungus in the receptacle area from where it can spread into the berry during ripening. During the development of berries until veraison, when the berries begin to soften, the berries are resistant to infection. The ripening process corresponds to a senescence process with a degradation of the berry tissues, especially activity allowing disease expression to occur. During this phase, the whole defence mechanisms controlling the pathogen loose their activity, allowing disease expression to occur. Grapevine tissues defend themselves against fungal attack by the accumulation of phytoalexins, like stilbenes, mostly in the green berries but stilbenes appears to be inactive during ripening (Pezet & Pont, 1992; Bais et al., 2000). After veraison the berries become increasingly susceptible to infection. At lower sugar content, less than 13 Brix, the so-called sour rot affects berries and leads very often to a complete loss of attached grapes. Sour rot is favourable with frequent rainfalls. At higher sugar content, attached berries can be processed normally but these forces growers to an earlier harvesting or to picking of moulded grapes. Infections of berries occur at temperature interval between 20 and 25 C and are accomplished by conidia. Germlings that developed from conidia enter grape berries through different pathways, namely through stigmata, pedicels, natural openings and wounds, or by direct penetration of the cuticle (Coertze *et al*., 2001; Holz *et al.*, 2003). Conidia are deposited on berry surphace by air, rain or insects. The most prominent symptom of the disease is found on the berries in the ripening period when the disease reaches its highest stage and lasts up to the end of harvest, being marked by softening and decay of grape berries. Infected berries are dark coloured and show the typical greyish, hairy mycelium all over their surface. Especially sporulating mycelium can be seen to grow along cracks or splits on the berries because tufts of condiophores with conida are protrude from stoma and peristomal cracks on the skin of the berry. The *B. cinerea* can also infect young leaves and relatively older leaves. Leaf infections occur occasionally during long rainy periods with continuous leaf wetness over 48 hours and temperature between 15 and 20 C. Heavy leaves infections are not very common because only long duration of leaf wetness allow mycelium to spread in the mesophyll. Therefore, leaves infections normally take place during rain spring. For the same reason in spring also young shoots can be infected from attached tendrils or small wounds. The quantitative relation between incidence of *B. cinerea* at critical stage in the growth of grapevines; pre-flowering (carry over), flowering and harvest was described by Nair et al. (1995). According to their observation the 50% incidence of *B. cinerea* monitored on grapevine tissues carried over from previous season during pre-flowering can predict 29% primary infections of flowers in the new season.

#### **2.1 Managing grey mould**

Although some prognostic models are developed based on etiology and epidemiology of grey mould disease the severity of the grey mould disease in vineyards cannot be easily predicted so therefore control based on prognosis may not be satisfactory. Effective control of grey mould in vineyard is usually based on preventive repeated fungicide applications during the season. Already the Romans used sulphur to control this disease. For the same purpose sulphur and potassium were recommended in 18th century. During the late 1950s fungicides were introduced in viticulture and until 1968 in many countries for *Botrytis* control were used: sulphamides (dichlofluanid), pthalimides (captan, captafol, folpet) and dithiocarbamate (thiram). At this point of time the efficacy of fungicidal treatments for *Botrytis* control ranged between 20 and 50 percent. All this fungicides were multi-site inhibitors, affecting many target sites in fungal cell and therefore acting as general enzyme inhibitors. In 1960s, first fungicides appeared which act primarily at one target site therefore referred to as single-site or site-specific and they more efficiently control pathogen. Today, several families of synthetic site-specific botryticides are available. They can be classified according to their biochemical modes of action into five categories: 1) anti-microtubule toxicants (benzimidazoles); 2) compounds affecting osmoregulation (dicarboximides, fludioxonil); 3) inhibitors of methionine biosynthesis (anilinopyrimidines) and 4) sterol biosynthesis inhibitors (fenhexamid); 5) fungicides affecting fungal respiration (fluazinam, boscalid and multi-site inhibitors). The era of sigle-site or site specific fungicides begun in late 1960s with introduction of benzimidazoles (benomyl, thiophanate-methyl, carbendazim) that improved *Botrytis* control (Dekker, 1977; Georgopoulos, 1979; Beever & O'Flaherty, 1985). Only a few years later the new group of dicarboximides become available and they shadowed all previously used ingredients. Dicarboximides were introduced into the market between 1975 and 1977 primarily for the control of *B. cinerea* in grapes (Beetz & Löcher, 1979). Due to good efficacy they were popularly named botryticides and it seemed that the problem of protection against *Botrytis* had been successfully solved. Dicarboximides or cyclic imides (e.g. chlozolinate, iprodione, procymidone, vinclozolin) are characterized by the presence of a 3,5-dichlorophenyl group. The activity of dicarboximides fungicides was first reported in the early 1970's with the three key commercial products being introduced within three years; iprodione in 1974 (Lacroix et al., 1974), vinclozolin in 1975 (Pommer & Mangold 1975) while procymidone was registered a year later (Hisada et al., 1976). They are typically protectant fungicides and although some claims to systemicity have been made (Hisada et al.*,* 1976) they are best regarded as protectant materials. In the mid-1990s a novel

young leaves and relatively older leaves. Leaf infections occur occasionally during long rainy periods with continuous leaf wetness over 48 hours and temperature between 15 and 20 C. Heavy leaves infections are not very common because only long duration of leaf wetness allow mycelium to spread in the mesophyll. Therefore, leaves infections normally take place during rain spring. For the same reason in spring also young shoots can be infected from attached tendrils or small wounds. The quantitative relation between incidence of *B. cinerea* at critical stage in the growth of grapevines; pre-flowering (carry over), flowering and harvest was described by Nair et al. (1995). According to their observation the 50% incidence of *B. cinerea* monitored on grapevine tissues carried over from previous season during pre-flowering can predict 29% primary infections of flowers in the

Although some prognostic models are developed based on etiology and epidemiology of grey mould disease the severity of the grey mould disease in vineyards cannot be easily predicted so therefore control based on prognosis may not be satisfactory. Effective control of grey mould in vineyard is usually based on preventive repeated fungicide applications during the season. Already the Romans used sulphur to control this disease. For the same purpose sulphur and potassium were recommended in 18th century. During the late 1950s fungicides were introduced in viticulture and until 1968 in many countries for *Botrytis* control were used: sulphamides (dichlofluanid), pthalimides (captan, captafol, folpet) and dithiocarbamate (thiram). At this point of time the efficacy of fungicidal treatments for *Botrytis* control ranged between 20 and 50 percent. All this fungicides were multi-site inhibitors, affecting many target sites in fungal cell and therefore acting as general enzyme inhibitors. In 1960s, first fungicides appeared which act primarily at one target site therefore referred to as single-site or site-specific and they more efficiently control pathogen. Today, several families of synthetic site-specific botryticides are available. They can be classified according to their biochemical modes of action into five categories: 1) anti-microtubule toxicants (benzimidazoles); 2) compounds affecting osmoregulation (dicarboximides, fludioxonil); 3) inhibitors of methionine biosynthesis (anilinopyrimidines) and 4) sterol biosynthesis inhibitors (fenhexamid); 5) fungicides affecting fungal respiration (fluazinam, boscalid and multi-site inhibitors). The era of sigle-site or site specific fungicides begun in late 1960s with introduction of benzimidazoles (benomyl, thiophanate-methyl, carbendazim) that improved *Botrytis* control (Dekker, 1977; Georgopoulos, 1979; Beever & O'Flaherty, 1985). Only a few years later the new group of dicarboximides become available and they shadowed all previously used ingredients. Dicarboximides were introduced into the market between 1975 and 1977 primarily for the control of *B. cinerea* in grapes (Beetz & Löcher, 1979). Due to good efficacy they were popularly named botryticides and it seemed that the problem of protection against *Botrytis* had been successfully solved. Dicarboximides or cyclic imides (e.g. chlozolinate, iprodione, procymidone, vinclozolin) are characterized by the presence of a 3,5-dichlorophenyl group. The activity of dicarboximides fungicides was first reported in the early 1970's with the three key commercial products being introduced within three years; iprodione in 1974 (Lacroix et al., 1974), vinclozolin in 1975 (Pommer & Mangold 1975) while procymidone was registered a year later (Hisada et al., 1976). They are typically protectant fungicides and although some claims to systemicity have been made (Hisada et al.*,* 1976) they are best regarded as protectant materials. In the mid-1990s a novel

new season.

**2.1 Managing grey mould** 

family of botryticides was arose, the anilinopyrimidines, with three representative ingredients: pyrimethanil, cyprodinil and mepanipyrim. Mepanipyrim and pyrimethanil exhibit a high activity against *B. cinerea,* while cyprodinil came in combination with fludioxonil (phenylpyrroles) in protection of grapes. Pyrimethanil and cyprodinil were introduced in French vineyards in 1994 (Leroux & Gredt, 1995) and in Switzerland they were registered since 1995 (Hilber & Hilber-Bodmer, 1998). In Italy cyprodinil was registered in 1997 (Liguori & Bassi, 1998.). Mepanipyrim was in 1995 registered in Switzerland, Japan and Israel (Muramatsu et al., 1996). Mixture of cyprodinil and fludioxonil was firstly introduced in Switzerland in 1995 (Zobrist & Hess, 1996). In Croatia pyrimethanil was acknowledged in 1997 under the commercial name Mythos and cyprodinil came as a mixture with fludioxonil named Switch while mepanipyrim was not registered at all (Topolovec-Pintarić & Cvjetković, 2003). Although anilinopyirimidines showed to be highly effective against *B. cinerea,* a high risk of resistance build up was already evident in the laboratory investigations at preregistration phase (Birchmore & Forster, 1996). In spite of that they have been registered in most European winegrowing countries since 1994 but with recommendations for restricted use: once per season when anilinopyirimidines are applied alone and a maximum of two applications per season is proposed for the mixture cyprodinil + fludioxonil (phenylpyrrol) (Fabreges & Birchmore, 1998; Leroux, 1995). Shortly after introduction of anilinopyrimidines in 1995 fludioxonil (phenylphyroles) start to be used in vineyards against *B. cinerea*. Fludioxonil is synthetic analouge of antibiotic pyrrolnitril (phenylphyrol), an antibiotic compound produced by a number of *Pseudomonas* spp. and is thought to play a role in biocontrol by these bacteria. Fludioxonil belong to class of fungicides affecting osmoregulation and is inhibitor of both spore germination and hyphal growth. In 1999 fluazinam (phenylpyridinamine) was introduced in French vineyards although in Japan has been used since 1990 against grey mould in various crops. Fluazinam belongs to group of fungicides that affecting fungal respiration so, it shows multi-site activity probably related to uncoupling of mitochondrial oxidative phosphorilation. It is highly toxic to spores and mycelia. Any shift of *B. cinerea* toward fluazinam in vineyards has still not revealed. In 1999, firstly in Switzerland, a botryticide with novel botryticidial action was registered, the fenhexamid (Baroffio et al., 2003). Early investigations on the fenhexamid mode of action suggested that it has different mechanism from than of all other botryticides (Rosslenbroich & Stuebler, 2000). Fenhexamid is a 1,4.hydroxyanilide with a high preventive activity against *B. cinerea*. It is easily degraded and therefore presents a favourable toxicological profile and environmental behaviour (Rosslenbroich et al., 1998; Rosslenbroich & Stuebler, 2000). It is characterized by a long duration action. Due to its lipophilic character it shows rapid uptake into the plant cuticle and within the upper tissue layer limited but significant locosystemic redistribution occurs (Haenssler & Pontzen, 1999) and as a result the rain fastness of fenhexamid is very pronounced. Fenhexamid suppresses the germination of spores only at relatively high concentrations but it is highly effective in inhibiting subsequent stages of infections. After the initiation of spore germination the fenhexamid inhibit the germ-tube elongations, germtubes collapse and die before they are able to penetrate plant surface. Also, treated hyphae frequently show a characteristic leakage of cytoplasm or cell wall associated material at the hyphal tip area (Haenssler & Pontzen, 1999; Debieu et al., 2001). It is sterol biosynthesis inhibitor and blocks the C4-demethylations (Rosslenbroich & Stuebler, 2000). The lastly released botryticide for use in grapevines, in 2004, is novel ingredient boscalid (syn. nicobifen). Boscalid from carboxamide group is systemic and is the only representative of new generation of fungal respiration inhibitors. It act as inhibitor of fungal respiration morover it is new generation of succinate dehydrogenase inhibitors (SDHIs) which inhibit respirations by blocking the ubiquinone-binding site of mitochondrial complex II. In the future, arrivial of new anilide is expected, still described under code SC-0858.

#### **3. Resistance to botryticides**

In *B. cinerea* the resistance phenomenon, as in other plant pathogenic fungi, becomes apparent with the site-specific fungicides. Site-specific or single-site fungicides act primarily at single target under responsibility of single major gene. Thus, just a single gene mutation can cause the target site to alter (monogenic resistance), so as to become much less vulnerable to the fungicide (Brent, 1995). Therefore, within few years of intensive use of such fungicide, in populations of polycyclic pathogen with high propagation rate, can be found a high frequency of resistant mutants. The most common mechanism of fungicide resistance is based on alternations in the fungicide target protein. The resistance to multi-site fungicides, which effect many target sites in fungal cell, has been rarely reported. Multi-site fungicides have been considered as low-risk fungicide from the resistance point of view because they interfere with numerous metabolic steps and cause alternation of cellular structures.

#### **3.1 Retrospective of botryticide resistance**

As it was mentioned earlier, the oldest multi-site fungicides used in vineyards against grey mould, were thiram (dithiocarbamate), captan, folpet (chloromethylmercaptan derivates) chlorotalonil (phthalonitrile) and dichlofluanid (phenylsulphamide). This ingredients react with thiol, SH and amino group inducing formation of thiophosgene and hydrogen disulphide. They block several thiol-containing enzymes involved in respiratory processes during spore germination and this multi-site action is believed to prevent the development of resistance (Leroux et al., 2002). Therefore, they have been considered low-risk fungicide from the resistance point of view. But, in the 1980's strains resistant to dichlofluanid and to the chemically related tolylfluanid, chlorthalonil and even to phthalimides, captan and folpet, have occasionally been reported (Malatrakis, 1989; Rewal et al., 1991; Pollastro et al., 1996). Moreover, cross-resistance among captan, thiram, chlorothalonil and related fungicides were identified (Barak & Edington, 1984). Resistance to dichlofluanid is determined by two major genes, named *Dic1* and *Dic2*, probably involved in a detoxifying mechanism and in glutathione regulation (Pollastro et al., 1996; Leroux et al., 2002). The mutation of this genes lead to the formations of two sensitive phenotypes Dic1S and Dic2S, two phenotypes with low level resistance Dic1LR and Dic2LR and one high leveled resistant phenotype Dic1HR. In practice only a few cases of control failure due to dichlofluanidresistant strains were noted. Although these ingredients are not at risk from resistance development and are still registered their practical use is restricted because they are weak botryticides and their residues can cause problems in vine making process (delay fermentation). First site-specific fungicide used in vineyards since the late 1960's was benzimidazole carbendazim or MBC. But, in the early 1970s, only a few years after commercialization loss of disease control due to resistance was reported in many crops especially in vineyards (Leroux et al., 1998). First report of surprisingly enhanced attacks of *B. cinerea*, rather then suppressed, after benzimidazole treatments was in Germany

new generation of fungal respiration inhibitors. It act as inhibitor of fungal respiration morover it is new generation of succinate dehydrogenase inhibitors (SDHIs) which inhibit respirations by blocking the ubiquinone-binding site of mitochondrial complex II. In the

In *B. cinerea* the resistance phenomenon, as in other plant pathogenic fungi, becomes apparent with the site-specific fungicides. Site-specific or single-site fungicides act primarily at single target under responsibility of single major gene. Thus, just a single gene mutation can cause the target site to alter (monogenic resistance), so as to become much less vulnerable to the fungicide (Brent, 1995). Therefore, within few years of intensive use of such fungicide, in populations of polycyclic pathogen with high propagation rate, can be found a high frequency of resistant mutants. The most common mechanism of fungicide resistance is based on alternations in the fungicide target protein. The resistance to multi-site fungicides, which effect many target sites in fungal cell, has been rarely reported. Multi-site fungicides have been considered as low-risk fungicide from the resistance point of view because they interfere with numerous metabolic steps and cause alternation of cellular

As it was mentioned earlier, the oldest multi-site fungicides used in vineyards against grey mould, were thiram (dithiocarbamate), captan, folpet (chloromethylmercaptan derivates) chlorotalonil (phthalonitrile) and dichlofluanid (phenylsulphamide). This ingredients react with thiol, SH and amino group inducing formation of thiophosgene and hydrogen disulphide. They block several thiol-containing enzymes involved in respiratory processes during spore germination and this multi-site action is believed to prevent the development of resistance (Leroux et al., 2002). Therefore, they have been considered low-risk fungicide from the resistance point of view. But, in the 1980's strains resistant to dichlofluanid and to the chemically related tolylfluanid, chlorthalonil and even to phthalimides, captan and folpet, have occasionally been reported (Malatrakis, 1989; Rewal et al., 1991; Pollastro et al., 1996). Moreover, cross-resistance among captan, thiram, chlorothalonil and related fungicides were identified (Barak & Edington, 1984). Resistance to dichlofluanid is determined by two major genes, named *Dic1* and *Dic2*, probably involved in a detoxifying mechanism and in glutathione regulation (Pollastro et al., 1996; Leroux et al., 2002). The mutation of this genes lead to the formations of two sensitive phenotypes Dic1S and Dic2S, two phenotypes with low level resistance Dic1LR and Dic2LR and one high leveled resistant phenotype Dic1HR. In practice only a few cases of control failure due to dichlofluanidresistant strains were noted. Although these ingredients are not at risk from resistance development and are still registered their practical use is restricted because they are weak botryticides and their residues can cause problems in vine making process (delay fermentation). First site-specific fungicide used in vineyards since the late 1960's was benzimidazole carbendazim or MBC. But, in the early 1970s, only a few years after commercialization loss of disease control due to resistance was reported in many crops especially in vineyards (Leroux et al., 1998). First report of surprisingly enhanced attacks of *B. cinerea*, rather then suppressed, after benzimidazole treatments was in Germany

future, arrivial of new anilide is expected, still described under code SC-0858.

**3. Resistance to botryticides** 

**3.1 Retrospective of botryticide resistance** 

structures.

(Ehrenhardt et al., 1973; Triphati & Schlosser, 1982; Bolton, 1976) but the outbreak of tolerant strains occurred simultaneously in many winegrowing countries in temperate climate. In Switzerland after only two years of use, in 1973, a complete loss of control by benzimidazole was observed and they were withdrawn (Schuepp & Küng, 1981). In Southern Europe where *B. cinerea* pressure is much lower, resistance appeared more slowly. In Mediterranean climate e.g. Italy satisfactory control was reported until 1977 (Bisiach et al., 1978). In Croatia benzimidazoles was used in protection of vineyards shortly from 1971 to 1974. Primarily they were redrawn from use in vineyards because of toxicological reason (negative residues in must and wine). A decrease in efficacy was in Hungary firstly observed in 1981 and it was confirmed by Kaptas & Dula (1984). In 1987 of special interest become mixture of carbendazime and dietophencarb owing to negatively correlated cross-resistance, allowing destruction of benzimidazole-resistant strains by dietophencarb. Soon, negatively correlated cross-resistance become positive as between 1988 and 1989 an overall increase of resistance from 4 to 22% to both components was detected. An explanation of the quick outcome of benzimidazole-resistance was the local existence of naturally resistant strains in the field population of *B. cinerea* before benzimidazole was introduced and their application acted as selected factor eliminating sensitive strains (Schuepp & Lauber, 1978). Benzimidazole carbendazim (MBC) does not affect spore germination but inhibit germ-tube elongation and mycelial growth at low concentrations. These anti-fungal impacts came from MBC binding to tubulin, which is the main protein in microtubules. Microtubules, one type of cytoskeleton filament, regulate organelle position and movement within the cell. Microtubules consist of long, hollow cylinders of repeating dimers of α- and ß-tubulin. MBC binding to tubulin leads to inhibition of the microtubule assembly (Leroux et al., 2002). Alterations in the binding sites on the ß-tubulin protein are related to benzimidazoleresistance (Leroux & Clearjeau, 1985). Approximately 10 mutations conferring resistance to MBC have been identified in the ß-tubulin gene in laboratory studies with a wide range of different fungi. Benzimidazole-resistance in *B. cinerea* is conferred by polyallelic major gene named *Mbc1* by Faretra & Polastro (1991) with at least four classes of alleles responsible for sensitivity or different levels of resistance variously accompanied by hypersensitivity to *N*phenylcarbamates (Faretra et al., 1989; Faretra & Pollastro, 1991, 1993a; Pollastro & Faretra, 1992; Yarden & Katan, 1993; Davidse & Ishii, 1995, De Guido et al., 2007). The presumed mutated locus encoded the structural gene for ß-tubulin and single base pair mutations occurred in codons 198 and 200. Two phenotypes exhibiting benzidimadozle-resistance were determined by Leroux et al (2002) in *B. cinerea* populations from French vineyards. Phenotype Ben R1 exhibit high resistance levels (greater then 250) to MBC is simultaneously more sensitive to phenylcarbamate dietophencarb then the wild type strains. The second phenotype Ben R2 was detected after introduction of the mixture carbendazime+ dietophencarb in 1987. Ben R2 is moderately resistant to MBC (levels 100-200) and insensitive to dietophencarb, just like strains sensitive to MBC. In both phenotypes resistance was conferred by alleles of the *Mbc1*: in Ben R1, at position 198 an alanine replaced a glutamate, whereas in Ben R2, at position 200 a tyrosine replaced a phenylalanine (Yarden & Katan, 1993). Resistance to the MBC is a type of 'qualitative' or 'single-step' resistance characterised by a sudden and marked loss of effectiveness, and by the presence of clear-cut sensitive and resistant pathogen populations with widely differing responses (Brent, 1995). Once developed, it tends to be stable, resistant strains have persisted after many years of non-use and sensitivity will usually not be restored by cessation of their use. Due to stable resistance in vineyards and also for toxicological reason (unwanted toxic residues in vine) MBI were redrawn from use in protection of vineyards.

Benzimidazole carbendazim was followed by dicarboximides which has been available since 1976 (Lorenz & Eichhorn, 1978). Owing to MBI resistance they were welcomed and become recognized as botryticides due to their efficacy superior to formerly used fungicides for that purpose. For almost a decade it seemed that the protection of vineyards against *B. cinerea* had been successfully solved. The appearance of resistance to dicarboximides did not come as so obvious and sudden loss of efficacy that gave first indication of resistance in the case of MBI. Dicarboximides efficacy was diminishing with time and protection slowly become insufficient. Therefore, resistance to dicarboximides, appears to involve slower shifts toward insensitivity because of multiple-gene involvement. As resistance management strategies were poorly understood at that time this inevitably led to dicarboximides overuse and resistance development. In spite of resistance development no total loss of control occurred so dicarboximides use was continued. Moreover, there were no alternative botryticides at the time and as consequence, the proportion of resistant strains in *B. cinerea* population increased considerably. Resistance to dicarboximides *in vitro* was achieved in 1976 (Leroux et al., 1977). Practical dicarboximides-resistance was firstly detected in 1978 in Switzerland (Schüepp & Küng, 1978). The first appearance of resistance in a particular fungicide-pathogen combination in one region has almost always been accompanied, or soon followed, by parallel behaviour in other regions where the fungicide is applied at a similar intensity (Brent, 1995). Thereby, resistance was determined in 1979 in Germany (Holz, 1979) and in Italy (Gulino & Garibaldi, 1979) and in 1982 in France (Leroux & Basselat, 1984; Leroux & Clerjeau, 1985). In Hungary dicarboximides were registered in 1978 and decrease in sensitivity was observed in 1988 and confirmed in 1994 (Dula & Kaptas, 1994). In Slovenian vineyads dicarboximides-resistance was reported (Maček, 1981). In Croatia dicarboximides were introduced in protection of vineyards in 1979. A decrease of efficacy was observed at the end of '80-ties and resistance was proved in 1990 (Cvjetković et al., 1994). Since the beginning of the 1980s, practical resistance to dicarboximides has been related to the selection of moderately resistant strains, named ImiR1 (Leroux & Clerjeau, 1985). Initial studies on dicarboximides-resistance management were started in Germany (Löcher et al., 1985) and France (Leroux & Clerjeau, 1985). To delay the selection of resistant strains during the vegetative period the use of dixarboximides was soon restricted to only two treatments after veraison in Europe (Besselat, 1984; Locher et al., 1987). Unfortunatelly, their efficacy seemed to decrease with infection pressure and goes under 40% and most of the dicarboximides-resistant strains also exhibited high simultaneous resistance to benzimidazoles (Schlamp, 1988). Dicarboximides disturb the synthesis of the cell wall of hyphae by inducing accumulation of glycerol, which burst eventually. A lot of effort was made to investigate primary mode of dicarboximides action. In 1977 was suggested that the primary effect of vinclozolin and iprodion is on DNA production and that lipid metabolism is also affected (Leroux et al., 1977). Following studies showed that dicarboximides have little effect on respiration or the biosynthesis of sterols, nucleic acids, proteins or chitin (Pappas & Fisher, 1979). Edlich & Lyr (1987) described that dicarboximides inactive enzymes are involved in electron transport, causing the production of reactive oxygen products (like O2- and H2O2) and initiate lipid peroxidation. Moreover, enhanced levels of catalase and superoxide dismutase recorded in some dicarboximides-resistant strains could be responsible for the detoxification of peroxy radicals although a conclusive correlation

Due to stable resistance in vineyards and also for toxicological reason (unwanted toxic

Benzimidazole carbendazim was followed by dicarboximides which has been available since 1976 (Lorenz & Eichhorn, 1978). Owing to MBI resistance they were welcomed and become recognized as botryticides due to their efficacy superior to formerly used fungicides for that purpose. For almost a decade it seemed that the protection of vineyards against *B. cinerea* had been successfully solved. The appearance of resistance to dicarboximides did not come as so obvious and sudden loss of efficacy that gave first indication of resistance in the case of MBI. Dicarboximides efficacy was diminishing with time and protection slowly become insufficient. Therefore, resistance to dicarboximides, appears to involve slower shifts toward insensitivity because of multiple-gene involvement. As resistance management strategies were poorly understood at that time this inevitably led to dicarboximides overuse and resistance development. In spite of resistance development no total loss of control occurred so dicarboximides use was continued. Moreover, there were no alternative botryticides at the time and as consequence, the proportion of resistant strains in *B. cinerea* population increased considerably. Resistance to dicarboximides *in vitro* was achieved in 1976 (Leroux et al., 1977). Practical dicarboximides-resistance was firstly detected in 1978 in Switzerland (Schüepp & Küng, 1978). The first appearance of resistance in a particular fungicide-pathogen combination in one region has almost always been accompanied, or soon followed, by parallel behaviour in other regions where the fungicide is applied at a similar intensity (Brent, 1995). Thereby, resistance was determined in 1979 in Germany (Holz, 1979) and in Italy (Gulino & Garibaldi, 1979) and in 1982 in France (Leroux & Basselat, 1984; Leroux & Clerjeau, 1985). In Hungary dicarboximides were registered in 1978 and decrease in sensitivity was observed in 1988 and confirmed in 1994 (Dula & Kaptas, 1994). In Slovenian vineyads dicarboximides-resistance was reported (Maček, 1981). In Croatia dicarboximides were introduced in protection of vineyards in 1979. A decrease of efficacy was observed at the end of '80-ties and resistance was proved in 1990 (Cvjetković et al., 1994). Since the beginning of the 1980s, practical resistance to dicarboximides has been related to the selection of moderately resistant strains, named ImiR1 (Leroux & Clerjeau, 1985). Initial studies on dicarboximides-resistance management were started in Germany (Löcher et al., 1985) and France (Leroux & Clerjeau, 1985). To delay the selection of resistant strains during the vegetative period the use of dixarboximides was soon restricted to only two treatments after veraison in Europe (Besselat, 1984; Locher et al., 1987). Unfortunatelly, their efficacy seemed to decrease with infection pressure and goes under 40% and most of the dicarboximides-resistant strains also exhibited high simultaneous resistance to benzimidazoles (Schlamp, 1988). Dicarboximides disturb the synthesis of the cell wall of hyphae by inducing accumulation of glycerol, which burst eventually. A lot of effort was made to investigate primary mode of dicarboximides action. In 1977 was suggested that the primary effect of vinclozolin and iprodion is on DNA production and that lipid metabolism is also affected (Leroux et al., 1977). Following studies showed that dicarboximides have little effect on respiration or the biosynthesis of sterols, nucleic acids, proteins or chitin (Pappas & Fisher, 1979). Edlich & Lyr (1987) described that dicarboximides inactive enzymes are involved in electron transport, causing the production of reactive oxygen

and H2O2) and initiate lipid peroxidation. Moreover, enhanced levels of

catalase and superoxide dismutase recorded in some dicarboximides-resistant strains could be responsible for the detoxification of peroxy radicals although a conclusive correlation

residues in vine) MBI were redrawn from use in protection of vineyards.

products (like O2-

between amounts of such enzymes and the levels of fungicide resistance has not been found when comparing many field strains and laboratory mutants of *B. cinerea* (Leroux et al., 2002; Edlich & Lyr, 1992). According to Edlich & Lyr (1992) the potential target site of dicarboximides might be a plasma-membrane-bound NADPH-dependent flavin enzyme, inhibition of which would initiate pathological oxidative processes. Therefore, components of glutathione system are targets of dicarboximides. Several findings suggest that they interfere with the osmotic signal transduction pathway consisting of histidine kinase and MAP kinase cascades. Therefore, their primary target sites could be protein kinases involved in the regulation of polyol biosynthesis (Leroux et al., 1999; Schumacher et al., 1997). Set up of target site dicarboximides affecting should enable confirmation of gene responsible for resistance. But, despite of many long-term investigations the mechanism of dicarboximides resistance is not elucidating yet. The most comprehensive data on the genetics of dicarboximides-resistance have been obtained from studies of F. Faretra whose work has clarified the sexual behaviour and matting system of *B. cinerea* and resulted in a reliable technique for obtaining ascospore progeny under laboratory conditions (Faretra & Antonaci, 1987). Resistance to dicarboximides is encoded by a single polyallelic major gene named *Daf1* (Faretra & Antonaci, 1987). Firstly, two alleles of *Daf1* have been recognized (Faretra & Pollastro, 1991): *Daf1* LR and *Daf1* HR responsible for low and high resistance to dicarboximides. Alleles *Daf1* HR also result in hypersensitivity to high osmotic pressure. In further studies conducted with field isolates and laboratory mutants general, was perceived that the resistance mechanism of field isolates differs from that of laboratory isolates. Dicarboximides resistant field isolates were designate as Imi R1 and laboratory mutants as Imi R4 (Leroux et al., 2002). Practical resistance to dicarboximides was only detected with Imi R1 strains (carrying *Daf1* LR alleles) and not with Imi R4 (carrying *Daf1* HR alleles) because of the absence of Imi R4 strains under field conditions. Most dicarboximidesresistant laboratory mutants (Imi R4) acquire high resistance to dicarboximides, but also to aromatic hydrocarbons (AHF) and pheylpyrolles and they are hypersensitive to osmotic stress. High-level dicarboximides-resistant strains of *B. cinerea* have seldom been obtained in the field whereas low- and moderate-level resistant strains (Imi R1) are normally associated with field isolates and are still capable of causing disease control failure. Furthermore, from the field only moderately resistant strains (Imi R1) without osmotic-sensitive phenotypes are recovered (wild type strains are tolerant to osmotic pressure). In addition, dicarboximidesresistant field isolates (Imi R1) show various levels of cross-resistance to aromatic hydrocarbons (AHF) (due to similarity of chemical structure because both have benzene ring in chemical structure) but not to phenylpyrolles (fludioxonil).

Fungicidal toxicity of phenylphyroles is reverted by piperonyl butoxide and α-tocopherol in *B. cinerea*. Different levels of dicarboximides-resistance variously accompanied by resistance to phenylpyrrole fungicides and reduced tolerance to high osmotic pressure point to polymorphism of *Daf1* and with time become evident that there are at least five classes of responsible alleles (Hilber et al., 1995; Faretra & Pollastro, 1991; Faretra & Pollastro, 1993a, 1993b; Vignutelli et al., 2002; Baroffio et al., 2003). Recent studies suggested that an amino acid substitution of serine for isoleucine in the second unit of tandem amino acid repeats on 86 codon of BcOS1p gene is responsible for dicarboximides resistance in the field (Oshima et al., 2002). Preliminary data show that all strains containing a mutation from isoleucine to serine are resistant to dicarboximides without exception. However, some isolates with isoleucine at codon 86 in the second unit are resistant to dicarboximides, suggesting the possibility of other types of resistant strains in the field. Furthermore, Oshima et al. (2002) suggest that most of the mutations within the *BcOS1* gene affect virulence or fitness in *B. cinerea* under field conditions owing to well known fact of dicarboximides-resistant strains rapid decreases after discontinues applications of dicarboximides. According to Leroux (Leroux et al., 2002) dicarboximides-resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Dicarboximides-resistant laboratory strains (Imi R4) contained a single base pare mutation on 325 codon in gene also responasble for histidine kinase. In addition, both field strains Imi R1 and laboratory resistant strains Imi R4 showed resistance to the aromatic hydrocarbon fungicides (AHF) and especially to dicloran which is effective against grey mould on lettuces and on fruits during storage. Other *B. cinerea* isolates, Imi R2 and Imi R3, with different patterns of cross-resistance, were also detected in French vineyards (Leroux et al., 1999). Dicarboximides-resistant strains Imi R2 show crossresistance to both phenylpyrroles and AHFs while Imi R3 are more resistant to dicarboximides then Imi R1 but are weakly resistant to pheylpyrroles. In some *B. cinerea* mutants, fungicide resistance was caused by a mutation in another gene, Daf2, which did not seem to be linked to the Daf1 gene (Faretra & Pollastro, 1993b). Although the primary target site of dicarboximides, phenylpyrroles and AHFs has not been clearly identified, these fungicides are the only commercial ones that seem so far to interfere with plant pathogens through the inhibition of a protein kinase (cit. Leroux et al., 2002). *B. cinerea* practical resistance to phenylpyrroles has not been demonstrated in the vineyards to date.

In the mid-1990s arise a novel family of botryticides, the anilinopyrimidines, with three representative ingredients: pyrimethanil, cyprodinil and mepanipyrim. Although anilinopyirimidines showed to be highly effective against *B. cinerea* a high risk of resistance was already evident in the first laboratory investigations (Birchmore & Forster, 1996) and therefore were put on the market with recommendations for restricted use. In the field pyrimethanil- and cyprodinil-resistant strains of *B. cinerea* were detected during preliminary testing in 1993 and 1994 in French (Leroux & Gredt, 1995) and Swiss vineyards (Forster & Staub, 1996). In Italy resistant strains were detected in 1996 even in vineyards where anilinopyrimidines have never been used before (Gullino & Garibaldi, 1979). Resistance to mepanipyrim was tested only in Japan and was not detected (Muramatsu & Miura, 1996). Organisation FRAC (Fungicide Resistance Action Committee at Global Crop Protection Federation (GCPF)) formed a new working group for anilinopyrimidine-resistance which in 1995 organised "ad hoc EPPO Workshop" in Switzerland and addressed to all winegrowing countries because of: "… *emergent and critical situation of B. cinerea resistance to anilinopyrimidines especially in vineyards*..." Even then was emphasize that efficacy of anilinopyrimidines can be saved and prolonged only with well organized monitoring and antiresistant strategy. Anilinopyrimidines exhibit some systemic translocation in plant tissues, and together with their image of pathogenesis inhibitors they possess protective activity and as it is said also some curative activity. Yet, in order to achieve satisfactory botryticidal effect it is recommended to use them preventively. They do not affect spore germination but germ tube elongation is inhibiting as well as mycelial growth at low concentrations. Under *in vitro* studies toxicity toward mycelial growth depends upon nutrition status of media and is greatly reduced on rich complex media. They posses ability to prevent fungal secretion of hydrolytic enzymes such as protease, cellulase, lipase or cutinase which play an important role in the infection and therefore they are considered as

possibility of other types of resistant strains in the field. Furthermore, Oshima et al. (2002) suggest that most of the mutations within the *BcOS1* gene affect virulence or fitness in *B. cinerea* under field conditions owing to well known fact of dicarboximides-resistant strains rapid decreases after discontinues applications of dicarboximides. According to Leroux (Leroux et al., 2002) dicarboximides-resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Dicarboximides-resistant laboratory strains (Imi R4) contained a single base pare mutation on 325 codon in gene also responasble for histidine kinase. In addition, both field strains Imi R1 and laboratory resistant strains Imi R4 showed resistance to the aromatic hydrocarbon fungicides (AHF) and especially to dicloran which is effective against grey mould on lettuces and on fruits during storage. Other *B. cinerea* isolates, Imi R2 and Imi R3, with different patterns of cross-resistance, were also detected in French vineyards (Leroux et al., 1999). Dicarboximides-resistant strains Imi R2 show crossresistance to both phenylpyrroles and AHFs while Imi R3 are more resistant to dicarboximides then Imi R1 but are weakly resistant to pheylpyrroles. In some *B. cinerea* mutants, fungicide resistance was caused by a mutation in another gene, Daf2, which did not seem to be linked to the Daf1 gene (Faretra & Pollastro, 1993b). Although the primary target site of dicarboximides, phenylpyrroles and AHFs has not been clearly identified, these fungicides are the only commercial ones that seem so far to interfere with plant pathogens through the inhibition of a protein kinase (cit. Leroux et al., 2002). *B. cinerea* practical resistance to phenylpyrroles has not been demonstrated in the vineyards to date. In the mid-1990s arise a novel family of botryticides, the anilinopyrimidines, with three representative ingredients: pyrimethanil, cyprodinil and mepanipyrim. Although anilinopyirimidines showed to be highly effective against *B. cinerea* a high risk of resistance was already evident in the first laboratory investigations (Birchmore & Forster, 1996) and therefore were put on the market with recommendations for restricted use. In the field pyrimethanil- and cyprodinil-resistant strains of *B. cinerea* were detected during preliminary testing in 1993 and 1994 in French (Leroux & Gredt, 1995) and Swiss vineyards (Forster & Staub, 1996). In Italy resistant strains were detected in 1996 even in vineyards where anilinopyrimidines have never been used before (Gullino & Garibaldi, 1979). Resistance to mepanipyrim was tested only in Japan and was not detected (Muramatsu & Miura, 1996). Organisation FRAC (Fungicide Resistance Action Committee at Global Crop Protection Federation (GCPF)) formed a new working group for anilinopyrimidine-resistance which in 1995 organised "ad hoc EPPO Workshop" in Switzerland and addressed to all winegrowing countries because of: "… *emergent and critical situation of B. cinerea resistance to anilinopyrimidines especially in vineyards*..." Even then was emphasize that efficacy of anilinopyrimidines can be saved and prolonged only with well organized monitoring and antiresistant strategy. Anilinopyrimidines exhibit some systemic translocation in plant tissues, and together with their image of pathogenesis inhibitors they possess protective activity and as it is said also some curative activity. Yet, in order to achieve satisfactory botryticidal effect it is recommended to use them preventively. They do not affect spore germination but germ tube elongation is inhibiting as well as mycelial growth at low concentrations. Under *in vitro* studies toxicity toward mycelial growth depends upon nutrition status of media and is greatly reduced on rich complex media. They posses ability to prevent fungal secretion of hydrolytic enzymes such as protease, cellulase, lipase or cutinase which play an important role in the infection and therefore they are considered as inhibitors of pathogenesis (Miura et al., 1994; Milling & Richardson, 1995). In *B. cinerea* anilinopyrimidines prevent secretion of laccase and in grape treated with pyrimethanil reduce laccase activity can be observed (Dubos et al., 1996). Laccasa is phenol oxidase and causes oxidation of must so reduction of their quantity by pyrimethanil is welcome effect. The exact mechanism of action in the protein secretory pathway is not yet understood; it has been hypothesized that the target of anilinopyrimidines could be a step involving the Golgi complex or a later stage (Milling & Rhichardson, 1995; Miura et al., 1994). Anilinopyrimidines are particularly inhibitors of methionine biosynthesis. Enzymes which are involved in methionine biosynthesis are cystathionine γ-sinthase and cystathionine βlyase. The late one might be their primary target site (Leroux et al., 2002). Biochemical studies showed that methionine and homocysteine (prior to methionine) were lower in mycelia after treatment by pyrimethanil while slight increase of precursor cystathionine was recored. However, recent enzymatic studies revealed only weak inhibition of cystathionine β-lyase by anilinopyrimidines (Leroux, 1994; Masner et al., 1994) and conclusive results with the isolated enzyme were not given. Moreover, the relevance of the inhibition of methionine biosynthesis in the fungus while it is invading plant tissue (that offers a rich source of methionine) has yet to be elucidated (Rosslenbroich & Stuebler, 2000). According to Rosslenbroich & Stuebler (2000) the inhibition of methionine biosynthesis and the secretion of hydrolytic enzymes may be associated with the mechanism of antifungal action of the anilinopyrimidines but might also be only a secondary effect. The discovery of *B. cinerea*  strains that exhibit *in vitro* and *in vivo* resistance to anilinopyrimidines suggests that they do not interfere with pathogenesis alone. Based on long-term monitoring conducted since 1993 in French vineyards Leroux (Leroux et al., 1999) distinguished two groups of strains resistant to anilinopyrimidines: I) highly resistant strains with EC 50 greater than 0.5 mg l-1 and II) less resistant strains with EC 50 lower than 0.4 mg l-1 . All AniR strains were *transposa* types but according to their response to other fungicides they proposed following three anilinopyrimidine-resistant phenotypes: AniR1, AniR2 and AniR3. Practical resistance was observed only with AniR1 strains. Phenotype AniR1 was found in most European countries. It is moderately to highly resistant to anilinopyrimidines and *in vitro* response to new experimental anilide SC-0858 and other fungicidal groups is similar to the wild-type strains. Phenotypes AniR2 and AniR3 are weakly resistant to anilinopyrimidines and resistance was mainly recorded at the germ-tube elongation stage. Most important was founding that AniR2 and AniR3 were cross-resistant to chemically unrelated fungicides: dicarboximides, phenylpyrroles, several inhibitors of sterol biosynthesis, fenhexamide, tolnaftate,14 αdemethylation (DMIs), anilide SC-0858 and cyclohexamide (Leroux et al., 1999; Chapeland et al, 1999). AniR3 isolates were also resistant to azole fungicides. Genetic analysis showed that fungicide resistance in phenotype AniR1 is encoded by one major gene (Chapeland et al., 1999; Hilber & Hilber-Bodmer, 1998) but AniR2 and AniR3 are encoded by two different single major genes. Chapeland et al (1999) hypothesize that AniR2 and AniR3 posse's mechanism of resistance which consist of reduced accumulation of fungicides within mycelium and could be mediated by excretion of toxic molecules. Mechanism is correlated with increased mRNA levels of specific transport genes. Phenomenon of reduced accumulation of fungicides is known as "pleiotropic drug resistance" (PDR) or "multi drug resistance" (MDR) which is discussed later in 3.2. Therefore, as these phenotypes are multidrug resistant (MDR) Chapeland et al. (1999) renamed AniR2 as MDR1 and determined them as anilniopyrimidine-resistant strains with considerable cross-resistance levels mainly towards fludioxonil, cyprodinil and tolnaftate. Ani R3 become MDR2 which is characterized by increased resistance to fenhexamid, tolnaftate, cycloheximide, and cyprodinil. A third MDR phenotype, MDR3, was first detected in 2001. It is characterized by the highest levels and broadest spectrum of resistance against most fungicides tested (Kretschmer et al., 2009) contraty to MDR1 and MDR2 which have overlapping but distinct profiles. The frequency of MDR strains in the Champagne steadily increased until 2008, when the three MDR phenotypes together represented 55% of the total population. In contrast to the Champagne in German Wine Road region between 2006 and 2009, increasing MDR populations were also observed, but the MDR1 phenotype was clearly dominating (Kretschmer et al., 2009). In Croatia the occurence of resistant phenotype of *B. cinerea* to pyrimethanil was determined for the first time in 1999 after three years of intensive use of pyrimethanil (Topolovec-Pintarić & Cvjetković, 2002). The cross-resistant strains to cyprodinil were detected also (Topolovec-Pintarić & Cvjetković, 2003). Pyrimethanil- and cyprodinil-resistant strains were also detected in vineyards where anilinopyrimidines had never been used and this strains seemed to be "naturally" resistant and could be of AniR1 type. The growing number of resistant phenotype from the first to the last year of the 3-years trial lead to the conclusion of the appearance of so called "acquired resistance". The testing was conducted *in vitro* by germ tube assay so the resistance was determined in the germ-tube elongation stage. All anilinopyrimidine-resistant strains were *transposa* (Topolovec-Pintarić et al., 2004) so they belong to AniR2 or AniR3. Some of isolates showed cross-resistance to unrelated vinclozolin and fenhexamid (Topolovec-Pintarić, 2009). Described profile imply that this strains could belong to MDR2 type.

In 1999 botryticide with a high preventive activity against *B. cinerea,* fenhexamide, was introduced into vineyards (Baroffio et al., 2003). Owing to its novel mode of action, differing from all other botryticides it was presumed that resistance to fenhexamid will not occur easily. Analyses of unsaponifiable compounds conducted by Debieu et al. (2001) revealed that fenhexamid inhibited sterol biosynthesis in *B. cinerea*. The major sterol constitutes in *B. cinerea*, as well as in most filamentous fungi, is ergosterol. In the presence of fenhexamide ergosterol is reduced while its precursors 4α-methyl and 4-desmethyl 3-keto compounds are accumulated. This indicates that fenhexamid inhibits the 3-keto reductase, one of the four proteins of the enzymatic complex implicated in sterol C-4 demethylation process (Debieu et al, 2001). Thus, inhibition of 3-keto reductase leads to sterol C-4 demethylation inhibition and as a result the 4α-fecosterone and fecosterone are produced. Subsequent isomerization of 4α-fecosterone and fecosterone would give 4α-methylepisterone and episterone. Sterone accumulation is linked to growth inhibition and therefore is responsible for fenhexamide fungi toxicity. High risk of resistance was already evident in the first laboratory investigations. The baseline sensitivity of *B. cinerea* towards fenhexamide was recorded in 1992 and afterwards resistant strains of *B. cinerea* were detected in French and Swiss vineyards but so far loss of the fungicide's effectiveness has never been observed (Leroux et al., 1999; Suty et al., 1997; Baroffio et al., 2003). In France were high level fenhexamidresistant strains collected even before use of fenhexamid. In Switzerland was obtained that fenhexamid-resistance can develop very rapidly, during 4 years from 0% up to 100% (Barofio et al., 2003). Knowledge on the risk of resistance to this fungicide is so far scant, although limiting the number of sprays per season is recommended (de Guido et al., 2007; Fungicide Resistance Action Committee [FRAC], 2006). It seems that fenhexamid resistance is not easily induced in *B. cinerea* because experiments towards selection of laboratory

towards fludioxonil, cyprodinil and tolnaftate. Ani R3 become MDR2 which is characterized by increased resistance to fenhexamid, tolnaftate, cycloheximide, and cyprodinil. A third MDR phenotype, MDR3, was first detected in 2001. It is characterized by the highest levels and broadest spectrum of resistance against most fungicides tested (Kretschmer et al., 2009) contraty to MDR1 and MDR2 which have overlapping but distinct profiles. The frequency of MDR strains in the Champagne steadily increased until 2008, when the three MDR phenotypes together represented 55% of the total population. In contrast to the Champagne in German Wine Road region between 2006 and 2009, increasing MDR populations were also observed, but the MDR1 phenotype was clearly dominating (Kretschmer et al., 2009). In Croatia the occurence of resistant phenotype of *B. cinerea* to pyrimethanil was determined for the first time in 1999 after three years of intensive use of pyrimethanil (Topolovec-Pintarić & Cvjetković, 2002). The cross-resistant strains to cyprodinil were detected also (Topolovec-Pintarić & Cvjetković, 2003). Pyrimethanil- and cyprodinil-resistant strains were also detected in vineyards where anilinopyrimidines had never been used and this strains seemed to be "naturally" resistant and could be of AniR1 type. The growing number of resistant phenotype from the first to the last year of the 3-years trial lead to the conclusion of the appearance of so called "acquired resistance". The testing was conducted *in vitro* by germ tube assay so the resistance was determined in the germ-tube elongation stage. All anilinopyrimidine-resistant strains were *transposa* (Topolovec-Pintarić et al., 2004) so they belong to AniR2 or AniR3. Some of isolates showed cross-resistance to unrelated vinclozolin and fenhexamid (Topolovec-Pintarić, 2009). Described profile imply that this strains could

In 1999 botryticide with a high preventive activity against *B. cinerea,* fenhexamide, was introduced into vineyards (Baroffio et al., 2003). Owing to its novel mode of action, differing from all other botryticides it was presumed that resistance to fenhexamid will not occur easily. Analyses of unsaponifiable compounds conducted by Debieu et al. (2001) revealed that fenhexamid inhibited sterol biosynthesis in *B. cinerea*. The major sterol constitutes in *B. cinerea*, as well as in most filamentous fungi, is ergosterol. In the presence of fenhexamide ergosterol is reduced while its precursors 4α-methyl and 4-desmethyl 3-keto compounds are accumulated. This indicates that fenhexamid inhibits the 3-keto reductase, one of the four proteins of the enzymatic complex implicated in sterol C-4 demethylation process (Debieu et al, 2001). Thus, inhibition of 3-keto reductase leads to sterol C-4 demethylation inhibition and as a result the 4α-fecosterone and fecosterone are produced. Subsequent isomerization of 4α-fecosterone and fecosterone would give 4α-methylepisterone and episterone. Sterone accumulation is linked to growth inhibition and therefore is responsible for fenhexamide fungi toxicity. High risk of resistance was already evident in the first laboratory investigations. The baseline sensitivity of *B. cinerea* towards fenhexamide was recorded in 1992 and afterwards resistant strains of *B. cinerea* were detected in French and Swiss vineyards but so far loss of the fungicide's effectiveness has never been observed (Leroux et al., 1999; Suty et al., 1997; Baroffio et al., 2003). In France were high level fenhexamidresistant strains collected even before use of fenhexamid. In Switzerland was obtained that fenhexamid-resistance can develop very rapidly, during 4 years from 0% up to 100% (Barofio et al., 2003). Knowledge on the risk of resistance to this fungicide is so far scant, although limiting the number of sprays per season is recommended (de Guido et al., 2007; Fungicide Resistance Action Committee [FRAC], 2006). It seems that fenhexamid resistance is not easily induced in *B. cinerea* because experiments towards selection of laboratory

belong to MDR2 type.

mutants resistant to fenhexamid (either spontaneous or UV-induced) produced only few mutants often with aberrant morphology and colony growth. Hence, there are an association between fenhexamid-resistance and reduced fitness (de Guido et al., 2007). Genetic analysis indicated that the resistant phenotypes are encoded by single major gene(s) which is/are not linked with *Mbc1* and *Daf1* (de Guido et al., 2007). Fenhexamid resistance is caused by mutations in the erg27 gene encoded 3-keto reductase according to Fillinger et al. (2008). Alberini et al. (2002) described four phenotypes according to their responses towards fenhexamid: HydS, HydR1, HydR2 and HydR3. A HydS type is wild type sensitive to fenhexamid. HydR1 is naturally fenhexamid-resistant strains with negative cross-resistance to other SBIs (sterol biosynthesis inhibitors) such as prochloraz (14α-demethylase inhibitor or DMI) and higher sensitivity to fenpropidin (Δ14-reductase inhibitor). HydR2 and HydR3 are insensitive to fenhexamid and are similar in lower sensitivity to SBIs fungicides and microtubule inhibitors (carbendazim and dietofencarb). They are representatives of acquired resistance differing in response toward fenhexamid in the stage of germ-tube elongation; HydR2 is weakly resistant while HydR3 is highly resistant to fenhexamid. This suggests that distinct mutation in the same locus or in different loci is involved. Because of their high resistance level Hyd R3 strains have to be considered relative to risk of resistance occurrence. But, their poor overwintering capacities suggesting that they probably do not impact fenhexamid field's efficacy as laboratory investigations as well as field trials indicated (Ziogas et al., 2003; Suty et al, 1997; Kretschmer & Hahn, 2008; Topolovec-Pintarić, 2009; Korolev et al., 2011). For example, in long term trial conducted in vineyard by Suty et al. (1999) fenhexamid was used for 3-4 sprays per year and no reduce of effectiveness was observed although *B. cinerea* isolate less sensitive than normal did appear in the field. The Albertini et al. (2002) analyzed gene CYP51 and determined its DNA sequence. The gene CYP51 was highly polymorphic and this allowed distinction among HydR1 and non-HydR1 strains. At HydR1 CYP51 gene show two non-silent mutations: at position 15 expressed is phenylalanin instead of isoleucin and a serine instead of asparagine at position 105. Recently, Billard et al (2011) described that the major mechanism responsible for fenhexamid-resistance at Hyd R2 and Hyd R3 is fenhexamid detoxification by cytochrome P450 named cyp68.4.

Recently released botryticidal ingredient for use in grapevines was boscalid (carboxamide). Preliminary survey of *B. cinerea* populations from Champagne vineyards did not detect any strains moderately or highly resistant to boscalid and showed the absence of cross-resistance with benzimidazoles, phenylcarbamates and anilinopyrimidines (Leroux et al*.*, 2010). The first resistant strains were found in 2006 in French and German vineyards and their number increased till 2008 (Lerox et al., 2010). Boscalid is the succinate dehydrogenase inhibitor and inhibits the fungal respiration by blocking the ubiquinone-binding site at mitochondrial complex II. Therefore, boscalid-resistance is caused by alterations in the respiratory succinate dehydrogenase (Avenot et al., 2008). The six phenotypes were characterized by Laleve et al (2011) according to their resistance pattern: CarR1-CarR4 with low to medium level of resistance and highly resistant CarR5 and CarR6. CarR1 and CarR2 are currently most frequent in France and Germany. For boscalid-resistance seemes to be responsible mutations in SDH proteins. For all except CarR2 phenotype, putative mutatuion occuring in the sdhB gene lead to a specific amino acid change in the *sdhB* gene. Strains of CarR2 phenotype may be distinguished in at least 3 sub-groups: I) point mutation in the *sdhB* gene, II) point mutation in the *sdhD* gene and III) no mutation in any of the four *sdh* genes. To summarize, *B. cinerea* resistant phenotypes in correlation to fungicides *in vitro* effect towards germ-tube elongation and mycelial growth can be separated into three main categories. Phenotypes which exibit resistance at both stages are: I) Ben R1 and Ben R2 resistant to antimicrotubule toxicants; II) Imi R1 resistant to dicarboximides, III) Ani R1 resistant to anilinopyrimidines and IV) Hyd R3 resistant to fenhexamid. Phenotypes which expressed resistance mainly at stage of germ-tube elongation are only anilinopyrimidine-resistant Ani R2 and Ani R3 (also MDR phenotypes). Finaly, only phenotype Hyd R2 expressed fenhexamid-resistance at mycelial growth stage. Leroux et al (2002) stated that only phenotypes whose exibiting in vitro resistance at both development stages of *B. cinerea* will probably lead to practical resistance. This found not to be true for Hyd R3 phenotype as pratical resistance to fenhexamide has never been recoreded naimly to the rarity of Hyd R3 strains in vineyards. Furthermore, three main mechanisms of *B. cinerea* resistance to botryticides are indentified: reduced penetration of toxicants, increased detoxification or decreased conversion to toxic metabolites and reduced sensitivity of the target site.

#### **3.2 Multi drug resistance phenomenon in** *Botrytis cinerea*

Multiple drug resistance (MDR) phenomenon imply simultaneous reduced sensitivity to several different unrelated compounds. MDR is known as common in human pathogenic microbes and even cancer cells. In agricultural practice obvious cases of MDR in field strains of plant pathogenic fungi are restricted. The classic example of an MDR phenotype in *B. cinerea* is the cross-resistance to aromatic hydrocarbons, dicarboximides and other fungicides. The actual mechanism for this type of B. cinerea mutants has never been elucidated although many putative mechanisms of resistance were suggested. Recently, de Waard et al. (2006) suggested that drug transporters may have played a role in this case of resistance. But, the first expression of *B. cinerea* MDR phenomenon in the vineyard was detected between 1993 and 1997 from French vineyards located in Alsace, Armagnac, Bordeaux, Champagne and Loire Valley and described by Leroux et al. (1999). In some anilinopyrimidine-resistant phenotypes, named AniR2 and AniR3, they observed resistance extending to dicarboximides, phenylpyrroles, sterol biosynthesis inhibitors (e. g. tolfante, prochloraz, tebuconazole), and finally to hydroxyanilide derivate, fenhexamide. In Italy in 1996 anilinopyrimidine-resistant strains were detected even in vineyards where ingredient from this botryticidal group were never used before. Moreover, one of the isolates showed to be resistant simultaneously to fludioxonil, dixarboximides and benzimidazoles although it's virulence was very low. According to Chapeland et al. (1999) three MDR phenotypes can be distinguished in *B. cinerea*: 1) MDR1 strains show reduced sensitivities against fludioxonil and cyprodinil, 2) MDR2 strains are less sensitive to fenhexamid, cyprodinil and iprodione, 3) MDR3 strains are MDR1xMDR2 recombinants and thus show further reduced fungicide sensitivity. Until today MDR strains with additional boscalid resistance were never observed. Recently description of MDR phenothypes was reported in Germany by Leroch et al. (2011). They found most dominant to be MDR1 phenotype but interestingly, they detected MDR2 in low frequencies (2 %) in 2006 but until 2009 their number escalated (up to 26.7 %). Furthermore, MDR2 strains were carbendasim resistant also. Their hypothesis is that MDR2 strains have migrated eastward from Champagne to Germany based on the investigations conducted by Kretschmeir et al. (2009). MDR monitoring data from various investigations indicate that fungicide resistance patterns in *B. cinerea* are following current fungicide treatments. Development of MDR in human pathogens is explained as

summarize, *B. cinerea* resistant phenotypes in correlation to fungicides *in vitro* effect towards germ-tube elongation and mycelial growth can be separated into three main categories. Phenotypes which exibit resistance at both stages are: I) Ben R1 and Ben R2 resistant to antimicrotubule toxicants; II) Imi R1 resistant to dicarboximides, III) Ani R1 resistant to anilinopyrimidines and IV) Hyd R3 resistant to fenhexamid. Phenotypes which expressed resistance mainly at stage of germ-tube elongation are only anilinopyrimidine-resistant Ani R2 and Ani R3 (also MDR phenotypes). Finaly, only phenotype Hyd R2 expressed fenhexamid-resistance at mycelial growth stage. Leroux et al (2002) stated that only phenotypes whose exibiting in vitro resistance at both development stages of *B. cinerea* will probably lead to practical resistance. This found not to be true for Hyd R3 phenotype as pratical resistance to fenhexamide has never been recoreded naimly to the rarity of Hyd R3 strains in vineyards. Furthermore, three main mechanisms of *B. cinerea* resistance to botryticides are indentified: reduced penetration of toxicants, increased detoxification or

decreased conversion to toxic metabolites and reduced sensitivity of the target site.

Multiple drug resistance (MDR) phenomenon imply simultaneous reduced sensitivity to several different unrelated compounds. MDR is known as common in human pathogenic microbes and even cancer cells. In agricultural practice obvious cases of MDR in field strains of plant pathogenic fungi are restricted. The classic example of an MDR phenotype in *B. cinerea* is the cross-resistance to aromatic hydrocarbons, dicarboximides and other fungicides. The actual mechanism for this type of B. cinerea mutants has never been elucidated although many putative mechanisms of resistance were suggested. Recently, de Waard et al. (2006) suggested that drug transporters may have played a role in this case of resistance. But, the first expression of *B. cinerea* MDR phenomenon in the vineyard was detected between 1993 and 1997 from French vineyards located in Alsace, Armagnac, Bordeaux, Champagne and Loire Valley and described by Leroux et al. (1999). In some anilinopyrimidine-resistant phenotypes, named AniR2 and AniR3, they observed resistance extending to dicarboximides, phenylpyrroles, sterol biosynthesis inhibitors (e. g. tolfante, prochloraz, tebuconazole), and finally to hydroxyanilide derivate, fenhexamide. In Italy in 1996 anilinopyrimidine-resistant strains were detected even in vineyards where ingredient from this botryticidal group were never used before. Moreover, one of the isolates showed to be resistant simultaneously to fludioxonil, dixarboximides and benzimidazoles although it's virulence was very low. According to Chapeland et al. (1999) three MDR phenotypes can be distinguished in *B. cinerea*: 1) MDR1 strains show reduced sensitivities against fludioxonil and cyprodinil, 2) MDR2 strains are less sensitive to fenhexamid, cyprodinil and iprodione, 3) MDR3 strains are MDR1xMDR2 recombinants and thus show further reduced fungicide sensitivity. Until today MDR strains with additional boscalid resistance were never observed. Recently description of MDR phenothypes was reported in Germany by Leroch et al. (2011). They found most dominant to be MDR1 phenotype but interestingly, they detected MDR2 in low frequencies (2 %) in 2006 but until 2009 their number escalated (up to 26.7 %). Furthermore, MDR2 strains were carbendasim resistant also. Their hypothesis is that MDR2 strains have migrated eastward from Champagne to Germany based on the investigations conducted by Kretschmeir et al. (2009). MDR monitoring data from various investigations indicate that fungicide resistance patterns in *B. cinerea* are following current fungicide treatments. Development of MDR in human pathogens is explained as

**3.2 Multi drug resistance phenomenon in** *Botrytis cinerea*

consequence of over expression of drug efflux transport proteins. In *B. cinerea*, as in other plant pathogenic fungi this is also one of the various mechanisms that allow them to survive toxic compunds in their environment such as plant defense compounds, antibiotics and fungicides (De Waard et al., 2006). Efflux transporters are plasma membrane factors with low substrate specificity and depended of energy. Mutations leading to over expression of individual transporters can result in increased export of drug molecules back into their outer environment and thereby reduced sensitivity to drug. Thereby, they prevent accumulation of drug up to fungictoxic concentrations at their target sites inside fungal cells, preventing or reducing their toxic action. The major types of drug efflux proteins are ATP binding cassette (ABC) and major facilitator superfamily (MFS) transporters. The role of ABC and MFS transporters in efflux of natural and synthetic toxicants is today well known (De Waard et al., 2006). The genome of *B. cinerea* encodes more than 40 ABC-type, and more than 100 putative MFS-type efflux transporters although most of these transporters are not yet functionally characterized. Several ABC transporter genes have been cloned with variant basal transcript level (Vermeulen et al., 2001): I) undetectable *BcatrC*, *Bcatr*J, II) low level *BactrA*, *BcatrB*, *BcatrE, BcatrG, BcatrK,* and III) high level *BcatrF, BcatrH* and *BcatrI. BcatrB* and *BctrD* are a true multidrug transporters (De Waard et al., 2006; Hayashi et al., 2002). *BacrB* is a determinant for the anilinopyrimidines, dicarboximides, phenyllpyroles fludioxonil and fenpiclonil and to antibiotic phenazines. *BctrD* affects the sensitivity of *B. cinerea* to azole fungicide oxpoconazol, dicarboximides and benzimidazoles as well as to the antibiotic cyclohexamide. Therefore, it is possible that *BcatrB* and *BctrD* function in the MDR1 and MDR2 isolates. Also, *BcatrK* is a determinant for orgnophosphorus fungicide iprobenfos and antibiotic polyoxin. The ABC transporter AtrB1 has been shown to transport a variety of natural and synthetic drugs (Stefanato et al., 2009). Transcription of *atrB* is induced by various drugs, and requires the zinc cluster transcription factor Mrr1. Factor Mrr is present in nucleus but remain inactive in the absence of inducing drugs. Permanent activation of Mrr1 due to mutations in *mrr1*, resulting in constitutive overexpression of *atrB*  and multidrug resistance (MDR) phenotypes. Kretschmer et al. (2009) identified that in MDR1 strains mutations in the transcription factor Mrr1 lead to over expression of the ABC transporters AtrB but in MDR2 strains a promoter rearrangement leads to over expression of the MFS transporters MfsM2.

#### **4. Why is** *Botytis cinerea* **prone to resistance building?**

*B. cinerea* is pathogen which can inflict an extreme number of plants without apparent specialisation which point to considerable variation. Already in 1922 Brierley nicely expressed that as follows: "*Botrytis cinerea is perhaps the commonest and best known fungus and has been the centre of mycological research since the time of de Bary. The species B. cinerea may be visualized as, at any one moment, a cluster of numerous races or strains morphologically congruent on the host plant but in vitro showing marked and constant cultural differences*". Genetic studies of nature and extent of genetic variability in *B. cinerea* were initiated to provide clues to the mechanisms and adaptability on different hosts. The resistance phenomenon intensified the genetic studies of *B. cinerea* assuming limited understanding of the genetic structure is reflecting in difficulties in managing the disease. Also, behavioral differences towards fungicides have provided additional markers for the wide genetic polymorphism encountered in *B. cinerea* (Giraud et al. 1999; Leroux et al. 1999). Genetic studies showed that this fungus has really great morphological variability and metabolic and genetic diversity but the genetic basis for this variability is not elucidated. As the sexual stages (*Botyotinia fuckeliana*) are rarely observed in vineyards it was generally accepted that sexual recombination played no role in somatic and genetic diversity of *B. cinerea* and that therefore diversity must be due to heterokaryosis and aneuploidy. Hence, the high genetic diversity as well as the equal distribution of the two matting types in field populations indicates that sexual reproduction is a major reason for the heterogeneity of *B. cinerea* population (Choquer et al., 2007). There are also other nonexclusive hypotheses under investigation: occurrence of microviruses among wild populations can be one source of variability and other source can be differences in gene content and gene variability among *B. cinerea* strains. The genetic diversity of *B. cinerea* vineyard populations is so opulent that always seems to exceed the sampling size that can be handled in investigations. Recent genetic analysis showed that one of the causes of *B.cinerea* genotype variability could be transposable elements. Investigation on *B.cinerea* populations in Champagne vineyard showed the existence of two sibling sympatric species which can be distinguished according to the content of transposable elements (Giraud et al., 1999). Transposable elements are parts of DNA molecule, jumping genes which can change their position inside genomes, which means to change their position or locus on the chromosomes causing appearances like: gene inactivation, reactivation of pseudogenes, gene expression disorder, and also mutation type: deletion, insertion, inversion and translocation (Daboussi 1996; Daboussi & Capy, 2003). These sympatric species have been described as *Transposa* and *Vacuma* by Giraud et al. (1999). *Transposa* contains two transposable elements: gypsi-like retrotransposon Boty (Diolez et al. 1995) and transposon Flipper while *Vacuma* strains are devoid of both elements (Levis et al., 1997). *Vacuma* is showing higher diversity than *Transposa*. Newly population studies and phylogenetic analyses in France (Fournier et al., 2003) consider *B. cinerea* as a complex of species containing two genetically distinct populations, namely "Group I" (referred as *Botrytis pseudocinerea*) and "Group II" (*B. cinerea sensu stricto*) that were proposed to be phylogenetic species (Choquer et al., 2007). Groups exhibit difference in ecology and their resistance pattern to fungicides but it should be emphasize that they are unable to cross with each other. Group I is characterized by *vacuma* isolates sensitive to fenhexamide which are genetically isolated from others and therefore form a true phylogenetic species. Group II are all fenhexamid-resistant *vacuma* strains and *transposa*. Therefore, they are three main genetic types: Group I, Group II *vacuma* and Group II *transposa*. In the vineyards, as on various host plant, evident are changes in *B. cinerea* population structure and dynamics between genetic types during season. Group I play only a minor role in the epidemiology of bunch rot and is found only sporadically mostly at flowering on leaves and blossoms. Group II *vacuma* reached maximum on senescing floral caps and then decrease significantly until harvest but increase from autumn to flowering. This type of isolates are mostly isolated from over wintering sclerotia and in order with this it is hypothesized that *vacuma* isolates will express a more ruderal life-strategy with greater saprophytic capability (Furnier et al., 2003). Furthermore, it is regarded as a mix of different migrant populations from other host (Giraud et al., 1999). Group II *transposa* is the most virulent one and seems well-adapted to the grapes cause it is dominant at every phenological stage and isolated from over wintering canes more then other types. As it's showing a peak occurrence in rotted grape berries an epidemic of bunch rot is dominated by Group II *transposa* (Matinez et al., 2005). Group II *transposa* it seams to represent commonly occurring population of *B. cinerea* in European vineyards (Kretschmer & Hahn, 2008; Furnier et al., 2003; Topolovec-Pintarić et al., 2004).

but the genetic basis for this variability is not elucidated. As the sexual stages (*Botyotinia fuckeliana*) are rarely observed in vineyards it was generally accepted that sexual recombination played no role in somatic and genetic diversity of *B. cinerea* and that therefore diversity must be due to heterokaryosis and aneuploidy. Hence, the high genetic diversity as well as the equal distribution of the two matting types in field populations indicates that sexual reproduction is a major reason for the heterogeneity of *B. cinerea* population (Choquer et al., 2007). There are also other nonexclusive hypotheses under investigation: occurrence of microviruses among wild populations can be one source of variability and other source can be differences in gene content and gene variability among *B. cinerea* strains. The genetic diversity of *B. cinerea* vineyard populations is so opulent that always seems to exceed the sampling size that can be handled in investigations. Recent genetic analysis showed that one of the causes of *B.cinerea* genotype variability could be transposable elements. Investigation on *B.cinerea* populations in Champagne vineyard showed the existence of two sibling sympatric species which can be distinguished according to the content of transposable elements (Giraud et al., 1999). Transposable elements are parts of DNA molecule, jumping genes which can change their position inside genomes, which means to change their position or locus on the chromosomes causing appearances like: gene inactivation, reactivation of pseudogenes, gene expression disorder, and also mutation type: deletion, insertion, inversion and translocation (Daboussi 1996; Daboussi & Capy, 2003). These sympatric species have been described as *Transposa* and *Vacuma* by Giraud et al. (1999). *Transposa* contains two transposable elements: gypsi-like retrotransposon Boty (Diolez et al. 1995) and transposon Flipper while *Vacuma* strains are devoid of both elements (Levis et al., 1997). *Vacuma* is showing higher diversity than *Transposa*. Newly population studies and phylogenetic analyses in France (Fournier et al., 2003) consider *B. cinerea* as a complex of species containing two genetically distinct populations, namely "Group I" (referred as *Botrytis pseudocinerea*) and "Group II" (*B. cinerea sensu stricto*) that were proposed to be phylogenetic species (Choquer et al., 2007). Groups exhibit difference in ecology and their resistance pattern to fungicides but it should be emphasize that they are unable to cross with each other. Group I is characterized by *vacuma* isolates sensitive to fenhexamide which are genetically isolated from others and therefore form a true phylogenetic species. Group II are all fenhexamid-resistant *vacuma* strains and *transposa*. Therefore, they are three main genetic types: Group I, Group II *vacuma* and Group II *transposa*. In the vineyards, as on various host plant, evident are changes in *B. cinerea* population structure and dynamics between genetic types during season. Group I play only a minor role in the epidemiology of bunch rot and is found only sporadically mostly at flowering on leaves and blossoms. Group II *vacuma* reached maximum on senescing floral caps and then decrease significantly until harvest but increase from autumn to flowering. This type of isolates are mostly isolated from over wintering sclerotia and in order with this it is hypothesized that *vacuma* isolates will express a more ruderal life-strategy with greater saprophytic capability (Furnier et al., 2003). Furthermore, it is regarded as a mix of different migrant populations from other host (Giraud et al., 1999). Group II *transposa* is the most virulent one and seems well-adapted to the grapes cause it is dominant at every phenological stage and isolated from over wintering canes more then other types. As it's showing a peak occurrence in rotted grape berries an epidemic of bunch rot is dominated by Group II *transposa* (Matinez et al., 2005). Group II *transposa* it seams to represent commonly occurring population of *B. cinerea* in European vineyards (Kretschmer & Hahn, 2008; Furnier et al., 2003; Topolovec-Pintarić et al., 2004). According to Martinez et al. (2005) differences in the saprotrophic and pathogenic ability of the *vacuma* and *transposa* combined with a switch in resource availability from dead to living tissues is the most likely mechanism accounting for the success of *transposa* isolates and the decline of *vacuma* isolates during the course of an epidemic. The isolates that had only Boty and therefore they were named Boty only and were detected in France (Giraud et al., 1999) and in Chile (Muñoz et al., 2002). The Boty only isolates were frequently isolated on kiwifruit and in lower frequency on grapes and tomato. It is hypothesized that Boty only isolates may be result of crosses between *vacuma* and *transposa* and the transposon Boty may even be invading isolates of *vacuma* gropus (Muñoz et al., 2002). The isolates containing only Flipper have been found by Albertini et al. (2002). These isolates were origin from France vineyard and from UK strawberry fields and were resistant to fenhexamide. Considering fact that transposones influence genetic changes, especially expression of genes and gene mutations, it was hypothesized about possible relation to fungicide resistance. Some data about possible influence of transposones on the resistance to botryticides were obtained recently (Giraud et al, 1999). Group I is characterized by natural resistance to fenhexamide (Leroux et al., 2002; Suty et al., 1999; Martinez et al., 2005). Group II t*ransposa* strains are multiresistant and frequently resistant to vinclozolin and diethofencarb (Giraud et al., 1999; Martinez et al., 2005). Alberini et al. (2002) described four phenotypes according to their responses towards fenhexamid: HydS, HydR1, HydR2 and HydR3. A HydS type is wild type sensitive to fenhexamid and is either *vacuma* or *transposa*. HydR1 is fenhexamidresistant with negative cross-resistance to other SBIs (sterol biosynthesis inhibitors) such as prochloraz (14α-demethylase inhibitor or DMI) and higher sensitivity to fenpropidin (Δ14 reductase inhibitor). Genetic analyses showed that they are mostly *vacuma* although few was Flipper only type. In comparison to other HydR types conidia of HydR1 are oversized and mycelial growth rate is higher. It should be emphasize that all HydR1 strains are compatible and mating obtains progeny but crosses between HydR1 and HydS failed to obtain progeny. Types HydR2 and HydR3 are of *transposa* type having smaller macroconidia (Giraud et al., 1999) and exhibit slower rates of mycelial extension when grown on highly nutritive agar media at different favorable temperatures (Martinez et al., 2005). They are similar in lower sensitivity to SBIs fungicides and microtubule inhibitors (carbendazim and dietofencarb). They differing in response toward fenhexamid in the stage of germ-tube elongation; HydR2 is weakly resistant while HydR3 is highly resistant to fenhexamid. The Albertini et al. (2002) analyzed gene CYP51 and determined its DNA sequence. The gene CYP51 was highly polymorphic and this allowed distinction among HydR1 and non-HydR1 strains rather then between *vacuma* and *transposa*. At HydR1 CYP51 gene show two non-silent mutations: at position 15 expressed is phenylalanin instead of isoleucin and a serine instead of asparagine at position 105. Absence of genetic exchange between HydR1 and non-HydR1 combined with morphological and somatic incompatibility suggest that these two groups are from distinct genetic entities and might even be non HydR two different species. As types in non HydR group are more variable it is probably composed of different subpopulations whose phylogenetic relationship is still not resolved. The *transposa* isolates resistant to fenhexamid, carbendazim and vinclozolin were detected in other investigations (Martinez et al., 2005; Giraud et al., 1999) and resistance appeared to be associated with an increased virulence. The *transposa* resistant profiles, according to Martinez et al. (2005) are most likely a consequence of population dynamics and are generated by the application scheme of fungicides. Therefore, *transposa* as predominant at veraison is exposed to greater selective pressure of vinclozoline, resulting in a greater frequency of vinclozoline-resistant strains. Also, with speculations about ability of *transposa* isolates to develop fungicide resistance, as well as increased virulence, arises a question if this ability could be based on MDR (multidrug resistance) systems. The MDR system allows efflux of various cytotoxic drugs such as plant defense compounds (e.g., phytoalexins) and botryticidal coumpound. The role of such mechanisms in the population dynamics of *B. cinerea* genetic types warrants further investigation. At the end, *B. cinerea* can be considered as actually a complex of several different entities (Albertini et al., 2002).

#### **5. Conclusion and future prospect**

In order to overcome the problem of *Botrytis* resistance there are continuous world efforts to develop new active ingredients. Monitoring methods were developed and again improving. Antiresistant strategies are applied. However, resistance to botryticides still pursuing to be economically significant problem in *B. cinerea* management as it will be in foreseeable future, mainly due to selection of MDR mutants with high levels of resistance. Ironically, selection of MDR mutant is favoured by some recommendation of antiresistant strategy. In most European vineyards is obey the recommendation to alternate the various groups of botryticides with restriction to one spray per year for each chemical group because they are single-site fungicides and to use fungicide mixtures. However, this antiresistant measure is impeded by the development of MDR phenotypes. This measure delay or prevent the evolution of mutant with target site modifications but presents a multi attack by fungicides which favours stepwise evolution of polygenic resistance related to ABC transporters. On the other hand, gained knowledge of MDR mechanisms could be used as new weapon against not only *B. cinerea* but other phytopathogenic fungi as well. Fungal mutants that lack drug transporters become hypersensitive and can be used as tester strains in the creating new fungicidal compounds. New antifungals perhaps can be compounds that inhibit ABC transporters. Analogues in human medicine exit as blockers or modulators of ABC transporters. Furthermore, these compounds can be synergist of existing fungicides or the ones that annul MDR in phytopathogenic fungus. Another line of new antifungals can be disease control compounds that even do not posses a direct fungitoxic activity and may be considered as inducers of plant defence mechanism. Such compounds can act as inhibitors of ABC transporters involved in fungal virulence which will lead to enhancement of plant defence compounds (phytoalexines, pathogenesis-related proteins) in fungal cells.

Yet after all, the basic for resistance prevention and management remains necessity of updating our knowledge about *B. cinerea,* although it is already abundad and comprehensive. For this goal the data about structure and dynamics of *B. cinerea* populations in commercial vineyards as well as the distribution of different types of fungicide resistance, including MDR types, should be obtained. More attentions should be given to today available alternatives to classical botryticides like biological control, or use of mineral salts and plant activators.

#### **6. Acknowledgements**

I am indebted to Edyta Đermić for her helpful contributions and valuable comments during the writing and revision of this test.

#### **7. References**

36 Fungicides – Beneficial and Harmful Aspects

pressure of vinclozoline, resulting in a greater frequency of vinclozoline-resistant strains. Also, with speculations about ability of *transposa* isolates to develop fungicide resistance, as well as increased virulence, arises a question if this ability could be based on MDR (multidrug resistance) systems. The MDR system allows efflux of various cytotoxic drugs such as plant defense compounds (e.g., phytoalexins) and botryticidal coumpound. The role of such mechanisms in the population dynamics of *B. cinerea* genetic types warrants further investigation. At the end, *B. cinerea* can be considered as actually a complex of several

In order to overcome the problem of *Botrytis* resistance there are continuous world efforts to develop new active ingredients. Monitoring methods were developed and again improving. Antiresistant strategies are applied. However, resistance to botryticides still pursuing to be economically significant problem in *B. cinerea* management as it will be in foreseeable future, mainly due to selection of MDR mutants with high levels of resistance. Ironically, selection of MDR mutant is favoured by some recommendation of antiresistant strategy. In most European vineyards is obey the recommendation to alternate the various groups of botryticides with restriction to one spray per year for each chemical group because they are single-site fungicides and to use fungicide mixtures. However, this antiresistant measure is impeded by the development of MDR phenotypes. This measure delay or prevent the evolution of mutant with target site modifications but presents a multi attack by fungicides which favours stepwise evolution of polygenic resistance related to ABC transporters. On the other hand, gained knowledge of MDR mechanisms could be used as new weapon against not only *B. cinerea* but other phytopathogenic fungi as well. Fungal mutants that lack drug transporters become hypersensitive and can be used as tester strains in the creating new fungicidal compounds. New antifungals perhaps can be compounds that inhibit ABC transporters. Analogues in human medicine exit as blockers or modulators of ABC transporters. Furthermore, these compounds can be synergist of existing fungicides or the ones that annul MDR in phytopathogenic fungus. Another line of new antifungals can be disease control compounds that even do not posses a direct fungitoxic activity and may be considered as inducers of plant defence mechanism. Such compounds can act as inhibitors of ABC transporters involved in fungal virulence which will lead to enhancement of plant

defence compounds (phytoalexines, pathogenesis-related proteins) in fungal cells.

Yet after all, the basic for resistance prevention and management remains necessity of updating our knowledge about *B. cinerea,* although it is already abundad and comprehensive. For this goal the data about structure and dynamics of *B. cinerea* populations in commercial vineyards as well as the distribution of different types of fungicide resistance, including MDR types, should be obtained. More attentions should be given to today available alternatives to classical botryticides like biological control, or use of

I am indebted to Edyta Đermić for her helpful contributions and valuable comments during

different entities (Albertini et al., 2002).

**5. Conclusion and future prospect** 

mineral salts and plant activators.

the writing and revision of this test.

**6. Acknowledgements** 


Choquer, M.; Fournier, E.; Kunz, C.; Levis, C.; Pradier, J.M.; Simon, A. & Viaud, M. (2007).

Coertze, S.; Holz, G. & Sadie, A. (2001). Germination and establishment of infection on grape

Cvjetković, B.; Topolovec-Pintarić, S. & Jurjević, Ž., (1994). Resistance of *Botrytis cinerea* Pers.

Daboussi, M. J. (1996). Fungal transposable elements: generators of diversity and genetic

Daboussi, M. J. & Capy, P. (2003). Transposable elements in filamentous fungi. *Annual* 

Davidse, L. & Ishii, T. (1995). Biochemical and molecular aspects of benzimidazoles, *N*-

Debieu, D.; Bach, J.; Hugon, M.; Malosse, C. & Leroux, P. (2001). The hydroxyanilide

De Guido, M.A. ; De Miccolis Angelini, R.M.; Pollastro, S.; Santomauro, A. & Faretra, F.

Dekker, J. (1977). Resistance, In: *Systemic Fungicides*, Marsh R.W., pp. 176-197. Longman

De Waard, M.A.; Andrade, A.C.; Hayashi, K.; Schoonbeek, H.J.; Stergiopoulos, I. & Zwiers,

Diolez, A.; Marches, F.; Fortini, D. & Brygoo,Y. (1995). Boty, a long terminal repeat

Dubos, B.; Roudet, J. & Lagouarde, P. (1996). The anti-laccase activity of pyrimethanil. Effect

Dula, T. & Kaptas, T. (1994). Monitoring study of the resistance of *Botrytis cinerea* to

Edlich, W. & Lyr H. (1987). Mechanism of action of dicarboximides fungicides. In: *Modern* 

Scientific &Technical, ISBN 0470572507, 9780470572504, London.

*Microbiology*, Vol. 61, No. 1, pp. 103-108, ISSN 0099-2240

Verlag J., pp. 107-118., Longman, London.

*Monograph*, *60: Fungicide resistance*, pp. 239-242. ISSN 0306-3941

tools. *Journal of Genetics,* No*.* 75, pp. 325-339, ISSN 0022-1333

*Review of Microbiology*, No. 57, pp. 275-299, ISSN 0066-4227

Gustav Fischer, ISBN-10: 3334604551, Jena, Germany.

No. 57, pp. 1060–1067, ISSN 1526-4998

1097

0929-1873

498X

0048-4091

677, ISSN 0191-2917

pp. 181-186, ISSN 0567-7572

*Botrytis cinerea* virulence factors :new insights into a necrotrophic and polyphageous pathogen. *FEMS Microbiologial Letters*, No. 277, pp. 1–10, ISSN 0378-

berries by single airborne conidia of *Botrytis cinerea*. *Plant Disease*, No. 85, pp. 668-

ex Fr. to dicarboximides in Croatian vineyards. *Atti Giornate Fitopatologiche*, No. 3,

phenylcarbamates and *N*-phenilformamidoxines and the mechanisms of resistance to these compounds in fungi. In: *Modern Selective Fungicides*, Lyr H., pp. 305-322,

fenhexamid, a new sterol biosynthesis inhibitor fungicide efficient against the plant pathogenic fungus *Botryotinia fuckeliana* (*Botrytis cinerea*). *Pest Management Science,*

(2007). Selection and genetic analysis of laboratory mutants of *Botryotinia fuckeliana*  resistant to fenhexamid. *Journal of Plant Pathology*, Vol. 89, No. 2, pp. 203-210, ISSN

L.H. (2006). Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence. *Pest Management Science,* No. 62, pp. 195–207, ISSN 1526-

retroelement in phytopathogenic fungus *Botrytis cinerea. Applied and Environmental* 

of the anti-*Botrytis* product on harvested grapes. *Phytoma*, No. 483, pp. 47-50, ISSN

benzimidazole nad dicarboximides fungicidies on grapes in Hungary. *BCPC* 

*selective fungicides: Properties, Applications, Mechanisms of action*, Lyr H., Fisher G.,


Gullino, M. L. & Garibaldi, A. (1979). Osservazioni sperimentali dalla resistence di

Haenssler, G. & Pontzen, R. (1999). Effect of fenhexamid on the development of *Botrytis* 

Hayashi, K.; Schoonbeek, H. & De Waard, M. A. (2002). Expression of the ABC transporter

Hilber, U. W.; Schwinn, F.J. & Schüepp, H. (1995). Comparative resistance patterns of

Hilber, U. W. & Hilber-Bodmer, M. (1998). Genetic basis and monitoring of resistance of

Hisada, Y.; Maeda, K.; Tottori, N. & Kawase, Y. (1976). Plant disease control by N-(3,5-

Holz, G. (1979). Űber eine resistenzerscheinung von *Botrytis cinerea* an Reben gegen die

Holz, G.; Gutschow, M.; Coertze, S. & Calitz, F. J. (2003). Occurrence of *Botrytis cinerea* and

Jarvis, W. R. (1980). Epidemiology, In: *The Biology of Botrytis,* Coley-Smith J.R., Verhoeff K. and Jarvis W.R. , pp. 219-250, ISBN 0-12-179850-X, Academic Press, London, UK Kaptas, T. & Dula, B. (1984). Benzimidazoltipusu fungicideekkel szembeni rezistens *Botrytis* 

Korolev, N.; Mamiev, M.; Zahavi, T. & Elad, Y. (2011). Screening of *Botrytis cinerea* isolates

Kretschmer, M. & Hahn, M. (2008). Fungicide resistance and genetic diversity of Botrytis

Kretschmer, M.; Leroch, M.; Mosbach, A.; Walker A. S.; Fillinger, S.; Mernkel, D.;

Lacroix, L.; Bic, C.; Burgaud, L.; Guillot, M.; Leblanc, R.; Riottot, R. & Sauli, M. (1974). Etude

341-350, ISSN 0391-4119

143. pp. 423-428 ISSN 0931-1785

pp. 18-25, ISSN 0508-2404

*Science*, No. 1, pp. 145-149, ISSN 1348-589X

grape. *Plant Disease,* No. 87, pp. 351-358, ISSN 0191-2917

*Pathology*, Vol. 129, No. 4, pp. 591-608, ISSN 0929-1873

(12), e1000696. doi:10.1371/journal.ppat.1000696

No. 115, pp. 214–219, ISSN 1861-3829

176, ISSN 0340-1723

3575

0191-2917

174-182

00318876

isolamenti Italiani di *Botrytis cinerea* a vinclozolin. *La difesa delle piante*, No. 6, pp.

*cinerea*. *Pflanzenschutz-Nachrichten Bayer (Bayer Crop Science journal)*, No. 52, pp. 158–

BcatrD from *Botrytis cinerea* reduces sensitivity to sterol demethylation inhibitor fungicides. *Pesticide Biochemistry and Physiology,* No. 73, pp. 110–121, ISSN 0048-

fludioxonil and vinclozolin in *Botryotinia fuckeliana*. *Journal of Phytopathology*, No.

*Botryotinia fuckeliana* to anilinopyrimidines, *Plant Disease,* No*.* 82, pp. 496-500, ISSN

dichlophenyl)-1,1-dimethyl-cyclopropane-1,2-dicarboxamide. *Journal of Pesticide* 

neuen Kontakt-Botrytizide im Gebiet der Mittelmosel. *Weinberg und Keller*, No. 26,

subsequent disease suppression at different positions on leaves and bunches of

*cinerea* Pers. törzs kialakulasa szölöben. (Resistance to benzimidazole fungicides built.up in strains of *Botrytis cinerea* Pers. in a vineyard). *Növenyvedelen*, No. 20, pp.

from vineyards in Israel for resistance to fungicides. *European Journal of Plat* 

cinerea isolates from a vineyard in Germany. *Journal of Plant Disease and Protecttion,*

Schoonbeek H. J.; Pradier, J. M.; Leroux, P.; De Waard, M. A. & Hahn, M. (2009). Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus *Botrytis cinerea*. *PLoS Pathogens*, Vol. 5, No.

des properties antifongiques d'une nouvelle famille de derives de l'hydantoine et an particulier du 26 019RP. *Phytiatrie-Phytopharmacie*, No. 23, pp. 165-174, ISSN


Löcher, F. J.; Lorenz, G. & Beetz, K. J. (1987). Resistance management strategies for

Maček, J. (1981). O odpornosti sive plesni iz dolenjskih vinogradov proti sistemičnim

Malatrakis, N. E. (1989). Resistance of *Botrytis cinerea* to dichlofluanid in greenhouse

Martinez, F.; Dubos, B. & Fermaud, M. (2005). The role of saprotrophy and virulence in the

Masner, P.; Muster, P. & Schmid, J. (1994). Possible methionine biosynthesis inhibition by pyrimidinamine fungicides. *Pesticide Sciience,* No. 42, pp.163-166, ISSN 0031-613X Milling, R. J. & Richardson, *C.* J., (1995). Mode of action of the anilinopyrimidine fungicide

Miura, I.; Kamakura, T.; Maeno, S.; Hayashi, S. & Yagamuchi, I., (1994). Inhibition of enzyme

Muñoz, G.; Hinrichsen, P.; Brygoo, Y. & Giraud, T. (2002). Genetic characterisation of

Muramatsu, N. & Miura, I. (1996). Methods for evaluating the sensitivity of *Botrytis cinerea*

Nair, N. G.; Guilbaud S.; Barchia I. & Emmett, R. (1995). Significance carryover inoculum,

Oshima, M.; Fujimura, M.; Banno, S.; Hashimoto, C.; Motoyama, T.; Ichiishi, A. &

Pappas, A. C. & Fisher D. J. (1979). A comparison of the mechanisms of action of vinclozolin,

Pezet, R. & Pont, V. (1992). Differing biochemical and histological studies of two grape

in Botrytis research. Pudoc Scientific Publishers, Wageningen, pp. 93-98. Pollastro, S. & Faretra, F. (1992). Genetic characterization of *Botryotinia fuckeliana* (*Botrytis* 

Pollastro, S.; Faretra, F.; Di Canio, V. & De Guido, A, (1996). Characterization and genetic

*Biochemistry and Physiology,* No. 48, pp. 222-228, ISSN 0048-3575

fungicidom. *Sodijsko kmetijstvo*, Vol. 14, No. 7/8, pp. 293-294,

vegetables. *Plant Disease,* No. 73, pp. 138–141 , ISSN 0191-2917

No. 6, pp. 139. ISSN 0261-2194

692-700, ISSN 0031-949X

45, pp. 43-48, ISSN 0031-613X

26, pp. 181-197, ISSN 0250-8052

10, pp. 239-246. ISSN 0031-613X

pp. 148-153, ISSN 0031-9465

*Agriculture*, No. 35, pp. 1177- 1180, ISSN 0816-1089

*Phytopathology*, No. 92, pp. 75-80, ISSN 0031-949X

601, ISSN 0953-7562

dicarboximides fungicides in grapes: Results of six years trial work. *Crop protection*,

population dynamics of *Botrytis cinerea* in vineyards. *Phytopathology,* No. 95, pp.

pyrimethanil. 2. Effects on enzyme excretion in *Botrytis cinerea. Pesticide Science,* No.

secretion in plant pathogens by mepanipyrim, a novel fungicide. *Pesticide* 

*Botrytis cinerea* populations in Chile, *Mycological Research*, Vol. 106, No. 5, pp. 594-

to mepanipyrim using cucumber cotyledons and paper discs. *Bulletin OEPP*, No.

flower infection and latency on the incidence of *B. cinerea* in berries of grapevines at harvest in New South wales, Australia. *Australian Journal of Experimental* 

Yamaguchi, I. (2002). A point mutation in the twocomponent histidine kinase *BcOS-1* gene confers dicarboximide resistance in field isolates of *Botrytis cinerea*.

procimidon, iprodion and prochloraz against *Botrytis cinerea*. *Pesticide Science*, No.

cultivars in the view of their respective susceptibility and resistance to Botrytis cinerea. In: Verhoeff, K., Malathrakis, N. E., Wiliamson, B. (Eds.): Recent advances

*cinerea*) field isolates coupling high resistance to benzimidazoles to insensitivity toward the *N*-phenylcarbamate Diethofencarb. *Phytopathologia Mediterranea,* No. 31,

analysis of field isolates of *Botryotinia fuckeliana* (*Botrytis cinerea*) resistant to

dichlofluanid. *European Journal of Plant Pathology*, No. 102, pp. 607–613, ISSN 0929- 1873


### **Multiple Fungicide Resistance in** *Botrytis***: A Growing Problem in German Soft-Fruit Production**

Roland W. S. Weber and Alfred-Peter Entrop *Esteburg Fruit Research and Advisory Centre, Jork Germany* 

#### **1. Introduction**

44 Fungicides – Beneficial and Harmful Aspects

Topolovec-Pintarić, S. & Cvjetković, B. (2003). *In vitro* sensitivity of *Botrytis cinerea* Pers.:Fr.

Topolovec-Pintarić, S.; Miličević, T. & Cvjetković, B., (2004). Genetic diversity and dynamic

Topolovec-Pintarić, S. (2009). Resistance risk to new botryticides in *Botrytis cinerea* Pers.:Fr.

Triphati, R. K. & Schlosser, E. (1982). The mechanism of resistance of *B. cinerea* to

Vermeulen, T.; Schoonbeek, H. & De Waard, M. A. (2001). The ABC transporter BcatrB from

Vignutelli, A.; Hilber-Bodmer, M. & Hilber, U. W. (2002). Genetic analysis of resistance to

*fuckeliana*. *Mycological Research*, No. 106, pp. 329-335, ISSN 0953-7562 Yarden, O. & Katan, T. (1993). Mutation leading to substitutions at aminoacids 198 and 200

of *Botrytis cinerea*. *Phytopathology,* No. 83, pp. 1478-1483, ISSN 0031-949X Ziogas, B.N.; Markoglou, A.N. & Malandrakis, A.A. (2003). Studies on the inherent

Zobrist, P. & Hess, E. (1996). Performance of a novel cyprodinil/fludioxonil mixture in an

*and Protection*, Vol. 110, No. 1, pp. 54-58, ISSN 1861-3829

*Management Science,* No. 57, pp. 393-402. ISSN 1526-498X

pp. 451-460, ISSN 1861-3829

2, pp. 73–77, ISSN 1861-3829

Vol. 89, No.., pp. 151-156, ISSN 1861-3829

No. 109, pp. 311–317, ISSN 0929-1873

Netherlands

to pyrimethanil and cyprodinil in some Croatian vineyards. *Journal of Plant Diseases* 

of pyrimethnil resistant phenotype in population of *Botrytis cinerea* Pers.:Fr. in one winegrowing area in Croatia. *Journal of Plant Diseases and Protection*, Vol 111, No. 5,

in vinegrowing areas in Croatia. *Journal of Plant Diseases and Protection*, Vol. 116, No.

methylbenzimidazol-2-yl-carbamate (MBC), *Journal of Plant Diseases and Protection*,

*Botrytis cinerea* is a determinant of the phenyllpyrole fungicide fludioxonil. *Pest* 

the phenylpyrrole fludioxonil and the dicarboximide vinclozolin in *Botryotinia* 

of beta-tubulin that correlated with Benomyl-resistance phenotypes of field strains

resistance risk to fenhexamid in *Botrytis cinerea. European Journal of Plant Pathology,*

integrated control and resistance management strategy for *Botrytis* in grapes; *Proceedings of XIth International Botrytis Symposium*, 23-27 June, Wageningen, The In the mild and humid climate of northwestern Europe, fungal pre-harvest rots are a major factor limiting the production of soft-fruits such as strawberries and raspberries. By far the most important fungal disease is grey mould caused by *Botrytis cinerea* Pers.:Fr., now known to be an aggregate of at least two distinct species (Fournier *et al.*, 2005; Giraud *et al.*, 1999). Primary infections are initiated at flowering, resulting in a quiescent colonisation of floral organs (Bristow *et al.*, 1986; Puhl & Treutter, 2008). Upon ripening of the infected fruit, a brown rot becomes apparent (Fig. 1A) which rapidly engulfs the entire fruit, culminating in the production of conidiophores (Fig. 1B). If mild and humid conditions prevail at harvest time, conidia released from fruits with primary infections may infect healthy fruits, causing a catastrophic grey mould epidemic (Fig. 1C). Repeated fungicide applications during flowering are therefore essential to control *B. cinerea* in soft-fruit production.

Fig. 1. Stages of *Botrytis cinerea* infections in soft-fruits. (A) Escape of a limited infection from quiescence in a ripening raspberry fruit. (B) Development of a spreading brown rot with the first crop of conidia on an infected strawberry fruit. (C) Occurrence of severe secondary infections during mild and humid weather in a strawberry field in early July 2007.

Northern Germany is a major soft-fruit producing region comprising some 4,000 ha strawberries, 1,500 ha blueberries, 100 ha raspberries and smaller areas dedicated to gooseberry, redcurrant and blackberry production. During the past five years, severe losses to grey mould were recorded by strawberry and raspberry growers. Damage was caused by pre-harvest rot as well as a reduced shelf-life of marketed produce. Although the weather conditions during harvest were often conducive to secondary infections by *B. cinerea*, several fruit farmers also raised concerns about a reduced efficacy of fungicides.

The past four decades have witnessed considerable changes in regional fungicide spray schedules against *Botrytis*. From about 1965 onwards, the sulphamide compounds dichlofluanid and tolylfluanid were widely used as contact (multi-site) fungicides. Although there are literature reports of the development of partial resistance of *B. cinerea* to sulphamides (Malathrakis, 1989; Pollastro *et al.*, 1996; Rewal *et al.*, 1991), no reductions of field efficacy were observed in Northern German soft-fruit production where sulphamides continued to be used as an alternative or complement to a range of specific, single-site fungicides such as benzimidazoles (infrequently used from 1971 to 1976) or dicarboximides (commonly used in 1979-2009). In February 2007, the registration of the last available multisite fungicide, tolylfluanid, was abruptly withdrawn due to environmental safety concerns. Since then, control of *B. cinerea* has been based solely on compounds with specific (singlesite) modes of action, current representatives being fenhexamid (registered since 1999), QoI fungicides (since 2001), boscalid (since 2009), anilinopyrimidines (since 1999) and fludioxonil (since 1999). At present, three widely used botryticides are Switch® (active ingredients cyprodinil + fludioxonil; Syngenta), Signum® (a.i. pyraclostrobin + boscalid; BASF), and Teldor® (a.i. fenhexamid; Bayer CropScience).

*Botrytis cinerea* is a high-risk pathogen for fungicide resistance development (Brent & Hollomon, 2007; Leroux, 2007), and Northern Germany is a potential 'hot spot' area according to the criteria of Brent & Hollomon (2007). We were concerned about the total reliance on single-site fungicides for grey mould control since 2007, and the lack of any information regarding the current resistance status of regional *Botrytis* populations. In order to address the latter problem, we carried out the surveys and experiments reported here and in previous publications (Weber, 2010b, 2011; Weber & Entrop, 2011; Weber & Hahn, 2011).

#### **2. Fungicide resistance in** *B. cinerea*

*Botrytis cinerea* has earned its reputation as a high-risk pathogen mainly because of its capacity to develop specific resistance to single-site fungicides based on target gene mutations. Specific resistance may emerge within a few years of release of a new fungicide group onto the market, and is usually associated with high resistance factors in laboratory tests. Specific resistance has been described e.g. to benzimidazoles such as benomyl, thiophanate-methyl and carbendazim (Yarden & Katan, 1993; Yourman & Jeffers, 1999), dicarboximides such as iprodione and vinclozolin (Northover & Matteoni, 1986; Yourman & Jeffers, 1999), QoI fungicides (strobilurins; Bardas *et al.*, 2010; Ishii *et al.*, 2009), anilinopyrimidines such as cyprodinil and pyrimethanil (Chapeland *et al.*, 1999; Myresiotis *et al.*, 2007), carboxamides/SDHIs (boscalid; Leroux *et al.*, 2010), and the hydroxyanilide compound fenhexamid (Fillinger *et al.*, 2008; Ziogas *et al.*, 2003). To the best of our knowledge, no reports of specific resistance to the phenylpyrrole compound fludioxonil have been published as yet.

In recent years there have been reports of a non-specific multiple drug resistance (MDR) mechanism based on the activity of ATP binding cassette (ABC) or major facilitator superfamily (MFS) transport proteins in *B. cinerea* (Kretschmer *et al.*, 2009; Vermeulen *et al.*, 2001). MDR phenotypes are usually associated with low or moderate resistance factors, the highest (up to 25-fold relative to baseline sensitivity) being associated with anilinopyrimidines and fludioxonil (Kretschmer *et al.*, 2009).

There is ample evidence that target mutations strongly reduce or even abolish the activity of a fungicide in the field (e.g. Ishii *et al.*, 2009; Myresiotis *et al.*, 2008; Yourman & Jeffers, 1999), and more tentative evidence that this may be the case for MDR phenotypes (Petit *et al.*, 2010; Weber, 2011). Further resistance mechanisms in *B. cinerea* are the metabolisation of fenhexamid (Billard *et al.*, 2011; Leroux *et al.*, 2002) or the role of alternative oxidases in QoI resistance (Wood & Hollomon, 2003), although the impact of either of these mechanisms on the field efficacy of the respective fungicides is far from clear (Leroux, 2007).

#### **2.1 Development of a resistance assay**

46 Fungicides – Beneficial and Harmful Aspects

to grey mould were recorded by strawberry and raspberry growers. Damage was caused by pre-harvest rot as well as a reduced shelf-life of marketed produce. Although the weather conditions during harvest were often conducive to secondary infections by *B. cinerea*, several

The past four decades have witnessed considerable changes in regional fungicide spray schedules against *Botrytis*. From about 1965 onwards, the sulphamide compounds dichlofluanid and tolylfluanid were widely used as contact (multi-site) fungicides. Although there are literature reports of the development of partial resistance of *B. cinerea* to sulphamides (Malathrakis, 1989; Pollastro *et al.*, 1996; Rewal *et al.*, 1991), no reductions of field efficacy were observed in Northern German soft-fruit production where sulphamides continued to be used as an alternative or complement to a range of specific, single-site fungicides such as benzimidazoles (infrequently used from 1971 to 1976) or dicarboximides (commonly used in 1979-2009). In February 2007, the registration of the last available multisite fungicide, tolylfluanid, was abruptly withdrawn due to environmental safety concerns. Since then, control of *B. cinerea* has been based solely on compounds with specific (singlesite) modes of action, current representatives being fenhexamid (registered since 1999), QoI fungicides (since 2001), boscalid (since 2009), anilinopyrimidines (since 1999) and fludioxonil (since 1999). At present, three widely used botryticides are Switch® (active ingredients cyprodinil + fludioxonil; Syngenta), Signum® (a.i. pyraclostrobin + boscalid;

*Botrytis cinerea* is a high-risk pathogen for fungicide resistance development (Brent & Hollomon, 2007; Leroux, 2007), and Northern Germany is a potential 'hot spot' area according to the criteria of Brent & Hollomon (2007). We were concerned about the total reliance on single-site fungicides for grey mould control since 2007, and the lack of any information regarding the current resistance status of regional *Botrytis* populations. In order to address the latter problem, we carried out the surveys and experiments reported here and in previous publications (Weber, 2010b, 2011; Weber & Entrop, 2011; Weber & Hahn, 2011).

*Botrytis cinerea* has earned its reputation as a high-risk pathogen mainly because of its capacity to develop specific resistance to single-site fungicides based on target gene mutations. Specific resistance may emerge within a few years of release of a new fungicide group onto the market, and is usually associated with high resistance factors in laboratory tests. Specific resistance has been described e.g. to benzimidazoles such as benomyl, thiophanate-methyl and carbendazim (Yarden & Katan, 1993; Yourman & Jeffers, 1999), dicarboximides such as iprodione and vinclozolin (Northover & Matteoni, 1986; Yourman & Jeffers, 1999), QoI fungicides (strobilurins; Bardas *et al.*, 2010; Ishii *et al.*, 2009), anilinopyrimidines such as cyprodinil and pyrimethanil (Chapeland *et al.*, 1999; Myresiotis *et al.*, 2007), carboxamides/SDHIs (boscalid; Leroux *et al.*, 2010), and the hydroxyanilide compound fenhexamid (Fillinger *et al.*, 2008; Ziogas *et al.*, 2003). To the best of our knowledge, no reports of specific resistance to the

In recent years there have been reports of a non-specific multiple drug resistance (MDR) mechanism based on the activity of ATP binding cassette (ABC) or major facilitator superfamily (MFS) transport proteins in *B. cinerea* (Kretschmer *et al.*, 2009; Vermeulen *et al.*, 2001).

fruit farmers also raised concerns about a reduced efficacy of fungicides.

BASF), and Teldor® (a.i. fenhexamid; Bayer CropScience).

phenylpyrrole compound fludioxonil have been published as yet.

**2. Fungicide resistance in** *B. cinerea* 

An assay of conidial germination and germ-tube growth on the surface of agar media was developed by Weber & Hahn (2011). For thiophanate-methyl, iprodione, fludioxonil and fenhexamid, 1% (w/v) malt extract agar was used. In order to exclude fenhexamidmetabolising isolates from being scored as false positives, germ-tube growth was also examined on tap-water agar containing a critical fenhexamid concentration (Weber, 2010a). For a test of the QoI fungicide trifloxystrobin, 1% malt extract agar was augmented with the alternative oxidase inhibitor salicyl hydroxamic acid (100 µg ml-1). For the boscalid and cyprodinil assays, 0.5% yeast extract agar or 0.5% sucrose agar (respectively) were used instead of malt extract agar.

Pure-culture isolates of *B. cinerea* were examined for their response to a wide range of fungicide concentrations in order to determine EC50 values under the chosen test conditions (Fig. 2; Table 1). For each fungicide Weber & Hahn (2011) also identified discriminatory concentrations at which highly sensitive or baseline (HS) and less sensitive (LS) isolates could be reliably distinguished from those showing moderate resistance (MR) and high resistance (HR). Examples of dose-response patterns to two fungicide groups are given in Fig. 2.

Fig. 2. Responses of selected isolates of *Botrytis cinerea* to trifloxystrobin (A) and iprodione (B), recorded as germ-tube length after 12 h incubation. Discriminatory concentrations for both assays are indicated by arrows. Adapted from Weber & Hahn (2011).

Based on two discriminatory concentrations for each fungicide (Table 1), a routine test was developed as a decision support tool for fruit farmers and their advisors wishing to optimise their fungicide spray sequence for the control of grey mould. This test had to fulfil certain criteria. Firstly, a relatively high throughput of *B. cinerea* isolates had to be possible in order to quantify the abundance of resistant strains in a field. Further, the test had to deliver fast results preferably prior to flowering, precluding lengthy isolation and cultivation periods. Thirdly, the test had to be simple so as to be usable with basic equipment and by nonspecialised technical staff associated with general microbiology laboratories.

By obtaining conidial suspensions directly from sporulating overwintered plant samples in early spring or from infected fruits at harvest, plating out 10-15 µl drops of suspension onto each of 20 different agar media, and examining germination and/or germ-tube growth with a light microscope after an overnight incubation (Fig. 3), the method was put to practical use during the 2010 and 2011 growing seasons. A high throughput was achieved by accommodating 20-24 drops of different spore suspensions on each agar plate. Samples were collected by the farmers themselves or by their advisors. Results were generated with this method for individual fields, and were communicated to the fruit farmers within 3-7 days of sampling.


1 Abbreviations: HR (highly resistant), HS (highly sensitive), LS (less sensitive), MEA (1% malt extract agar), MR (moderately resistant), SA (0.5% sucrose agar), SHAM (100 ppm salicyl hydroxamic acid), YEA (0.5% yeast extract agar)

2 The HR phenotype of the fenhexamid test on MEA was confirmed by full germ-tube growth on tapwater agar containing 10 ppm fenhexamid.

Table 1. Sensitivity categories and their EC50 values in the conidial germination assay of 7 fungicides (summarised from Weber & Hahn, 2011).

Based on two discriminatory concentrations for each fungicide (Table 1), a routine test was developed as a decision support tool for fruit farmers and their advisors wishing to optimise their fungicide spray sequence for the control of grey mould. This test had to fulfil certain criteria. Firstly, a relatively high throughput of *B. cinerea* isolates had to be possible in order to quantify the abundance of resistant strains in a field. Further, the test had to deliver fast results preferably prior to flowering, precluding lengthy isolation and cultivation periods. Thirdly, the test had to be simple so as to be usable with basic equipment and by non-

By obtaining conidial suspensions directly from sporulating overwintered plant samples in early spring or from infected fruits at harvest, plating out 10-15 µl drops of suspension onto each of 20 different agar media, and examining germination and/or germ-tube growth with a light microscope after an overnight incubation (Fig. 3), the method was put to practical use during the 2010 and 2011 growing seasons. A high throughput was achieved by accommodating 20-24 drops of different spore suspensions on each agar plate. Samples were collected by the farmers themselves or by their advisors. Results were generated with this method for individual fields, and were communicated to the fruit farmers within 3-7

> **EC50 [ppm] HS** <sup>1</sup> **LS MR HR** <sup>2</sup>

> 0.229 - - >100

16.1

9.17- 23.7

0.056 - >100

0.346 - 3.43-

0.748- 1.44

0.301-

0.647 -

28.0- 436

43.9- 96.1

54.5

1.89- 22.7

0.166 - 4.67-

0.976- 3.51

0.019-

0.116-

0.267- 0.482

0.104- 0.178

0.647

0.011

0.099

0.094

0.059

specialised technical staff associated with general microbiology laboratories.

**centration [ppm]** 

+SHAM 0, 0.1, 10 0.007-

1 Abbreviations: HR (highly resistant), HS (highly sensitive), LS (less sensitive), MEA (1% malt extract agar), MR (moderately resistant), SA (0.5% sucrose agar), SHAM (100 ppm salicyl hydroxamic acid),

2 The HR phenotype of the fenhexamid test on MEA was confirmed by full germ-tube growth on tap-

Table 1. Sensitivity categories and their EC50 values in the conidial germination assay of 7

days of sampling.

Thiophanate-

Trifloxystrobin

YEA (0.5% yeast extract agar)

water agar containing 10 ppm fenhexamid.

fungicides (summarised from Weber & Hahn, 2011).

**Fungicide Medium Fungicide con-**

MEA

methyl MEA 1 0, 1, 100 0.047-

Iprodione MEA 0, 5, 50 0.399-

Fenhexamid MEA 0, 1, 50 0.058-

Boscalid YEA 0, 1, 50 0.041-

Cyprodinil SA 0, 1, 25 0.028-

Fludioxonil MEA 0, 0.1, 10 0.013-

Fig. 3. Outline of the conidial germination assay used for resistance monitoring.

#### **2.2 Reproducibility of the assay method**

Overwintered raspberry primocanes infected by *B. cinerea* were collected from selected fields, and incubated in a damp chamber for 2 days. For each cane, conidial suspensions were harvested from three germinated sclerotia separated by a distance of at least 5 cm, and assayed on separate days as described in Fig. 3. The results revealed considerable variations between suspensions obtained from different canes, whereas in most cases the three suspensions collected from any one cane were identical to one another in their fungicide responses (Fig. 4). Further, the observed fungicide resistance phenotypes appeared to be randomly distributed across the field. From these and other results, it was concluded that at least 10 but ideally 15 infected samples should be collected from different parts of the field, and that it was sufficient to analyse one spore suspension from each sample.

Altogether, 93.8% of 1224 conidial suspensions obtained directly from sporulating grey mould lawns on strawberry fruits, raspberry fruits or raspberry canes produced an entirely homogeneous germ-tube growth response to each fungicide, a further 3.4% showing merely minor ambiguities in which less than 10% of conidia differed in their fungicide response from the rest (Weber & Hahn, 2011). The most obvious explanation for such a strong between-sample variation in the near-absence of within-sample variation is that local *B. cinerea* populations may comprise a high genetic diversity with respect to fungicide resistance (Leroch *et al.*, 2011), and that each individual infection is caused by a single germinating conidium.

Fig. 4. Reproducibility of the *B. cinerea* resistance assay with 7 fungicides. In April 2010, 30 overwintered infected raspberry canes were collected from a Northern German field at the positions indicated. For each cane, test results of three conidial suspensions obtained from individual germinating sclerotia are shown in columns.

#### **3. Temporal and spatial distribution of fungicide resistance**

In a large-scale survey carried out in Northern Germany in 2010, Weber (2011) analysed 353 representative *B. cinerea* conidial suspensions from strawberries, raspberries and other softfruits and found that resistance to all 7 fungicides was present at high levels (Table 2). Pure cultures of isolates representing all observed resistance responses to each fungicide were collected. Conidia from MR and/or HR isolates showed a significantly enhanced ability to cause fruit rot in apples pre-treated with the respective fungicides at commercially used concentrations (Weber, 2011), in comparison with sensitive (HS or LS) isolates. In spite of the limitations of the apple inoculation test, these results indicate the possibility that all MR and HR phenotypes identified in the resistance assay might compromise the efficacy of currently registered botryticides in soft-fruit production.


Table 2. Occurrence of fungicide resistance in Northern German soft-fruit production during 2010 (*n* = 353 isolates tested). From Weber (2011).

#### **3.1 The dimension of time**

50 Fungicides – Beneficial and Harmful Aspects

Altogether, 93.8% of 1224 conidial suspensions obtained directly from sporulating grey mould lawns on strawberry fruits, raspberry fruits or raspberry canes produced an entirely homogeneous germ-tube growth response to each fungicide, a further 3.4% showing merely minor ambiguities in which less than 10% of conidia differed in their fungicide response from the rest (Weber & Hahn, 2011). The most obvious explanation for such a strong between-sample variation in the near-absence of within-sample variation is that local *B. cinerea* populations may comprise a high genetic diversity with respect to fungicide resistance (Leroch *et al.*, 2011), and that each individual infection is caused by a single

Fig. 4. Reproducibility of the *B. cinerea* resistance assay with 7 fungicides. In April 2010, 30 overwintered infected raspberry canes were collected from a Northern German field at the positions indicated. For each cane, test results of three conidial suspensions obtained from

individual germinating sclerotia are shown in columns.

germinating conidium.

In Northern Germany, raspberry fields are being cropped for up to 10 years, rendering them useful objects for long-term studies of resistance development. Results of repeated sampling at two commercial sites are presented in Fig. 5, comparing field A under normal management (3-4 annual fungicide applications during flowering) *versus* field B under an intensive crop protection regime with 6-8 applications (Fig. 5A and Fig. 5B, respectively). Both fields had received 1-2 annual applications of fenhexamid during flowering up to and including the year 2008, but none thereafter. Whilst the share of fenhexamid-resistant isolates collapsed in field A, it remained at a high level in field B. On a regional scale, the percentage of fenhexamid resistance in Northern German soft-fruit production has risen from 18.5% in 2009 (Weber, 2010b) to 45.0% in 2010 (Weber, 2011).

Boscalid resistance is another striking case. This compound was not registered for grey mould control in Northern Germany before the 2009 vegetation period, yet by the end of 2010, *i.e.* its second season of use, there was already a 21.5% share of highly resistant isolates across the region (Weber, 2011). Almost simultaneously, boscalid resistant isolates of *B. cinerea* have been described from other regions and other crops (Bardas *et al.*, 2010; Kim & Xiao, 2010; Leroch *et al.*, 2011).

Fig. 5. Percentage of combined moderately and highly fungicide-resistant strains of *B. cinerea* in two Northern German raspberry orchards, one receiving 3-4 annual fungicide sprays at flowering (A), the other 6-8 such treatments (B). In both orchards, fenhexamid was last used in May 2008.

#### **3.2 Selection of resistance by fungicide applications**

Tentative correlations between the frequency of fungicide applications and the resulting percentage of resistant strains may be drawn from the results of the 2010 monitoring (Weber, 2011). Unfortunately, only spray schedules for the flowering period immediately preceding sampling were made available by fruit farmers. Nevertheless, for all chemical

Fig. 5. Percentage of combined moderately and highly fungicide-resistant strains of *B. cinerea* in two Northern German raspberry orchards, one receiving 3-4 annual fungicide sprays at flowering (A), the other 6-8 such treatments (B). In both orchards, fenhexamid was last used

Tentative correlations between the frequency of fungicide applications and the resulting percentage of resistant strains may be drawn from the results of the 2010 monitoring (Weber, 2011). Unfortunately, only spray schedules for the flowering period immediately preceding sampling were made available by fruit farmers. Nevertheless, for all chemical

**3.2 Selection of resistance by fungicide applications** 

in May 2008.

classes except anilinopyrimidines (cyprodinil, pyrimethanil, mepanipyrim) there was a clear trend towards an elevated share of MR and HR isolates in fields which had received several treatments with the respective fungicides at flowering, as compared to those with the lowest number of applications (Table 3). More detailed analyses incorporating up to 3 years' spray history are being conducted in the course of our 2011 resistance survey.


Table 3. Percent of fungicide-resistant isolates in comparison with the number of fungicide applications during the flowering period preceding sampling. Evaluation of data collated by Weber (2011).

In order to examine the relationship between fungicide application frequency and resistance development in greater depth, we are performing a series of forced selection experiments (see Brent & Hollomon, 2007). A preliminary trial was carried out in a raspberry orchard during the 2010 season. Variants included (1) a fungicide-free control, (2) four successive sprays of Teldor® as the sole fungicide during flowering, and (3) the farmer's standard spray sequence (Signum® – Switch® – Signum® – Switch®). Resistance tests were conducted with *B. cinerea* isolates obtained from infected fruits at harvest (Fig. 6).

With only 5.7% grey mould-affected fruits at harvest, the farmer's strategy of alternating treatments with two combination products was successful in terms of crop protection relative to the untreated control (18.8% fruit loss to grey mould). However, as indicated in Fig. 6 there was a significant enrichment of *B. cinerea* strains with boscalid resistance (16%) as compared to the other two variants (2.3-4.0%). This result substantiates preliminary observations (Table 3) and literature reports of the unusually rapid spread of boscalid resistance following the release of this fungicide in 2009 (see Section 3.1).

Four fenhexamid applications at flowering provided an acceptable control of grey mould (8.2% diseased fruits at harvest) but resulted in a significant enrichment of HR strains (26%) as compared to the other two variants (1.1-3.5%). A sequence of four successive fenhexamid applications may seem excessive but would have been in line with the manufacturer's original recommendation when this fungicide was first registered for the control of grey mould in raspberries (Rosslenbroich, 1999). Clearly, therefore, a strategy based on repeated treatments with the same single-site fungicide(s) may provide good crop protection during the first year(s) of fungicide use but could turn out to be non-sustainable due to the rapid enrichment of resistant *B. cinerea* strains*.* In Northern German horticulture there is a veritable history of such boom-and-bust cycles of resistance development within 2-4 years of excessive applications of newly registered fungicides. The best-known examples concern the apple-scab fungus *Venturia inaequalis* which developed resistance to QoI fungicides in 1999 during their third season of intensive use (Palm *et al.*, 2004), and to benzimidazoles in 1974 during their fourth season (Vagt, 1975). Obviously being aware of contemporary reports of benzimidazole resistance in *B. cinerea* from other horticultural situations (Bollen & Scholten, 1971), in a remarkable act of foresight Vagt (1975) cautioned against the continued use of this chemical group for grey mould control in regional soft-fruit production.

Fig. 6. Forced selection experiment in an 8-year-old raspberry orchard in which plots were left untreated at flowering, or treated with four applications of Teldor® (a.i. fenhexamid) or with an alternating sequence of two Signum® and two Switch® applications. Data are shown as percentage of isolates with MR and HR phenotypes to each of the 7 fungicides tested. Error bars indicate standard deviations of four replicates per treatment. Percentages of fruits with grey mould at harvest are indicated. Altogether 290 isolates were assayed at harvest.

#### **3.3 The issue of multiple fungicide resistance**

An important difference between fields A and B shown in Fig. 5 was that strains with multiple resistance were much less common in the former. Thus, only 22.9% of fenhexamidresistant isolates from field A possessed resistance to more than two of the other four currently used fungicides (QoI, boscalid, cyprodinil and fludioxonil) whereas that share was 49.4% in field B. A situation is therefore conceivable in which strains with resistance to a given fungicide may be enriched or maintained in a field even if that compound is no longer used. Such interactions must be taken into consideration when evaluating the 'fitness' of different fungicide resistance phenotypes.

In general terms, there seems to be a trend in *B. cinerea* towards accumulating resistance, as shown also in the large-scale survey by Weber (2011) who grouped his isolates according to multiples of resistance (0 to 5) to the five currently used fungicide groups, *i.e.* fenhexamid,

apple-scab fungus *Venturia inaequalis* which developed resistance to QoI fungicides in 1999 during their third season of intensive use (Palm *et al.*, 2004), and to benzimidazoles in 1974 during their fourth season (Vagt, 1975). Obviously being aware of contemporary reports of benzimidazole resistance in *B. cinerea* from other horticultural situations (Bollen & Scholten, 1971), in a remarkable act of foresight Vagt (1975) cautioned against the continued use of

Fig. 6. Forced selection experiment in an 8-year-old raspberry orchard in which plots were left untreated at flowering, or treated with four applications of Teldor® (a.i. fenhexamid) or with an alternating sequence of two Signum® and two Switch® applications. Data are shown as percentage of isolates with MR and HR phenotypes to each of the 7 fungicides tested. Error bars indicate standard deviations of four replicates per treatment. Percentages of fruits with grey mould at harvest are indicated. Altogether 290 isolates were assayed at harvest.

An important difference between fields A and B shown in Fig. 5 was that strains with multiple resistance were much less common in the former. Thus, only 22.9% of fenhexamidresistant isolates from field A possessed resistance to more than two of the other four currently used fungicides (QoI, boscalid, cyprodinil and fludioxonil) whereas that share was 49.4% in field B. A situation is therefore conceivable in which strains with resistance to a given fungicide may be enriched or maintained in a field even if that compound is no longer used. Such interactions must be taken into consideration when evaluating the 'fitness' of

In general terms, there seems to be a trend in *B. cinerea* towards accumulating resistance, as shown also in the large-scale survey by Weber (2011) who grouped his isolates according to multiples of resistance (0 to 5) to the five currently used fungicide groups, *i.e.* fenhexamid,

**3.3 The issue of multiple fungicide resistance** 

different fungicide resistance phenotypes.

this chemical group for grey mould control in regional soft-fruit production.

QoI compounds, boscalid, anilinopyrimidines, and fludioxonil. There was a significant positive correlation between the number of resistances to current fungicides and the proportion of strains harbouring resistance to iprodione or thiophanate-methyl, two compounds no longer in use. Furthermore, all isolates with quintuple resistance (MR or HR) to current fungicides were obtained from fields with a history of unusually heavy fungicide use.

It is of interest to re-interpret the combinations of resistances in individual isolates (Weber, 2011) in terms of their likely impact on the field efficacy of the commercial products Switch®, Signum® and Teldor®. Thus, even when adding up all fully sensitive isolates as well as those with sensitivity to at least one of the two active ingredients in Signum® or Switch®, merely 38% of all isolates obtained in the 2010 monitoring would have been susceptible to all three products, 40.5% to two, 15% to one, and 6.5% to none of them (Table 4). These data should ring alarm bells with fruit farmers, advisory services and regulatory authorities, especially if a trend towards a further increase in resistance development can be recognised in the results of the ongoing 2011 survey.


1 Meaning sensitivity to at least one of the active ingredients of the commercial product 2 Total = 353 isolates analysed

Table 4. Proportions of *Botrytis* isolates from Northern German soft-fruit fields with putative field sensitivity to the commercial fungicides Switch® (a.i. fludioxonil and cyprodinil), Signum® (a.i. pyraclostrobin and boscalid) and Teldor® (a.i. fenhexamid), as extrapolated from the resistance tests of Weber (2011).

#### **3.4 Factors favouring the spread of fungicide resistance in** *B. cinerea*

From this chapter as well as previous publications (Weber, 2010b, 2011), it is apparent that *B. cinerea* populations associated with Northern German strawberry and raspberry fields harbour an exceptionally high proportion of MR- and/or HR-type resistance against all currently registered fungicide classes, and that resistance development may be on the increase. We are particularly concerned about the possible spread of strains with multiple fungicide resistance because such a development questions the sustainability of the current crop protection practices.

There are several possible reasons to account for the spread of fungicide-resistant strains of *B. cinerea*. Firstly, since February 2007 grey mould control has been based solely on singlesite fungicides, all of which except fludioxonil are known to be susceptible to specific resistance development based on target gene mutations. Secondly, the number of fungicide applications made by fruit farmers during flowering has often exceeded regional recommendations (Weber & Entrop, 2011; see Fig. 5). Autumn-cropping strawberries are of particular concern in this respect because their prolonged flowering periods necessitate more numerous fungicide treatments. Thirdly, the short permissible pre-harvest intervals of 3-7 days for the registered fungicides Switch®, Signum® and Teldor® may have encouraged farmers to spray them against ongoing grey mould epidemics between successive fruit pickings at harvest. Fourthly, restrictions by food retail chains concerning the maximum permissible number of detectable pesticide residues in soft-fruits have forced fruit farmers to abandon resistance management strategies in favour of repeated applications of only one or two of the three main botryticides Switch®, Signum® and Teldor®. As we have seen, there is preliminary experimental evidence that such a strategy may lead to a specific build-up of resistance in selected fields within a single vegetation period (Fig. 6). Fifthly, promises of high degrees of efficacy of these botryticides by agrochemical companies may have encouraged fruit farmers to neglect non-chemical crop protection measures related to sanitation, irrigation, aeration and fertilisation.

#### **4. Recommendations**

There are several ways to support the activity of fungicides in grey mould control. Most of these complementary approaches are matters of basic horticultural knowledge (see e.g. Sutton, 1998; Weber & Entrop, 2011). All of the suggestions made below may not be applicable to all fruit farmers, but most farmers should be able to implement some of them.

#### **4.1 Fungicide applications**

The number of fungicide applications during flowering should be limited to 3-4 sprays at intervals of approx. 7 days. Additional applications do not generally lead to a higher efficacy of grey mould control under Northern German conditions (Faby, 2009) and are counterproductive in terms of resistance management. All three available botryticides should be used, unless site-specific tests have indicated the presence of resistant strains at frequencies at or above 25% (Forster & Staub, 1996; Petit *et al.*, 2010; Weber, 2010a). Switch®, Signum® and Teldor® should not be applied at harvest, as post-harvest treatments e.g. of cane diseases, or indeed at any time of year other than flowering. In view of the widespread resistance to QoI fungicides in *B. cinerea* and their limited efficacy against grey mould, this fungicide class should not be applied singly during flowering.

#### **4.2 Registration of new fungicides**

In general terms, broad-spectrum fungicides such as tolylfluanid, chlorothalonil, folpet or thiram possess a lesser efficacy against *B. cinerea* than single-site fungicides (Gullino *et al.*, 1989; Singh & Milne, 1974; Weber & Entrop, 2011), but they are also less susceptible to resistance development (Leroux, 2007). Future experiments should focus on examining their impact on the development of resistance against the current single-site fungicides when applied as tank mixtures. Detailed experiments under regional conditions are required because previous workers have obtained heterogeneous results (Gullino *et al.*, 1989; Hunter *et al.*, 1987; Northover & Matteoni, 1986). If a suitable broad-spectrum fungicide can be found, this should be registered for grey mould control with some urgency.

Similar trials should also be performed with alternative control agents such as inducers of systemic acquired resistance or biological control organisms. As with broad-spectrum fungicides, efficacy should be evaluated as an effect on the spread of fungicide resistance, rather than (or in addition to) grey mould control *per se.*

#### **4.3 Issues of cultivation**

56 Fungicides – Beneficial and Harmful Aspects

There are several possible reasons to account for the spread of fungicide-resistant strains of *B. cinerea*. Firstly, since February 2007 grey mould control has been based solely on singlesite fungicides, all of which except fludioxonil are known to be susceptible to specific resistance development based on target gene mutations. Secondly, the number of fungicide applications made by fruit farmers during flowering has often exceeded regional recommendations (Weber & Entrop, 2011; see Fig. 5). Autumn-cropping strawberries are of particular concern in this respect because their prolonged flowering periods necessitate more numerous fungicide treatments. Thirdly, the short permissible pre-harvest intervals of 3-7 days for the registered fungicides Switch®, Signum® and Teldor® may have encouraged farmers to spray them against ongoing grey mould epidemics between successive fruit pickings at harvest. Fourthly, restrictions by food retail chains concerning the maximum permissible number of detectable pesticide residues in soft-fruits have forced fruit farmers to abandon resistance management strategies in favour of repeated applications of only one or two of the three main botryticides Switch®, Signum® and Teldor®. As we have seen, there is preliminary experimental evidence that such a strategy may lead to a specific build-up of resistance in selected fields within a single vegetation period (Fig. 6). Fifthly, promises of high degrees of efficacy of these botryticides by agrochemical companies may have encouraged fruit farmers to neglect non-chemical crop protection measures related to

There are several ways to support the activity of fungicides in grey mould control. Most of these complementary approaches are matters of basic horticultural knowledge (see e.g. Sutton, 1998; Weber & Entrop, 2011). All of the suggestions made below may not be applicable to all fruit farmers, but most farmers should be able to implement some of them.

The number of fungicide applications during flowering should be limited to 3-4 sprays at intervals of approx. 7 days. Additional applications do not generally lead to a higher efficacy of grey mould control under Northern German conditions (Faby, 2009) and are counterproductive in terms of resistance management. All three available botryticides should be used, unless site-specific tests have indicated the presence of resistant strains at frequencies at or above 25% (Forster & Staub, 1996; Petit *et al.*, 2010; Weber, 2010a). Switch®, Signum® and Teldor® should not be applied at harvest, as post-harvest treatments e.g. of cane diseases, or indeed at any time of year other than flowering. In view of the widespread resistance to QoI fungicides in *B. cinerea* and their limited efficacy against grey mould, this

In general terms, broad-spectrum fungicides such as tolylfluanid, chlorothalonil, folpet or thiram possess a lesser efficacy against *B. cinerea* than single-site fungicides (Gullino *et al.*, 1989; Singh & Milne, 1974; Weber & Entrop, 2011), but they are also less susceptible to resistance development (Leroux, 2007). Future experiments should focus on examining their impact on the development of resistance against the current single-site fungicides when applied as tank mixtures. Detailed experiments under regional conditions are required

sanitation, irrigation, aeration and fertilisation.

fungicide class should not be applied singly during flowering.

**4. Recommendations** 

**4.1 Fungicide applications** 

**4.2 Registration of new fungicides** 

Crop cultivation should aim to maximise yields as well as create conditions which optimise fungicide efficacy. Nitrogen fertilisation of strawberries should be reduced to about 60 kg ha-1 *per annum* which is sufficient to ensure a good yield whilst avoiding soft plant tissues susceptible to *B. cinerea* infections. Periods of leaf wetness should be reduced to a minimum by using drip irrigation if possible, or alternatively by using rather than extending natural periods of leaf wetness for overhead irrigation. Strawberry or raspberry crops grown in poly-tunnels or under fleece for early cropping should be aerated regularly in order to reduce periods of leaf wetness. Likewise, the planting distance should be sufficient to ensure a good ventilation of leaves and flowers.

Hygiene measures aimed at reducing inoculum should be given a high priority. Thus, rotting or mouldy fruits should be collected separately and removed from the field especially during the first pickings of the season. Likewise, infected raspberry canes should be pruned and removed from the field before bud burst.

#### **4.4 Outlook**

In contrast to pome fruit farming, there is no commercially relevant organic soft-fruit production in Germany, the chief reason being dramatic pre- and post-harvest losses to grey mould. Clearly, non-chemical crop protection measures alone are insufficient to provide acceptable control of *B. cinerea* in Northern Germany. However, as we have discussed in the present chapter, a similar situation may soon hold for the current crop protection strategy based on specific fungicides. Fungicide resistance development in *B. cinerea* should catalyse a change of paradigm towards a truly integrated crop protection concept embracing both chemical and supplementary non-chemical measures. There should be ample rewards for fruit farmers willing to embark on such a concept because of a stable demand for fresh and regionally produced soft-fruits on the market.

#### **5. References**


Bollen, G.J. & Scholten, G. (1971). Acquired resistance to benomyl and some other systemic

Brent, K.J. & Hollomon, D.W. (2007). *Fungicide Resistance: The Assessment of Risk* (second

Bristow, P.R., McNicol, R.J. & Williamson, B. (1986). Infection of strawberry flowers by

Chapeland, F., Fritz, R., Lanen, C., Gredt, M. & Leroux, P. (1999). Inheritance and

Faby, R. (2009). Termine und Häufigkeit der Botrytis-Anwendungen in Erdbeeren.

Fillinger, S., Leroux, P., Auclair, C., Barreau, C., al Hajj, C. & Debieu, D. (2008). Genetic

Forster, B. & Staub, T. (1996). Basis for use strategies of anilinopyrimidine and

Fournier, E., Giraud, T, Albertini, C. & Brygoo, Y. (2005). Partition of the *Botrytis cinerea*

Giraud, T., Fortini, D., Levis, C., Lamarque, C., Leroux, P., LoBuglio, K. & Brygoo, Y. (1999).

Gullino, M.L., Aloi, C. & Garibaldi, A. (1989). Influence of spray schedules on fungicide

Hunter, T., Brent, K.J., Carter, G.A. & Hutcheon, J.A. (1987). Effects of fungicide spray

Ishii, H., Fountaine, J., Chung, W.-H., Kansako, M., Nishimura, K., Takahashi, K. & Oshima,

Kim, Y.K. & Xiao, C.L. (2010). Resistance to pyraclostrobin and boscalid in populations of

Kretschmer, M., Leroch, M., Mosbach, A., Walker, A.-S., Fillinger, S., Mernke, D.,

*Plant Pathology* Vol.95, Suppl.1, pp.87-94, ISSN 0028-2944.

www.frac.info/frac/publication/anhang/FRAC\_Mono2\_2007.pdf

*Pathology* Vol.77, No.3, pp.83-90, ISSN 0028-2944.

*Biology* Vol.109, No.3, pp.545-554, ISSN 1744-7348.

ISSN 0048-3575.

ISSN 0178-2916.

ISSN 0066-4804.

ISSN 0031-949X.

4746.

4998.

pp.529-537, ISSN 0261-2194.

pp.1251-1267, ISSN 0027-5514.

pp.604-612, ISSN 0191-2917.

fungicides in a strain of *Botrytis cinerea* in cyclamen. *Netherlands Journal of Plant* 

edition). Croplife International, Brussels, Belgium. Avaliable from http://

*Botrytis cinerea* and its relevance to grey mould development. *Annals of Applied* 

mechanisms of resistance to anilinopyrimidine fungicides in *Botrytis cinerea* (*Botryotinia fuckeliana*). *Pesticide Biochemistry and Physiology* Vol.64, No.2, pp.85-100,

*Mitteilungen des Obstbauversuchsringes des Alten Landes* Vol.64, No.4, pp.148-153,

analysis of fenhexamid-resistant field isolates of the phytopathogenic fungus *Botrytis cinerea. Antimicrobial Agents and Chemotherapy* Vol.52, No.11, pp.3933-3940,

phenylpyrrole fungicides against *Botrytis cinerea*. *Crop Protection* Vol.15, No.6,

complex in France using multiple gene genealogies. *Mycologia* Vol.97, No.6,

Two sibling species of the *Botrytis cinerea* complex, *transposa* and *vacuma*, are found in sympatry on numerous host plants. *Phytopathology* Vol.89, No.10, pp.967-973,

resistant populations of *Botrytis cinerea* Pers. on grapevine. *Netherlands Journal of* 

regimes on incidence of dicarboximide resistance in grey mould (*Botrytis cinerea*) on strawberry plants. *Annals of Applied Biology* Vol.110, No.3, pp.515-525, ISSN 0003-

M. (2009). Characterisation of QoI-resistant field isolates of *Botrytis cinerea* from citrus and strawberry. *Pest Management Science* Vol.65, No.8, pp.916-922, ISSN 1526-

*Botrytis cinerea* from stored apples in Washington State. *Plant Disease* Vol.94, No.5,

Schoonbeek, H.-J., Pradier, J.-M., Leroux, P., De Waard, M.A. & Hahn, M. (2009).

Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus *Botrytis cinerea*. *PLoS Pathogens* Vol.5, No.12, e1000696.


### **Fenhexamid Resistance in the** *Botrytis* **Species Complex, Responsible for Grey Mould Disease**

A. Billard1,2 et al\* *1INRA UR 1290 BIOGER-CPP Thiverval-Grignon, 2Bayer SAS, Bayer CropScience, Research Center La Dargoire, Lyon, France* 

#### **1. Introduction**

60 Fungicides – Beneficial and Harmful Aspects

Rewal, N., Coley-Smith, J.R. & Sealy-Lewis, H.M. (1991). Studies on resistance to

Rosslenbroich, H.-J. (1999). Efficacy of fenhexamid (KBR2738) against *Botrytis cinerea* and

Singh, G. & Milne, K.S. (1974). Field evaluation of fungicides for the control of

Sutton, J.C. (1998). Botrytis fruit rot (gray mold) and blossom blight. In: *Compendium of* 

Vagt, W. (1975). Die Schorfsituation 1974 und unsere Spritzempfehlungen für 1975.

Vermeulen, T., Schoonbeek, H. & de Waard, M. (2001). The ABC transporter BcatrB from

Weber, R.W.S. (2011). Resistance of *Botrytis cinerea* to multiple fungicides in Northern

Weber, R.W.S. & Entrop, A.-P. (2011). Auftreten, Bedeutung und Vermeidung von Fungizid-

Wood, P.M. & Hollomon, D.W. (2003). A critical evaluation of the role of alternative oxidase

*Botrytis cinerea. Phytopathology* Vol.83, No.12, pp.1478-1483, ISSN 0031-949X. Yourman, L.F. & Jeffers, S.N. (1999). Resistance to benzimidazole and dicarboximide

Ziogas, B.N., Markoglou, A.N. & Malandrakis, A.A. (2003). Studies on the inherent

pp.554-560, ISSN 1365-3059.

89053-194-9, St. Paul, USA.

179, ISSN 1861-3829.

Vol.2, No.2, pp.185-188, ISSN 0301-5521.

ISSN 0340-1723.

0178-2916.

0191-2917.

ISSN 1861-3829.

pp.569-575, ISSN 0191-2917.

Vol.109, No.4, pp.311-317, ISSN 0929-1873.

dichlofluanid and other fungicides in *Botrytis cinerea. Plant Pathology* Vol.40, No.4,

related fungal pathogens. *Pflanzenschutz-Nachrichten Bayer* Vol.52, No.2, pp.127-144,

chrysamthemum flower blight. *New Zealand Journal of Experimental Agriculture*

*Strawberry Diseases* (second edition), J.L. Maas (Ed.), pp.28-31, APS Press, ISBN 0-

*Mitteilungen des Obstbauversuchsringes des Alten Landes* Vol.30, No.3, pp.76-80, ISSN

*Botrytis cinerea* is a determinant of the activity of the phenylpyrrole fungicide fludioxonil. *Pest Management Science* Vol.57, No.5, pp.393-402, ISSN 1526-4998. Weber, R.W.S. (2010a). Schnelle und einfache Methode zum Nachweis der Fenhexamid-Resistenz bei *Botrytis. Erwerbs-Obstbau* Vol.52, No.1, pp.27-32, ISSN 0014-0309. Weber, R.W.S. (2010b). Occurrence of Hyd R3 fenhexamid resistance among *Botrytis* isolates

in Northern Germany. *Journal of Plant Diseases and Protection* Vol.117, No.4, pp.177-

German small fruit production. *Plant Disease* Vol.95, No.10, pp.1263-1269, ISSN

Resistenzen bei *Botrytis* an Erdbeeren und Himbeeren. *Mitteilungen des Obstbauversuchsringes des Alten Landes* Vol.66, No.5, pp.136-144, ISSN 0178-2916. Weber, R.W.S. & Hahn, M. (2011). A rapid and simple method for determining fungicide

resistance in *Botrytis*. *Journal of Plant Diseases and Protection* Vol.118, No.1, pp.17-25,

in the performance of strobilurin and related fungicides acting at the Qo site of complex III. *Pest Management Science* Vol.59, No.5, pp.499-511, ISSN 1526-4998. Yarden, O. & Katan, T. (1993). Mutations leading to substitutions at amino acids 198 and 200

of beta-tubulin that correlate with benomyl-resistance phenotypes of field strains of

fungicides in greenhouse isolates of *Botrytis cinerea. Plant Disease* Vol.83, No.6,

resistance risk to fenhexamid in *Botrytis cinerea*. *European Journal of Plant Pathology*

#### **1.1 Chemical control of grey mould in French vineyards**

The three major fungal pests of grapevine, powdery and downy mildew and grey mould are mostly controlled through the application of fungicides. Some of those are particularly active against the grey mould agent *Botrytis cinerea*. The panel of fungicides authorized in France comprise since many years anilinopyrimidines, benzimidazoles, dithiocarbamates, dicarboximides, phenylpyrroles and pyridinamines. Lately, the panel has been completed by fenhexamid, a sterol biosynthesis inhibitor (SBI) and the pyridine boscalid, a succinate dehydrogenase inhibitor (SDHI). In addition, a biofungicide based on the *Bacillus subtilis* strain QST 713 (Serenade) has been authorized in 2010 against grey mould in vineyards. The compounds authorized against grey mould in French vineyards are listed in Table 1.

Since grey mould may infect grapevine from flowering until harvest, optimal protection needs to be obtained during this period. Nowadays up to three treatments per season are recommended in vineyards, corresponding to the stages A-C (A: flower cap falling – B: bunch closure – C: veraison). To reduce pesticide applications along with the general trend of reduction of chemical inputs in agriculture chemical treatments against grey mould are positioned according to epidemiological and meteorological parameters. Grey mould not only affects quantity of harvest but also the wine quality. Therefore, treatments also depend on the economic value of the wine. The number of treatments is variable between regions and years according to the factors cited above.

In those regions with regular applications of anti-*Botrytis* fungicides, especially in the Northern regions, resistant strains have been selected which can ultimately lead to treatment failure. In order to reduce the risk of specific resistance development each anti-*Botrytis* mode-of-action is limited to one application/season in France since the 90's, involving alternations of different chemical families to combat grey mould in the vineyards.

 \* S. Fillinger1, P. Leroux1, J. Bach1, C. Lanen1, H. Lachaise2, R. Beffa3 and D. Debieu1

*<sup>1</sup> INRA UR 1290 BIOGER-CPP Thiverval-Grignon, France,* 

*<sup>2</sup> Bayer SAS, Bayer CropScience, Research Center La Dargoire, Lyon, France,* 

*<sup>3</sup> Bayer CropScience AG, Frankfurt, Germany.* 


Table 1. Active substances approved against grey mould of grapevine in France and recommended stages of applications (French National *Botrytis* Note - Vigne 2010). For further explanations, see text.

#### **1.2 Fenhexamid: A single mode of action**

Fenhexamid is a narrow spectrum fungicide (Rossenbloich and Stuebler*,* 2000) belonging to the chemical family of hydroxyanilides inhibiting sterol biosynthesis (SBI)(Debieu *et al.,*  2001). Bayer CropScience has introduced it to the French fungicide market more than 10 years ago as a specific anti-*Botrytis*. It is principally used in vineyards, but also on tomato and strawberry cultures up to two treatments per season (Couteux *&* Lejeune, 2003). Fenhexamid differs from other known SBIs (allylamines, azoles, imidazoles and morpholines/amines) by the enzymatic step that it inhibits. Sterols are essential lipid compounds found in all eukaryotes. They are principally localized to cytoplasmic or endomembranes, although the latter are composed predominantly of phospholipids. Sterols participate in membrane permeability and rigidity, in the formation of lipid rafts and, eventually, in post-translational control of membrane proteins (Hac-Wydro *et al.,* 2007; Yeagle, 1990; Epand, 2008; Gilbert, 2010; Paila *&* Chattopadhyay, 2010). The major fungal sterol is ergosterol and specific to this kingdom. Therefore, many attempts to develop targets for antifungal treatments have been orientated towards the ergosterol biosynthesis pathway. Noticeably, all SBIs used against fungal diseases target enzymatic steps that are conserved among all kingdoms and not fungal specific.

The major class of SBIs used in agriculture represented by azoles, imidazoles and triazolinethions inhibits the 14-demethylase enzyme therefore called DMIs (demethylation inhibitors), amines inhibit the ∆14-reductase and/or the ∆8∆7-isomerase. The youngest SBI used against grey mould, fenhexamid, blocks the demethylation at C4 of sterols. This important process involves three different enzymes: Erg25 (C4-methyle oxidase), Erg26 (C3 dehydrogenase, C4-decarboxylase) and Erg27 (3-ketoreductase). The first two enzymes eliminate methyl groups at C4 leading to a keto group at C3. The Erg27 enzyme then reduces this keto group to a hydroxyl (Gachotte *et al.,* 1999) leading to a functional sterol.

Chemical families Fungicides Timing of application

A, B or C

applications/year

Pyrimethanil, Mepanipyrim, Cyprodinil

Benzimidazoles Thiophanate-methyl A or B Carboxamides Boscalid A, B or C

Hydroxyanilides Fenhexamid A, B or C Dicarboximides Iprodione A, B or C Phenylpyrroles Fludioxonil A or B Pyridinamines Fluazinam A, B or C

(biofungicide) Serenade up to 8

Table 1. Active substances approved against grey mould of grapevine in France and recommended stages of applications (French National *Botrytis* Note - Vigne 2010).

Fenhexamid is a narrow spectrum fungicide (Rossenbloich and Stuebler*,* 2000) belonging to the chemical family of hydroxyanilides inhibiting sterol biosynthesis (SBI)(Debieu *et al.,*  2001). Bayer CropScience has introduced it to the French fungicide market more than 10 years ago as a specific anti-*Botrytis*. It is principally used in vineyards, but also on tomato and strawberry cultures up to two treatments per season (Couteux *&* Lejeune, 2003). Fenhexamid differs from other known SBIs (allylamines, azoles, imidazoles and morpholines/amines) by the enzymatic step that it inhibits. Sterols are essential lipid compounds found in all eukaryotes. They are principally localized to cytoplasmic or endomembranes, although the latter are composed predominantly of phospholipids. Sterols participate in membrane permeability and rigidity, in the formation of lipid rafts and, eventually, in post-translational control of membrane proteins (Hac-Wydro *et al.,* 2007; Yeagle, 1990; Epand, 2008; Gilbert, 2010; Paila *&* Chattopadhyay, 2010). The major fungal sterol is ergosterol and specific to this kingdom. Therefore, many attempts to develop targets for antifungal treatments have been orientated towards the ergosterol biosynthesis pathway. Noticeably, all SBIs used against fungal diseases target enzymatic steps that are

The major class of SBIs used in agriculture represented by azoles, imidazoles and triazolinethions inhibits the 14-demethylase enzyme therefore called DMIs (demethylation inhibitors), amines inhibit the ∆14-reductase and/or the ∆8∆7-isomerase. The youngest SBI used against grey mould, fenhexamid, blocks the demethylation at C4 of sterols. This important process involves three different enzymes: Erg25 (C4-methyle oxidase), Erg26 (C3 dehydrogenase, C4-decarboxylase) and Erg27 (3-ketoreductase). The first two enzymes eliminate methyl groups at C4 leading to a keto group at C3. The Erg27 enzyme then reduces this keto group to a hydroxyl (Gachotte *et al.,* 1999) leading to a functional sterol.

Dithiocarbamates Thiram A

Anilinopyrimidines

*Bacillus subtilis* 

For further explanations, see text.

**1.2 Fenhexamid: A single mode of action** 

conserved among all kingdoms and not fungal specific.

This last step is the target of fenhexamid. Its mode-of-action has been uncovered in 2001, by the characterization of sterol composition of fenhexamid treated *B. cinerea* strains. Debieu and co-workers, observed an accumulation of compounds with a keto group at C3, suggesting the inhibition of the 3-ketoreductase step (Debieu *et al.,* 2001). Since this enzymatic step is conserved in all euascomycetes, one may expect fenhexamid to be a large spectrum fungicide as are the DMIs instead of its narrow spectrum (see paragraph 2.3).

#### **1.3 Resistance to fenhexamid in the** *Botrytis* **species complex**

The activity spectrum of a new fungicide is generally established by biological tests on diverse fungal species allowing distinguishing sensitive and tolerant species. While naturally resistant - or tolerant species - are insensitive to a given fungicide prior to its introduction and defining the fungicide's spectrum of activity, sensitive fungal species can evolve and select less susceptible - or resistant - isolates. Natural polymorphism or randomly occurring mutations may confer a certain level of resistance to some strains. Through the application of the corresponding fungicide, these isolates are preferentially selected and their proportion within the population will increase (selection pressure of the fungicide). The differentiation between natural and acquired resistance can sometimes lead to the identification of new fungal species; e.g. *Oculimacula yallundae* and *O. acuformis:* the second species is naturally resistant to triazole SBIs. Another example is *Rhizoctonia solani* which is subdivided into different anastomosis groups some of which are tolerant to pencycuron, an anti-microtubular agent (Campion *et al.,* 2005).


a concentration of fenhexamid (mg l-1) inhibiting growth at 50%.

Table 2. Sensitivities of resistant phenotypes towards fenhexamid on germ tube elongation and mycelium growth stages. HydS= sensitive to fenhexamid; HydR= resistant to fenhexamid; MDR= multidrug resistant.

Also in the case of grey mould, it became clear that the species later on named *Botrytis pseudocinerea* a close relative of *B. cinerea,* was naturally resistant to fenhexamid at the stage of mycelial growth (Fournier *et al.,* 2005, Walker *et al.,* 2011). Later, resistant isolates were selected from the *B. cinerea sensu stricto* populations, initially sensitive to fenhexamid. Until now, four different fenhexamid resistance categories could be identified among grey mould populations (Table 2): HydR1, corresponding to the naturally resistant species *B. pseudocinerea* and the *B. cinerea* phenotypes MDR, HydR2 and HydR3 (Leroux *et al*, 2002).

For the phenotypes HydR1 (*B. pseudocinerea*) and HydR2 (*B. cinerea*), high to moderate resistance levels respectively, are observed nearly exclusively at the level of mycelial growth, whereas the other phenotypes display similar resistance levels at both developmental stages, germ tube elongation and mycelial growth. In addition, *B. pseudocinerea* (HydR1) presents increased sensitivities to other fungicides, such as DMIs, fenproprimorph, fenpropidine as well as to some SDHIs (Leroux *et al*., 2002). Only few HydR2 strains have been isolated and to our knowledge, they have not yet been found in French vineyard populations. On the opposite, HydR3 phenotypes are regularly found since 2003. This category has been subdivided into two sub-classes, HydR3 or HydR3+ with moderate or high resistance levels respectively.

MDR2 (and MDR3) phenotypes display weak to moderate resistance levels (resistance factor <15) to various unlinked mode-of-actions including fenhexamid, SDHIs, dicroboximides, anilinopyrimidines etc. This **m**ulti**d**rug **r**esistance (MDR) is linked to the over expression of a membrane transporter leading to increased drug-efflux outside the fungal cell (Kretschmer *et al.,* 2009). Anti-resistance strategies involving systematic alterations of different mode-ofactions seem to favor the selection of these MDR phenotypes instead of specific resistances, in particular in regions with more than one treatment per season such as the Champagne wine region.

#### **2. Acquired resistance to fenhexamid in** *Botrytis cinerea*

#### **2.1 Target alterations cause reduced affinity of fenhexamid and are responsible for resistance**

In agriculture, acquired resistance towards agrochemical products is frequently conferred by target site modifications leading to decreased inhibitor affinities. Target overproduction is rarely found. Pesticide detoxification is a mechanism particularly important for insecticide resistance involving diverse enzymes such as hydrolases, cytochrome P450 monooxygenases, glutathione-S-transferases or glycosyl transferases. In rare cases, resistance results from nonactivation of pro-pesticides. Concerning antifungal compounds, the principal resistance mechanisms are target site modifications and increased efflux, in the agronomical field as well as in the medical field (Ma & Michaelides, 2005; Sanglard *et al.*, 2009).

With respect to the *B. cinerea* HydR phenotypes, target site modifications were found in the HydR3 (+ and -) isolates, but not in HydR2 phenotypes (Albertini & Leroux, 2004; Fillinger *et al.,* 2008). In *B. pseudocinerea* the fenhexamid target encoding gene, *erg27* shows a high degree of polymorphism compared to the sensitive *B. cinerea* allele. Its implication in fenhexamid resistance is described in paragraph 3.1.

Eight different amino acid replacements were found in *erg27* alleles from HydR3- field strains either as single or as double mutations (L195F, N196Y, V309M, A314V, S336C, N369D, L400F/S, et L501W). HydR3+ field isolates display a single mutation of the phenylalanine at positions 412 (F412S, F412I or F412V) (Fig. 1; Fillinger *et al.,* 2008).

Other *erg27* mutations have been found in fenhexamid resistant strains selected after chemical mutagenesis (G23S, C53R, T63I, K73E, H105Y, K159N, L195S, T273A, S310P, I397V, I411V, H423R, A452P, Q495R and C516R) (Saito *et al.,* 2010). However, their involvement in resistance to fenhexamid remains to be shown.

For the phenotypes HydR1 (*B. pseudocinerea*) and HydR2 (*B. cinerea*), high to moderate resistance levels respectively, are observed nearly exclusively at the level of mycelial growth, whereas the other phenotypes display similar resistance levels at both developmental stages, germ tube elongation and mycelial growth. In addition, *B. pseudocinerea* (HydR1) presents increased sensitivities to other fungicides, such as DMIs, fenproprimorph, fenpropidine as well as to some SDHIs (Leroux *et al*., 2002). Only few HydR2 strains have been isolated and to our knowledge, they have not yet been found in French vineyard populations. On the opposite, HydR3 phenotypes are regularly found since

MDR2 (and MDR3) phenotypes display weak to moderate resistance levels (resistance factor <15) to various unlinked mode-of-actions including fenhexamid, SDHIs, dicroboximides, anilinopyrimidines etc. This **m**ulti**d**rug **r**esistance (MDR) is linked to the over expression of a membrane transporter leading to increased drug-efflux outside the fungal cell (Kretschmer *et al.,* 2009). Anti-resistance strategies involving systematic alterations of different mode-ofactions seem to favor the selection of these MDR phenotypes instead of specific resistances, in particular in regions with more than one treatment per season such as the Champagne

**2.1 Target alterations cause reduced affinity of fenhexamid and are responsible for** 

In agriculture, acquired resistance towards agrochemical products is frequently conferred by target site modifications leading to decreased inhibitor affinities. Target overproduction is rarely found. Pesticide detoxification is a mechanism particularly important for insecticide resistance involving diverse enzymes such as hydrolases, cytochrome P450 monooxygenases, glutathione-S-transferases or glycosyl transferases. In rare cases, resistance results from nonactivation of pro-pesticides. Concerning antifungal compounds, the principal resistance mechanisms are target site modifications and increased efflux, in the agronomical field as well

With respect to the *B. cinerea* HydR phenotypes, target site modifications were found in the HydR3 (+ and -) isolates, but not in HydR2 phenotypes (Albertini & Leroux, 2004; Fillinger *et al.,* 2008). In *B. pseudocinerea* the fenhexamid target encoding gene, *erg27* shows a high degree of polymorphism compared to the sensitive *B. cinerea* allele. Its implication in

Eight different amino acid replacements were found in *erg27* alleles from HydR3- field strains either as single or as double mutations (L195F, N196Y, V309M, A314V, S336C, N369D, L400F/S, et L501W). HydR3+ field isolates display a single mutation of the

Other *erg27* mutations have been found in fenhexamid resistant strains selected after chemical mutagenesis (G23S, C53R, T63I, K73E, H105Y, K159N, L195S, T273A, S310P, I397V, I411V, H423R, A452P, Q495R and C516R) (Saito *et al.,* 2010). However, their involvement in

phenylalanine at positions 412 (F412S, F412I or F412V) (Fig. 1; Fillinger *et al.,* 2008).

or HydR3+ with

2003. This category has been subdivided into two sub-classes, HydR3-

**2. Acquired resistance to fenhexamid in** *Botrytis cinerea*

as in the medical field (Ma & Michaelides, 2005; Sanglard *et al.*, 2009).

fenhexamid resistance is described in paragraph 3.1.

resistance to fenhexamid remains to be shown.

moderate or high resistance levels respectively.

wine region.

**resistance** 

Fig. 1. **Protein structure and functional domains of the 3-ketoreductase Erg27 of** *B. cinerea***.** The active site catalyzing the formation of a hydroxyl group on position C3 of sterols is located near the transmembrane domain allowing the anchorage of the enzyme to the endoplasmic reticulum. The position of the mutations detected in HydR3 resistant isolates are indicated by black arrows and concerning HydR3+ isolates by a red one.

We analyzed the involvement of the natural HydR3 mutations in fenhexamid resistance by site directed mutagenesis. The *erg27* allele - encoding 3-ketoreductase – was replaced in a sensitive strain by the *erg27* allele of a HydR3 strain by homologous recombination. Two mutations identified from HydR3- strains (L195F et V309M) in addition to the three HydR3+ alleles were studied. The fenhexamid sensitivity profiles of the generated mutants were similar to those from the field strains (Table 3). Indeed the transgenic *erg27L195F* or *erg27V309M* mutants display low to moderate resistance levels according to the developmental stage, whereas the transgenic HydR3*+* mutants (F412S, F412I, F412V) present high resistance levels at both growth stages, as do the highly resistant HydR3+ field strains.


Table 3. **Sensitivity to fenhexamid of transgenic mutants**. LR=low resistance; MR=moderate resistance; HR=high resistance

In addition, the inhibition of 3-ketoreductase activity by fenhexamid is reduced in the artificial mutants (Billard *et al*., unpublished), strongly suggesting a decrease in fenhexamid's affinity to its target 3-ketoreductase in HydR3 mutants. These results prove the direct correlation between the amino acid substitution and the specific resistance to fenhexamid in the *B. cinerea* HydR3+ and HydR3- phenotypes. In addition they show target site modification as sole resistance mechanism in these isolates.

In contrast to HydR3 phenotypes, neither target site modification nor over expression seem to be responsible for fenhexamid resistance in HydR2 phenotypes. The only hint towards the potential resistance mechanism is an observed synergy between fenhexamid and DMIs suggesting fenhexamid metabolization involving a cytochrome P450 monooxygenase enzyme. Which mechanism precisely is at work in HydR2 strains needs yet to be identified.

We have modeled the *B. cinerea* Erg27 protein after alignment with the peptide sequences of various dehydrogenases/reductases whose crystal structures have been established. The homology-based model presented in Fig. 2 monitors the position of the HydR3 mutations relative to the substrate- and cofactor binding sites. None of the mutations are in the vicinity of these docking sites, although it cannot be excluded that the L195F and N369D replacements modify the Erg27 protein structure leading to different substrate- and cofactor affinities. Mutations of F412 however, potentially interfere with the helical structure of the transmembrane helix.

Fig. 2. Predicted 3D structure of the 3-ketoreductase (Erg27) of *Botrytis cinerea* based on the alignement with Salutaridine Reductase From *Papaver Somniferum* (Higashi *et al.,* 2011). The cofactor (NADHP) binding site and the active sites are highlighted in cyan and purple respectively. The green ribbon represents the transmembrane domain. The mutated residues responsible for specific fenhexamid resistance (HydR3) are highlighted in red. The blue circle designates the extension of *Botrytis* and *Sclerotinia* Erg27 proteins compared to other fungal orthologs.

In contrast to HydR3 phenotypes, neither target site modification nor over expression seem to be responsible for fenhexamid resistance in HydR2 phenotypes. The only hint towards the potential resistance mechanism is an observed synergy between fenhexamid and DMIs suggesting fenhexamid metabolization involving a cytochrome P450 monooxygenase enzyme. Which mechanism precisely is at work in HydR2 strains needs yet to be identified. We have modeled the *B. cinerea* Erg27 protein after alignment with the peptide sequences of various dehydrogenases/reductases whose crystal structures have been established. The homology-based model presented in Fig. 2 monitors the position of the HydR3 mutations relative to the substrate- and cofactor binding sites. None of the mutations are in the vicinity of these docking sites, although it cannot be excluded that the L195F and N369D replacements modify the Erg27 protein structure leading to different substrate- and cofactor affinities. Mutations of F412 however, potentially interfere with the helical structure

Fig. 2. Predicted 3D structure of the 3-ketoreductase (Erg27) of *Botrytis cinerea* based on the alignement with Salutaridine Reductase From *Papaver Somniferum* (Higashi *et al.,* 2011). The cofactor (NADHP) binding site and the active sites are highlighted in cyan and purple respectively. The green ribbon represents the transmembrane domain. The mutated residues responsible for specific fenhexamid resistance (HydR3) are highlighted in red. The blue circle designates the extension of *Botrytis* and *Sclerotinia* Erg27 proteins compared to other

of the transmembrane helix.

fungal orthologs.

#### **2.2 Why fenhexamid has a narrow spectrum?**

Fenhexamid is the sole SBI with a narrow antifungal spectrum. Its activity is restricted to close relatives of *B. cinerea*, e.g. *Botrytis* and *Monilinia spp.*, *Sclerotinia sclerotiorum* (Rosslenbroich & Stuebler, 2000). We therefore raised the question if 3-ketoreductase of ascomycetes naturally resistant to fenhexamid is insentitive to inhibition exerted by fenhexamid. The alignment of Erg27 to orthologous peptide sequences show that both 3 ketoreductase sequences, those from *B. cinerea* and *S. sclerotiorum*, harbor three additional fragments compared to the other fungal proteins, including one extension of 34 amino acids (Albertini *&* Leroux, 2004; Fillinger, unpublished; Fig. 2). In addition, we have shown by biochemical assays that 3-ketoredcutase activity of insensitive fungal species, is barely inhibited by fenhexamid, suggesting that fenhexamid has a strong affinity towards 3 ketoreductase of *Botrytis* related species only (Debieu *et al*., submitted). In how far the above mentioned protein extensions and/or specific residues of the *B. cinerea* Erg27 protein sequence are involved in this affinity remains to be established.

#### **2.3 The HydR3<sup>+</sup> resistance acquisition entails a cost in controlled conditions**

The fitness corresponds to the capacity of one individual (strain) to survive among others under the same conditions. In the absence of selective pressure exerted by the application of fungicides, the relative fitness determines the persistence of a resistant isolate among a natural fungal population. Acquiring a fungicide resistance linked to a high fitness cost is a clear disadvantage for the fungal strain, as it is counter selected in absence of the corresponding fungicide from a mixed population with sensitive and resistant strains. In natural grey mould populations, this seems to be the case for the phenylpyrrol fungicides for which no (or only few) specifically resistant strains were detected up to now (Leroux *et al,* 2002; Moyano *et al.,*  2004). The efficacy of such fungicides can be maintained over long periods. On the opposite, resistant mutations that are not associated to a fitness cost, may threaten the "life-time" of the corresponding fungicide. Indeed, such kind of resistances without associated fitness costs were obtained after few years only with the strobilurins used against cereal fungal pathogens or - on different crops and different pathogens - with the benzimidazoles. Resistance against either or both fungicides is generalized in many pests.

Concerning fenhexamid, the frequency of the HydR3+ strains of the greatest concern in practice, is in progression since 2003 and has reached now non-negligible levels, e.g. 27% in Champagne. This increase remains weak in regions despite annual applications and not comparable to the rapid generalization of strobilurin resistance. This evolution indicates that probably an effective anti-resistance strategy combined to a potential fitness cost linked to high levels of fenhexamid resistance limits the progression of HydR3+ frequencies among grey mould populations.

In order to evaluate the fitness cost linked to the HydR3+ phenotypes, we measured divers physiological parameters under controlled conditions. The general problem for fitness measurements in natural *B. cinerea* strains is the phenotypic variability between strains. Different growth parameters cannot be easily correlated to resistance due to different genetic backgrounds. We circumvented this problem by the construction of HydR3+ (F412S, F412I and F412V) mutants using site directed mutagenesis. The strains were isogenic except for the *erg27* allele providing the ideal material for fitness analyses. Particular attention was paid to temperature and nutritional factors in the measurement of the fitness parameters as well as the quantification of developmental stages important for survival and dispersion (sclerotia and conidia respectively). Statistically significant decreases were observed in the mutants for mycelial growth – especially in extreme conditions (restricted nutrients at low temperatures), for conidia- and sclerotia-production – the last parameter especially at low temperatures, and for mycelial outgrowth from frozen sclerotia. Theses results obtained with isogenic strains, like those reported by Ziogas *et al* (2003) and Saito *et al.* (2010) for chemically resistant mutants of *B. cinerea*, indicate that the acquisition of high-level specific resistance to fenhexamid is associated with a decrease in fitness (Billard *et al.,* submitted). Our data show that *in vitro* pathogenicity is not affected (measured on detached bean leaves), whereas stages of the fungal life cycle during which there is no selection pressure seem to be the most affected.

Fig. 3. **Life cycle of** *Botrytis cinerea***.** In laboratory conditions, a fitness cost of the HydR3+ resistance was observed in isogenic mutants at different phases of the asexual cycle (in red) of *B. cinerea*. However, the pathogenicity of these mutants, measured on beans, remained unchanged from the parental strain sensitive to fenhexamid (green).

quantification of developmental stages important for survival and dispersion (sclerotia and conidia respectively). Statistically significant decreases were observed in the mutants for mycelial growth – especially in extreme conditions (restricted nutrients at low temperatures), for conidia- and sclerotia-production – the last parameter especially at low temperatures, and for mycelial outgrowth from frozen sclerotia. Theses results obtained with isogenic strains, like those reported by Ziogas *et al* (2003) and Saito *et al.* (2010) for chemically resistant mutants of *B. cinerea*, indicate that the acquisition of high-level specific resistance to fenhexamid is associated with a decrease in fitness (Billard *et al.,* submitted). Our data show that *in vitro* pathogenicity is not affected (measured on detached bean leaves), whereas stages of the fungal life cycle during

Fig. 3. **Life cycle of** *Botrytis cinerea***.** In laboratory conditions, a fitness cost of the HydR3+ resistance was observed in isogenic mutants at different phases of the asexual cycle (in red) of *B. cinerea*. However, the pathogenicity of these mutants, measured on beans, remained

unchanged from the parental strain sensitive to fenhexamid (green).

which there is no selection pressure seem to be the most affected.

These findings for isogenic *erg27F412* mutants suggest that the HydR3+ phenotype does not persist in the absence of the fungicide and that this phenotype has a moderate impact on the efficacy of fenhexamid treatments for controlling grey mould disease in vineyards. However long-term studies in vineyards are required to confirm these laboratory observations.

#### **2.4 Fenhexamid monitoring in fields: A molecular approach**

Monitoring of this resistance is a crucial area of research where our knowledge of the field distribution, evolution and impact of fungicide resistance depends on. In most cases, the degree of sensitivity of fungal populations to one or more fungicides is assessed by biological methods. These bioassays conducted, *in vitro* or *in vivo*, have been miniaturized (i.e. microtiter plate methods), but nonetheless consume considerable resources and remain time-consuming. When the molecular mechanisms of resistance are known (e.g. target mutation, target over expression, increased drug efflux), and particularly when the underlying DNA polymorphisms (single-nucleotide polymorphism (SNPs), deletions or insertions) have been defined, various molecular methods can be used to monitor antimicrobial resistance. The principal methods for quantifying resistance are based on realtime PCR (polymerase chain reaction) technology. Generally, studies have been made on isolated and purified strains using qualitative methods like RFLP, AS PCR or Real time PCR (Benzimidazole resistance in *Botrytis cinerea* Banno *et al*., 2008, *Venturia inequaelis* Koenraadt *et al.,* 1992, *Rhyncosporium secalis* Wheller *et al*., 1995, *Helminthosporium solani* McKay *et al.,* 1998; Strobilurines resistance on *Erisiphe graminis* Baümler *et al.,* 2003, *Plasmopara viticola* Furuya *et al*., 2009, *Alternaria spp*. Ma *&* Michailides, 2003, *Mycosphaerella graminicola* Fraaije *et al.*, 2007, *Mycosphaerella fijiensis* Sierotzki *et al.,* 2000) and on the opposite some of them on populations, where resistance allele is quantify in gDNA pools by using Real time PCR technologies (Benzimidazole resistance in *Sclerotinia sclerotiorum* Chen *et al.,* 2009 ; Strobilurins resistance on *Blumeria graminis* Fraaije *et al*., 2002 and *Pyrenophora teres* Kianianmomeni *et al*., 2007). However, one limitation of this method concerns the nonspecific amplification of alleles, which may affect precision. This limitation does not generally hinder detection of the polymorphism, but it may affect quantification capacity, particularly for mutated alleles with a low abundance.

Taking these results into account, we investigated the development of a new technique for quantifying, with a high precision, the three different *erg27* alleles from the HydR3+ phenotype. The underlying DNA polymorphism is the modification of the TTC codon encoding the F412 residue, which is converted into a TCC (serine), GTC (isoleucine) or ATC (valine) (Fillinger *et al.,* 2008) codon. The best result was obtained with a non multiplexed method combining four allele-specific MGB Taqman® probes and four mismatched specific primers. This technique was named the *Allele Specific Probe and Primer Amplification Assay* (ASPPAA PCR), (Fig. 4; Billard *et al*., submitted).

The sensitivity of ASPPAA PCR is sufficiently good to quantify a SNP at a rate of 1% in a DNA pool. In the future, the multiplexing, in the same run, of the analysis of several polymorphisms at different genomic loci with probes picked up in different fluorophore channels is conceivable and would be expected to decrease the time required for monitoring, and its cost, significantly.

The principle disadvantage of these molecular quantification methods is that they do not allow to detect emerging resistances in contrast to biological methods. One alternative molecular method to be considered is the HRM (high resolution melt curve) analysis (Pasay *et al.,* 2008*)*. HRM offers a fast and convenient method of assessing the presence of mutations without sequencing in a short (< 400 bp) defined genomic region. Using this tool, the identification of new mutations in the *erg27* gene is possible.

Fig. 4. **Principle of ASPPAA PCR for quantitative SNP detection.** The high robustness of this new allele quantification method using real-time PCR is caused by the specificity of amplification of the mutation by allele specific probes and primers present on both DNA strands.

#### **3. Natural resistance to fenhexamid in** *Botrytis pseudocinerea*

The genus *Botrytis* (Ascomycota) contains 22 highly specialised species and one hybrid. A multiple-gene gene genealogy study recently showed that this genus could be subdivided into two categories, one consisting of *Botrytis* species acting as pest on monocots, and the other containing *Botrytis* species acting as parasites on eudicots (Staats *et al.,* 2005). Within this second category, *Botrytis cinerea* has the widest host range, being able to infect more than 220 types of eudicot, including grapevine and many fruit and vegetable crops. However, *B. cinerea* has recently been shown to be a complex of two sibling species living in sympatry. *B. cinerea sensu stricto* is the predominant species (Fournier *et al.*, 2005). The other species, called *Botrytis pseudocinerea*, has been found at low frequency in French populations. This species is morphologically indistinguishable from *B. cinerea sensu* stricto, but Walker *et*  al (2011) established several molecular markers to distinguish both species. In addition *B. pseudocinerea* has a different pattern of fungicide susceptibility (phenotype HydR1), displaying natural resistance to fenhexamid and hypersensitivity to fenpropidin and edifenphos (Leroux *et al*, 2002).

In contrast to the *B. cinerea* fenhexamid resistant phenotypes (HydR2 and HydR3), *B. pseudocinerea* was resistant to fenhexamid prior to its introduction, therefore displaying a natural resistance to fenhexamid.

#### **3.1 Role of the natural target polymorphism in fenhexamid resistance**

The genetic polymorphism of *B. pseudocinerea* compared to *B. cinerea* is also visible on the 3 ketoredcutase encoding gene *erg27*. Twelve codon modifications lead to amino acid replacements in the Erg27 protein (N93V, D146L, I211V, I215V, M218T, V234A, I235V, D261G, S264T, P269L, A285T and Q354K) (Albertini *&* Leroux, 2004). None of theses substitutions corresponds to any of the HydR3 mutations. In order to evaluate the impact of the *B; pseudocinerea erg27* allele in fenhexamid resistance we replaced the *erg27wt* allele in a

molecular method to be considered is the HRM (high resolution melt curve) analysis (Pasay *et al.,* 2008*)*. HRM offers a fast and convenient method of assessing the presence of mutations without sequencing in a short (< 400 bp) defined genomic region. Using this tool, the

Fig. 4. **Principle of ASPPAA PCR for quantitative SNP detection.** The high robustness of this new allele quantification method using real-time PCR is caused by the specificity of amplification of the mutation by allele specific probes and primers present on both DNA

The genus *Botrytis* (Ascomycota) contains 22 highly specialised species and one hybrid. A multiple-gene gene genealogy study recently showed that this genus could be subdivided into two categories, one consisting of *Botrytis* species acting as pest on monocots, and the other containing *Botrytis* species acting as parasites on eudicots (Staats *et al.,* 2005). Within this second category, *Botrytis cinerea* has the widest host range, being able to infect more than 220 types of eudicot, including grapevine and many fruit and vegetable crops. However, *B. cinerea* has recently been shown to be a complex of two sibling species living in sympatry. *B. cinerea sensu stricto* is the predominant species (Fournier *et al.*, 2005). The other species, called *Botrytis pseudocinerea*, has been found at low frequency in French populations. This species is morphologically indistinguishable from *B. cinerea sensu* stricto, but Walker *et*  al (2011) established several molecular markers to distinguish both species. In addition *B. pseudocinerea* has a different pattern of fungicide susceptibility (phenotype HydR1), displaying natural resistance to fenhexamid and hypersensitivity to fenpropidin and

In contrast to the *B. cinerea* fenhexamid resistant phenotypes (HydR2 and HydR3), *B. pseudocinerea* was resistant to fenhexamid prior to its introduction, therefore displaying a

The genetic polymorphism of *B. pseudocinerea* compared to *B. cinerea* is also visible on the 3 ketoredcutase encoding gene *erg27*. Twelve codon modifications lead to amino acid replacements in the Erg27 protein (N93V, D146L, I211V, I215V, M218T, V234A, I235V, D261G, S264T, P269L, A285T and Q354K) (Albertini *&* Leroux, 2004). None of theses substitutions corresponds to any of the HydR3 mutations. In order to evaluate the impact of the *B; pseudocinerea erg27* allele in fenhexamid resistance we replaced the *erg27wt* allele in a

**3.1 Role of the natural target polymorphism in fenhexamid resistance** 

**3. Natural resistance to fenhexamid in** *Botrytis pseudocinerea*

identification of new mutations in the *erg27* gene is possible.

strands.

edifenphos (Leroux *et al*, 2002).

natural resistance to fenhexamid.

sensitive *B. cinerea* strain by that of a HydR1 "resistant" *B. pseudocinerea* strain. The gene replacement strategy is explained in Fig. 5.

Fig. 5. Gene replacement strategy of the *erg27* allele by that of *B. pseudocinerea* in a sensitive strain of *B. cinerea*. The *hph* gene conferring resistance to hygromycine was used as transformation selection marker.

The transformants harboring the *B. pseudocinerea* allele instead of the *B. cinerea* allele showed a slightly increased resistance to fenhexamid (RF<10) compared to the parental *B. cinerea* strain (Billard *et al*., unpublished). Those results indicate that the major part of fenhexamid resistance in *B. pseudocinerea* is conferred by a mechanism independent of the target.

#### **3.2 Characterization of an unusual resistance mechanism towards fungicides: Detoxification**

Indications about the second mechanism were obtained more than ten years ago by two studies. Suty and co-workers (1999) performed fenhexamid metabolization assays on *B. cinerea* and *B. pseudocinerea* and observed that naturally resistant species displayed a metabolization pattern different from *B. cinerea* suggesting fenhexamid degradation as possible resistance mechanism. These results were supported by the synergy between DMIs and fenhexamid observed only for *B. pseudocinerea* (Leroux *et al.,* 2002). Together these results suggested for *B. pseudocinera* an enzyme(s) similar to Cyp51 (the target of DMIs) to degrade fenhexamid and therefore conferring resistance to fenhexamid.

We have searched the *B. cinerea* genome database (Amselem *et al.,* 2011) (http://urgi.versailles.inra.fr/Species/Botrytis) for cytochrome P450 proteins similar to Cyp51 and compared their expression profiles between *B. cinerea* and *B. pseudocinerea*. The candidate gene showing the highest similarity to *cyp51* (19 % protein identity, 37 % protein similarity) showed increased expression in *B. pseudocinerea* (Billard *et al.*, unpublished). According to the nomenclature of P450s, after phylogenic analysis against all P450s of the ascomycete *Aspergillus nidulans* (Kelly *et al*., 2009) the corresponding gene was named *cyp684*. We created *cyp684* deletion mutants in a *B. pseudocinerea* strain through a gene replacement strategy. The *∆cyp684* mutants showed a 200 fold reduction in fenhexamid resistance and a simultaneous reduction in DMI-fenhexamid synergy (Table 4, Fig. 6). Although the *B. pseudocinerea ∆cyp684*  mutants do not reach *B. cinerea* sensitivity levels, theses results show that the cytochrome P450 encoding gene *cyp684* is responsible for the biggest part of *B. pseudocinerea's* natural resistance to fenhexamid. Natural polymorphism between the *B. cinerea/B. pseudocinerea cyp684* orthologues may account for the different phenotypes. It remains to be shown if *cyp684* overexpression and/or differential enzyme parameters are involved.

In *B. pseudocinerea* however the *cyp684* deletion abolishes the hypersensitivity to the phosphothiolate edifenphos in addition to the loss of fenhexamid resistance (Table 6)


suggesting that Cyp684 could have a similar enzyme activity on fenhexamid as on edifenphos. Phosphorothiolates (e.g. edifenphos) are profungicides, nearly exclusively used against the rice-blast disease caused by *Magnaporthe oryzae*.

RL= resistance levels, S= sensitive, HS=hypersensitive, HR=highly resistant, MR=moderately resistant, Ind.= independent, Syn.= synergistic

Table 4. Sensitivity profiles to fenhexamid and edifenphos (phosphorothiolate) of *B. cinerea, B. pseudocinerea* and *Δcyp684* mutants of *B. pseudocinerea*.

Fig. 6. Joint action between fenhexamid (vertical) and prochloraz or tebuconazole (horizontal strip) on the *B. pseudocinerea B900* strain and a B900*∆cyp684* deletion mutant. The curves around paper crosses indicate strong synergism between chemicals (in the left panel). In the *B. pseudocinerea ∆cyp684* mutant growth inhibition is typical of independent actions (right panel).

suggesting that Cyp684 could have a similar enzyme activity on fenhexamid as on edifenphos. Phosphorothiolates (e.g. edifenphos) are profungicides, nearly exclusively used

(Hydroxyanilides) S HR **MR**

Fenhexamid+DMI Ind. Syn. Ind.

(Phosphorothiolates) S HS S

RL= resistance levels, S= sensitive, HS=hypersensitive, HR=highly resistant, MR=moderately resistant,

Table 4. Sensitivity profiles to fenhexamid and edifenphos (phosphorothiolate) of *B. cinerea,* 

Fig. 6. Joint action between fenhexamid (vertical) and prochloraz or tebuconazole (horizontal strip) on the *B. pseudocinerea B900* strain and a B900*∆cyp684* deletion mutant. The curves around paper crosses indicate strong synergism between chemicals (in the left panel). In the *B. pseudocinerea ∆cyp684* mutant growth inhibition is typical of independent actions (right panel).

*B. pseudocinerea (RL)*

*wt Δcyp684* 

against the rice-blast disease caused by *Magnaporthe oryzae*.

*B. pseudocinerea* and *Δcyp684* mutants of *B. pseudocinerea*.

Fenhexamid

edifenphos

Ind.= independent, Syn.= synergistic

*B. cinerea* 

These compounds need to be modified in order to become active. In the case of edifenphos, the active metabolite is produced by the cleavage of a phosphor-sulfur bridge (Uesugi, 2001). As for *B. pseudocinerea,* a strong synergy was observed between phosphorothiolates and DMIs. Moreover negative cross-resistance between phosphorothiolates and phosphoroamidates in *M. oryzae* allowed the identification of the biochemical reactions involved (Uesugi & Takenake, 1992 ; Fig. 7). The enzymes involved are yet unknown, but negative cross resistance between BPA and edifenphos on one hand, and the synergy between edifenphos and DMIs on the other, suggest the involvement of a cytochrome P450 accepting as a substrate edifenphos as well as BPA. The comparable situation observed in *B. pseudocinerea* is in favor of a comparable enzymatic reaction involved in the detoxification of fenhexamid. We are currently analyzing the metabolites produced by *B. pseudocinerea* from fenhexamid in order to unravel the reaction mechanism*.*

Fig. 7. Metabolic pathways of phosphorothiolates (edifenphos) and phosphoroamidates (BPA) in susceptible and resistant strains of *Magnaporthe oryzae.* (from Uesugi *&* Takenaka, 1992, modified).

#### **4. Conclusions**

We and others have identified and characterized various phenotypes displaying reduced sensitivity to fenhexamid among natural grey mould populations. In the case of the phenotypes with low resistance at the spore germination stage (HydR1, HydR2, MDR2 et MDR3 phenotypes), Petit *et al.,* 2010 have shown these have only a limited impact on fenhexamid's efficacy in artificial inoculation studies and in vineyards studies. In the same line, monitoring started before the introduction of fenhexamid, show that the variations in frequencies of *B. pseudocinerea* – naturally resistant to fenhexamid – in grey mould populations, seem to be independent of selection by fenhexamid. *B. cinerea* and *B. pseudocinerea* are found in sympatry on the same hosts, but clearly differ in their phenology, demonstrating differences in ecological niche (Walker *et al.,* 2011): although present at low frequencies (0-15 %), *B. pseudocinerea* is predominantly found in spring populations and has only a reduced impact on grey mould epidemics.

Finally, the HydR3 phenotype comprises strains with moderate (HydR3-) to high (HydR3+) resistance levels at all growth stages. This resistance is conferred by target site modifications. HydR3+ strains are the predominant strains resistant to fenhexamid in French vineyards and those of greatest concern in practice, because they threaten the sustainability of fenhexamid in combating grey mould. Frequencies of HydR3+ strains are slowly but steadily increasing in French vineyard populations. Even if their mean frequencies stayed moderately low (e.g. 27 % in Champagne in 2010, Fig. 8), their proportion within same plots has strongly increased in the last years. Although one can observe over 50 % highly resistant strains at the end of the season after fenhexamid treatment, our multi-annual monitoring survey did not reveal sites with such important frequencies over several years. In addition, we are not aware of any loss-of-efficacy observed for fenhexamid due to the selection of resistant populations. Several hypotheses can be put forward to explain the situation: (1) the use of fenhexamid limited to one application per year (as for other botryticides), (2) the fitness cost of the HydR3+ mutations and (3) the predominance of MDR strains at least in Champagne (55 % mean frequencies). Indeed, strains combining MDR (multiple drug resistance) and specific resistance to fenhexamid are rarely found.

Fig. 8. Evolution of the HydR3 phenotype in Champagne since the introduction of fenhexamid (source : CIVC, AS Walker INRA).

We have developed a molecular tool allowing the rapid detection and quantification of the three major HydR3 alleles from a mixture of *B. cinerea* spores. (ASPPAA PCR). This tool will make large scale field assays realistic to evaluate the percentage of HydR3*<sup>+</sup>* strains among populations subjected to different fungicide pressures in order to precise the best strategy for fenhexamid treatment and the best state for its application. Moreover, this method may monitor frequencies of HydR3+ strains after stopping fenhexamid treatments and therefore allows to analyze the persistence of such resistance phenotype under real conditions.

#### **5. References**

74 Fungicides – Beneficial and Harmful Aspects

phenotypes with low resistance at the spore germination stage (HydR1, HydR2, MDR2 et MDR3 phenotypes), Petit *et al.,* 2010 have shown these have only a limited impact on fenhexamid's efficacy in artificial inoculation studies and in vineyards studies. In the same line, monitoring started before the introduction of fenhexamid, show that the variations in frequencies of *B. pseudocinerea* – naturally resistant to fenhexamid – in grey mould populations, seem to be independent of selection by fenhexamid. *B. cinerea* and *B. pseudocinerea* are found in sympatry on the same hosts, but clearly differ in their phenology, demonstrating differences in ecological niche (Walker *et al.,* 2011): although present at low frequencies (0-15 %), *B. pseudocinerea* is predominantly found in spring populations and has

Finally, the HydR3 phenotype comprises strains with moderate (HydR3-) to high (HydR3+) resistance levels at all growth stages. This resistance is conferred by target site modifications. HydR3+ strains are the predominant strains resistant to fenhexamid in French vineyards and those of greatest concern in practice, because they threaten the sustainability of fenhexamid in combating grey mould. Frequencies of HydR3+ strains are slowly but steadily increasing in French vineyard populations. Even if their mean frequencies stayed moderately low (e.g. 27 % in Champagne in 2010, Fig. 8), their proportion within same plots has strongly increased in the last years. Although one can observe over 50 % highly resistant strains at the end of the season after fenhexamid treatment, our multi-annual monitoring survey did not reveal sites with such important frequencies over several years. In addition, we are not aware of any loss-of-efficacy observed for fenhexamid due to the selection of resistant populations. Several hypotheses can be put forward to explain the situation: (1) the use of fenhexamid limited to one application per year (as for other botryticides), (2) the fitness cost of the HydR3+ mutations and (3) the predominance of MDR strains at least in Champagne (55 % mean frequencies). Indeed, strains combining MDR (multiple drug

only a reduced impact on grey mould epidemics.

resistance) and specific resistance to fenhexamid are rarely found.

Fig. 8. Evolution of the HydR3 phenotype in Champagne since the introduction of

We have developed a molecular tool allowing the rapid detection and quantification of the three major HydR3 alleles from a mixture of *B. cinerea* spores. (ASPPAA PCR). This tool will

fenhexamid (source : CIVC, AS Walker INRA).


Fournier, E.; Giraud, T.; Albertini, C. & Brygoo, Y. (2005). Partition of the *Botrytis cinerea* complex in France using multiple gene genealogies. *Mycologia*. 97(6): 1251-1267. Fraaije, B.A.; Butters, J.A.; Coelho, J.M.; Jones, D.R. & Hollomon, D.W. (2002). Following the

Fraaije, B.A.; Cools, H.J.; Kim, S.H.; Motteram, J.; Clark, W.S. & Lucas, J.A. (2007). A novel

Furuya, S.; Suzuki, S.; Kobayashi, H.; Saito, S. & Takayanagi, T. (2009). Rapid method for

Gachotte, D.; Sen, S.E.; Eckstein, J.; Barbuch, R.; Krieger, M.; Ray, B.D. & Bard, M. (1999).

Higashi, Y.; Kutchan, TM.; Smith, T.J. (2011). Atomic structure of salutaridine reductase

Ishii, H. (2010). Fungicide resistance in rice. *Modern Fungicides and Antifungal Compounds VI,* 

H.B. Deising, U. Gisi, K. H. Kuck, P.E. Russell, H. Lyr (Eds.), *in press.* Kelly DE, Krasevec N, Mullins J, Nelson DR (2009) The CYPome (Cytochrome P450 complement) of Aspergillus nidulans. *Fungal Genetics and Biology* 46: S53-S61 Kianianmomeni, A.; Schwarz, G.; Felsenstein, F.G. & Wenzel, G. (2007). Validation of a real-

*bc1* gene of *Pyrenophora teres. Pest Management Science*. 63(3): 219–224. Koenraadt, H.; Somerville, S.C. & Jones, A.L. (1992). Characterization of mutations in the

Kretschmer, M.; Leroch, M.; Mosbach, M.; Walker, A.S.; Fillinger, S.; Mernke, D.;

populations of the Grey Mould Fungus *Botrytis cinerea*. *PLoS Pathogen* 5(12). Leroux, P.; Debieu, D.; Albertini, C.; Arnold, A.; Bach, J.; Chapeland, F.; Fournier, E.; Fritz,

plant pathogenic fungi. *Phytopathology*. 82(11): 1348–1354.

Gilbert, R.J.C. (2010). Cholesterol-dependent cytolysins. *Adv. Exp. Med. Biol*. 677: 56-66. Hac-Wydro, K.; Wydro, P.; Jagoda, A. & Kapusta, J. (2007). The study on the interaction

SYBR Green I. *Plant pathology.* 51(1): 45–54.

8(3): 245–254.

12655-12660.

150(1): 22-34.

6532-6541.

Germany.

*Manag Sci* 65:840–843

dynamics of strobilurin resistance in *Blumeria graminis* f. sp. *tritici* using quantitative allele-specific real-time PCR measurements with the fluorescent dye

substitution I381V in the sterol 14alpha-demethylase (CYP51) of *Mycosphaerella graminicola* is differentially selected by azole fungicides. *Molecular Plant Pathology*.

detecting resistance to a QoI fungicide in *Plasmopara viticola* populations. *Pest* 

Characterization of the *Saccharomyces cerevisiae* ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation. *Proc. Natl. Acad. Sci. U.S.A*. 96(22):

between phytosterols and phospholipids in model membranes. *Chem. Phys. Lipids*.

from the Opium Poppy (*Papaver somniferum*). *Journal of Biological Chemistry* 286:

Proceedings *16th International Reinhardsbrunn Symposium* 2010 Eds H.W. Dehne,

time PCR for the quantitative estimation of a G143A mutation in the cytochrome

beta-tubulin gene of benomyl-resistant field strains of *Venturia inaequalis* and other

Schoonbeek, H.J.; Pradier, J.M.; Leroux, P.; De Waard, M.A. & Hahn, M. (2009). Fungicide-driven evolution and molecular basis of multidrug resistance in field

R.; Gredt, M.; Giraud, T.; Hugon, M.; Lanen, C.; Malosse, C. & Thebaud. G. (2002). The Hydroxyanilide Botryticide Fenhexamid/ Mode of Action and Mechanism of Resistance. *Modern Fungicides and Antifungal Compounds III*. Proceedings *13th International Reinhardsbrunn Symposium* 2001: 29-40. Eds H.W. Dehne, H.B. Deising, U. Gisi, K. H. Kuck, P.E. Russell, H. Lyr (Eds.)*,* AgroConcept GmbH, Bonn,


### **Impact of Fungicide Timing on the Composition of the** *Fusarium* **Head Blight Disease Complex and the Presence of Deoxynivalenol (DON) in Wheat**

Kris Audenaert1,2, Sofie Landschoot1,2, Adriaan Vanheule1,2, Willem Waegeman3, Bernard De Baets3 and Geert Haesaert1,2 *1Associated Faculty of Applied Bioscience Engineering, Ghent University College, Ghent 2Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent University, Ghent 3KERMIT, Department of Mathematical Modelling, Statistics and Bioinformatics, Ghent University, Ghent Belgium* 

#### **1. Introduction**

78 Fungicides – Beneficial and Harmful Aspects

Uesugi, Y. (2001). Fungal choline biosynthesis – a target for controlling rice blast. *Pesticide* 

Uesugi, Y. & Takenata, M. (1992). The mechanisms of action of phosphorothiolates

Walker, A.S., Gautier, A.; Confais, C.; Martinho, D.; Viaud, M.; Le Pêcheur, P.; Dupont, J.;

Yeagle, P.L. (1990). Frontiers of membrane research: lipid-protein complexes in membranes;

Zhang, C.Q.; Zhu, G.N.; Ma, Z.H. & Zhou, M. G. (2006). Isolation, characterization and

Ziogas, B.N.; Markoglou, A.N. and Malandrakis, A.A. (2003). Studies on the inherent resistance risk to fenhexamid in *Botrytis cinerea*. *Eur J Plant Pathol* 109: 311–317.

Blast Fungus, *Magnaporthe grisea*. *J. Phytopathology* 154, 392–397

Phytomedical Society Series, Stuttgart, Germany.

*EPPO Bulletin* 25, Issue 1-2, pages 113–116,

membrane fusion. *Prog. Clin. Biol. Res*. 343: 15-28.

fungicides. In Proceeding of the 10th International Symposium on Systemic Fungicides and Antifungal Compounds, eds H. Lyr and C. Polter, 159-164, German

Fournier, J. (2011). *Botrytis pseudocinerea*, a new cryptic species causing grey mould in French vineyards in sympatry with *Botrytis cinerea*. *Phytopathology*, in press Wheeler, I.; Kendall, S.; Butters, J. & Hollomon, D. (1995). Detection of benzimidazole

resistance in *Rhynchosporium secalis* using allele-specific oligonucleotide probe.

preliminary genetic analysis of laboratory tricyclazole-resistant mutants of the Rice

*Outloo*k, February.

#### **1.1** *Fusarium* **head blight: A multi-faceted agricultural problem**

*Fusarium* Head Blight (FHB) is one of the most important diseases in wheat, caused by a complex of up to 17 *Fusarium* species. The main causal agents of FHB in Europe are *Fusarium graminearum*, *Fusarium culmorum*, *Fusarium avenaceum*, *Fusarium poae* and *Microdochium nivale* (Audenaert *et al*. 2009; Brennan *et al*. 2003; Leonard & Bushnell 2003; Mudge *et al*. 2006; Parry *et al*. 1995). There is extensive work on the effect of FHB on grain yields of cereals. For example, in breeding programs aiming to generate resistant cultivars, yield losses have been observed ranging from 6 up to 74% (Snijders 1990; Snijders & Perkowski 1990). Symptoms of *Fusarium* occur just after anthesis. The partly white and partly green heads are diagnostic for the disease in wheat (Figure 1C). The fungus also may infect the peduncle immediately below the head, causing a brown/purplish discoloration of the stem tissue. Additional indications of FHB infection are pink to salmon-orange spore masses of the fungus often seen on the infected spikelets and glumes. Infected kernels are shriveled, lightweight and dull grayish or pinkish. These kernels sometimes are called "tomb-stones" because of their chalky, lifeless appearance. If infection occurs late in kernel development, *Fusarium*-infected kernels may be normal in size, but have a dull appearance or a pink discoloration.

Although FHB may cause wheat yield losses, the interest in FHB is primarily fuelled by the ability of *Fusarium* species to produce mycotoxins. FHB pathogens produce a diversified spectrum of mycotoxins depending on the species (Bennett & Klich 2003). Trichothecenes, zearalenon, moniliformin and fumonisins are the most important mycotoxins produced by *Fusarium* fungi. Among the trichothecenes, deoxynivalenol (DON) is the predominant mycotoxin throughout Europe and is mainly produced by *F. graminearum*. These secondary fungal metabolites can accumulate to significant doses and as such cause a serious impediment for human and animal health. Moreover, the European concern for several *Fusarium* mycotoxins has been concretized in regulations for maximum levels for human and animal consumption. These regulations provide an extra economic motive for farmers to prevent FHB infection and mycotoxin accumulation in small grain cereals such as wheat.

The prevention of DON and *Fusarium* in wheat is not easy, since the disease is primarily associated with weather conditions during anthesis of the crop. It is generally accepted that rainfall just before and during anthesis, which is situated in the month of June, favours the FHB pathogens and can cause serious yield losses. Conidia present on crop residues reach the ears by splashed rain droplets. A recent study by Landschoot *et al*. (2011a+b) fine-tuned the influence of weather conditions. These authors demonstrated nicely that also weather conditions during the vegetative growth of the crop in winter and spring are important parameters determining the disease incidence and DON level (Table 1). This conclusion is remarkable since infection with *Fusarium* starts in the months of June and July. A possible explanation for this remarkable findings may come from the influence of weather conditions during winter on the survival of the primary inoculums in soil, weeds and crop residues. In cold winters, survival of *Fusarium* conidia is poor, resulting in a lower primary inoculums pressure in June.

Until now, no absolute FHB resistance encoded by single dominant resistance genes has been characterized in wheat. Consequently, it is difficult to implement *Fusarium* resistance into breeding programs. Two major sources for resistance have been characterized. Type I resistance stops the pathogen at the level of penetration while type II resistance is involved the inhibition of fungal spread within the infected ear (Ban & Suenaga 2000; Singh *et al*. 1995). However, the implementation of quantitative trait loci associated with resistance into commercial wheat varieties is not for tomorrow because of economic drawbacks.


Table 1. A. Highest pearson correlation coefficients for weather variables (Days with Frost, Air Pressure, Total Rainfall, Relative humidity (RH) Temperature, Dew point and wind speed) and the DI, 10%P, 25%P, 75%P, 90%P, respectively mean 10%, 25%, 75%, 90% percentiles. B. Highest pearson correlation coefficients for weather variables (Days with Frost, Air Pressure, Total Rainfall, Relative humidity (RH) Temperature, Dew point and wind speed) and DON content in grain, 10%P, 25%P, 75%P, 90%P, respectively mean 10%, 25%, 75%, 90% percentiles.

Although good agricultural practices certainly help to reduce the risk for *Fusarium* epidemics, the application of fungicides remains the most important control measure to reduce *Fusarium* symptoms. Although there are a limited number of active ingredients with good control activity for FHB, the chemical control of this pathogenic disease complex remains a serious issue. The short vulnerable period of the pathogen, the fact that it is an ear pathogen, ands the fact that it mainly infects under wet conditions all hamper an efficient control of the FHB complex.

#### **1.2 The Belgian situation**

80 Fungicides – Beneficial and Harmful Aspects

and animal consumption. These regulations provide an extra economic motive for farmers to prevent FHB infection and mycotoxin accumulation in small grain cereals such as wheat. The prevention of DON and *Fusarium* in wheat is not easy, since the disease is primarily associated with weather conditions during anthesis of the crop. It is generally accepted that rainfall just before and during anthesis, which is situated in the month of June, favours the FHB pathogens and can cause serious yield losses. Conidia present on crop residues reach the ears by splashed rain droplets. A recent study by Landschoot *et al*. (2011a+b) fine-tuned the influence of weather conditions. These authors demonstrated nicely that also weather conditions during the vegetative growth of the crop in winter and spring are important parameters determining the disease incidence and DON level (Table 1). This conclusion is remarkable since infection with *Fusarium* starts in the months of June and July. A possible explanation for this remarkable findings may come from the influence of weather conditions during winter on the survival of the primary inoculums in soil, weeds and crop residues. In cold winters, survival of *Fusarium* conidia

Until now, no absolute FHB resistance encoded by single dominant resistance genes has been characterized in wheat. Consequently, it is difficult to implement *Fusarium* resistance into breeding programs. Two major sources for resistance have been characterized. Type I resistance stops the pathogen at the level of penetration while type II resistance is involved the inhibition of fungal spread within the infected ear (Ban & Suenaga 2000; Singh *et al*. 1995). However, the implementation of quantitative trait loci associated with resistance into

commercial wheat varieties is not for tomorrow because of economic drawbacks.

**A. Month Variable negative Variable positive November** Days with frost -0.24 75%P Air pressure 0.47 **December** Days with frost -0.53 Average Dew point 0.68 **January** 90%P Air pressure -0.28 75%P Dew point 0.69 **February** Days with frost -0.53 Median Dew point 0.74 **March** Days with rainfall -0.28 Average Dew point 0.62 **April** Days with rainfall -0.65 75%P Dew point 0.51 **May** Average Air pressure -0.58 Median wind speed 0.34 **June** 25%P Air pressure -0.69 Days with RH >80% 0.66 **July** 75%P Temperature -0.58 Total Rainfall 0.63 **B. Month Variable negative Variable positive November** 25%P RH -0.24 75%P Air pressure 0.23 **December** Median RH -0.19 90%P Temperature 0.33 **January** Days with frost -0.28 75%P Temperature 0.35 **February** Days with frost -0.34 75%P Temperature 0.48 **March** 90%P Dew point -0.23 10%P Temperature 0.39 **April** Days with rainfall -0.31 90%P Air pressure 0.36 **May** Average Air pressure -0.32 25%P Temperature 0.34 **June** Average Air pressure -0.42 25%P RH 0.34 **July** 10%P Temperature -0.31 10%P RH 0.28 Table 1. A. Highest pearson correlation coefficients for weather variables (Days with Frost, Air Pressure, Total Rainfall, Relative humidity (RH) Temperature, Dew point and wind speed) and the DI, 10%P, 25%P, 75%P, 90%P, respectively mean 10%, 25%, 75%, 90% percentiles. B. Highest pearson correlation coefficients for weather variables (Days with Frost, Air Pressure, Total Rainfall, Relative humidity (RH) Temperature, Dew point and wind speed) and DON content in grain, 10%P, 25%P, 75%P, 90%P, respectively mean 10%, 25%, 75%, 90% percentiles.

is poor, resulting in a lower primary inoculums pressure in June.

In order to get a better view on the FHB problem in Flanders, a region situated in the North of Belgium, an intensive survey started in 2002. Pursuing a combined approach of symptom evaluation, DON measurement and genetic characterization of the population, a comprehensive dataset was obtained. This dataset comprised data of ten growing seasons, on at least ten locations throughout Flanders. On each location 12 cultivars of wheat were sown in a complete randomized block design with four replications. An overview of the obtained results have previously been published (Audenaert *et al*., 2009; Landschoot *et al*., 2011a+b) and are presented in Figure 1. From this extensive survey, several solid conclusions could be drawn.

First, it was clear that the FHB population in Flanders is very dynamic evolving from a *F. graminearum/F. culmorum* dominated population in 2002 and 2003 towards a *F. poae/F. graminearum* population in 2008 and 2009 (Figure 1A). In addition, a correlative study on all variables elucidated some clear population characteristics. First, *F. poae* was shown to be a pathogen that is often occurring in association with other members of the disease complex. This is illustrated in the heat map presented in Figure 2A where *F. poae* clearly clusters with other species such as *F. avenaceum* and *M. nivale*.

A second layer of complexity is the link between DON level and the DON-producing species *F. graminearum* and *F. culmorum*. As illustrated in Figure 1B, the presence of DON was not really correlated with the presence of DON-producing species since it clustered separately in a different branch of the tree. In addition, the presence of *F. graminearum* and *F. culmorum* was rather linked to low disease classes such as Dc1 and Dc2 while the presence of the other species was linked with the higher disease classes Dc2, Dc3 and Dc4. Finally, the presence of DON was also associated with the higher disease classes. Nevertheless, although this link was apparent, no clear linear correlation was observed between quantitative DON presence and disease symptoms (Audenaert *et al*., 2009).

#### **2. Fungicides to control FHB and associated mycotoxins**

#### **2.1 Fungicides to control fungal growth**

Several active ingredients such as triazoles and strobilurins have been reported for their efficiency against several species of the *Fusarium* complex. Triazoles are known inhibitors of the ergosterol biosynthesis in fungi while strobilurin fungicides inhibit mitochondrial electron transport by binding on the Qo site of the cytochrome BC1 complex. Where the effectiveness of triazole fungicides against *Fusarium* spp. is a certainty, the activity of strobilurins against *Fusarium* spp. is doubtable. A considerable amount of evidence shows that strobilurins are mainly active against *M. nivale.* Laboratories around the globe have devoted considerable efforts to develop a coherent view of the activity of fungicides against the FHB causing species. A comprehensive overview is illustrated in Table 2.

Fig. 1. Results of an extensive field survey in Flanders wheat fields on the FHB population, DON content and disease symptoms. A. shows the evolution of the population from 2002- 2009. B shows the links between disease symptoms (Dc1 = disease class 1; Dc2=disease class 2; Dc3= disease class 3; Dc4 = disease class 4; Dc5 = disease class 5). C shows typical FHB symptoms at anthesis (two left ears) compared to asymptomatic ears (two right ears).

Some exceptions notwithstanding no real contradictory reports are mentioned although Zhang *et al*. (2009b) and Pirgozliev *et al*. (2002) obtained different results on the effect of azoxystrobin to control *F. graminearum* and *F. culmorum*. Possibly, these differences originate from different environmental conditions under which experiments were carried out. In line with this assumption Magan *et al*. (2002) clearly highlighted the importance of the aw value in the efficiency of fungicides. Similarly, other researchers showed the importance of wheat cultivar and isolate aggressiveness for control of *Fusarium* using fungicides (Mesterhazy *et al*. 2003).

#### **2.2 Fungicides to control mycotoxin production**

82 Fungicides – Beneficial and Harmful Aspects

Fig. 1. Results of an extensive field survey in Flanders wheat fields on the FHB population, DON content and disease symptoms. A. shows the evolution of the population from 2002- 2009. B shows the links between disease symptoms (Dc1 = disease class 1; Dc2=disease class 2; Dc3= disease class 3; Dc4 = disease class 4; Dc5 = disease class 5). C shows typical FHB symptoms at anthesis (two left ears) compared to asymptomatic ears (two right ears).

Where the effect of fungicides on fungal outgrowth is quite straightforward, reports on the effect of fungicides on the production of mycotoxins is rather contradictory and information is fragmentary. Indeed, to date, no studies are available describing the effect of fungicides to the broad array of mycotoxins that can be produced by *Fusarium*. Most studies focus on just one or two mycotoxins.

For tebuconazole it is generally accepted that it causes a reduction in the biosynthesis or DON level (Edwards *et al*. 2001; Haidukowski *et al*. 2005; Ioos *et al*. 2005; Paul *et al*. 2008; Simpson *et al*. 2001; Zhang *et al*. 2009a) and the trichothecene nivalenol (NIV) (Ioos *et al*. 2005). Information on another triazole fungicide propiconazole is contradictionary. Application of propiconazole resulted in decreased DON levels in a study by Paul *et al*. (2008), while other studies reported increased levels of DON upon propiconazole application (Magan *et al*. 2002).

Application of the triazole metconazole generally results in decreased DON levels in grain samples. This observation was corroborated by several scientific reports (Edwards *et al*. 2001; Paul *et al*. 2008; Pirgozliev *et al*. 2002). Finally for prothioconazole Paul *et al*. (2008) mentioned decreased DON levels. Consonant with this observation, Audenaert *et al*. (2010) described reduced DON levels upon application of field doses of prothioconazole in an *in vitro* assay. However, these authors added another layer of complexity in developing a coherent view on the effect of prothioconazole on DON biosynthesis. Sub lethal application of prothioconazole resulted in increased DON levels (Audenaert *et al*. 2010). This induction was shown to be orchestrated through a reactive oxygen mediated pathway. Indeed, using an *in vitro* approach the former authors succeeded to demonstrate that sub lethal application of prothioconazole results in the prompt induction of H2O2 which preceded the DON accumulation. In addition, elimination of H2O2 using catalase inhibited the production of DON.

The effect of the strobilurin fungicide azoxystrobin on DON varies from a proliferated DON biosynthesis (Zhang *et al*. 2009; Magan *et al*. 2002; Simpson *et al*. 2001; Gaurilcikiene *et al*. 2011) towards reduced DON levels (Pirgozliev *et al*. 2002). It is tempting to speculate on this observation. The fact that strobilurins often result in increased DON levels might be explained by the pathogen spectrum of strobilurin which mainly targets *M. nivale* while being less effective against *F. graminearum*. It is not unlikely that the niches that are not longer occupied by *M. nivale* are taken by *F. graminearum* which consequently lead to increased DON levels.

Although this kind of research is mainly carried out on the mycotoxin DON, the focus is also shifted to other mycotoxins. Gaurilcikiene *et al*. (2011) demonstrated increased T-2 levels upon azoxystrobin application. A similar result was obtained for NIV (Ioos *et al*. 2005).


Table 2. Effect of several fungicides on *Fusarium* spp. and corresponding mycotoxin production. ↑: proliferated growth/production; ↓: reduced growth/production; ─: no effect; NR: not relevant; ND: not detected;\*: effect dependent on the aw value.

Finally, some other fungicides namely carbendazim and thiram were tested for their efficiency to reduce DON in grain samples. However, no clear effect was observed (Zhang *et al*. 2009). A nice study with *F. verticiloides* showed decreased fumonisin levels upon application of respectively quintozene and fludioxynil+metalaxyl-N.

Although the above mentioned examples are not meant to provide a complete and extensive literature review on the use of fungicides against FHB, they clearly demonstrate that the infield control of FHB symptoms does not completely cover control of myctoxin production. We can conclude that when fungicides are not sprayed optimally, conditions which might be conducive for mycotoxin production might be created in the field. This conclusion will hopefully encourage further research in this scientific field.

### **3. Effect of fungicides on the fungal metabolome**

Recently, interest in the effect of fungicides on the fungal metabolome has increased. Primarily fueling this interest in the interaction between sub lethal fungical concentrations and the fungus is that in practice, fungicidal treatments cannot always be carried out under optimal conditions. Consequently, the fungicide concentrations encountered by the pathogen are often lower than one would expect.

#### **3.1 Short term effects**

84 Fungicides – Beneficial and Harmful Aspects

TRIAZOLES

STROBILURINS

OTHERS

species effect on species mycotoxin effect on mycotoxin reference

*Fusarium spp.* ↓ DON ↓ Edwards *et al* . (2001) *F. culmorum* ↓ DON ↓ Simpson *et al* . (2001) *F. avenaceum* ↓ ND ND Simpson *et al* . (2001) *M. nivale* ─ NR NR Paul *et al* . (2008) *Fusarium spp.* ↓ DON ↓ Haidukowski *et al* . (2005) *F. culmorum* ↓ NIV ─ Ioos *et al* . (2005)

*Fusarium spp.* ↓ DON ↓ Paul *et al* . (2008)

*F. graminearum* ↓ DON ↓ Pirgozliev *et al* . (2002) *Fusarium spp.* ↓ DON ↓ Edwards *et al* . (2001) *Fusarium spp.* ↓ DON ↓ Paul *et al* . (2008)

*F. graminearum* ↓ DON ↑ Audenaert *et al* . (2010)

*F. culmorum* ─ DON ↑ Magan *et al* . (2002) *F. culmorum* ↓ DON ↓ Pirgozliev *et al* . (2002) *F. graminearum* ↓ DON ↓ Pirgozliev *et al* . (2002) *M. nivale* ↓ NR NR Simpson *et al* . (2001) *Fusarium spp.* ↑ DON ↑ Simpson *et al* . (2001) *Fusarium spp.* ─ DON ─ Edwards *et al* . (2001) *F. poae* ↑ T2 ↑ Gaurilcikiene *et al.* (2011) *F. culmorum* ↑ DON ↑ Gaurilcikiene *et al.* (2011) *F. poae* ↓ T2 ↑ This work *F. culmorum* ─ NIV ─ / ↑ Ioos *et al* . (2005)

Tebuconazole *F.graminearum* ↓ DON ↓ Zhang *et al* . (2008)

Propiconazole *F. culmorum* ↓\* DON ↑ Magan *et al* . (2002)

Metconazole *F. culmorum* ↓ DON ↓ Pirgozliev *et al* . (2002)

Prothioconazole *Fusarium spp.* ↓ DON ↓ Paul *et al* . (2008)

Azoxystrobin *F. graminearum* ─ DON ↑ Zhang *et al* . (2008)

Table 2. Effect of several fungicides on *Fusarium* spp. and corresponding mycotoxin

NR: not relevant; ND: not detected;\*: effect dependent on the aw value.

production. ↑: proliferated growth/production; ↓: reduced growth/production; ─: no effect;

Fludioxynil+metalaxyl-N *F. verticillioides* ↓ Fum ↓ Falcao *et al.* (2011)

Carbendazim *F. graminearum* ─ DON ─ Zhang *et al.* (2008) Thiram *F. graminearum* ─ DON ─ Zhang *et al.* (2008) Quintozene *F. verticillioides* ↓ Fum ↓ Falcao *et al* . (2011) When *Fusarium* encounters fungicide concentrations that are not lethal, a complex spectrum of metabolic changes occurs. The full range of these metabolic changes is still not well dissected although the first steps have been taken to use genome wide approaches to disentangle transcriptional changes in *Fusarium* upon fungicide treatments (Liu *et al*. 2010).

Although the majority of these metabolic changes remain elusive, a fast growing number of papers focus on the oxidative stress induced by fungicides. In an *in vitro* approach it was demonstrated that exposing *F. graminearum* to sub lethal doses of prothioconazole resulted in proliferated production of DON. In addition, an increase in H2O2 which preceded the DON accumulation was observed. Addition of catalase, an H2O2 scavenger, resulted in loss of DON production. Similar results were obtained in a study using *F. graminearum* and *M. nivale*. This study provided evidence that H2O2 was produced by *F. graminearum* and *M. nivale* upon azoxystrobin application (Kaneko & Ishii 2009). However, this phenomenon is possibly isolate or species dependent. In a study by Covarelli *et al*. (2004), tebuconazole was shown to have a negative effect on the expression of the Tri5 gene, an indication for DON bioynthesis in *F. culmorum*.

#### **3.2 Long term effects**

The ability of fungi to adapt to stress is pivotal to their survival in the environment, and this adaptation ability is one of the key factors leading to mutations or adaptations that can give rise to more aggressive crop pathogens in an agricultural setting. In a recent study, evidence was brought forward showing that the initial efficacy of the triazole epoxiconazole eroded resulting in increasing EC50 values with a factor of approximately 1.4 (Klix *et al*. 2007). An interesting study illustrated that mutations in a β-tubulin conferred resistance of *F. graminearum* to benzimidazole fungicides (Chen *et al*. 2009). In addition, a benzimidazole binding site on the β-tubulin gene was suggested to be mutated conferring strains resistant to benzimidazole fungicides such as carbendazim (Qiu *et al*. 2011). An even more interesting observation in carbendazim resistant *F. graminearum* strains was that a proliferated production of DON was observed (Zhang *et al*. 2009b).

Typically for triazole fungicides, a slowly evolving fungicide resistance has been observed. Decreases in azole sensitivity can be caused by (i) point mutations in the target gene, (ii) overexpression of the target gene, (iii) alterations in ergosterol biosynthesis, (iv) enhanced efflux of toxic compounds, and (v) increased copy numbers of target genes or genes for efflux pumps (Becher *et al*. 2010). Similarly as in the carbendazim story, isolates displaying increased resistance to tebuconazole showed increased mycotoxin production.

Finally, for azoxystrobin, at least 27 fungal species are listed to be resistant. The majority of the resistance types are correlated with the G143A substitution in the quinol oxidation site of cytochrome b, the target for strobilurins. Also for members of the FHB complex, this type and other types of resistance towards strobilurins have been described in respectively *M. nivale* (Walker *et al*. 2009) and *F. graminearum* (Dubos *et al*. 2011). The results and examples given in the previous paragraphs clearly peeled away several layers of complexity in the chemical control of FHB. The divergence of fungicide effectiveness both at species and mycotoxin level hamper a simple control of this disease complex. Still, a better insight into the effect of fungicide application on *Fusarium* in the field is needed. European legislation for several *Fusarium* mycotoxins has been established. This legislation provides an extra economic motive for farmers to prevent FHB infection and mycotoxin accumulation in small grain cereals such as wheat.

A detailed study on the effect of fungicides at a population level will certainly contribute to new insights in the adaptive dynamics of a *Fusarium* population upon fungicide application. In addition, shifts in the population might have its consequences for the mycotoxin profiles present in these fields. In the present study, results from fungicide field trials from 2002- 2010 are presented with regard to the effect of triazole and strobilurin fungicides on symptoms, population composition and the presence of the trichothecene mycotoxin DON in the field. These data provide new insights into the effect of fungicides on FHB both at a species- and mycotoxin level.

#### **4. Experimental setup of this study**

#### **4.1 Experimental field trials**

From 2002 to 2010, different field trials of winter wheat throughout Belgium were followed up for at least ten locations that were located in the most important wheat regions characterized by different growth conditions and crop husbandry measurements. The winter wheat area of Belgium is situated in the centre of the wheat growing region in Europe. Each year at each location, commercial wheat varieties were sown in a complete randomized block design with four replications. For all locations the normal crop husbandry measures were taken. Depending on the experiment, several fungicides and fungicide combinations were used and were applied at various Zadoks growth stages (GS39, GS55 up to GS65) of the crop. In this way both the effect of the active ingredient and the time of application was monitored. The wheat cultivars were sown in common crop rotation systems which lead to different previous crops, both host crops (maize or wheat) as well as non-host crops for *Fusarium* spp. (beans, sugar beets, onions or chicory).

From GS71 to GS75 the experiments were evaluated for the presence of *Fusarium* symptoms. Both the FHB incidence and the FHB severity (disease classes 1-5 with 0, 25, 50, 75 or 100 % bleached ear surface, respectively) were scored. To take into account both assessments for 100 randomly chosen ears per plot, the disease index (DI) was computed as follows: DI = (0n1 + 1n2 + 2n3 + 3n4 + 4n5)/4n x 100%; with "n" the number of evaluated ears and "ni" the number of ears in disease class i.

In order to assess the composition of the FHB population, wheat ears were plated on PDA medium (potato dextrose agar, Oxoid, Belgium) for further species identification. Seeds were surface-sterilized for 1 minute in 1% NaOCl, washed for 1 min with 70% EtOH, washed with distilled sterile water, dried for 5 min and subsequently put on PDA plates. After five days of incubation at 20°C, outgrowing mycelium was transferred to a new PDA plate. For species determination, five mycelium plugs randomly taken from the fully grown PDA plates were transferred to liquid GPY-broth (10 g glucose, 1 g yeast extract and 1 g peptone, Oxoid, Belgium) and incubated for five days at 20°C. After five days, mycelium was transferred to eppendorf tubes, centrifuged for 10 min at 12,000 rpm and then freezedried for 6 h at -10°C and 4 h at -50°C (Christ Alpha 1-2 LD Plus, Osterode, Deutschland). DNA extraction was performed as described by Audenaert *et al*. (2009), based on the CTAB (hexadecyl trimethyl ammonium bromide) method (Saghai-Maroof *et al*., 1984). PCR for single species detection was performed in a 25 µl reaction mixture (Demeke *et al*., 2005).

DNA amplification was performed in an Applied Biosystems GeneAmp PCR system 97000 PCR. Amplicons were separated on 1.5% (wt/vol) agarose gels stained with 0.1 µl ethidium bromide. PCR was validated by including reference strains obtained from the MUCL/BCCM collection in each PCR run: *F. graminearum* MUCL 42841; *F. culmorum* MUCL 555; *F. poae* MUCL 6114; *M. nivale* MUCL 15949; *F. avenaceum* MUCL 6130 (Audenaert *et al*., 2009, Isebaert *et al*., 2009).

At harvest, DON levels was analyzed by enzyme-linked immunosorbent assay (ELISA) (Veratox DON 5/5 kit Biognost - Neogen). A subsample was taken from each field out of the randomized block design. All DON results are the average of at least four DON measurements per treatment.

#### **4.2** *In vitro* **trials**

86 Fungicides – Beneficial and Harmful Aspects

observation in carbendazim resistant *F. graminearum* strains was that a proliferated

Typically for triazole fungicides, a slowly evolving fungicide resistance has been observed. Decreases in azole sensitivity can be caused by (i) point mutations in the target gene, (ii) overexpression of the target gene, (iii) alterations in ergosterol biosynthesis, (iv) enhanced efflux of toxic compounds, and (v) increased copy numbers of target genes or genes for efflux pumps (Becher *et al*. 2010). Similarly as in the carbendazim story, isolates displaying

Finally, for azoxystrobin, at least 27 fungal species are listed to be resistant. The majority of the resistance types are correlated with the G143A substitution in the quinol oxidation site of cytochrome b, the target for strobilurins. Also for members of the FHB complex, this type and other types of resistance towards strobilurins have been described in respectively *M. nivale* (Walker *et al*. 2009) and *F. graminearum* (Dubos *et al*. 2011). The results and examples given in the previous paragraphs clearly peeled away several layers of complexity in the chemical control of FHB. The divergence of fungicide effectiveness both at species and mycotoxin level hamper a simple control of this disease complex. Still, a better insight into the effect of fungicide application on *Fusarium* in the field is needed. European legislation for several *Fusarium* mycotoxins has been established. This legislation provides an extra economic motive for farmers to prevent FHB infection and mycotoxin accumulation in small

A detailed study on the effect of fungicides at a population level will certainly contribute to new insights in the adaptive dynamics of a *Fusarium* population upon fungicide application. In addition, shifts in the population might have its consequences for the mycotoxin profiles present in these fields. In the present study, results from fungicide field trials from 2002- 2010 are presented with regard to the effect of triazole and strobilurin fungicides on symptoms, population composition and the presence of the trichothecene mycotoxin DON in the field. These data provide new insights into the effect of fungicides on FHB both at a

From 2002 to 2010, different field trials of winter wheat throughout Belgium were followed up for at least ten locations that were located in the most important wheat regions characterized by different growth conditions and crop husbandry measurements. The winter wheat area of Belgium is situated in the centre of the wheat growing region in Europe. Each year at each location, commercial wheat varieties were sown in a complete randomized block design with four replications. For all locations the normal crop husbandry measures were taken. Depending on the experiment, several fungicides and fungicide combinations were used and were applied at various Zadoks growth stages (GS39, GS55 up to GS65) of the crop. In this way both the effect of the active ingredient and the time of application was monitored. The wheat cultivars were sown in common crop rotation systems which lead to different previous crops, both host crops (maize or wheat) as

well as non-host crops for *Fusarium* spp. (beans, sugar beets, onions or chicory).

increased resistance to tebuconazole showed increased mycotoxin production.

production of DON was observed (Zhang *et al*. 2009b).

grain cereals such as wheat.

species- and mycotoxin level.

**4.1 Experimental field trials** 

**4. Experimental setup of this study** 

In the present study, fluoxastrobin+prothioconazole was tested for its efficiency to control several field isolates of *F. poae*. The field dose of the fungicide was the point of departure for the *in vitro* assay. The field dose mounted to 0.5 g/l + 0.5 g/l for fluoxastrobin+prothioconazole, A dilution series of the fungicide was prepared to obtain a final concentration of 1 mg/l, 5 mg/l, 10 mg/l and 50 mg/l in the 24-well plates in which the assay was executed. In these wells, 250 μl of conidial suspension was added and amended with 250 μl of the fungicide. The final concentration of the microconidia was 106 conidia per ml. These wells were incubated at 22°C. Two repetitions were done per dilution and the experiment was repeated two times independently in time. Control treatments consisted of 250 μl of spore suspension and 250 μl of distilled water. T-2 production kinetics were monitored using an ELISA (Veratox T-2 kit, Biognost-Neogen).

At each time point (4 h, 24 h, 48 h) after inoculation, the percentage of germinated conidia were counted. At each time point, three repetitions per treatment were counted.

#### **5. Results**

#### **5.1** *In vitro* **effect of fluoxastrobin+prothioconazole on** *F. poae*

From previous work it is known that fluoxastrobin+prothioconazole provides good protection against *F. graminearum*. For several isolates it was shown that a dose of 50 mg/l of this fungicide resulted in a reduction in germination rate of 95% (Audenaert *et al*. 2010). Based on these results, we wanted to focus on the sensitivity of *F. poae* to this fungicide. *F. poae* was an underestimated species for long time since it was described being a weak pathogen. However, throughout Europe a steadily increase of this species has been observed. Yet, the basis for this increased importance remains elusive. To date research on this pathogen remains limited although research groups around the globe tend to initiate research initiatives with regard to this pathogens (Stenglein, 2009). From Figure 2 it is clear that a diversified spectrum of susceptibility can be observed in *F. poae* depending on the isolate. Most importantly, some of the isolated strains showed residual germination levels ranging from 20% up to 80% at fungicide concentrations three times the field dose (data not shown). In addition, among isolates very diverse reaction patterns were observed in the dilution series. This result highlights the high diversity of *F. poae* with regard to fungicide resistance.

Fig. 2. Effect of a dilution series of fluoxastrobin+prothioconazole on the conidia germination of four different *F. poae* isolates (A,B,C, and D). Each panel shows the germination of a different isolate.

The results obtained in this work are rather contradictory with what has been described in literature. Generally it is accepted that *F. graminearum* is more resistant to fungicides than *F. poae*. Using several fungicides such as diphenoconazole, tebuconazole, iprodione,… it was demonstrated that the sensitivity of *F. graminearum* was lower compared to *F. poae* (Hudec, 2007; Mullenborn *et al*. 2008). Till now, we have no clear explanation for this discrepancy, however, possibly the isolate, the incubation temperature, the culture conditions might influence the sensitivity response in both species.

From previous work it is known that fluoxastrobin+prothioconazole provides good protection against *F. graminearum*. For several isolates it was shown that a dose of 50 mg/l of this fungicide resulted in a reduction in germination rate of 95% (Audenaert *et al*. 2010). Based on these results, we wanted to focus on the sensitivity of *F. poae* to this fungicide. *F. poae* was an underestimated species for long time since it was described being a weak pathogen. However, throughout Europe a steadily increase of this species has been observed. Yet, the basis for this increased importance remains elusive. To date research on this pathogen remains limited although research groups around the globe tend to initiate research initiatives with regard to this pathogens (Stenglein, 2009). From Figure 2 it is clear that a diversified spectrum of susceptibility can be observed in *F. poae* depending on the isolate. Most importantly, some of the isolated strains showed residual germination levels ranging from 20% up to 80% at fungicide concentrations three times the field dose (data not shown). In addition, among isolates very diverse reaction patterns were observed in the dilution series. This result

**5.1** *In vitro* **effect of fluoxastrobin+prothioconazole on** *F. poae*

highlights the high diversity of *F. poae* with regard to fungicide resistance.

Fig. 2. Effect of a dilution series of fluoxastrobin+prothioconazole on the conidia germination of four different *F. poae* isolates (A,B,C, and D). Each panel shows the

The results obtained in this work are rather contradictory with what has been described in literature. Generally it is accepted that *F. graminearum* is more resistant to fungicides than *F. poae*. Using several fungicides such as diphenoconazole, tebuconazole, iprodione,… it was demonstrated that the sensitivity of *F. graminearum* was lower compared to *F. poae* (Hudec, 2007; Mullenborn *et al*. 2008). Till now, we have no clear explanation for this discrepancy, however, possibly the isolate, the incubation temperature, the culture conditions might

% conidia germinated

D

% germinated conidia

B

control 1 mg/l 5 mg/l 10 mg/l 50 mg/l prothioconazole+fluoxastrobin

control 1 mg/l 5 mg/l 10 mg/l 50 mg/l prothioconazole+fluoxastrobin

4 h 24 h 48 h

germination of a different isolate.

influence the sensitivity response in both species.

control 1 mg/l 5 mg/l 10 mg/l 50 mg/l prothioconazole+fluoxastrobin

control 1 mg/l 5 mg/l 10 mg/l 50 mg/l prothioconazole+fluoxastrobin

**5. Results** 

% germinated conidia

C

% germinated conidia

A

In a second step, we wanted to investigate the interaction between stress induced by prothioconazole+fluoxastrobin and the toxin production by the *F. poae* isolates. Surprisingly, using an LC-MSMS approach, the *F. poae* isolates were characterized as T-2 chemotype. In addition, several other mycotoxins were produced such as diacetylscirpenol and nivalenol (data not shown). The ability of *F. poae* to produce T-2 is rather exceptional. Indeed, the ability of *Fusarium* to produce T-2 toxin has been described to be mainly restricted to *F. langsethiae* and *F. sporotrichoides*. A reason for this apparent discrepancy originates from the fact that no structural genetic information is available regarding the toxic metabolome of *F. poae*. Therefore, majority of the studies use artificial media in search for toxins of *F. poae*. However, recent research in our laboratory clearly illustrates that *F. poae* does not produce toxins on all media. There is some evidence that the nitrogen source and eventual amino acids or polyamines could play a role in the induction of T-2 production by *F. poae*. In Figure 2, the production kinetics of T-2 upon prothioconazole+fluoxastrobin is shown.

Similar to the results on the conidia germination (Figure 3), the effect of the fungicide prothioconazole+fluoxastrobin differed clearly depending on the isolate. Some isolates did not show a consistent proliferated T-2 production upon fungicide stress (Figure 3 A and C) others did (Figure 3B). Remarkably, the isolate that was extremely resistant to the fungicide prothioconazole+fluoxastrobin also showed extremely high basal levels of T-2 production which even increased upon fungicide application. Although these results are very preliminary, they pinpoint T-2 production as a possible protective mechanism upon fungicide application. Similar results were previously obtained with DON. Using a tri5 knockout mutant, it was demonstrated that DON-negative mutants of *F. graminearum*  became hypersusceptible to fungicide application (Audenaert *et al*., 2011).

Fig. 3. Effect of sub lethal prothioconazole+fluoxastrobin concentrations on T-2 production on four different isolates (A,B,C and D).

#### **5.2 Control of FHB by fungicide application at growth stage GS55**

#### **5.2.1 Effect of fungicides on DON content**

In order to peel away the layers of complexity regarding control of *Fusarium* and corresponding DON contamination, a fungicide trial using prothioconazole, epoxyconazole+metconazole and prothioconazole+tebuconazole was set up during the growing seasons of 2007-2008, 2008-2009 and 2009-2010. Pursuing a combined approach of symptom scoring and DON measurement, we aimed to disentangle the effect of triazole fungicides on *Fusarium* development and mycotoxin production. For all treatments, fungicide applications resulted in a clear reduction in symptom development which was the same for all tested active ingredients (data not shown).

More interesting is the effect of these treatments on the DON level. In Figure 4, the effect of triazole application on the DON levels is displayed. By these results, evidence is brought forward demonstrating that the efficiency of triazole fungicides to reduce DON levels is depending on the background level of DON observed in the control treatments. Under conditions of low DON levels, fungicides do not result in decreases in DON level. On the contrary, several fungicide treatments resulted in increased DON levels compared to the control. This detrimental effect of fungicide treatments with respect to DON content has previously been described by other authors (Magan *et al*., 2002).

Fig. 4. Levels of DON after application of prothioconazole (▲), epoxyconazole+metconazole (□) and prothioconazole+tebuconazole (▬) in function of the DON levels present in the untreated control fields. All data points are the result of four independent repetitions.

In order to peel away the layers of complexity regarding control of *Fusarium* and corresponding DON contamination, a fungicide trial using prothioconazole, epoxyconazole+metconazole and prothioconazole+tebuconazole was set up during the growing seasons of 2007-2008, 2008-2009 and 2009-2010. Pursuing a combined approach of symptom scoring and DON measurement, we aimed to disentangle the effect of triazole fungicides on *Fusarium* development and mycotoxin production. For all treatments, fungicide applications resulted in a clear reduction in symptom development which was the

More interesting is the effect of these treatments on the DON level. In Figure 4, the effect of triazole application on the DON levels is displayed. By these results, evidence is brought forward demonstrating that the efficiency of triazole fungicides to reduce DON levels is depending on the background level of DON observed in the control treatments. Under conditions of low DON levels, fungicides do not result in decreases in DON level. On the contrary, several fungicide treatments resulted in increased DON levels compared to the control. This detrimental effect of fungicide treatments with respect to DON content has

Fig. 4. Levels of DON after application of prothioconazole (▲), epoxyconazole+metconazole (□) and prothioconazole+tebuconazole (▬) in function of the DON levels present in the untreated control fields. All data points are the result of four independent repetitions.

012345

DON (mg/kg)

**5.2 Control of FHB by fungicide application at growth stage GS55** 

**5.2.1 Effect of fungicides on DON content** 

same for all tested active ingredients (data not shown).

previously been described by other authors (Magan *et al*., 2002).




10

30

% reduction of DON level compared to control

50

70

90

Surprisingly, our experimental field trials show similar efficiencies in function of the DON concentration for the three triazoles: prothioconazole, epoxyconazole+metconazole and prothioconazole+tebuconazole applied at GS55 were all the most efficient at DON concentrations between 1 mg/kg up to 2 mg/kg while the efficiency reduced to about 50 % for higher DON concentrations.

These results suggest that although fungicides have been described to be very effective to control *Fusarium* symptoms in the field, their efficiency to reduce DON seems to be limited. In addition, Figure 4 illustrates the usefulness of fungicides for fields with DON levels that are situated around the DON threshold values set by the European Commision: with reductions of the DON level from 50% to 90% this implies that samples that would exceed the European threshold limits drop below these limits when triazole fungicides are applied.

#### **5.2.2 Effect of fungicides on the population structure**

Besides the effect of fungicide application on FHB symptoms and DON levels, the impact of fungicides on the population constitution was monitored as well. The five predominant species in Flanders wheat fields were monitored i.e. *F. graminearum, F. poae, F.culmorum, F. avenaceum* and *M. nivale*. Results from these field trials clearly subscribe that fungicide application clearly influences the species distribution within the population (Figure 5).

Fig. 5. Effect of prothioconazole, epoxiconazole+metconazole and prothioconazole+tebuconazole on the population of *Fusarium* species.

In fields treated with prothioconazole at GS55, *F. poae* became the predominant species whereas in the untreated fields, the population was initially dominated by *F. culmorum* and *F. graminearum*. This phenomenon was to a lesser extend also observed in the other treatments with prothioconazole+tebuconazole and epoxiconazole+metconazole. This result is in concordance with the *in vitro* experiments shown in Figure 1 which already suggested that a considerable portion of the *F. poae* isolates possesses a considerable level of resistance towards triazole fungicides. Finally, it was consistently surprising that this shift within the population towards an increase of *F. poae* was mainly at the expense of *F. culmorum*. Surprisingly, application of triazole fungicides did not result in a consistent increase of *M. nivale* in the population. *M. nivale* has previously been described for its resistance towards triazole fungicides but this could not be pinpointed in the present field study. No clear explanation for this observation can be found.

#### **5.3 Fungicide application timing and control of FHB**

#### **5.3.1 Effect of fungicide timing on FHB symptoms and DON content**

Another layer of complexity in developing a coherent view of the effects of fungicide application versus *Fusarium* and its mycotoxins is the timing of the application. In order to investigate the effect of timings for chemical control of *Fusarium*, several triazole fungicides were applied at different growth stages during wheat growth. One series of trials involved a *Fusarium* treatment at GS55 and GS65. Figure 5A clearly illustrates that application of triazole fungicides at GS65 does not result in reduced *Fusarium* symptoms. At the other hand, application of triazole fungicides at GS55 clearly reduces the impact of *Fusarium* at the level of the symptoms. The results for the concomitant DON levels are slightly different. Although chemical control of *Fusarium* at GS65 is inefficient at the level of symptom development, a consistent effect was observed at the level of DON (Figure 6C). This result came as a surprise since several authors report that suboptimal fungicide application can result in proliferated DON production (Audenaert *et al*., 2010; Magan *et al*., 2002). We assume that this late fungicide application at GS65 and at the normal application rate comes too late to avoid symptom development, however this application is still in time to decrease DON levels to some extent. It has been described by several authors that DON is a crucial virulence/pathogenicity factor at later stages of infection to facilitate migration of the pathogen in the ear (Mudge *et al*. 2006).

In a second series of time trials, application of prothioconazole was performed at GS39, GS65 and a combination at GS39+GS65. Results are presented in Figure 6B+D. Similar as in Figure 6A, application of prothioconazole at GS65 results in a small decreased number of symptoms, although this reduction was not significant. Remarkably, application of prothioconazole at GS39 resulted in a significant decrease in *Fusarium* symptoms. In addition, a combined application at GS39+GS65 had a synergistic effect at the level of symptoms. For DON, results were quite similar. Application of prothioconazole at GS39 resulted in reduced DON levels compared to untreated plots and plots treated at GS65. A combined application of prothioconazole did not result in further reduction of the DON level. This result confirms that the ability of fungicides to reduce DON levels in *Fusarium* infected fields is limited to reductions of about 50%.

Scientific research on the effects of fungicide timing with regard to *Fusarium* symptom development and DON levels are scarce. Previously, Wiersma and Motteberg (2005) reported

In fields treated with prothioconazole at GS55, *F. poae* became the predominant species whereas in the untreated fields, the population was initially dominated by *F. culmorum* and *F. graminearum*. This phenomenon was to a lesser extend also observed in the other treatments with prothioconazole+tebuconazole and epoxiconazole+metconazole. This result is in concordance with the *in vitro* experiments shown in Figure 1 which already suggested that a considerable portion of the *F. poae* isolates possesses a considerable level of resistance towards triazole fungicides. Finally, it was consistently surprising that this shift within the population towards an increase of *F. poae* was mainly at the expense of *F. culmorum*. Surprisingly, application of triazole fungicides did not result in a consistent increase of *M. nivale* in the population. *M. nivale* has previously been described for its resistance towards triazole fungicides but this could not be pinpointed in the present field study. No clear

Another layer of complexity in developing a coherent view of the effects of fungicide application versus *Fusarium* and its mycotoxins is the timing of the application. In order to investigate the effect of timings for chemical control of *Fusarium*, several triazole fungicides were applied at different growth stages during wheat growth. One series of trials involved a *Fusarium* treatment at GS55 and GS65. Figure 5A clearly illustrates that application of triazole fungicides at GS65 does not result in reduced *Fusarium* symptoms. At the other hand, application of triazole fungicides at GS55 clearly reduces the impact of *Fusarium* at the level of the symptoms. The results for the concomitant DON levels are slightly different. Although chemical control of *Fusarium* at GS65 is inefficient at the level of symptom development, a consistent effect was observed at the level of DON (Figure 6C). This result came as a surprise since several authors report that suboptimal fungicide application can result in proliferated DON production (Audenaert *et al*., 2010; Magan *et al*., 2002). We assume that this late fungicide application at GS65 and at the normal application rate comes too late to avoid symptom development, however this application is still in time to decrease DON levels to some extent. It has been described by several authors that DON is a crucial virulence/pathogenicity factor at later stages of infection to facilitate migration of the

In a second series of time trials, application of prothioconazole was performed at GS39, GS65 and a combination at GS39+GS65. Results are presented in Figure 6B+D. Similar as in Figure 6A, application of prothioconazole at GS65 results in a small decreased number of symptoms, although this reduction was not significant. Remarkably, application of prothioconazole at GS39 resulted in a significant decrease in *Fusarium* symptoms. In addition, a combined application at GS39+GS65 had a synergistic effect at the level of symptoms. For DON, results were quite similar. Application of prothioconazole at GS39 resulted in reduced DON levels compared to untreated plots and plots treated at GS65. A combined application of prothioconazole did not result in further reduction of the DON level. This result confirms that the ability of fungicides to reduce DON levels in *Fusarium*

Scientific research on the effects of fungicide timing with regard to *Fusarium* symptom development and DON levels are scarce. Previously, Wiersma and Motteberg (2005) reported

explanation for this observation can be found.

pathogen in the ear (Mudge *et al*. 2006).

infected fields is limited to reductions of about 50%.

**5.3 Fungicide application timing and control of FHB** 

**5.3.1 Effect of fungicide timing on FHB symptoms and DON content** 

GS60 as ideal for control of FHB. In addition, using tebuconazole, these authors checked the efficiency to control FHB and DON levels but regarding *Fusarium* symptoms, they could not come up with solid conclusions regarding the timing of application. For the efficiency of tebuconazole to reduce DON levels, applications at GS39 and GS60 performed equally.

Fig. 6. Effect of timing of fungicide application on *Fusarium* symptom development and DON content. A+C: effect of prothioconazole, tebuconazole and epoxyconazole applied at GS65 and GS55 on symptoms (A) and DON level (C). B+D: effect of prothioconazole applied at GS39, GS65 and GS39+GS65 on *Fusarium* symptom development (B) and DON level (D).

#### **5.3.2 Fungicide timing and population structure**

The timing of fungicide application to control *Fusarium* clearly had an effect on the composition of the population. When we compare the population in the untreated control of the experimental field trial presented in Figure 7 with the untreated control in Figure 4, the huge differences in population composition is obvious. This enormous elasticity of the FHB population depending on field location has previously been described by Audenaert *et al*. (2009).

Application of prothioconazole at GS55 clearly favoured *F. poae* which was not present in the control fields but which popped up in the prothioconazole treatment (Figure 7A). For the other triazole fungicides, a consistent reduction of *F. avenaecum* was observed. In addition, the niches seemed to solely colonized by *F. graminearum*. In the other field trial, where prothioconazole was applied at GS39, GS65 and GS39+GS65, major shifts in the population were observed although no consistent changes were observed between the treatments. At first sight this might be unexpected, however both application of GS39 and GS65 can be considered to be apsecific for FHB. Therefore, the population causing disease symptoms has not yet been established (at GS39) or is already fading (GS65). This might explain the less consistent results in this experiments compared to the results obtained in the experiment where fungicides were applied at GS55 (Figure 5).

Fig. 7. Effect of timing of fungicide application on the FHB population. A: effect of prothioconazole, tebuconazole and epoxyconazole applied at GS65 and GS55. B: effect of prothioconazole applied at GS39, GS65 and GS39+GS65.

It was remarkable that the increased portion of *F. graminearum* in the fungicide treatments did not result is increased DON levels. This result provides indirect evidence that the presence of DON-producing chemotypes within the population does not necessarily result in a proliferated DON level in the field. This finding underscores the work by Landschoot *et al*. (2011a+b).

#### **6. Conclusions**

The aim of this study was to disentangle the effect of fungicide application on the FHB disease complex. Pursuing a combined approach of *in vitro* and *in vivo* field trials, some very interesting conclusions could be drawn. First, it is important to state that fungicides applied at normal rate under optimal conditions are effective in controlling FHB disease. However, working in the field often implies working under suboptimal conditions, and then, problems regarding FHB presence of concomitant mycotoxins might occur. First, it is important to highlight that not all species comprised in the FHB disease show the same susceptibility towards fungicide application. The data presented in the present work provide a compelling amount of evidence illustrating that *F. poae* is more resistant to triazoles than the other members of the disease complex. This was illustrated in *in vitro*  trials which showed that a considerable amount of isolates were able to grow at field doses of fungicide applications. Consonant with these *in vitro* trials field trials demonstrated that triazole application generally resulted in a shift in the FHB population in favour of *F. poae.* 

At the level of symptom development, all tested triazole fungicides when optimally applied at GS55 resulted in similar disease symptoms reduction. When varying the timing of application, it was obvious that triazole application at GS65 came too late to efficiently reduce FHB symptoms. On the contrary, fungicide spraying at GS39 resulted in a significant reduction of disease symptoms. We suggest that these treatments reduce the primary inoculums present in the vegetative crop. A combined application at GS39+GS65 resulted in a synergistic effect of the treatments.

With regard to DON, several lines of evidence corroborate a role for timing of fungicide application. The results obtained in the present study demonstrate nicely that where complete control of FHB can be obtained at the level of disease symptoms, there seems to be some sort of threshold efficiencies that cannot be exceeded when DON levels are higher than 2 mg/kg. In all the field trials comprised in the present study, a maximum DON reduction of 50% was obtained compared to the untreated control fields. In addition, where application of triazoles was not affecting disease symptoms when applied at GS65, a minor reduction of DON levels was observed. Contrary to observations on the symptom level, no additional effect was observed when performing a combined application of triazoles at GS39+GS65 compared to single applications at GS39 or GS65.

In conclusion, this study peeled away several layers of complexity in the chemical control of FHB in wheat. We are convinced that fungicide use is an important hurdle that can be included in crop management systems to prevent FHB and concomitant mycotoxin present. However, it is clearly an oversimplification to pretend that fungicide use is the "holy grail" in the control of FHB disease. The disease is far too complex and too multifaceted to draw this conclusion. It will take all branches of scientific research to keep this problem under control. The use of wheat varieties with high levels of resistance, the use of good culture practices such as broad crop rotations and intelligent soil tillage will certainly contribute

#### **7. Acknowledgment**

94 Fungicides – Beneficial and Harmful Aspects

For the other triazole fungicides, a consistent reduction of *F. avenaecum* was observed. In addition, the niches seemed to solely colonized by *F. graminearum*. In the other field trial, where prothioconazole was applied at GS39, GS65 and GS39+GS65, major shifts in the population were observed although no consistent changes were observed between the treatments. At first sight this might be unexpected, however both application of GS39 and GS65 can be considered to be apsecific for FHB. Therefore, the population causing disease symptoms has not yet been established (at GS39) or is already fading (GS65). This might explain the less consistent results in this experiments compared to the results obtained in the

Fig. 7. Effect of timing of fungicide application on the FHB population. A: effect of prothioconazole, tebuconazole and epoxyconazole applied at GS65 and GS55. B: effect of

epoxyconazoleGS55

It was remarkable that the increased portion of *F. graminearum* in the fungicide treatments did not result is increased DON levels. This result provides indirect evidence that the presence of DON-producing chemotypes within the population does not necessarily result in a proliferated DON level in the field. This finding underscores the work by Landschoot

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

control

prothioconazoleGS65

prothioconazoleGS39

prothioconazoleGS39+GS65

The aim of this study was to disentangle the effect of fungicide application on the FHB disease complex. Pursuing a combined approach of *in vitro* and *in vivo* field trials, some very interesting conclusions could be drawn. First, it is important to state that fungicides applied at normal rate under optimal conditions are effective in controlling FHB disease. However, working in the field often implies working under suboptimal conditions, and then, problems regarding FHB presence of concomitant mycotoxins might occur. First, it is important to highlight that not all species comprised in the FHB disease show the same susceptibility towards fungicide application. The data presented in the present work provide a compelling amount of evidence illustrating that *F. poae* is more resistant to triazoles than the other members of the disease complex. This was illustrated in *in vitro* 

prothioconazole applied at GS39, GS65 and GS39+GS65.

*et al*. (2011a+b).

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

*F. poae M. nivale F. graminearum F. culmorum F. avenaceum*

control

tebuconazoleGS65

prothioconazoleGS65

epoxyconazoleGS65

A B

tebuconazoleGS55

prothioconazoleGS55

**6. Conclusions** 

experiment where fungicides were applied at GS55 (Figure 5).

Kris Audenaert is a postdoctoral fellow of the Ghent University Research Fund. Sofie Landschoot and Adriaan Van Heule are PhD students financed by the Ghent University College research fund. We greatly acknowledge the help of Bernard De Baets and Willem Waegeman from Ghent University, research group KERMIT, Belgium for the nice collaboration in the data analysis. Secondly, our gratitude goes to Lies Willaert, Melvin Berten and Daniël Wittouck from the Landbouw Centrum Granen for helping to establish all field trials troughout Flanders during the last 10 years. Sarah De Saeger and Sofie Monbaliu, from the faculty of Pharmaceutical Sciences, Ghent University helped with the mycotoxin analysis using LC-MSMS.

Part of this work was financially supported by the Flemish Institute for the Stimulation of Scientific - Technological Research in Industry project 70575 (IWT, Brussels, Belgium).

#### **8. References**


Audenaert, K., Callewaert, E., Hofte, M., De Saeger, S., & Haesaert, G. (2010). Hydrogen

Audenaert, K., Van Broeck, R., Bekaert, B., De Witte, F., Heremans, B., Messens, K., Hofte,

Becher, R., Hettwer, U., Karlovsky, P., Deising, H.B., & Wirsel, S.G.R. (2010). Adaptation of

Bennett, J.W., & Klich, M. (2003) Mycotoxins. *Clinical Microbiology Reviews,* vol.16, pp. 497-

Brennan, J.M., Fagan, B., van Maanen, A., Cooke, B.M., & Doohan, F.M. (2003). Studies on in

Chen, C.J., Yu, J.J., Bi, C.W., Zhang, Y.N., Xu, J.Q., Wang, J.X., & Zhou, M.G. (2009).

Covarelli, L., Turner, A.S., & Nicholson, P. (2004). Repression of deoxynivalenol

Demeke, T., Clear, R.M., Patrick, S.K., & Gaba, D. (2005). Species-specific PCR-based assays

Dubos, T., Pasquali, M., Pogoda, F., Hoffmann, L., & Beyer, M. (2011). Evidence for natural

Edwards, S.G., Pirgozliev, S.R., Hare, M.C., & Jenkinson, P. (2001). Quantification of

Falcao, V.C., Ono, M.A., Vizoni, E., de Avila Miguel, T., Hirooka, E. Y., & Ono, E.Y. (2011).

on in vitro inhibition of mycelial growth. *Mycopathologia*, vol.171, pp. 77-84. Gaurilcikiene, I., Mankeviciene, A., & Suproniene, S. (2011). The effect of fungicides on rye

*Applied and Environmental Microbiology*, vol.67, pp. 1575-1580.

wheat varieties. *European Journal of Plant Pathology*, vol.125, pp. 445-458. Ban, T., & Suenaga, K. (2000). Genetic analysis of resistance to *Fusarium* head blight caused

production by *Fusarium graminearum*. *BMC Microbiology,* vol.10, pp.1-10. Audenaert, K., De Schuyffeleer, N., Maene, P., Monbaliu, S., Vekeman, F., De Saeger, S.,

kernels. Submitted to *Food control*.

*Plant Pathology,* vol.109, pp. 577-587.

*vitro*. *Plant Pathology* vol.53, pp. 22-28.

*Plant Pathology*, vol.130, pp. 239-248.

vol.100, pp. 444-453.

vol.103, pp 271-284.

vol.98, pp. 19-26.

512.

cultivar Saikai 165. *Euphytica*, vol.113, pp. 87-99.

Fungicides. *Phytopathology*, vol.99, pp. 1403-1411.

peroxide induced by the fungicide prothioconazole triggers deoxynivalenol (DON)

Haesaert, G., & Eeckhout, M. (2011). Efficacy of neutral electrolyzed water to reduce *Fusarium* spp and deoxynivalenol (DON) production in vitro and on wheat

M., & Haesaert, G. (2009). *Fusarium* head blight (FHB) in Flanders: population diversity, inter-species associations and DON contamination in commercial winter

by *Fusarium graminearum* in Chinese wheat cultivar Sumai 3 and the Japanese

*Fusarium* graminearum to tebuconazole yielded descendants diverging for levels of fitness, fungicide resistance, virulence, and mycotoxin production. *Phytopathology*,

vitro growth and pathogenicity of European *Fusarium* fungi. *European Journal of* 

Mutations in a beta-Tubulin Confer Resistance of *Gibberella zeae* to Benzimidazole

accumulation and expression of Tri genes in *Fusarium culmorum* by fungicides *in* 

for the detection of *Fusarium* species and a comparison with the whole seed agar plate method and trichothecene analysis. *International Journal of Food Microbiology*,

resistance towards trifloxystrobin in *Fusarium* graminearum. *European Journal of* 

trichothecene-producing *Fusarium* species in harvested grain by competitive PCR to determine efficacies of fungicides against *Fusarium* head blight of winter wheat.

*Fusarium verticillioides*: evaluation of fumonisin production and effect of fungicides

and triticale grain contamination with *Fusarium* fungi and mycotoxins. *Zemdirbyste*,

**8. References** 


### **Inoculation of Sugar Beet Seed with Bacteria**  *P. fluorescens***,** *B. subtilis* **and** *B. megaterium* **– Chemical Fungicides Alternative**

Suzana Kristek, Andrija Kristek and Dragana Kocevski *University of J. J. Strossmayer, Faculty of Agriculture Croatia* 

#### **1. Introduction**

98 Fungicides – Beneficial and Harmful Aspects

Paul, P.A., Lipps, P.E., Hershman, D.E., McMullen, M.P., Draper, M.A., & Madden, L.V.

Pirgozliev, S.R., Edwards, S.G., Hare, M.C., & Jenkinson, P. (2002). Effect of dose rate of

Qiu, J.B., Xu, J.Q., Yu, J.J., Bi, C.W., Chen, C.J., & Zhou, M.G. (2011). Localisation of the

Simpson, D.R., Weston, G.E., Turner, J.A., Jennings, P., & Nicholson, P. (2001). Differential

Singh, R.P., Ma, H., & Rajaram, S. (1995). Genetic analysis of resistance to scab in spring

Snijders, C.H.A. (1990). *Fusarium* head blight and mycotoxin contamination of wheat, a

Snijders, C.H.A., & Perkowski, J. (1990). Effects of head blight caused by *Fusarium culmorum* on toxin content and weight of wheat kernels. *Phytopathology*, vol.80, pp. 566-570. Stenglein, S.A. (2009). *Fusarium poae*: a pathogen that needs more attention. *Journal of Plant* 

Walker, A.S., Auclair, C., Gredt, M., & Leroux, P. (2009). First occurrence of resistance to

Zhang, Y.J., Fan, P.S., Zhang, X. , Chen, C.J., & Zhou, M.G. Quantification of *Fusarium* 

Zhang, Y.J., Fan, P.S., Zhang, X., Chen, C.J., & Zhou, M.G. (2009a). Quantification of *Fusarium*

Zhang, Y.J., Yu, J.J., Zhang, Y.N., Zhang, X., Cheng, C.J., Wang, J.X., Hollomon, D.W., Fan,

and yield of winter wheat.(2008.) *Phytopathology*, vol.99, pp.95-100.

and Yield of Winter Wheat. *Phytopathology*, vol.99, pp. 95-100.

strobilurin fungicides in *Microdochium nivale* and *Microdochium majus* from French naturally infected wheat grains. *Pest Management Science*, vol.65, pp. 906-915. Wiersma, J.J., & Motteberg, C.D. (2005). Evaluation of five fungicide timings for the control

of leaf - spot diseases and *Fusarium* head blight in hard red spring wheat. *Canadian* 

*graminearum* in harvested grain by Real-Time Polymerase Chain Reaction to assess efficacies of fungicides on Fusarium head blight, deoxynivalenol contamination,

*graminearum* in Harvested Grain by Real-Time Polymerase Chain Reaction to Assess Efficacies of Fungicides on *Fusarium* Head Blight, Deoxynivalenol Contamination,

P.S., Zhou, M.G. (2009b). Effect of Carbendazim Resistance on Trichothecene Production and Aggressiveness of *Fusarium graminearum*. *Molecular Plant-Microbe* 

site-directed mutagenesis. *Pest Management Science*, vol.67, pp. 191-198. Saghai-Maroof, M.A., Soliman, K.M., Jorgensen, R.A., & Allard, R.W. (1984). Ribosomal

*Academic of Sciences of the USA*, vol.81, pp. 8014-8018.

wheat cultivar Frontana. *Plant Disease,* vol.79, pp. 238-240.

review. *Netherlands Journal of Plant Pathology*, vol.96, pp. 187-198.

vol*.*98, pp. 999-1011.

421-431.

*Pathology*, vol.108, pp. 469-478.

*Pathology*, vol. 91, pp. 25-36.

*Journal of Plant Pathology,* vol. 27, pp. 25-37.

*Interactions*, vol.22, pp. 1143-1150.

(2008). Efficacy of triazole-based fungicides for *Fusarium* head blight and deoxynivalenol control in wheat: A multivariate meta-analysis. *Phytopathology,* 

azoxystrobin and metconazole on the development of *Fusarium* head blight and the accumulation of deoxynivalenol (DON) in wheat grain. *European Journal of Plant* 

benzimidazole fungicide binding site of *Gibberella zeae* beta(2)-tubulin studied by

DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. *Proceedings of the National* 

control of head blight pathogens of wheat by fungicides and consequences for mycotoxin contamination of grain. *European Journal of Plant Pathology*, vol.107, pp.

> During the process of sugar beet cultivation, depending on weather conditions, the degree of soil infection with pathogenic microorganisms, and the application of soil management, the presence of sugar beet root rot is frequently observed. Pathogens are numerous, inflicting serious damage.

> According to the occurrence time of root rot symptoms, the pathogens can be divided into three major groups: pathogens occurring during the emergence phase, pathogens occurring in the growing season, and ones that cause sugar beet root rot during the period of storage.

> The most frequent decay pathogens on sugar beet seedlings are the fungi *Aphanomyces cochlioides*, *Pythium ultimum* and *Pythium debarianum*, as well as *Rhizoctonia solani.* In the growing season plant decay is most frequently caused by the fungi *Rhizoctonia solani*, *Fusarium* spp., and by the bacteria *Erwinia* spp. From the root rot pathogens most damage is inflicted by *Rhizoctonia solani.* Besides weather conditions that influence this type of root decay, the inadequate soil management – irregular soil cultivation resulting in damaged soil structure and rise in soil acidity – is a contributory factor.

> Apart from the root pathogens occurring during cultivation in the field, certain pathogens cause sugar beet decay in the period of storage, and before processing. Storage conditions, as well as plant damages caused when digging are contributory factors in the occurrence of fungal diseases.

> The most frequent pathogenic microorganisms observed are fungi, such as *Rhizoctonia solani*, *Botrytis cinerea, Penicillium* spp., *Aspergillus* spp. ... The pathogenic fungus *Rhizoctonia solani,* if compared with all other sugar beet root rot pathogens, is able to infect the plant from the emergence phase, during growing season, until the process of sugar beet storage.

> Fungicidal treatment of seeds, use of tolerant hybrids, or proper soil management does not ensure full plant protection against this pathogen. Moreover, heavily infected soils and weather conditions favourable for development of the pathogen, contribute to the infection to higher or lower degree.

Agronomists are faced with a growing problem of evolving resistance of soil pathogens to chemical fungicides. Also, there is an increasing sensitivity to the importance of health food production and rising awareness for environmental protection. Namely, all pesticides which are not biodegraded in the soil are being washed out in deeper layers causing eutrophication of underground waters. Therefore, natural resources and processes in the soil are widely used as an alternative to chemical fungicides.

Seed inoculation with the bacteria showing antagonistic activity against pathogenic fungi such as *Rhizoctonia solani, Pythium ultimum, P. debarianum, Phoma betae* and *Aphanomyces cochlioides* account for an alternative to chemical fungicides and an option of solving problem of disease control, not only in sugar beet, but in other, mainly vegetable crops. Many authors (Koch et al., 2002; Sorensen et al., 2001; Thrane et al., 2000; Whipps, 2001) have studied control of these pathogens by the beneficial bacterium *Pseudomonas fluorescens* which shows pronounced antagonistic activity against the fungi. Similarly, results of their studies proved positive effect of the bacterium *Bacillus megaterium* against the same root decay agents of sugar beet (Asaka & Shoda, 1996; Choudhary & Johri, 2009; Thrane et al., 2000). However, some authors proved antagonistic effect of certain beneficial fungi and yeast (*Trichoderma harzianum, Candida valida, Rhodotula glutinis, …*) against these pathogenic fungi (Abada, 1994; El-Tarabily & Sivasithamparam, 2006).

*Pseudomonas fluorescens* belongs to rod-shaped, asporogenous, gram-negative bacteria that as saprophytes prevalent in soil and water belong to a group of soil microorganisms crucial to the process of soil denitrification.

Due to the production of antimicrobial agents, these bacteria also show a distinguished antibiosis against pathogens causing diseases on arable crops, by inactivating their growth and reproduction (Whipps, 2001). For the capacity to synthesize toxic cyclic lipopeptides (Andersen et al., 2003; Koch et al., 2002; Thrane et al., 2000) they are used as effective biological control agents (Sorensen et al., 2001). Many authors have reported that these purified lipopeptides show an antagonistic activity against certain fungi pathogenic on sugar beet roots such as *Rhizoctonia solani* (Andersen et al., 2003; Nielsen et al., 2000, 2002), *Aphanomyces cochlioides* (Raaijmakers et al., 2010; Sorensen et al., 2001), *Pythium ultimum* and *Pythium debarianum* (Andersen et al., 2003; Gorlach-Lira & Stefaniak, 2009; Lee et al., 2000; Nielsen et al., 2000; Thrane et al., 2000). This suggests that bacteria producing lipopeptides could have a potential role in the biocontrol of fungal diseases, which was approved in both laboratory and field trials (Thrane et al., 2000, 2001). Lipopeptides may also function as biosurfactants (Desai et al., 1997) which can facilitate bacterial growth on water – insoluble carbon sources (Koch et al., 1991; Ron & Rosenberg, 2001) or their interaction with hydrophobic surfaces (Neu, 1996), e. g., surface motility (Engelhardt et al., 2009; Lindum et al., 1998).

*Bacillus subtilis* and *Bacillus megaterium* form symbiotic relationship with the root system of field crops, and like *P. fluorescens* show antagonistic activity against these pathogens. In this study we have investigated synergistic effect of the bacteria in order to achieve as better results as possible.

*Bacillus* is a genus of Gram-positive rod-shaped bacteria and a member of the division *Firmicutes*. *Bacillus* species can be obligate aerobes or facultative anaerobes. The species *Bacillus subtilis* and *Bacillus megaterium* participate in nitrogen cycle, and in the soil processes of organic matter humification and mineralization of humus, especially in mineralization of phosphorus and potassium converting them into plant-accessible forms. These bacteria belong to biological control agents, i. e. they produce antimicrobial substances that exhibit antagonistic activity against soilborne pathogens, agents of diseases in arable crops (Sorensen et al., 2001).

Therefore, seed inoculation with bacteria showing antagonism against pathogenic fungi is an acceptable alternative to chemical pesticide application (Andersen et al., 2003).

Moreover, considering heavily infected soils with the pathogen *R. solani* and the fact that beneficial bacteria *Pseudomonas fluorescens*, *B. subtilis* and *B. megaterium* are not sensitive to a low dose fungicide (Pedersen et al., 2002), it is possible to treat the seeds combining low doses of the chemical agents aiming to control growth and reproduction of the pathogenic fungi, which in consequence, produce positive effect on all the parameters of sugar beet yield and quality.

#### **2. Materials and methods**

100 Fungicides – Beneficial and Harmful Aspects

Agronomists are faced with a growing problem of evolving resistance of soil pathogens to chemical fungicides. Also, there is an increasing sensitivity to the importance of health food production and rising awareness for environmental protection. Namely, all pesticides which are not biodegraded in the soil are being washed out in deeper layers causing eutrophication of underground waters. Therefore, natural resources and processes in the

Seed inoculation with the bacteria showing antagonistic activity against pathogenic fungi such as *Rhizoctonia solani, Pythium ultimum, P. debarianum, Phoma betae* and *Aphanomyces cochlioides* account for an alternative to chemical fungicides and an option of solving problem of disease control, not only in sugar beet, but in other, mainly vegetable crops. Many authors (Koch et al., 2002; Sorensen et al., 2001; Thrane et al., 2000; Whipps, 2001) have studied control of these pathogens by the beneficial bacterium *Pseudomonas fluorescens* which shows pronounced antagonistic activity against the fungi. Similarly, results of their studies proved positive effect of the bacterium *Bacillus megaterium* against the same root decay agents of sugar beet (Asaka & Shoda, 1996; Choudhary & Johri, 2009; Thrane et al., 2000). However, some authors proved antagonistic effect of certain beneficial fungi and yeast (*Trichoderma harzianum, Candida valida, Rhodotula glutinis, …*) against these pathogenic

*Pseudomonas fluorescens* belongs to rod-shaped, asporogenous, gram-negative bacteria that as saprophytes prevalent in soil and water belong to a group of soil microorganisms crucial to

Due to the production of antimicrobial agents, these bacteria also show a distinguished antibiosis against pathogens causing diseases on arable crops, by inactivating their growth and reproduction (Whipps, 2001). For the capacity to synthesize toxic cyclic lipopeptides (Andersen et al., 2003; Koch et al., 2002; Thrane et al., 2000) they are used as effective biological control agents (Sorensen et al., 2001). Many authors have reported that these purified lipopeptides show an antagonistic activity against certain fungi pathogenic on sugar beet roots such as *Rhizoctonia solani* (Andersen et al., 2003; Nielsen et al., 2000, 2002), *Aphanomyces cochlioides* (Raaijmakers et al., 2010; Sorensen et al., 2001), *Pythium ultimum* and *Pythium debarianum* (Andersen et al., 2003; Gorlach-Lira & Stefaniak, 2009; Lee et al., 2000; Nielsen et al., 2000; Thrane et al., 2000). This suggests that bacteria producing lipopeptides could have a potential role in the biocontrol of fungal diseases, which was approved in both laboratory and field trials (Thrane et al., 2000, 2001). Lipopeptides may also function as biosurfactants (Desai et al., 1997) which can facilitate bacterial growth on water – insoluble carbon sources (Koch et al., 1991; Ron & Rosenberg, 2001) or their interaction with hydrophobic surfaces (Neu, 1996), e. g., surface motility (Engelhardt et al., 2009; Lindum et

*Bacillus subtilis* and *Bacillus megaterium* form symbiotic relationship with the root system of field crops, and like *P. fluorescens* show antagonistic activity against these pathogens. In this study we have investigated synergistic effect of the bacteria in order to achieve as better

*Bacillus* is a genus of Gram-positive rod-shaped bacteria and a member of the division *Firmicutes*. *Bacillus* species can be obligate aerobes or facultative anaerobes. The species *Bacillus subtilis* and *Bacillus megaterium* participate in nitrogen cycle, and in the soil processes

soil are widely used as an alternative to chemical fungicides.

fungi (Abada, 1994; El-Tarabily & Sivasithamparam, 2006).

the process of soil denitrification.

al., 1998).

results as possible.

The experiment was set up on two types of the soil: Mollic Gleysols (FAO, 1998) and Eutric Cambisols.

On Mollic Gleysols soil type in the period of 2005 – 2009 pathogenic fungus *R. solani* (FAO, 1998) was determined, whilst on Eutric Cambisols (Table 1) soil type in the same period *P. debarianum* was determined.


Table 1. Soil characteristics

In 2007, 2008 and 2009 the experiment was set up by completely randomised block design in 4 repetitions and 8 various seed treating variants: 1. control (untreated seed); 2. Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed); 3. seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha); 4. seed inoculated with *B. subtilis* No 2109 (1.2 x 1010 bacteria/ha); 5. seed inoculated with *B. megaterium* No 2894 (1.2 x 1010 bacteria/ha); 6. seed inoculated with *B. subtilis* No 2109 (0.6 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.6 x 1010 bacteria/ha); 7. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha); 8. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed).

Bacteriological cultures applied in the experiment were from German Collection of Microorganisms and Cell Cultures (DSMZ). *P. fluorescens* No 8569 were transferred from lyophilised into vegetative form, and multiplied on Caso agar (Merck 105458) having following composition: peptone from casein 15.0 g, peptone from soymeal 5.0 g, NaCl 5.0 g, agar 15.0 g, distilled water 1000.0 ml. For *B. megaterium* No 2894 and *B. subtilis* No 2109 nutrient agar of the following composition was used: peptone 5.0 g, meat extract 3.0 g, agar 15.0 g, MnSO4 x H2O 10.0 mg, distilled water 1000.0 ml.


Table 2. Weather conditions in experimental years and years – long average in Osijek. (Meteorological and hydrological service of Croatia (DHMZ). Agrometeorological bureau in Osijek)

Hybrid of Belinda (KWS) sugar beet was used in the sowing. In the three trial years the sowing was conducted in the second decade of March. The row spacing was 50 cm, with 20 cm within row. Adequate plant stand was obtained with no corrections necessary.

Percentage of the plants infected with pathogenic fungi *R. solani* and *P. debarianum* as well as percentage of decayed plants was stipulated in 2 - 4 true leaves phase. The sugar beet digging conducted in the middle of October was followed by determination of root yield (t/ha), sugar content (%), sugar in molasses (%) and sugar yield (t/ha).

Weather conditions appeared to affect sugar beet growth. Data obtained from the Agrometeorological bureau in Osijek, Croatia (Table 2) were used in weather analysis. Weather conditions in 2007 were characterized by the increase in air temperature of more than 10C above multiyear average. Precipitation rates in the growing season measured 74% out of the average, with rainfall deficiency in June and July, and excessive rainfall in September and October which was unfavourable for sugar beet growth. 2008 and 2009 were also characterized by higher air temperatures in comparison with multiyear average, which was unfavourable for sugar beet growth due to the values significantly (2.5oC in 2008; and 3.0oC in 2009) above the optimum. Precipitation rates in the growing season 2008 were level with the multiyear average, with rainfall deficiency in August and excessive rainfall in September. Considering precipitation amount in the growing season (April – September), 2009 was dry. Only 59 % out of the average precipitation rates were measured, with rainfall deficiency throughout the year except for June. Such weather conditions were favourable for sugar beet growth until July, and then followed by the unfavourable conditions until the end of the growing season.

Results were processed by modern statistical methods (ANOVA) using the computer program StatSoft Inc. (2001) STATISTICA (data analysis software system), version 6.

#### **3. Results and discussion**

102 Fungicides – Beneficial and Harmful Aspects

Bacteriological cultures applied in the experiment were from German Collection of Microorganisms and Cell Cultures (DSMZ). *P. fluorescens* No 8569 were transferred from lyophilised into vegetative form, and multiplied on Caso agar (Merck 105458) having following composition: peptone from casein 15.0 g, peptone from soymeal 5.0 g, NaCl 5.0 g, agar 15.0 g, distilled water 1000.0 ml. For *B. megaterium* No 2894 and *B. subtilis* No 2109 nutrient agar of the following composition was used: peptone 5.0 g, meat extract 3.0 g, agar

**Precipitation amounts Air temperatures** 

**<sup>91</sup> 2007 2008 2009** 

**<sup>91</sup> 2007 2008 2009 Necessity 1901-**

April 40 56 3 50 19 - 11.2 13.3 12.5 12.4 May 50 63 56 67 39 14.2 16.8 18.3 18.1 16.5 June 50 88 33 76 63 18.0 19.4 22.2 21.4 20.3 July 80 66 27 79 12 18.5 21.2 23.9 21.8 23.2 August 65 61 46 46 61 18.2 20.4 22.2 21.8 21.7 September 35 46 65 86 10 14.0 16.8 14.5 15.6 15.6 October 40 56 94 30 55 - 11.1 10.3 13.0 9.1 *Total 360 436 324 434 259 - -* - - *Average* - - - - - *- 16.7 17.8 17.7 17.0* 

Table 2. Weather conditions in experimental years and years – long average in Osijek. (Meteorological and hydrological service of Croatia (DHMZ). Agrometeorological bureau in

cm within row. Adequate plant stand was obtained with no corrections necessary.

(t/ha), sugar content (%), sugar in molasses (%) and sugar yield (t/ha).

Hybrid of Belinda (KWS) sugar beet was used in the sowing. In the three trial years the sowing was conducted in the second decade of March. The row spacing was 50 cm, with 20

Percentage of the plants infected with pathogenic fungi *R. solani* and *P. debarianum* as well as percentage of decayed plants was stipulated in 2 - 4 true leaves phase. The sugar beet digging conducted in the middle of October was followed by determination of root yield

Weather conditions appeared to affect sugar beet growth. Data obtained from the Agrometeorological bureau in Osijek, Croatia (Table 2) were used in weather analysis. Weather conditions in 2007 were characterized by the increase in air temperature of more than 10C above multiyear average. Precipitation rates in the growing season measured 74% out of the average, with rainfall deficiency in June and July, and excessive rainfall in September and October which was unfavourable for sugar beet growth. 2008 and 2009 were also characterized by higher air temperatures in comparison with multiyear average, which was unfavourable for sugar beet growth due to the values significantly (2.5oC in 2008; and 3.0oC in 2009) above the optimum. Precipitation rates in the growing season 2008 were level with the multiyear average, with rainfall deficiency in August and excessive rainfall in September. Considering precipitation amount in the growing season (April – September), 2009 was dry. Only 59 % out of the average precipitation rates were measured, with rainfall deficiency throughout the year except for June. Such weather conditions were favourable for sugar beet growth until July, and then followed by the unfavourable conditions until the

15.0 g, MnSO4 x H2O 10.0 mg, distilled water 1000.0 ml.

**Necessity 1901-**

**Month** 

Osijek)

end of the growing season.

#### **3.1 Percentage of infected and decayed plants**

During the three - year research into the influence of beneficial bacteria and chemical fungicide Thiram 42-S on the intensity of infection and decay of sugar beet plants caused by the parasitic fungi *Rhizoctonia solani* on Mollic Gleysols, and by the parasitic fungi *Pythium debarianum* on Eutric Cambisols significant influence of the bacteria and the chemical fungicide was determined in all tested variants when compared with the control (Table 3).

On Mollic Gleysols in the three trial years the best results were recorded in the variant 7 seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha). All other variants of treated seeds obtained highly significant larger number (p < 0.01) of infected and decayed plants due to the attack of the parasitic fungi *Rhizoctonia solani* if compared with all other tested variants. Among the variants 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed), 3 - seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha) and 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed) no significant differences were determined (p > 0.01), though the smallest number of the infected and decayed plants was recorded in the variant 8 in the three trial years. Average number of infected plants in treated variants in the three trial years was 10.86% or 58.17% lower than the three-year average in the control variants which was 25.96%. The difference in decayed plants was 74.70%.

The results obtained are in agreement with those of Whipps (2001) who reported that the plants inoculated with bacteria *P. fluorescens* were characterized with rapid initial growth that enabled fast undergoing through the most vulnerable phase when the damage of the pathogen affecting was the most pronounced. Due to the pronounced antagonism of the bacterium to the root rot agent of sugar beet – fungi *R. solani* high survival percentage of inoculated plants was obtained in comparison with the non-inoculated ones and decrease in the damage of infected plants, which consequently, influenced the sugar beet root yield. Kristek et al. (2007) in their research results of the effects of the beneficial bacterium *P. fluorescens* on the pathogenic fungus *R. solani* recorded obvious decrease in the number of infected and decayed plants in variants treated with the bacterium *P. fluorescens* when compared with the control variants. They also reported significant difference in the number of infected and decayed plants between tolerant hybrids compared with the hybrids sensitive to the pathogenic fungi. With tolerant hybrids in treated variants 4.10 % smaller number of infected plants was obtained than in the same variants of sensitive hybrids. The difference in the number of decayed plants was 3.86 %.

On Eutric Cambisols in the three trial years the best results were recorded in the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no significant difference (p > 0.01) was determined between the above variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed). All other variants obtained highly significant (p < 0.01) larger number of infected and decayed plants due to the attack of the parasitic fungi *P. debarianum*. Average number of infected plants in the three trial years in treated variants was 11.70% or 56.16% lower than in the three-year average in the control variants which was 26.69%. The difference in decayed plants was 70.97%.


1. control (untreated seed); 2. Thiram 42-S fungicide treated seed (48% Tiram, 600 ml/100 kg seed); 3. seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha); 4. seed inoculated with *B. subtilis* No 2109 (1.2 x 1010 bacteria/ha); 5. seed inoculated with *B. megaterium* No 2894 (1.2 x 1010 bacteria/ha); 6. seed inoculated with *B. subtilis* No 2109 (0.6 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.6 x 1010 bacteria/ha); 7. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha); 8. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Tiram, 200 ml/100 kg seed)

Table 3. Percentage of the infected and decayed plants as a consequence of *R. solani* (Mollic Gleysols)*, P. debarianum* (Mollic Gleysols) infection in the 2–4 true leaves stage

(0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no significant difference (p > 0.01) was determined between the above variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed). All other variants obtained highly significant (p < 0.01) larger number of infected and decayed plants due to the attack of the parasitic fungi *P. debarianum*. Average number of infected plants in the three trial years in treated variants was 11.70% or 56.16% lower than in the three-year average

*Average*

**2007 2008 2009 2007 2008 2009** 

**LSD0.05** 0.24 0.15 0.13 0.16 0.11 0.12 0.10 0.10 **LSD0.01** 0.43 0.27 0.24 0.28 0.21 0.23 0.19 0.18

**LSD0.05** 0.14 0.18 0.13 0.15 0.09 0.08 0.06 0.07 **LSD0.01** 0.26 0.34 0.25 0.27 0.17 0.15 0.13 0.14

1. control (untreated seed); 2. Thiram 42-S fungicide treated seed (48% Tiram, 600 ml/100 kg seed); 3. seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha); 4. seed inoculated with *B. subtilis* No 2109 (1.2 x 1010 bacteria/ha); 5. seed inoculated with *B. megaterium* No 2894 (1.2 x 1010 bacteria/ha); 6. seed inoculated with *B. subtilis* No 2109 (0.6 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.6 x 1010 bacteria/ha); 7. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha); 8. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894

(0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Tiram, 200 ml/100 kg seed)

Gleysols)*, P. debarianum* (Mollic Gleysols) infection in the 2–4 true leaves stage

Table 3. Percentage of the infected and decayed plants as a consequence of *R. solani* (Mollic

1 25.90 18.40 23.66 *22.65* 25.98 21.04 24.18 *23.73*  2 5.61 5.27 5.28 *5.39* 6.70 5.99 6.24 *6.31*  3 5.70 5.19 5.33 *5.41* 7.11 6.22 6.57 *6.63*  4 7.12 6.33 6.90 *6.78* 7.98 7.08 7.85 *7.64*  5 7.06 6.38 6.84 *6.76* 8.03 7.12 7.78 *7.64*  6 6.19 5.65 5.92 *5.92* 7.65 6.79 7.20 *7.21*  7 5.03 4.11 4.80 *4.65* 7.04 6.13 6.48 *6.55*  8 5.48 4.95 5.11 *5.18* 6.56 5.96 6.16 *6.23*

1 29.46 20.78 27.65 *25.96* 28.46 23.51 28.10 *26.69*  2 11.39 9.59 10.43 *10.47* 11.29 10.55 10.96 *10.93*  3 11.35 9.66 10.39 *10.47* 11.71 10.96 11.44 *11.37*  4 12.97 11.59 12.21 *12.26* 13.18 12.68 12.73 *12.86*  5 12.85 11.53 12.29 *12.22* 12.99 12.43 12.65 *12.69*  6 11.14 10.27 10.94 *10.78* 12.35 11.55 11.97 *11.96*  7 10.25 8.90 9.66 *9.60* 11.64 10.89 11.39 *11.31*  8 10.99 9.46 10.27 *10.24* 11.13 10.41 10.92 *10.82*

**Eutric Cambisols**  *(Pythium debarianum)*

*Average*

**Year**

in the control variants which was 26.69%. The difference in decayed plants was 70.97%.

**Mollic Gleysols**  *(Rhizoctonia solani)*

**Year**

**Investigated parameter Variants** 

**Infected plants** 

**Decayed plants (%)** 

**(%)** 

Similar results in examining the effect of the bacterium *B. megaterium* (a constituent of the applied microbiological preparation) in the control of pathogenic fungi *P. ultimum* and *P. debarianum* were reported in the studies on the same parameters by Evačić et al. (2008).

It is evident that weather conditions in the growing season of sugar beet influenced intensity of the infection by pathogenic fungi *Rhizoctonia solani* and *Pythium debarianum*. Namely, in the second trial year precipitation rates and air temperatures were favourable for crop development which enabled faster undergoing of young sugar beet plants through the most vulnerable phase of the growth. Consequently, intensity of the infection and the number of decayed plants as the result of the pathogen attack were both decreased which was recorded in the controls. Furthermore, there was obvious difference in the number of infected and decayed plants on different types of the soil. As it did not concern the same pathogen, it could be higher sensitivity of the hybrid to *Pythium debarianum* than to *Rhizoctonia solani*. Nevertheless, pathogenic fungi *Rhizoctonia solani* usually cause more economic damage in the middle and eastern European countries than pathogenic fungi *Pythium debarianum*. Belinda hybrid was chosen due to the high sensitivity to the fungi *Rhizoctonia solani*. Therefore chemical and microbiological soil properties could be presumed to have great importance in plant development and resistance against the infection caused by the pathogenic fungi. Namely, Mollic Gleysols has chemical (Table 1) and microbiological (Figure 1, 2, 3, 4, 5) properties of better quality.

(▲ – Mollic Gleysols; ■ – Eutric Cambisols)

Fig. 1. Average number of total Bacteria in soil during three trial years

(▲ – Mollic Gleysols; ■ – Eutric Cambisols)

Fig. 2. Average number of total Fungi in soil during three trial years

Fig. 3. Average number of total Actinomycetes in soil during three trial years

 **Month**

**IV V VI VII VIII**

 **Month**

Fig. 3. Average number of total Actinomycetes in soil during three trial years

**IV V VI VII VIII**

(▲ – Mollic Gleysols; ■ – Eutric Cambisols)

**Actinomycetes (x 103 CFU g-1 of soil)**

(▲ – Mollic Gleysols; ■ – Eutric Cambisols)

Fig. 2. Average number of total Fungi in soil during three trial years

**Fungi (x103 CFU g-1 of soil)**

Fig. 4. Average number of *Bacillus* spp. in soil during three trial years

Fig. 5. Average number of *Pseudomonas fluorescens* in soil during three trial years

It is the soil of neutral reaction with high portion of organic matter and large supply of phosphorus and potassium. The soil is abundant in bacteria and actinomycetes that participate in the processes of soil organic matter humification and mineralization of humus. Large portion in microbial mass is comprised of non-symbiotic nitrogen fixers and cellulolytic microorganisms. The presence of the large number of the bacteria of the *Pseudomonas fluorescens* species, as well as *Bacillus* spp., showing antagonistic activity against the soilborne pathogenic fungi is a factor responsible for smaller number of infected plants in the controls if compared with the same on Eutric Cambisols, a soil of poorer chemical and microbiological properties. Average number of infected plants was in highly significant positive correlation with average number of decayed plants (r=0.965; p<0.01).

#### **3.2 Root yield**

Sugar beet root yield depended on soil types, or on chemical and microbiological soil properties, and on trial years.

On Mollic Gleysols in the three trial years the highest average sugar beet root yield was recorded in the variant 7 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha). All other variants obtained statistically highly significant lower average root yield (Table 4). Average sugar beet root yield in treated variants was 80.63 t/ha, or 21.12% higher than average root yield in the control variant (66.57 t/ha). Since the variant with the strains of beneficial bacteria being applied together obtained significantly higher average root yield than the variant with the strains applied one at a time, it can be concluded that there was highly positive synergistic activity between them.

On Eutric Cambisols in the three trial years the highest average sugar beet root yield was recorded in the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no significant difference (p > 0.01) was determined between this variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed). All other variants obtained highly significant (p < 0.01) lower sugar beet root yield. Average sugar beet root yield in treated variants was 69.45 t/ha or 21.08 % higher than the average root yield in the control variant (57.02 t/ha).

It is evident that on the soil type of poorer microbiological properties the best results were obtained with the application of chemical fungicides. It is also obvious that between variant 3 with application of the bacteria *P. fluorescens*, and variant 7 with application of

*P. fluorescens*, *B. subtilis* and *B. megaterium* no significant differences were determined. Therefore, it can be concluded that the bacteria *P. fluorescens* played a crucial role in the control of pathogenic fungi *P. debarianum*.

Significantly better results in sugar beet production by applying plant growth – promoting rhizobacteria *P. fluorescens* in the control of pathogenic fungi *R. solani* were reported by Esh & El-Kholi (2005), as well as by Kiewnick et al. (2001) in the control of fungi *Rhizoctonia crown* by combination of beneficial bacteria.

*P. fluorescens* shows distinguished biosurfactant properties (Raaijmakers et al., 2010; Ron & Rosenberg, 2001) and improves general plants condition resulting in arable crops yield and quality. As the results of their research studies some authors stated that *P. fluorescens* stimulate nutrients uptake (Duijff et al., 1993; Loper & Buyer, 1991), increases photosynthesis intensity (Zhang et al., 2002) as well as solubility of phosphorus inorganic forms. The aforesaid improves plants vigour and increases yield by 15-20% (Whipps, 2001).

#### **3.3 Sugar content**

108 Fungicides – Beneficial and Harmful Aspects

It is the soil of neutral reaction with high portion of organic matter and large supply of phosphorus and potassium. The soil is abundant in bacteria and actinomycetes that participate in the processes of soil organic matter humification and mineralization of humus. Large portion in microbial mass is comprised of non-symbiotic nitrogen fixers and cellulolytic microorganisms. The presence of the large number of the bacteria of the *Pseudomonas fluorescens* species, as well as *Bacillus* spp., showing antagonistic activity against the soilborne pathogenic fungi is a factor responsible for smaller number of infected plants in the controls if compared with the same on Eutric Cambisols, a soil of poorer chemical and microbiological properties. Average number of infected plants was in highly significant positive correlation with average number of decayed plants (r=0.965;

Sugar beet root yield depended on soil types, or on chemical and microbiological soil

On Mollic Gleysols in the three trial years the highest average sugar beet root yield was recorded in the variant 7 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha). All other variants obtained statistically highly significant lower average root yield (Table 4). Average sugar beet root yield in treated variants was 80.63 t/ha, or 21.12% higher than average root yield in the control variant (66.57 t/ha). Since the variant with the strains of beneficial bacteria being applied together obtained significantly higher average root yield than the variant with the strains applied one at a time, it

On Eutric Cambisols in the three trial years the highest average sugar beet root yield was recorded in the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no significant difference (p > 0.01) was determined between this variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed). All other variants obtained highly significant (p < 0.01) lower sugar beet root yield. Average sugar beet root yield in treated variants was 69.45 t/ha

It is evident that on the soil type of poorer microbiological properties the best results were obtained with the application of chemical fungicides. It is also obvious that between variant

*P. fluorescens*, *B. subtilis* and *B. megaterium* no significant differences were determined. Therefore, it can be concluded that the bacteria *P. fluorescens* played a crucial role in the

Significantly better results in sugar beet production by applying plant growth – promoting rhizobacteria *P. fluorescens* in the control of pathogenic fungi *R. solani* were reported by Esh & El-Kholi (2005), as well as by Kiewnick et al. (2001) in the control of fungi *Rhizoctonia* 

can be concluded that there was highly positive synergistic activity between them.

or 21.08 % higher than the average root yield in the control variant (57.02 t/ha).

3 with application of the bacteria *P. fluorescens*, and variant 7 with application of

p<0.01).

**3.2 Root yield** 

properties, and on trial years.

control of pathogenic fungi *P. debarianum*.

*crown* by combination of beneficial bacteria.

This parameter was also influenced by weather conditions in the growing season of sugar beet. Namely, higher sugar content in sugar beet root (%) was recorded in the second trial year (Table 4).

On Mollic Gleysols in the three trial years the highest average sugar content in sugar beet was recorded in the variant 7 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha). All other variants obtained statistically highly significant lower (p<0.01) average sugar content. Average sugar content in sugar beet in treated variants was 15.27% or 7.01% higher than sugar content in the control (14.27%).

No significant differences among the variants 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed), 3 - seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha) and 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed) were determined. Due to the fact that variant 3, as well as variant 7- the one which obtained the best results, were not treated with a chemical fungicide, it can be left out from application in this case.

Fungicide treated seed on finishing is obviously neither sufficient nor safe measure since the fungi fast develop resistance to these pesticide active agents (Cooke et al., 1999; Durrant et al., 1998). Furthermore, fungicides are exposed to washing out, hence the possibility of underground waters eutrophication, i.e. direct danger for a human health and environment. For its physical characteristics, this beneficial bacterium represents an acceptable alternative to chemical fungicides application (Kristek et al., 2005, 2006; Thrane et al., 2000).

On Eutric Cambisols in the three trial years the highest average sugar content in sugar beet was recorded in the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed). Between this variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed) no significant difference was determined. High sugar content in sugar beet was obtained in variant 3 - seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha) and variant 7 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) with no statistical significance determined. Average sugar content in sugar beet in treated variants was 14.44 % or 4.94 % higher than sugar content in the control (13.76 %).


Similar results were reported in the studies of Esh & El-Kholi (2005), Kristek et al. (2006, 2007) and Pedersen et al. (2002).

1. control (untreated seed); 2. Thiram 42-S fungicide treated seed (48% Tiram, 600 ml/100 kg seed); 3. seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha); 4. seed inoculated with *B. subtilis* No 2109 (1.2 x 1010 bacteria/ha); 5. seed inoculated with *B. megaterium* No 2894 (1.2 x 1010 bacteria/ha); 6. seed inoculated with *B. subtilis* No 2109 (0.6 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.6 x 1010 bacteria/ha); 7. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha); 8. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Tiram, 200 ml/100 kg seed)

Table 4. Root yield (t/ha) and sugar content (%) on two soil types during three trial years

#### **3.4 Sugar in molasses**

Sugar content in molasses depended, respectively, on seed treated variant, hybrid properties (tolerant, sensitive), soil type tested, and trial year.

Similar results were reported in the studies of Esh & El-Kholi (2005), Kristek et al. (2006,

*Average*

**2007 2008 2009 2007 2008 2009** 

**LSD0.05** 0.75 0.99 0.81 0.84 0.84 0.72 0.97 0.68 **LSD0.01** 1.42 1.86 1.51 1.48 1.56 1.39 1.83 1.22

**LSD0.05** 0.08 0.17 0.14 0.11 0.11 0.17 0.20 0.08 **LSD0.01** 0.14 0.31 0.25 0.20 0.20 0.29 0.31 0.14 1. control (untreated seed); 2. Thiram 42-S fungicide treated seed (48% Tiram, 600 ml/100 kg seed); 3. seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha); 4. seed inoculated with *B. subtilis* No 2109 (1.2 x 1010 bacteria/ha); 5. seed inoculated with *B. megaterium* No 2894 (1.2 x 1010 bacteria/ha); 6. seed inoculated with *B. subtilis* No 2109 (0.6 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.6 x 1010 bacteria/ha); 7. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha); 8. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) +

1 14.08 14.53 14.21 *14.27* 13.46 14.28 13.55 *13.76*  2 14.73 16.01 15.65 *15.46* 14.27 15.71 14.51 *14.83*  3 14.68 15.84 15.59 *15.37* 13.99 15.29 14.09 *14.46*  4 14.31 15.26 14.85 *14.81* 13.70 14.68 13.82 *14.07*  5 14.37 15.32 14.62 *14.77* 13.69 14.70 13.90 *14.10*  6 14.50 15.55 15.12 *15.06* 13.85 15.05 13.87 *14.26*  7 15.26 16.45 16.08 *15.93* 13.82 15.36 14.11 *14.43*  8 14.75 16.08 15.73 *15.52* 14.30 15.90 14.59 *14.93*

1 63.40 70.21 66.10 *66.57* 50.65 68.30 52.10 *57.02*  2 73.91 95.50 78.92 *82.77* 65.97 90.24 70.66 *75.62*  3 72.96 94.63 79.45 *82.35* 58.79 87.10 64.98 *70.29*  4 64.28 89.45 70.55 *74.76* 53.20 75.45 59.75 *62.80*  5 65.96 88.16 69.18 *74.43* 54.88 76.21 60.30 *63.80*  6 68.50 93.10 74.80 *78.80* 56.90 80.20 62.50 *66.53*  7 78.10 98.35 86.54 *87.66* 60.25 86.47 65.10 *70.61*  8 74.13 96.45 80.24 *83.61* 66.80 90.55 72.18 *76.51*

**Eutric Cambisols**  *(Pythium debarianum)*

*Average*

**Year**

**Mollic Gleysols**  *(Rhizoctonia solani)*

**Year**

Thiram 42-S fungicide treated seed (48% Tiram, 200 ml/100 kg seed)

(tolerant, sensitive), soil type tested, and trial year.

Table 4. Root yield (t/ha) and sugar content (%) on two soil types during three trial

Sugar content in molasses depended, respectively, on seed treated variant, hybrid properties

2007) and Pedersen et al. (2002).

**Investigated parameter** Variants

**Root yield** 

**(t/ha)** 

**Sugar content (%)** 

years

**3.4 Sugar in molasses** 

The lowest average sugar content in molasses in the three year trial (Table 5) was recorded on Mollic Gleysols in the variant 7 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha). Average sugar content in molasses in treated variants was 2.60% or 14.75% lower than the average sugar content in molasses in the control variants (3.05%).


1. control (untreated seed); 2. Thiram 42-S fungicide treated seed (48% Tiram, 600 ml/100 kg seed); 3. seed inoculated with *P. fluorescens* No 8569 (1.2 x 1010 bacteria/ha); 4. seed inoculated with *B. subtilis* No 2109 (1.2 x 1010 bacteria/ha); 5. seed inoculated with *B. megaterium* No 2894 (1.2 x 1010 bacteria/ha); 6. seed inoculated with *B. subtilis* No 2109 (0.6 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.6 x 1010 bacteria/ha); 7. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha); 8. seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Tiram, 200 ml/100 kg seed)

Table 5. Sugar in molasses (%) and sugar yield (t/ha) on two soil types during three trial years

By drawing comparison among the variants with the strains of the beneficial bacteria applied one at a time, the best results in this parameter as well were recorded with the bacteria *P. fluorescens.* The variants comprising the bacteria *B. subtilis* and *B. megaterium*, being applied one at a time, or together, obtained highly significant (p<0.01) lower values in all tested parameters.

This is in agreement with the results of Lifshitz et al. (1987) and Whipps (2001) who stated that *P. fluorescens* as plant growth – promoting bacteria increased the solubility of phosphorus in inorganic forms around the active root zone. By mobilizing soil phosphorus plant vigour improves and yield increases by 15–20% (great root availability reduce negative nitrogen effect on sugar beet achieving balanced plant nutrition and reduced production of alpha - amino nitrogen, potassium and sodium i.e. decrease in the amount of sugar which cannot be isolated in the production process but turns into molasses).

The lowest average sugar content in molasses in the three year trial was recorded on Eutric Cambisols with the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed). Between this variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed) no statistical significance (p>0.05) was determined. All other variants obtained highly significant (p<0.01) higher values of sugar in molasses. Average sugar content in molasses in treated variants was 3.39% or 14.61% lower than average sugar content in molasses in the control variants (3.97%).

#### **3.5 Sugar yield**

On Mollic Gleysols the highest average sugar yield in the three trial years was recorded in the variant 7 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha). All other variants obtained statistically highly significant lower average sugar yield. Average sugar yield in treated variants was 10.31 t/ha or 37.83% higher than average sugar yield in the control (7.48 t/ha). It is evident that in this parameter as well, the best results were obtained in the second year of the trial (Table 5).

On Eutric Cambisols the highest average sugar yield in the three trial years was recorded in the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no statistical significant difference (p > 0.05) was determined between the above variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed). All other variants obtained highly significant (p < 0.01) larger number of infected and decayed plants. Average sugar yield in treated variants was 7.78 t/ha or 38.18% higher than average sugar yield in the control variant (5.63 t/ha).

Sugar yield was in very significant positive correlation with root yield (r=0.975; p<0.01) and sugar content (r=0.946; p<0.01).

#### **4. Conclusion**

112 Fungicides – Beneficial and Harmful Aspects

By drawing comparison among the variants with the strains of the beneficial bacteria applied one at a time, the best results in this parameter as well were recorded with the bacteria *P. fluorescens.* The variants comprising the bacteria *B. subtilis* and *B. megaterium*, being applied one at a time, or together, obtained highly significant (p<0.01) lower values in

This is in agreement with the results of Lifshitz et al. (1987) and Whipps (2001) who stated that *P. fluorescens* as plant growth – promoting bacteria increased the solubility of phosphorus in inorganic forms around the active root zone. By mobilizing soil phosphorus plant vigour improves and yield increases by 15–20% (great root availability reduce negative nitrogen effect on sugar beet achieving balanced plant nutrition and reduced production of alpha - amino nitrogen, potassium and sodium i.e. decrease in the amount of sugar which cannot be isolated in the production process but turns into

The lowest average sugar content in molasses in the three year trial was recorded on Eutric Cambisols with the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed). Between this variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed) no statistical significance (p>0.05) was determined. All other variants obtained highly significant (p<0.01) higher values of sugar in molasses. Average sugar content in molasses in treated variants was 3.39% or 14.61% lower than average sugar content in molasses in the control variants

On Mollic Gleysols the highest average sugar yield in the three trial years was recorded in the variant 7 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha). All other variants obtained statistically highly significant lower average sugar yield. Average sugar yield in treated variants was 10.31 t/ha or 37.83% higher than average sugar yield in the control (7.48 t/ha). It is evident that in this parameter as well, the best results were obtained in the second year of the trial

On Eutric Cambisols the highest average sugar yield in the three trial years was recorded in the variant 8 - seed inoculated with *P. fluorescens* No 8569 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. subtilis* No 2109 (0.4 x 1010 bacteria/ha) + seed inoculated with *B. megaterium* No 2894 (0.4 x 1010 bacteria/ha) + Thiram 42-S fungicide treated seed (48% Thiram, 200 ml/100 kg seed), though no statistical significant difference (p > 0.05) was determined between the above variant and variant 2 - Thiram 42-S fungicide treated seed (48% Thiram, 600 ml/100 kg seed). All other variants obtained highly significant (p < 0.01) larger number of infected and decayed plants. Average sugar yield in treated variants was

7.78 t/ha or 38.18% higher than average sugar yield in the control variant (5.63 t/ha).

Sugar yield was in very significant positive correlation with root yield (r=0.975; p<0.01) and

all tested parameters.

molasses).

(3.97%).

(Table 5).

sugar content (r=0.946; p<0.01).

**3.5 Sugar yield** 

Inoculation of sugar beet seed with the bacteria *Pseudomonas fluorescens*, *Bacillus subtilis* and *Bacillus megaterium* affected root decay agents *Rhizoctonia solani* and *Pythium debarianum* and showed highly significant influence on all the elements of sugar beet yield and quality in the three trial years.

The best results in all elements of the research on both soil types were recorded in the second trial year (2008) due to the more favourable weather conditions (air temperatures, precipitation values) in the growing season 2008 than in the dry 2007 and 2009.

Better results in all tested parameters in the three trial years were obtained on Mollic Gleysols. The reason is in chemical and microbiological properties of much better quality than on Eutric Cambisols. In Mollic Gleysols total number of the bacteria and actinomycetes is higher enabling more intensive humification of organic matter and mineralization of humus in the plant-accessible mineral compounds. Significantly higher number of beneficial bacteria in the soil (*P. fluorescens*, *Bacillus* spp.) that show antagonism against soilborne pathogens is evident.

On Mollic Gleysols the best results in all tested parameters were recorded in the variant treated with the beneficial bacteria (*P. fluorescens*, *B. subtilis*, *B. megaterium*), whereas all other variants obtained significantly (p<0.01) lower values. Due to the beneficial bacteria in the soil, population of pathogenic microorganisms was reduced, making application of the beneficial bacteria in the sowing period sufficient to reduce or control the infection with pathogenic fungi *R. solani*. Among the beneficial bacteria *P. fluorescens*, *B. subtilis* and *B. megaterium* highly positive synergism is evident.

On Eutric Cambisols the best results in all tested parameters were reached in the variant treated with chemical fungicide and the beneficial bacteria (*P. fluorescens*, *B. subtilis*, *B. megaterium*). Nevertheless, in most cases no statistically significant difference (p>0.05) was determined between this variant and the variant being treated solely with chemical fungicide. The reason is in poorer quality of chemical and microbiological soil properties which is favourable for reproduction of pathogenic fungi *P. debarianum* in the soil.

If necessary measures of improving chemical soil properties are to be carried out, neutralization of soil acidity in the first place, microbiological soil properties will be improved, and the beneficial soil microorganisms will increase in number. Due to the antagonism of beneficial microorganisms against soilborne plant pathogens the number of pathogenic microorganisms will be reduced as well as the infection of arable crops. In this case the application of beneficial microorganisms will reach satisfactory level without application of chemical pesticides. By introducing beneficial microorganisms microbiological soil properties will be consequently improved and number of pathogens in the forthcoming growing season will be decreasing. The reduction in the application of chemical pesticides is the matter of great importance from the both economic and ecological point of view.

#### **5. Acknowledgment**

I would like to show my gratitude to Ministry of Science, Education and Sports of the Republic of Croatia for financial support of our project research (079-0791843-1933). Results presented in this chapter were obtained during this project research.

#### **6. References**


Andersen, J.B.; Koch, B.; Nielsen, T.H.; Sørensen, D.; Hansen, M.; Nybroe, O.;

Asaka, O. & Shoda, M. (1996). Biocontrol of *Rhizoctonia solani* damping-off of tomato with

Choudhary, D.K. & Johri, B.N. (2009). Interactions of *Bacillus* spp. and plants-with special

Cooke, D.A.; Dewar, A.M. & Asher, M.J.C. (1989). Pests and diseases of sugar beet, In: *Pest* 

Desai, J.D. & Banat, I. M. (1997). Microbial production of surfactants and their commercial

Duijff, B.J.; Meijer, J.W.; Bakker, P.A.H.M. & Schippers, B. (1993). Siderophore mediated

Durrant, M.J.; Payne, P.A.; Prince, J.W.F. & Fletcher, R. (1988). Thiram steep seed treatment

Engelhardt, S.; Lee, J.; Gäbler, Y.; Kemmerling, B.; Haapalainen, M.L.; Li, C.M.; Wei, Z.;

Esh, A.M.H. & El-Kholi, M.M. (2005). Effect of *Pseudomonas fluorescens* extra cellular

Evačić, M.; Kristek, S. & Stanisavljević, A. (2008). Use of the bacteria *Bacillus megaterium* in

Gorlach-Lira, K. & Stefaniak, O. (2009): Antagonistic activity of bacteria isolated from crops

Kiewnick, S.; Barry, J.J.; Braun-Kiewnick, A.; Eckhoff, L.A. & Bergman, J.W. (2001).

*Crop protection,* Vol.7, No.5, (October 1988), pp. 319-326, ISSN 0261-2194 El-Tarabily, K.A. & Sivasithamparam, K. (2006). Potential of yeasts as biocontrol agents of

Vol.149, No. 1, (January 2003), pp. 37-46, ISSN 1350-0872

Protection Council, ISBN 0-948404-28-0, Thornton Heath, UK

(November 1996), pp. 4081-4085, ISSN 0099-2240

(September 2009), pp. 493-513, ISSN 0944-5013

6, (September 1993), pp. 277-289, ISSN 0028-2944

FAO. World Reference Base for Soil Resources, (1998). FAO Roma

ISSN 0960-7412

ISSN 1110-0338

5632

Vol.47, No. 1, (February 2006), pp. 25-35, ISSN 1340-3540

Christophersen, C.; Sørensen, J.; Molin, S. & Giskov, M. (2003). Surface motility in *Pseudomonas sp*. DSS73 is required for efficient biological containment of the root – pathogenic microfungi *Rhizoctonia solani* and *Pythium ultimum*. *Microbiology*,

*Bacillus subtilis* RB14. *Applied and Environmental Microbiology*, Vol.62, No.11,

reference to induced systemic resistance (ISR). *Mirobiological research,* Vol.164, No.5,

*and Disease Control Handbook,* N. Scopes & L. Stables, (Ed.), 241-259, British Crop

potential. *Micr. Mol. Biol. Rev.* Vol.61, No.1, (March 1997), pp. 47-64, ISSN 1092-2172

competition for iron and induced disease resistance in the suppression of *Fusarium wilt* of carnation by fluorescent *Pseudomonas* spp. *Neth. J. Plant Pathol.,* Vol.99, No.5-

to control *Phoma betae* and improve the establishment of the sugar beet plant stand.

soil-borne fungal plant pathogens and as plant growth promoters. *Mycoscience*,

Keller, H.; Joosten, M. & Taira, S. (2009). Separable roles of the *Pseudomonas syringae*  pv. *phaseolicola* accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity. *Plant J.*, Vol.57, No.4, (February 2009), pp. 706-717,

enzymes and secondary metabolites on *Rhizoctonia solani* the causal of sugar beet damping off disease. *Zagazig Journal of Agricultural Research,* Vol.32, pp. 1537-1557,

the control of sugar beet root decay agents – *Pythium ultimum* and *Pythium debarianum. Cereal Resear.Comm.*, Vol.36, Suppl.5, Part1, pp. 383-386, ISSN 0133-3720

cultivated in a rotation system and a monoculture against *Pythium debaryanum* and *Fusarium oxysporum*. *Folia microb*., Vol.54, No.5, (July 2009), pp. 447-450, ISSN 0015-

Integrated control of *Rhizoctonia crown* and root rot of sugar beet with fungicides

**6. References** 

and antagonistic bacteria. *Plant Disease*, Vol.85, No.7, (July 2001), pp. 718-722, ISSN 0191-2917


## **State of the Art and Future Prospects of Alternative Control Means Against Postharvest Blue Mould of Apple: Exploiting the Induction of Resistance**

Simona Marianna Sanzani and Antonio Ippolito *Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari "Aldo Moro" Italy* 

#### **1. Introduction**

116 Fungicides – Beneficial and Harmful Aspects

Pedersen, H.C.; Weiergang, I.; Pontoppidan, M.M.; Jørgensen, L. & Svingel, A. (2002).

Raaijmakers, J.M.; De Brujin, I.; Nybroe, O. & Ongena, M. (2010). Natural functions of

Ron, E.Z. & Rosenberg, E. (2001). Natural roles of biosurfactants. *Environmental* 

Sørensen, D.; Nielsen, T.H.; Christophersen, C.; Sørensen, J. & Gajhede, M. (2001). Cyclic

Thrane, C.; Nielsen, T.H.; Nielsen, M.N.; Sørensen, J. & Olsson, S. (2000). Viscosinamide –

Zhang, S.; Reddy, M.S. & Kloepper, J.W. (2002). Development of assays for assessing

Whipps, J.M. (2001). Microbial interactions and biocontrol in the rhizosphere. *Journal of Experimental Botany*, Vol.52, Suppl.1, (March 2001), pp. 487-511, ISSN 0022-0957

FEMS *Microb. Reviews,* Vol.34, No.6, pp.1037-1062, ISSN 1574-6976

*Microbiol*ogy, Vol.3, No.4, (April 2001), pp. 229-236, ISSN 1462-2912

(September 2001), pp. 1123 – 1124, ISSN 0108-2701

(August 2000), pp. 139-146, ISSN 0168-6496

Denmark

1049-9644

www.statsoft.com

Danisco Seed, Seed Technology Dept., Hoejbygaardvej 31, DK – 4960 Holeby,

lipopeptides from *Bacillus* and *Pseudomonas*: more than surfactants and antibiotics.

lipoundecapeptide amphisin from *Pseudomonas* sp. Strain Dss73. *Acta Crystallographica Section C Crystal Structure Communications,* Vol.57, No.9,

producing *Pseudomonas fluorescens* DR54 exerts a biocontrol effect on *Pythium ultimum* in sugar beet rhizosphere. *FEMS Microbiology Ecology,* Vol.33, No.2,

induced systemic resistance by plant growth-promoting rhizobacteria against blue mold of tobacco. *Biological Control*, Vol.23, No.1, (January 2002), pp. 79-86, ISSN

> Fresh fruit and vegetables supply essential nutrients, such as vitamins and minerals, and are a major source of complex carbohydrates, antioxidants, and anticarcinogenic substances which are important to human health and well being (Arul, 1994). Being aware of the advantages potentially coming from their use, the consumers' demand for fresh products has greatly increased during recent years. On the other end, the possible presence of chemical residues, mycotoxins and other contaminants of fruit and vegetables (Paster et al., 1995), creates great concern among consumers for safety issues.

> Therefore, aim of an adequate storage is to help the harvested fruit and vegetables to arrive at their destination fresh, disease-free, and safe for consumers, despite the complexity of treatments they have to undergo prior to or during storage, and the long period between harvest and consumption. All the means and methods with the power to aid in preserving the quality of the harvested products and in protecting them from decay agents during storage and shelf-life, are aimed at this objective.

#### **2. Apple: Origin, distribution and economic importance**

Apples **(***Malus domestica* Borkh.**)** belongs to the *Pomoideae* subfamily *Rosaceae*, along with pear (*Pyrus communis* L.), apricot (*Prunus armeniaca* L.), prune (*Prunus domesticus* L.), cherry (*Prunus avium* L.) and peach (*Prunus persica* L.). Actually there is a debate about whether *M. domestica* originated from hybridization among various wild species or from a single species, still growing on the Northern slopes of the Tien Shan mountains at the border between northwest China and the former Soviet Republic of Kazakhstan (Phipps *et al.*, 1990; Juniper & Mabberley, 2006). Apples were consumed by human beings since the Stone Age, as documented by numerous Neolithic finds discovered in Northern Europe. In the medieval times monasteries were responsible for selection, propagation, and perpetuation of hundred of different cultivar types. In the Nineteenth Century these types became the ideal stock for horticulturists to develop techniques to cross desirable selections. From that time *M. domestica* cultivars spread throughout the world, particularly in North America, Russia, Australia, and Japan. Over 7500 apple cultivars are known, but even though nowadays breeders worldwide create new selections annually, only very few of them are widely produced (Janick, 1996).

In 2008 apple constituted the third fruit most cultivated in the world with and area of 4,696,472 hectares and a production of 69,304,442 tonnes (Food and Agriculture Organization of the United Nations [FAO], 2011). Moreover, Italy represented in the world the sixth highest producing country (more than 2 million tonnes), and the first exporting one (683 thousand tonnes); whereas, apple consumption was estimated to be 47.78 g/day/capita (FAO, 2011). The apple production is concentrated in Northern Italy: 80% of the crop, in fact, comes from Trentino-Alto Adige (46%), Emilia-Romagna (17%), and Veneto (14%) regions. Golden Delicious and Granny Smith are two of the most popular apple cultivars among consumers. In Italy apples are mainly consumed as fresh commodity, whereas in Northern Europe and America they are used for juices and cider production.

Apples are commonly harvested from the end of August till mid-October, although, because of the postharvest practices (pre-refrigeration, controlled atmosphere, etc.), they can be stored for long times and so are available on commerce all through the year.

#### **3. Apple pathogens and diseases**

The surface of fruit or vegetables is covered by fungal and bacterial propagules that they have acquired from the air during their development on the parent plant, or with which they have come in contact during picking or any of the subsequent stages of handling. However, most fungal and bacterial propagules that reach the harvested product do not cause decay, even when conditions suitable for penetration and development are present.

Harvested fruit and vegetables are naturally attacked by a relatively small group of pathogens: approximately forty species. However, each fruit or vegetable has its own typical pathogens out of this particular group. Eckert & Ogawa (1988) divided the major postharvest pathogens of pome fruits into two groups: (a) those that cause quiescent infections of lenticels, including *Phlyctema vagabunda* Desm. (syn. *Gloeosporium album*)*, G. perennans* Zeller & Childs (syn. *Cryptosporiopsis curvispora*) and *Nectria galligena* Bresad; and (b) those that preferably enter through wounds after harvest, including *Penicillium expansum* Link*, Botrytis cinerea* Pers. ex Fr.*, Monilinia* spp*.*, *Mucor* spp., *Rhizopus* spp., *Alternaria alternata* (Fr.) Keissl*, Stemphylium botryosum* Wallr, and *Cladosporium herbarum* (Pers.) Link*.* The rots in the lenticels are initiated in the orchard in the late summer, and are a major problem for apples grown in areas with late summer rainfall such as the United Kingdom and Northern European Countries (Edney, 1983). In drier apple production areas, main problems are caused by wound pathogens that invade the fruit after harvest through injuries sustained during harvesting and handling and via puncture wounds, bruised lenticels, etc. Other important pathogens of pome fruits are species of *Phytophthora*, that may become a serious problem during rainy seasons for fruit from orchards with heavy soils (Edney, 1978), and *Colletotrichum gloeosporioides* Penz (syn. *Gloeosporium fructigenum),* the bitter rot fungus, which is capable of direct penetration of the intact skin (Brook, 1977). *Botryosphaeria* spp., the black and white rot fungi, are of importance in several areas of the USA (Snowdon, 1990). Finally, pathogens of minor importance that may occasionally be found on harvested apples and pears include *Trichothecium roseum* Link*,* and species of *Phomopsis, Nigrospora, Fusarium, Epicoccum, Aspergillus,* and *Trichoderma* (Snowdon, 1990).

#### **3.1** *Penicillium expansum*

118 Fungicides – Beneficial and Harmful Aspects

throughout the world, particularly in North America, Russia, Australia, and Japan. Over 7500 apple cultivars are known, but even though nowadays breeders worldwide create new

In 2008 apple constituted the third fruit most cultivated in the world with and area of 4,696,472 hectares and a production of 69,304,442 tonnes (Food and Agriculture Organization of the United Nations [FAO], 2011). Moreover, Italy represented in the world the sixth highest producing country (more than 2 million tonnes), and the first exporting one (683 thousand tonnes); whereas, apple consumption was estimated to be 47.78 g/day/capita (FAO, 2011). The apple production is concentrated in Northern Italy: 80% of the crop, in fact, comes from Trentino-Alto Adige (46%), Emilia-Romagna (17%), and Veneto (14%) regions. Golden Delicious and Granny Smith are two of the most popular apple cultivars among consumers. In Italy apples are mainly consumed as fresh commodity, whereas in Northern

Apples are commonly harvested from the end of August till mid-October, although, because of the postharvest practices (pre-refrigeration, controlled atmosphere, etc.), they can be

The surface of fruit or vegetables is covered by fungal and bacterial propagules that they have acquired from the air during their development on the parent plant, or with which they have come in contact during picking or any of the subsequent stages of handling. However, most fungal and bacterial propagules that reach the harvested product do not cause decay, even when conditions suitable for penetration and development are present. Harvested fruit and vegetables are naturally attacked by a relatively small group of pathogens: approximately forty species. However, each fruit or vegetable has its own typical pathogens out of this particular group. Eckert & Ogawa (1988) divided the major postharvest pathogens of pome fruits into two groups: (a) those that cause quiescent infections of lenticels, including *Phlyctema vagabunda* Desm. (syn. *Gloeosporium album*)*, G. perennans* Zeller & Childs (syn. *Cryptosporiopsis curvispora*) and *Nectria galligena* Bresad; and (b) those that preferably enter through wounds after harvest, including *Penicillium expansum* Link*, Botrytis cinerea* Pers. ex Fr.*, Monilinia* spp*.*, *Mucor* spp., *Rhizopus* spp., *Alternaria alternata* (Fr.) Keissl*, Stemphylium botryosum* Wallr, and *Cladosporium herbarum* (Pers.) Link*.* The rots in the lenticels are initiated in the orchard in the late summer, and are a major problem for apples grown in areas with late summer rainfall such as the United Kingdom and Northern European Countries (Edney, 1983). In drier apple production areas, main problems are caused by wound pathogens that invade the fruit after harvest through injuries sustained during harvesting and handling and via puncture wounds, bruised lenticels, etc. Other important pathogens of pome fruits are species of *Phytophthora*, that may become a serious problem during rainy seasons for fruit from orchards with heavy soils (Edney, 1978), and *Colletotrichum gloeosporioides* Penz (syn. *Gloeosporium fructigenum),* the bitter rot fungus, which is capable of direct penetration of the intact skin (Brook, 1977). *Botryosphaeria* spp., the black and white rot fungi, are of importance in several areas of the USA (Snowdon, 1990). Finally, pathogens of minor importance that may occasionally be found on harvested apples and pears include *Trichothecium roseum* Link*,* and species of *Phomopsis, Nigrospora, Fusarium, Epicoccum,* 

selections annually, only very few of them are widely produced (Janick, 1996).

Europe and America they are used for juices and cider production.

**3. Apple pathogens and diseases** 

*Aspergillus,* and *Trichoderma* (Snowdon, 1990).

stored for long times and so are available on commerce all through the year.

The blue mould rot, caused by *P. expansum,* is one of the most common and destructive rots of harvested apples and pears, but it can also be found on sweet cherries and other commodities such as apricots, grapes, blueberries, peaches, strawberries, walnuts, pecans, hazelnuts, and acorns (Andersen *et al.*, 2004; Murphy *et al.*, 2006). Blue mould is a worldwide severe disease, even in production areas where the most advanced storage technologies are available. In Northern Europe the estimated incidence of this disease varies between seasons and cultivars, ranging from 5 to 20% (Mari *et al.*, 2002). *P. expansum* is primarily a necrotroph and a wound parasite, most frequently gaining entrance through fresh mechanical injuries such as stem punctures, bruises and insect injuries, finger-nail scratches by pickers and necrotic tissues of diverse origin, for instance due to infections by other pathogens, such as *Gloeosporium* spp.*, Phytophthora* spp., and *Mucor* spp. (Snowdon, 1990). Thus, resistance of the epidermis to breakage may be an important factor in the resistance of apple cultivars to decay. Studying the force required to break the epidermis of several cultivars, as a criterion for resistance to wound pathogens, Spotts *et al.* (1999) found that the epidermis of Golden Delicious and Jonagold was more easily broken than that of other cultivars, while the epidermal tissues of Fuji and Granny Smith were the most resistant to puncture. Sometimes infections may occur through normal stems, open calyx canals or lenticels, especially when they are damaged by cracking after a sudden abundant supply of water following a period of dryness, or after bruising late in the storage season when fruit have been weakened by ripening and aging (Janisiewicz *et al.*, 1991).

The fungus produces pale brown to brown soft-watery spots that enlarge rapidly under shelf life conditions. Under humid conditions, conidia-bearing conidiophores group to form coremia on the surface of the lesion. As the conidia mature, they turned from nearly snow white to blue-green and form masses which give the decay its typical colour. Another characteristics, important in the recognition of *P. expansum,* is the earthy, musty odour.

Since decay development is favoured by high humidity, the blue mould is a particularly serious problem on fruits stored or shipped in plastic film liners (Hall & Scott, 1989). Decay can progress, albeit slowly, during cold storage; rapid development begins when the fruits are transferred to warmer conditions. The fungus can spread during the postharvest handling, since the blue mold spores are long-lived and may easily survive from season to season on contaminated bins, where the fungus can grow and produce copious amounts of spores. Contamination with these spores may come from various other sources including orchard soil present on bins, decaying fruit or air. Inoculation of the fruit going into storage is believed to occur mainly from the diphenylamine (DPA) drenching solution used for protection against superficial scald, where the spore concentration increases with each successively drenched bin and may reach high levels if solutions are not changed regularly. Inoculation can also occur during fruit handling in water contaminated with the fungus in packing houses. A single decayed fruit may contain enough spores to contaminate water of the entire packing line (Janisiewicz *et al.*, 1991). At present few chemicals are permitted in Europe to be used in the postharvest phase: for instance, only thiabendazole and pyrimethanilare are allowed in Italy for postharvest control of blue mold. Moreover, during the past few decades thiabendazole has lost efficacy due to the establishment of resistant pathogen populations (Baraldi *et al.*, 2003).

Finally, it should be considered that contamination of fruits by *P. expansum* not only results in economic losses during storage and shelf-life, but it also has a potential public health significance, since some strains of *P. expansum* produces the mycotoxin patulin.

#### **3.2 Patulin**

Thousands of mycotoxins exist, but only a few represent significant food safety challenges. The most important mycotoxins impacting food production and manufacturing are: patulin, produced by *Penicillium* species (mainly from *P. expansum*); ochratoxin, which is present in a large variety of foods as it is produced by several fungal strains of *Penicillium* and *Aspergillus* species; zearalenone, found prevalently in grains infected by *Fusarium graminearum* Rank; aflatoxins, mainly produced by *Aspergillus flavus* Link and *A. parasiticus* Speare; trichothecene, whose production is known for several *Fusarium* species; and, finally, fumonisins mainly produced by the maize pathogens *Fusarium verticillioides* (formerly *F. moniliforme*) (Sacc.) Nirenberg and *F. proliferatum* (Matsush.) Nirenberg ex Gerlach and Nirenberg.

Of special interest to postharvest pathology is the production of patulin by *P. expansum* in pome fruits. Patulin {4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one} is a polar compound transferred in fruit juices if rotten fruits, especially apples, are not picked up during fruit juice processing. There is no clear evidence that patulin is carcinogenic, however, it has been shown to cause immunotoxic effects (Pacoud *et al.*, 1990) and to be neurotoxic in animals (Deveraj *et al.*, 1982), so that the European Commission in 2006 established the maximum levels of patulin permitted in foodstuffs (European Commission, 2006). The amount of patulin produced by *P. expansum* may vary greatly according to the strain involved. Sommer *et al.* (1974) found that patulin production in different strains of *P. expansum* in Golden Delicious apples ranged from 2 to 100 µg gram-1 of tissue. On the other hand, other constitutive and environmental parameters, such as the cultivar and the storage temperature may influence patulin accumulation. Paster *et al.* (1995) demonstrated that while more patulin was produced in Starking apples than in Spadona pears held at 0-17°C, higher toxin levels were produced in pears than in apples held at 25°C. Furthermore, a consistent reduction in patulin production was observed when inoculated apples were held in a 3%CO2/2%O2 atmosphere. Thus, it can be concluded that the ability to produce patulin and the amounts of patulin produced depend on fungal strain, fruit cultivar, storage temperature, and composition of storage atmosphere (Lovett *et al.*, 1975; Paster *et al.*, 1995). Finally, it has to be considered that, patulin, as many fungal secondary metabolites, has an antibiotic activity. This ability is particularly useful because the secondary products may represent competitive weapons in nature (Sutton, 1996).

#### **4. Alternative control means**

The development of resistance, together with the increasing concern about possible adverse effects on human health and environment caused by fungicides, have contributed to arouse interest in the development of alternative means for controlling plant pathogens, capable of integrating, if not totally replacing, synthetic fungicides. Substantial progress has been made in finding alternatives to synthetic fungicides for the control of postharvest diseases of fruit and vegetables (Ippolito *et al.*, 2004; Palou *et al.*, 2008; Sanzani *et al.*, 2009a; Schena *et al.*, 2007; Sharma *et al.*, 2009; Zhang *et al.*, 2009).

Emerging postharvest biocontrol technology employs different approaches, such as use of physical means, natural biocides, antagonistic microorganisms and their products, or the intensification of natural defense mechanisms. Treatments that have been evaluated for effectiveness against *P. expansum* on apples include chlorine dioxide administration in aqueous environment (Okull *et al.*, 2006); surface application of cinnamon oil or potassium sorbate (Ryu & Holt, 1993); fumigation with acetic acid vapor (Sholberg *et al.*, 2000); immersion in electrolyzed oxidizing water (Okull & LaBorde, 2004). A wash treatment with ≥ 2% acetic acid for more than 1 min proved to be effective in completely inhibiting *P. expansum* growth and relevant patulin production on apples destined for cider (Chen *et al.*, 2004). Hot water immersion inhibited decay development in *P. expansum-*inoculated apples (Fallik, 2010).

Several examples of success in preventing *P. expansum* rots during postharvest phase of fruits by using yeasts antagonists (Ippolito *et al.*, 2000; Zhang *et al.*, 2009), bacteria (Morales *et al.*, 2008) and biologically active natural products (Mari *et al.*, 2002), have been reported. The antifungal effectiveness of an antagonist can be increased by addition of substances, as in the case of sodium bicarbonate in combination with a strain of *Metschnikowia pulcherrima* (Spadaro *et al.*, 2004). Finally the controlled atmosphere (CA), used for the long-term storage of apples, may also influence the efficacy of biocontrol agents by affecting their vitality or by altering the physiological status of the treated fruits (Chalutz & Droby, 1997).

#### **4.1 Host protection and defence mechanisms**

120 Fungicides – Beneficial and Harmful Aspects

Thousands of mycotoxins exist, but only a few represent significant food safety challenges. The most important mycotoxins impacting food production and manufacturing are: patulin, produced by *Penicillium* species (mainly from *P. expansum*); ochratoxin, which is present in a large variety of foods as it is produced by several fungal strains of *Penicillium* and *Aspergillus* species; zearalenone, found prevalently in grains infected by *Fusarium graminearum* Rank; aflatoxins, mainly produced by *Aspergillus flavus* Link and *A. parasiticus* Speare; trichothecene, whose production is known for several *Fusarium* species; and, finally, fumonisins mainly produced by the maize pathogens *Fusarium verticillioides* (formerly *F. moniliforme*) (Sacc.)

Of special interest to postharvest pathology is the production of patulin by *P. expansum* in pome fruits. Patulin {4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one} is a polar compound transferred in fruit juices if rotten fruits, especially apples, are not picked up during fruit juice processing. There is no clear evidence that patulin is carcinogenic, however, it has been shown to cause immunotoxic effects (Pacoud *et al.*, 1990) and to be neurotoxic in animals (Deveraj *et al.*, 1982), so that the European Commission in 2006 established the maximum levels of patulin permitted in foodstuffs (European Commission, 2006). The amount of patulin produced by *P. expansum* may vary greatly according to the strain involved. Sommer *et al.* (1974) found that patulin production in different strains of *P. expansum* in Golden Delicious apples ranged from 2 to 100 µg gram-1 of tissue. On the other hand, other constitutive and environmental parameters, such as the cultivar and the storage temperature may influence patulin accumulation. Paster *et al.* (1995) demonstrated that while more patulin was produced in Starking apples than in Spadona pears held at 0-17°C, higher toxin levels were produced in pears than in apples held at 25°C. Furthermore, a consistent reduction in patulin production was observed when inoculated apples were held in a 3%CO2/2%O2 atmosphere. Thus, it can be concluded that the ability to produce patulin and the amounts of patulin produced depend on fungal strain, fruit cultivar, storage temperature, and composition of storage atmosphere (Lovett *et al.*, 1975; Paster *et al.*, 1995). Finally, it has to be considered that, patulin, as many fungal secondary metabolites, has an antibiotic activity. This ability is particularly useful because the secondary products may

The development of resistance, together with the increasing concern about possible adverse effects on human health and environment caused by fungicides, have contributed to arouse interest in the development of alternative means for controlling plant pathogens, capable of integrating, if not totally replacing, synthetic fungicides. Substantial progress has been made in finding alternatives to synthetic fungicides for the control of postharvest diseases of fruit and vegetables (Ippolito *et al.*, 2004; Palou *et al.*, 2008; Sanzani *et al.*, 2009a; Schena *et al.*, 2007;

Emerging postharvest biocontrol technology employs different approaches, such as use of physical means, natural biocides, antagonistic microorganisms and their products, or the intensification of natural defense mechanisms. Treatments that have been evaluated for effectiveness against *P. expansum* on apples include chlorine dioxide administration in aqueous environment (Okull *et al.*, 2006); surface application of cinnamon oil or potassium sorbate (Ryu

Nirenberg and *F. proliferatum* (Matsush.) Nirenberg ex Gerlach and Nirenberg.

represent competitive weapons in nature (Sutton, 1996).

**4. Alternative control means** 

Sharma *et al.*, 2009; Zhang *et al.*, 2009).

**3.2 Patulin** 

Induced disease resistance has been adopted as a general term and defined as 'the process of active resistance, dependent on the physical or chemical barriers of the host plant, activated by biotic or abiotic agents (inducing agents)' (Kloepper *et al.*, 1992). In compatible plantfungus interactions resistance mechanisms may be activated too slowly to be effective or be suppressed by the invading pathogen. So the level of basic resistance may simply not be sufficient to halt infection and prevent extensive tissue colonization and symptom development. Whereas, in induced tissues the balance may be shifted in favour of the plant, by an earlier and quicker response, that can be effective in limiting tissue colonization to various extents, depending on the specific plant-pathogen relationship.

Plants respond to invasion by pathogens with an array of biochemical and genetic changes, including the production of reactive oxygen species (ROS), antimicrobial compounds, antioxidants, and signalling molecules such as salicylic acid (SA), ethylene and jasmonic acid (JA) (Mahalingam *et al.*, 2003). They also respond by the localized activation of a celldeath program, designated "hypersensitive response (HR)", and by the systemic activation of cellular and molecular defences, termed "systemic acquired resistance (SAR)" (Ryals *et al.*, 1996). There is evidence for commonalities between plant responses to pathogens (referred to as defence responses) and environmental stresses (referred to as stress responses). However, a plant response to each environmental challenge is unique and tailored to increasing the plant ability to survive the inciting stress.

Most plant antimicrobial natural products have relatively broad spectrum activity, and specificity is often determined by whether or not a pathogen has the enzymatic machinery to detoxify a particular host product. Accumulation of inducible antimicrobial compounds is often orchestrated through signal-transduction pathways linked to perception of the pathogen by host receptors. The simplest functional definitions recognize phytoalexins as compounds that are synthesized *de novo* and phytoanticipins as pre-formed infectional inhibitors. However, the distinction between phytoalexin and phytoanticipin is not always obvious, as some compounds may be phytoalexins in one species and phytoanticipins in others (Dixon, 2001).

Phytoalexins are low-molecular-weight toxic compounds mainly produced in the host tissue in response to initial infection by microorganisms (Harborne, 1999). In other words, in order to overcome an attack by the pathogen, the host is induced to produce antifungal compounds that would prevent pathogen development. However, the accumulation of phytoalexins does not depend on infection only. Such compounds may be elicited by microbial metabolites, mechanical damage, plant constituents released after injury, a wide diversity of chemical compounds, or by low temperature, irradiation, and other stress conditions. Phytoalexins are, thus, considered to be general stress-response compounds, produced after biotic or abiotic stress. The most available evidence on the role of phytoalexins shows that disruption of cell membranes is a central factor in their toxicity (Smith, 1996), and that the mechanism is consistent with the lipophilic properties of most phytoalexins (Arnoldi & Merlini, 1990).

Earlier studies by Müller & Borger (1940) already provided strong evidence that resistance of potato to *Phytophthora infestans* (Mont.) de Bary is based on the production of fungitoxic compounds by the host. A terpenoid compound, rishitin, produced in potato tubers following infection by *P. infestans,* was first isolated by Tomiyama *et al.* (1968) from resistant potatoes inoculated with the fungus. It accumulates rapidly in the tuber and reaches levels much higher than those required to prevent fungal development. The relationship between the accumulation of rishitin in the tuber and its resistance to late blight may point to its role in resistance development (Kuc, 1976). Other sesquiterpenoids that have been found in potatoes may also play a role in tuber disease resistance; they include rishitinol (Katsui *et al.*, 1972), lubimin (Katsui *et al.*, 1974), oxylubimin (Katsui *et al.*, 1974), solavetivone (Coxon *et al.*, 1974), and others. These sesquiterpenoids proved also to be effective in suppressing mycelial growth of the potato pathogen *P. infestans* on a defined medium (Engstrom *et al.*, 1999). Moreover, several phytoalexins, such as umbelliferone, scopoletin, and esculetin, are produced in sweet potato roots infected by the fungus *Ceratocystis fimbriata* Ellis and Halsted; it was noted that these compounds accumulate more rapidly in roots resistant to this fungus than in sensitive roots (Minamikawa *et al.*, 1963).

Further studies with celery (Afek *et al.*, 1995a) indicate that (+) marmesin, the precursor of linear furanocoumarins in this crop, is the major compound involved in celery resistance to pathogens, indeed increased susceptibility of stored celery to pathogens is accompanied by a decrease in (+) marmesin concentration. Indeed treatment of celery prior to storage with gibberellic acid (GA3), a naturally occurring phytohormone in juvenile plant tissue, resulted in decay suppression during 1 month of storage at 2°C, although GA3 does not have any effect on fungal growth *in vitro* (Barkai-Golan & Aharoni, 1976). It was suggested that the phytohormone retards celery decay during storage by slowing down the conversion of (+) marmesin to psoralens, thereby maintaining high level of (+) marmesin and low levels of psoralens and, thus increasing celery resistance to storage pathogens (Afek *et al.*, 1995b). Another phytoalexin found in celery tissue is columbianetin, which probably also plays a more important role than psoralens in celery resistance to decay (Afek *et al.*, 1995c). This hypothesis is derived from the following facts: (a) columbianetin exhibits strong activity against *A. alternata, B. cinerea,* and *Sclerotinia sclerotiorum* (Lib.) de Bary*,* the main postharvest pathogens of celery; (b) the concentration of columbianetin in the tissue is close to that required for their suppression; (c) increased sensitivity of celery to pathogens during storage occurred in parallel with the decrease in the concentration of columbianetin.

The phytoalexin capsidiol is a sesquiterpenoid compound produced by pepper fruits in response to infection by a range of fungi (Stoessl *et al.*, 1972). Pepper fruits inoculated with

to overcome an attack by the pathogen, the host is induced to produce antifungal compounds that would prevent pathogen development. However, the accumulation of phytoalexins does not depend on infection only. Such compounds may be elicited by microbial metabolites, mechanical damage, plant constituents released after injury, a wide diversity of chemical compounds, or by low temperature, irradiation, and other stress conditions. Phytoalexins are, thus, considered to be general stress-response compounds, produced after biotic or abiotic stress. The most available evidence on the role of phytoalexins shows that disruption of cell membranes is a central factor in their toxicity (Smith, 1996), and that the mechanism is consistent with the lipophilic properties of most

Earlier studies by Müller & Borger (1940) already provided strong evidence that resistance of potato to *Phytophthora infestans* (Mont.) de Bary is based on the production of fungitoxic compounds by the host. A terpenoid compound, rishitin, produced in potato tubers following infection by *P. infestans,* was first isolated by Tomiyama *et al.* (1968) from resistant potatoes inoculated with the fungus. It accumulates rapidly in the tuber and reaches levels much higher than those required to prevent fungal development. The relationship between the accumulation of rishitin in the tuber and its resistance to late blight may point to its role in resistance development (Kuc, 1976). Other sesquiterpenoids that have been found in potatoes may also play a role in tuber disease resistance; they include rishitinol (Katsui *et al.*, 1972), lubimin (Katsui *et al.*, 1974), oxylubimin (Katsui *et al.*, 1974), solavetivone (Coxon *et al.*, 1974), and others. These sesquiterpenoids proved also to be effective in suppressing mycelial growth of the potato pathogen *P. infestans* on a defined medium (Engstrom *et al.*, 1999). Moreover, several phytoalexins, such as umbelliferone, scopoletin, and esculetin, are produced in sweet potato roots infected by the fungus *Ceratocystis fimbriata* Ellis and Halsted; it was noted that these compounds accumulate more rapidly in roots resistant to

Further studies with celery (Afek *et al.*, 1995a) indicate that (+) marmesin, the precursor of linear furanocoumarins in this crop, is the major compound involved in celery resistance to pathogens, indeed increased susceptibility of stored celery to pathogens is accompanied by a decrease in (+) marmesin concentration. Indeed treatment of celery prior to storage with gibberellic acid (GA3), a naturally occurring phytohormone in juvenile plant tissue, resulted in decay suppression during 1 month of storage at 2°C, although GA3 does not have any effect on fungal growth *in vitro* (Barkai-Golan & Aharoni, 1976). It was suggested that the phytohormone retards celery decay during storage by slowing down the conversion of (+) marmesin to psoralens, thereby maintaining high level of (+) marmesin and low levels of psoralens and, thus increasing celery resistance to storage pathogens (Afek *et al.*, 1995b). Another phytoalexin found in celery tissue is columbianetin, which probably also plays a more important role than psoralens in celery resistance to decay (Afek *et al.*, 1995c). This hypothesis is derived from the following facts: (a) columbianetin exhibits strong activity against *A. alternata, B. cinerea,* and *Sclerotinia sclerotiorum* (Lib.) de Bary*,* the main postharvest pathogens of celery; (b) the concentration of columbianetin in the tissue is close to that required for their suppression; (c) increased sensitivity of celery to pathogens during storage

occurred in parallel with the decrease in the concentration of columbianetin.

The phytoalexin capsidiol is a sesquiterpenoid compound produced by pepper fruits in response to infection by a range of fungi (Stoessl *et al.*, 1972). Pepper fruits inoculated with

phytoalexins (Arnoldi & Merlini, 1990).

this fungus than in sensitive roots (Minamikawa *et al.*, 1963).

*B. cinerea* and *Phytophthora capsici* Leon contain only small quantities of capsidiol, whereas fruits inoculated with saprophytic species or with weak pathogens may produce higher concentrations of the phytoalexin, which inhibit spore germination and mycelial growth. Inoculating peppers with *Fusarium* species results in increased capsidiol concentration from 6 to 12 h after inoculation. In such cases, the capsidiol accumulation in the tissue is rapid, whereas in other cases it is quickly oxidized to capsenone, which is characterized by a much weaker toxic effect. When unripe pepper fruits were inoculated with *Glomerella cingulata* Stonem*,* the causal agent of anthracnose, a phytoalexin was readily identified in tissue extracts (Adikaram *et al.*, 1982). This compound, possibly related to capsidiol but much less water soluble, has been named capsicannol (Swinburne, 1983).

The resistance of unripe banana to anthracnose incited by *Colletotrichum musae* Berk and Curt has been attributed to the accumulation of five fungitoxic phytoalexin compounds that were not present in healthy tissue. As the fruit ripened these compounds diminished and, at a progressive stage of disease development, no phytoalexins were detected (Brown & Swinburne, 1980). Elicitors composed of a glucan-like fraction of the cell walls of hyphae and conidia of C. *musae* elicited both necrosis and the accumulation of the two major phytoalexins found in naturally infected tissues.

The apple main phytoalexins are phloretin, naringenin, quercetin, (+)-catechin and benzoic acid (Burse *et al.*, 2004; Gottstein *et al.*, 1992; Iwashina, 2003). Benzoic acid is produced in apples as a result of infection by *Nectria galligena* and other pathogens. Fruit resistance to this pathogen at the beginning of a long storage period was attributed to the formation of this phytoalexin (Swinburne, 1973). *Nectria* penetrates apples via wounds or lenticels prior to picking, but its development in the fruit is very limited. Benzoic acid is the compound isolated from the limited infected area. The elicitor of benzoic acid synthesis was found to be a protease produced by the pathogen (Swinburne, 1975). This protease is a non-specific elicitor and a number of proteases from several sources may elicit the same response. On the other hand*, P. expansum, B. cinerea, Sclerotinia fructigena* Pers.*,* and *Aspergillus niger* van Tieghem*,* which do not produce protease in the infected tissue and do not induce the accumulation of benzoic acid, can rot immature fruit (Swinburne, 1975).

The *in vivo* levels of the principal phenolic compounds found in olive plants infected by *Phytophthora megasperma* Drechsler and *Cylindrocarpon destructans* (Zinssm.) Scholten differed from the levels observed in non-infected plants. When the antifungal activity of these compounds against both fungi was studied *in vitro*, the most active were quercetin and luteolin aglycons, followed by rutin, oleuropein, *p*-coumaric acid, luteolin-7-glucoside, tyrosol, and catechin (Baidez *et al.*, 2006). Moreover, it has been reported that the antifungal activity of aqueous neem leaf extract against *P. expansum* was related to the presence of highly bioactive compounds including quercetin (Allameh *et al.*, 2001; Mossini *et al.*, 2004).

Inoculating citrus fruits with their specific pathogens *Penicillium digitatum* Pers. and *Penicillium italicum* Wehm results in the accumulation of the phytoalexins scopoletin, scoparone, and umbelliferone. The induced compounds have a greater toxic effect than that of the preformed antifungal compounds naturally found in the fruit tissue, such as citral and limetin, as indicated by the inhibition of *P. digitatum* spore germination (Ben-Yehoshua *et al.*, 1992). The antifungal activity of both scoparone and scopoletin against and *P. digitatum* was observed in UV-C irradiated grapefruits (D'hallewin *et al.*, 2000; Rodov *et al.*, 1992). Indeed, a correlation has been drawn between the level of phytoalexin accumulated in the flavedo of irradiated fruits and its increased resistance. In particular, decay reduction was achieved when irradiation was applied to the fruit prior to its inoculation, and therefore without any direct exposure of the pathogen to the radiation; this finding led to speculate that disease inhibition stems from increased resistance of the fruit to infection and not from the direct fungicidal effect of UV on the pathogen (Rodov *et al.*, 1992). Moreover, Kim *et al.* (1991) reported that the increased concentration of scoparone in heat-treated lemon fruits was in good correlation with their increasing resistance to *P. digitatum* and enhanced antifungal activity of the fruits extract. Similarly, Afek *et al.* (1999) reported that umbelliferone accumulated in the albedo of pathogen-challenged grapefruit played a role in defence mechanisms of immature grapefruit against wound pathogens such as *P. digitatum*.

Biosynthesis of toxic compounds as a result of wounding or other stress conditions, is a ubiquitous phenomenon in various plant tissues. An example of such a synthesis is the production of the toxic compound 6-methoxymellein in carrot roots in response to wounding or to ethylene application (Chalutz *et al.*, 1969; Coxon *et al.*, 1973); the application of *B. cinerea* conidia and other fungal spores to the wounded area was found to stimulate the formation of this compound (Coxon *et al.*, 1973). This 6-methoxymellein probably has an important role in the resistance of fresh carrots to infection. Carrots that have been stored for a long period at a low temperature lose the ability to produce this compound and, in parallel, their susceptibility to pathogens increases.

Resveratrol is a phenolic substance present in both grape skin and wines in response to various fungal infections, UV radiation, or chemicals (Adrian *et al.*, 1997; Jeandet *et al.*, 1995; Langcake, 1981) and it is involved in grey mould resistance (Celotti *et al.*, 1996; Gonzalez Ureña *et al.*, 2003).

The garlic and strawberry phytoalexin esculetin showed a strong activity against fungal strains, especially *Trichophyton mentagrophytes* Malmsten and *Rhizoctonia solani* J.G. Kühn (Cespedes *et al.*, 2006), whereas a moderate antifungal activity against *Fusarium* spp. was reported for ferulic acid (Walker *et al.*, 2003).

#### **4.2 A case study: The flavonoid quercetin**

Enhanced protection of host plant tissue during periods of susceptibility through induced/acquired resistance is considered a preferred strategy for achieving integrated pest management (Kuć, 2000). Luckey (1980) reported that natural disease resistance may be induced by low or sub-lethal doses of an elicitor/agent, such as a chemical inducer or a physical stress. For instance, chemical activators could act by modifying the plant-pathogen interaction so that it resembles an incompatible interaction with defence-related mechanisms induced prior to or after challenge (Sticher *et al.*, 1997).

Flavonoids is the general name of compounds that have a fifteen-carbon skeleton, which consists of two phenyl rings connected by a three-carbon bridge. They are potent dietary antioxidants that are found in several plant materials. They are also thought to improve human health and this effect seems related, at least partially, to their antioxidant effect (Nijveldt *et al.*, 2001). Ingham *et al.* (1972) have reviewed the role of flavonoid phytoalexins and other natural products as factors in plant disease resistance. Among these compounds, quercetin was considered the most prominent (Bock, 2003). It consists of 3 rings and 5 hydroxyl groups and occurs in food (i.e. apple, tea, onion, nuts, berries, cauliflower and

irradiated fruits and its increased resistance. In particular, decay reduction was achieved when irradiation was applied to the fruit prior to its inoculation, and therefore without any direct exposure of the pathogen to the radiation; this finding led to speculate that disease inhibition stems from increased resistance of the fruit to infection and not from the direct fungicidal effect of UV on the pathogen (Rodov *et al.*, 1992). Moreover, Kim *et al.* (1991) reported that the increased concentration of scoparone in heat-treated lemon fruits was in good correlation with their increasing resistance to *P. digitatum* and enhanced antifungal activity of the fruits extract. Similarly, Afek *et al.* (1999) reported that umbelliferone accumulated in the albedo of pathogen-challenged grapefruit played a role in defence

mechanisms of immature grapefruit against wound pathogens such as *P. digitatum*.

parallel, their susceptibility to pathogens increases.

reported for ferulic acid (Walker *et al.*, 2003).

**4.2 A case study: The flavonoid quercetin** 

mechanisms induced prior to or after challenge (Sticher *et al.*, 1997).

Ureña *et al.*, 2003).

Biosynthesis of toxic compounds as a result of wounding or other stress conditions, is a ubiquitous phenomenon in various plant tissues. An example of such a synthesis is the production of the toxic compound 6-methoxymellein in carrot roots in response to wounding or to ethylene application (Chalutz *et al.*, 1969; Coxon *et al.*, 1973); the application of *B. cinerea* conidia and other fungal spores to the wounded area was found to stimulate the formation of this compound (Coxon *et al.*, 1973). This 6-methoxymellein probably has an important role in the resistance of fresh carrots to infection. Carrots that have been stored for a long period at a low temperature lose the ability to produce this compound and, in

Resveratrol is a phenolic substance present in both grape skin and wines in response to various fungal infections, UV radiation, or chemicals (Adrian *et al.*, 1997; Jeandet *et al.*, 1995; Langcake, 1981) and it is involved in grey mould resistance (Celotti *et al.*, 1996; Gonzalez

The garlic and strawberry phytoalexin esculetin showed a strong activity against fungal strains, especially *Trichophyton mentagrophytes* Malmsten and *Rhizoctonia solani* J.G. Kühn (Cespedes *et al.*, 2006), whereas a moderate antifungal activity against *Fusarium* spp. was

Enhanced protection of host plant tissue during periods of susceptibility through induced/acquired resistance is considered a preferred strategy for achieving integrated pest management (Kuć, 2000). Luckey (1980) reported that natural disease resistance may be induced by low or sub-lethal doses of an elicitor/agent, such as a chemical inducer or a physical stress. For instance, chemical activators could act by modifying the plant-pathogen interaction so that it resembles an incompatible interaction with defence-related

Flavonoids is the general name of compounds that have a fifteen-carbon skeleton, which consists of two phenyl rings connected by a three-carbon bridge. They are potent dietary antioxidants that are found in several plant materials. They are also thought to improve human health and this effect seems related, at least partially, to their antioxidant effect (Nijveldt *et al.*, 2001). Ingham *et al.* (1972) have reviewed the role of flavonoid phytoalexins and other natural products as factors in plant disease resistance. Among these compounds, quercetin was considered the most prominent (Bock, 2003). It consists of 3 rings and 5 hydroxyl groups and occurs in food (i.e. apple, tea, onion, nuts, berries, cauliflower and cabbage) as the aglycone (attached to a sugar molecule) of many plant glycosides. Quercetin can scavenge superoxide and hydroxyl radicals and reduce lipid peroxidation. In addition, it has been reported that quercetin reduces the biosynthesis of heat shock proteins, by reducing the heat shock factor which is the transcriptional factor contributing to their expression (Ishida *et al.*, 2005). However, nothing is known about its possible mode of action in reducing blue mould incidence and severity in apple fruit.

In recent publications the flavonoid quercetin, commonly available on commerce, even as dietary supplements, was tested both *in vitro* and *in vivo* against blue mould and relevant patulin accumulation on apples (Sanzani *et al.*, 2009b). The *in vitro* trials on amended and non-amended PDA plates revealed that although quercetin, at the tested concentrations (100 µg/dish), exerted a slight reduction on fungal growth, it proved to be effective in reducing patulin accumulation, in a dose-dependent way. These results were not surprising, as the antitoxigenic properties of phenolic antioxidants with any interference on fungal growth has already been reported (Kim *et al.*, 2008). However, since quercetin proved to be much more stable in acidic conditions (S.M. Sanzani, unpublished data), as those typically present in apple tissues, and considering that the *in vitro* evaluation of the antifungal activity of a compound is just the first step to test its suitability in preventing the growth and development of a phytopathogenic microorganism, quercetin was further tested *in vivo* by adding it to wounds (100 µg/wound) of Golden Delicious and Granny Smith apples*.* The compound not only confirmed the suppressive activity on toxin accumulation, but proved to be effective in controlling blue mould incidence and severity. In particular, the best results were observed on Golden Delicious, the apple cultivar considered more resistant to diseases, and on disease severity control, i.e. practically slowing down disease development. The chance of an application by dipping was investigated; however, results were weaker since the wider surface to be treated was not taken into account.

Considering that, although slightly active on *in vitro* fungal growth, quercetin was effective in consistently controlling *in vivo* blue mould, it was hypothesized that quercetin could act by enhancing host natural defence response. A further clue came from a paper by Sanzani *et al.* (2010) in which quercetin proved to exert its activity even in lack of direct contact with the pathogen, particularly when *P. expansum* was inoculated 24-48 h after quercetin application. Thus, to try to gain much insight into the mode of action of quercetin, a molecular technique, called Suppression Subtractive Hybridization (SSH), was applied to identify apple genes putatively induced by its application. Results revealed that a substantial number of enzymes or proteins, which have a function in the adaptation process to oxidative or more general stresses, seemed to be up-regulated after quercetin application. In particular, the antifungal activity seemed to be mainly associated to pathogenesis-related proteins family 10, expressed also in response to challenge with *Venturia inaequalis*, and to PhzC/PhzF proteins. These proteins are tightly linked to structural genes and enzymes from the shikimic acid and tryptophan biosynthetic pathway, known to be involved in the biosynthesis of all the plant phenolic compounds, including phytoalexins and lignin. As a consequence of this response, the fruit should be prepared for a successful defence against pathogens.

Concerning quercetin effect on patulin production, the good results obtained independently from the effect on the fungal growth, suggested that the compound might act directly on toxin biosynthetic pathway, which is mainly associated with fungal secondary metabolism. In a study on the topic Sanzani *et al.* (2009c) proved that the expression level of selected genes, known to code enzymes involved in patulin biosynthesis (i.e. the 6-methylsalicilic acid synthase, the isoepoxydon dehydrogenase, an ABC transporter and two cytocrome P450 monoxygenases), was determined by quantitative real-time PCR. Results evidenced that quercetin down-regulated the expression of two P450 monooxygenases involved in oxygen activation. These results are consistent with quercetin antioxidant properties and with a similar study in which it is reported that the application of the antioxidant phenolic compound caffeic acid reduced aflatoxin production and down-regulated P450 monooxygenases (Kim *et al.*, 2008). Therefore, the initial hypothesis about a presumed direct activity of quercetin on patulin biosynthetic pathway, particularly at the transcriptional level, seems to be confirmed.

#### **5. Conclusions**

From this review it appears that some significant progress has been made toward alternative control of postharvest diseases on fruit. Some biofungicides are already on the market in a few countries, and will probably become more widely available as they are registered in more areas. For instance, quercetin might represent an interesting alternative to synthetic fungicides to be applied in the postharvest phase of apples against blue mould and relevant patulin accumulation. Indeed, on the basis of new available information on the mode of action, further studies might lead to the determination of proper conditions to improve their applicability on a commercial scale.

Under field conditions, many alternative control agents have provided limited success, which is often attributed to "uncontrollable" environmental conditions. However, the likelihood of success greatly increases during the postharvest phase due to better environmental control. Moreover, it is often easier to effectively apply alternative control agents while commodities are being processed after harvest.

The success of alternative control greatly depends on influencing the consumer to prefer inner quality to outward appearance. Indeed, alternative control means might represent an important if not essential component of an integrated disease management scheme aimed to reduce economic losses and risks for consumers' health.

#### **6. References**


known to code enzymes involved in patulin biosynthesis (i.e. the 6-methylsalicilic acid synthase, the isoepoxydon dehydrogenase, an ABC transporter and two cytocrome P450 monoxygenases), was determined by quantitative real-time PCR. Results evidenced that quercetin down-regulated the expression of two P450 monooxygenases involved in oxygen activation. These results are consistent with quercetin antioxidant properties and with a similar study in which it is reported that the application of the antioxidant phenolic compound caffeic acid reduced aflatoxin production and down-regulated P450 monooxygenases (Kim *et al.*, 2008). Therefore, the initial hypothesis about a presumed direct activity of quercetin on patulin biosynthetic pathway, particularly at the transcriptional level, seems to be confirmed.

From this review it appears that some significant progress has been made toward alternative control of postharvest diseases on fruit. Some biofungicides are already on the market in a few countries, and will probably become more widely available as they are registered in more areas. For instance, quercetin might represent an interesting alternative to synthetic fungicides to be applied in the postharvest phase of apples against blue mould and relevant patulin accumulation. Indeed, on the basis of new available information on the mode of action, further studies might lead to the determination of proper conditions to improve their

Under field conditions, many alternative control agents have provided limited success, which is often attributed to "uncontrollable" environmental conditions. However, the likelihood of success greatly increases during the postharvest phase due to better environmental control. Moreover, it is often easier to effectively apply alternative control

The success of alternative control greatly depends on influencing the consumer to prefer inner quality to outward appearance. Indeed, alternative control means might represent an important if not essential component of an integrated disease management scheme aimed to

Adikaram, N.K.B., Brown, A.E., & Swinburne, T.R. 1982. Phytoalexin involvement in the

Adrian, M., Jeandet, P, Veneau, J., Weston, L.A., & Bessis, R. 1997. Biological activity of

Afek, U., Aharoni, N., & Carmeli, S. 1995a. The involvement of marmesin in celery

Afek, U., Aharoni, N., & Carmeli, S. 1995b. Increasing celery resistance or pathogens during

*American Society for Horticultural Science*, 120, pp. 562-565, ISSN 0003-1062

*Physiological Plant Pathology,* 21, pp. 161-170, ISSN 0048-4059

concentration. *Phytopathology,* 85, pp. 1033-1103, ISSN 0031949X

latent infection of *Capsicum annuum* L. fruit by *Glomerella cingulata* (Stonem.).

resveratrol, a stilbenic compound from grapevines, against *Botrytis cinerea*, the causal agent for gray mould. *Journal of Chemical Ecology,* 23, pp. 1689-1702, ISSN

resistance to the pathogens during storage and the effect of temperature on its

storage and reducing high-risk psoralen concentration by treatment with GA3.

**5. Conclusions** 

**6. References** 

0098-0331

applicability on a commercial scale.

agents while commodities are being processed after harvest.

reduce economic losses and risks for consumers' health.


Céspedes, C.L., Avila, J.G., Martínez, A., Serrato, B., Calderón-Mugica, J.C., & Salgado-

Chalutz, E., Devay, J.E., & Maxie, E.C. 1969. Ethylene induced isocoumarin formation in

Chalutz, E., & Droby, S. 1997. Biological control of postharvest disease. In: *Plant–microbe* 

Chen, L., Ingham B.H., & Ingham, S.C. 2004. Survival of *Penicillium expansum* and patulin

Coxon, D.T., Curtis, R.F., Price, K.R., & Levett, G. 1973. Abnormal metabolites produced by

Coxon, D.T., Pwuce, K.R., Howard, B., Osman, S.F., Kaan, E.B., Zacharius, R.M. 1974 Two

D'hallewin, G., Schirra, M., Pala, M., & Ben-Yehoshua, S. 2000. Ultraviolet C irradiation at

Deveraj, H., Shanmugasundaram, K.R., & Shanmugasundaram, E.R.B. 1982. Neurotoxic

Dixon, R.A. 2001. Natural products and plant disease resistance. *Nature*, 411, pp. 843-847,

Eckert, J.W., & Ogawa, J.M. 1988. The chemical control of postharvest diseases: deciduous

Edney, K.L. 1978. The infection of apples by *Phytophthora syringae. Annals of Applied Biology*,

Edney, K.L. 1983. Top fruit. In: *Post-Harvest Pathology of Fruits and Vegetables,* Dennis, C., pp.

Engstrom, K., Widmark, A.K., Brishammar, S., & Helmersson, S. 1999. Antifungal activity to

European Commission, 2006. Commission Regulation (EC) no. 1881/2006 setting maximum

Fallik, E. 2010. Hot Water Treatments of Fruits and Vegetables for Postharvest Storage. In:

FAO Statistical Database (FAOSTAT), 2011. Statistics on agriculture, nutrition, fisheries,

tubers. *Tetrahedron Letters,* 34, pp. 2921-2924, ISSN 0040-4039

carrot root tissue. *Plant Physiology*, 44, pp. 235-241, ISSN 1532-2548

0021-8561

Marcel Dekker Inc., New York, USA

48, pp. 4571-4575, ISSN 0021-8561

pp. 433-469, ISSN 0066-4286

88, pp. 31-36, ISSN 0003-4746

364, pp.5-24, ISSN 1725-2555

http://faostat.fao.org/

43-71, Academic Press, London, UK

*Research,* 42, pp. 43-50, ISSN 0014-3065

USA, doi: 10.1002/9780470872376.ch5

C669-C675, ISSN 1750-3841

1885, ISSN 0031-9422

5189

ISSN 0028-0836

Garciglia, R. 2006. Antifungal and Antibacterial Activities of Mexican Tarragon (*Tagetes lucida*). *Journal of Agricultural and Food Chemistry*, 54, 10, pp. 3521-3527, ISSN

*interactions and biological control*. Boland, G.J., & Kuykendall, L.D., pp. 157–170,

production on stored apples after wash treatments. *Journal of Food Science*, 69, pp.

*Daucus carota* roots stored under conditions of stress. *Phytochemistry*, 12, pp. 1881-

new vetispirane derivatives: stress metabolites from potato (*Solanum tuberosum*)

0.5 kJâm-2 reduces decay without causing damage or affecting postharvest quality of Star Ruby grapefruit (*C. paradisi* Macf.). *Journal of Agricultural and Food Chemistry*,

effect of patulin. *Indian journal of experimental biology,* 20, pp. 230-231, ISSN:0019-

fruits, berries, vegetables and root/tuber crops. *Annual Review of Phytopathology*, 26,

*Phytophthora infestants* of sesquiterpenoids from infected potato tubers. *Potato* 

levels for certain contaminants in foodstuffs. *Official Journal of the European Union,* L,

*Horticultural Reviews* (volume 38), Janick, J., John Wiley & Sons, Inc., Hoboken, NJ,

forestry, food aid, land use and population. Retrieved from:


Kim, J.H., Yu, J., Mahoney, N., Chan, K.L., Molyneux, R.J., Varga, J., Bhatnagar, D.,

Kuc, J. 2000. Development and future direction of induced systemic acquired resistance in

Langcake, P. 1981. Disease resistance of *Vitis* spp. and the production of the stress

Lovett, J., Thompson, R.G., & Boutin B.K. 1975. Patulin production in apples stored in a

Mahalingam, R., Gomez-Buitrago, A.M., Eckardt, N., Shah, N., Guevara-Garcia, A., Day, P.,

Mari, M., Leoni, O., Iori, R., & Cembali, T. 2002. Antifungal vapour-phase activity of ally-

Minamikawa, T., Akazawa, T., & Utitani, I. 1963. Analytical study of umbelliferone and

Morales, H., Sanchis, V., Usall, J., Ramos, A.J., & Marin, S. 2008. Effect of biocontrol agents

Mossini, S.A.G., De Oliveira K.P., & Kemmelmeier, K. 2004. Inhibition of patulin production

Muller, K., & Borger, H. 1940. Experimentelle Untersuchungen uber die *Phytophthora* 

Murphy, P.A., Hendrich, S., Landgren, C., & Bryant C.M. 2006. Food Mycotoxins: An

Nijveldt, R.J., van Nood, E., van Hoorn, D.E.C., Boelens, P.G., van Norren, K., & van

Okull, D.O., & LaBorde, L.F. 2004. Activity of electrolyzed oxidizing water against

Update. *Journal of Food Science* 71 (5), pp. R51-R65, ISSN 0022-1147

Luckey, T.D. 1980. *Hormesis with Ionizing Radiation*. CRC Press, Boca Raton, FL, USA

plants. *Crop Protection,* 19, pp. 859–861, ISSN 0261-2194

*Molecular Plant Pathology*, 18, pp. 213–226, ISSN 0885-5765

of Arabidopsis. *Genome Biology,* 4, pp. R20, ISSN 1465-6914

*Physiology*, 38, pp. 493-497, ISSN 1532-2548

*of Basic Microbiology,* 44, pp. 106-113, ISSN 1521-4028

*International Journal of Food Microbiology*, 122, pp. 49–60, ISSN 0168-1605 Kloepper, J.W., Tuzun, S., & Kuc, J.A., 1992. Proposed definitions related to induced disease resistance. *Biocontrol Science and Technology,* 2, pp. 349–351, ISSN 1360-0478 Kuc, J. 1976. Phytoalexins. In: *Encyclopedia of Plant Physiology, New Sen Vol. 4, Physiological* 

Heidelberg, New York, USA

912-914, ISSN 0004-5756

ISSN 1365-3059

ISSN 0168-1605

0077-961

1938-3207

69, 23-27, ISSN 0022-1147

Cleveland, T.E., Nierman, W.C., & Campbell, B.C. 2008. Elucidation of the functional genomics of antioxidant-based inhibition of aflatoxin biosynthesis.

*Plant Pathology*. Heitefuss, R., & Williams, P.H., pp. 632-652, Springer-Verlag Berlin

metabolites resveratrol, ε-viniferin, α-viniferin and pterostilbene. *Physiological and* 

controlled atmosphere. *Journal of the Association of Official Analytical Chemists*, 58, pp.

Raina, R., & Fedoroff, N.V. 2003, Characterizing the stress/defense transcriptome

isothiocyanate against *Penicillium expansum* on pears. *Plant Pathology*, 51, 231-236,

scopoletin synthesis in sweet potato roots infected by *Ceratocystis fimbriata. Plant* 

*Candida sake* and *Pantoea agglomerans* on *Penicillium expansum* growth and patulin accumulation in apples. *International Journal of Food Microbiology,* 122, 1-2, pp. 61-67,

by *Penicillium expansum* cultured with neem (*Azadirachta indica*) leaf extracts. *Journal* 

Resistenz der Kartoffel. *Arb Biol Reichsanst Land Forstwirtsch*, 23, pp. 189-231, ISSN

Leeuwen P.A.M. 2001. Flavonoids: a review of probable mechanisms of action and potential applications. *American Journal of Clinical Nutrition*, 74, pp.418–425, ISSN

*Penicillium expansum* in suspension and on wounded apples *Journal of Food Science,*


### **Combined Effects of Fungicides and Thermotherapy on Post-Harvest Quality of Horticultural Commodities**

Maurizio Mulas *University of Sassari Italy* 

#### **1. Introduction**

132 Fungicides – Beneficial and Harmful Aspects

Sholberg, P., Haag, P., Hocking, R., & Bedford, K. 2000. The use of vinegar vapor to reduce

Smith, C.J. 1996. Accumulation of phytoalexins: defence mechanism and stimulus response

Snowdon, A.L. 1990. *Post-Harvest Diseases and Disorders of Fruits and Vegetables, Vol. 1. General Introduction and Fruits*, CRC Press, Inc., Boca Raton, FL, USA. Sommer, N.F., Buchanan, J.R., & Fortlage, R.J. 1974. Production of patulin by *Penicillium expansum. Applied and Environmental Microbiology*, 28, pp. 589-593, ISSN 1098-5336. Spadaro, D., Garibaldi, A., & Gullino, M.L. 2004. Control of *Penicillium expansum* and *Botrytis* 

Spotts, R.A., Cervantes, L.A., & Mielke, E.A. 1999. Variability in postharvest decay among

Sticher, L., Mauch-Mani, B., & Métraux J.P. 1997. Systemic acquired resistance. *Annual* 

Stoessl, A., Unwin, C.H., & Ward, E.W.B. 1972. Post-infectional inhibitor from plants. I.

Sutton, B.C. 1996. *A century of mycology*. British Mycological Society Century Symposium.

Swinburne, T.R. 1973. The resistance of immature Bramley's seedling apples to rotting by

Swinburne, T.R. 1983. Quiescent infections in postharvest diseases. In: *Postharvest Pathology of Fruits and Vegetables*, Dennis, C., pp. 1-21, Academic Press, London, UK Tomiyama, K., Sakuma, T., Ishizaka, N., Katsui, N., Takasugi, M., & Masamune, T. 1968. A

Walker, T.S., Bais, H.P., Halligan, K.M., Stermitz, F.R., & Vivanco, J.M. 2003. Metabolic

Zhang, H., Wang, L., Ma, L., Dong, Y., Jiang, S., Xu, B., & Zheng, X., 2009. Biocontrol of

apple cultivars. *Plant Disease,* 83, pp. 1051-1054, ISSN 0191-2917

*Review of Phytopathology*, 35, pp. 235–270, ISSN 0066-4286

Ed. Cambridge University Press, UK, ISBN 9780521050197

apples. *Phytopathology Z*., 82, pp.152-162, ISSN 0031-9481

pathogens. *Phytopathology,* 58, pp. 115-116, ISSN 0031-949X

system. *New phytologist*, 132, pp. 1-45, ISSN 0028-646X

*Technology*, 33, 2, pp. 141-151, ISSN 0925-5214

pp. 141-152, ISSN 0031-9481

51, pp. 2548-2554, ISSN 0021-8561

0862-867X.

postharvest decay of harvested fruit. Horticultural Science, 35, pp. 898–903, ISSN

*cinerea* on apple combining a biocontrol agent with hot water dipping and acibenzolar-S-methyl, baking soda, or ethanol application. *Postharvest Biology and* 

Capsidiol, an antifungal compound from *Capsicum frutescens. Phytopathology Z*., 74,

*Nectria galligena* Bres. In*: Fungal Pathogenicity and the Plant's Response*. Byrde, R.J.W., & Cutting, C.V., pp. 365-382, Academic Press, London, UK, ISBN 0121488500 Swinburne, T.R. 1975. Microbial proteases as elicitors of benzoic acid accumulation in

new antifungal substance isolated from resistant potato tuber tissue infected by

Profiling of Root Exudates of *Arabidopsis*. *Journal of Agricultural and Food Chemistry,*

major postharvest pathogens on apple using *Rhodotorula glutinis* and its effects on postharvest quality parameters. *Biological Control*, 48, 1, pp. 79-83, ISSN 1049-9644

Production, market and consumption of fresh fruit and vegetables (Fig. 1) are continuously increasing in the world (FAOSTAT, 2011). This increase is not only linked to the growth of the world population and to the consequent demand of food but also to the continuous improving of the food quality in the emerging countries. Immediately after the satisfaction of the energy needs, in fact, more lipids are introduced in the diet with the aim of improving the organoleptic quality of the food. Subsequently, a wide variety of ingredients, including fresh fruit and vegetables are requested to integrate the daily diet. Cereals are absolutely fundamental as they provide energy, proteins, lipids and minerals. However, fibre, minerals, vitamins, sugars, acids, aminoacids, and other compounds, like polyphenols and other antioxidants of particular importance because of their nutritional and nutraceutical value are provided by fresh fruits and vegetables (Schreiner & Huyskens-Keil, 2006).

In spite of the large variability of horticultural commodities marketed today in the world, not all the regions of the globe showed possibilities to give high yields of many crops. Often geographical reasons made impossible the production of any commodity, like in the extreme latitude North and South, or in arid and desert areas. Technology advances may contribute to extend the growing area of some crops, but not always there is a real economic convenience in this effort. Furthermore, in other lands poverty and lack of professional abilities make difficult cultivation even when the natural environment is potentially favourable. For these reasons the international market of horticultural commodities is very big and diversified. The most developed countries manage the highest part of exchanges, while a part of producer countries showed an increasing activity with the aim of taking a part of the international market management due to quality of commodities and competitive prices. Some of these "emerging" countries have also an internal market absolutely wide, and an increasing demand which is the market base to export commodities of excellent and selected quality. This is the case of Brazil, South Africa, Mexico, India, and China. Some other advanced countries have in the export of horticultural commodities a point of excellence of their economy, like Spain, Italy, Turkey, Israel, and Greece. Moreover, other "emerging" countries are small producers and have a small internal market, but have found a good specialization in the export of high value horticultural commodities thus obtaining an interesting added value from this activity: like Cyprus, Chile, Cuba, Costa Rica and Morocco.

The big quantity of fresh horticultural commodities that are the object of import/export on the international markets, but also on the big internal markets, is characterized by quality and nutritional properties that should be maintained until the final consumer (Kader, 2003). Many difficulties operate against this objective. The first is the physiological weight loss of horticultural commodities determined by both water loss, due to transpiration of living plant tissues, and by dry matter loss due to product respiration. This weight and value loss may be comprised between 10 and 20%. Other losses are determined by some physiological disorders like the chilling injury which can occur during and after the cold storage. The cold storage is often a need in order to preserve the product quality by minimizing the physiological losses and the mould decay. The losses for chilling injury are comprised between 10 and 15%, mainly for tropical and subtropical fruits and vegetables. Finally, the biggest losses are caused by specific postharvest diseases. Fungi and other microorganisms may be the responsible of 15-25% decay of horticulture commodities during storage and transport (Barkai-Golan, 2001).

Since the first experiences of the fruit and vegetable postharvest export industry, fungicide treatments have been applied to horticultural commodities in order to reduce mould decay in the best effective way. However, approved fungicides for postharvest treatments have been always a small number of compounds because of the short period of tolerance between application and product consumption. Severity in the control of fungicides residues is also usual in technological advanced importing countries. Moreover, the amounts of fungicides applied to horticultural commodities during postharvest process are small respect to the applied on the field, and the cost for the authorization of a new product is very high, thus determining low interest in the development of new fungicides by the specialized chemical industries (Narayanasamy, 2006).

Fig. 1. World yield of fruits and vegetables in the last decade (data source: FAOSTAT 2011).

The big quantity of fresh horticultural commodities that are the object of import/export on the international markets, but also on the big internal markets, is characterized by quality and nutritional properties that should be maintained until the final consumer (Kader, 2003). Many difficulties operate against this objective. The first is the physiological weight loss of horticultural commodities determined by both water loss, due to transpiration of living plant tissues, and by dry matter loss due to product respiration. This weight and value loss may be comprised between 10 and 20%. Other losses are determined by some physiological disorders like the chilling injury which can occur during and after the cold storage. The cold storage is often a need in order to preserve the product quality by minimizing the physiological losses and the mould decay. The losses for chilling injury are comprised between 10 and 15%, mainly for tropical and subtropical fruits and vegetables. Finally, the biggest losses are caused by specific postharvest diseases. Fungi and other microorganisms may be the responsible of 15-25% decay of horticulture commodities during storage and

Since the first experiences of the fruit and vegetable postharvest export industry, fungicide treatments have been applied to horticultural commodities in order to reduce mould decay in the best effective way. However, approved fungicides for postharvest treatments have been always a small number of compounds because of the short period of tolerance between application and product consumption. Severity in the control of fungicides residues is also usual in technological advanced importing countries. Moreover, the amounts of fungicides applied to horticultural commodities during postharvest process are small respect to the applied on the field, and the cost for the authorization of a new product is very high, thus determining low interest in the development of new fungicides by the specialized chemical

Fig. 1. World yield of fruits and vegetables in the last decade (data source: FAOSTAT 2011).

transport (Barkai-Golan, 2001).

industries (Narayanasamy, 2006).

As a consequence of the repeated and exclusive use of the few admitted fungicides for postharvest treatments of fresh horticultural commodities in a very limited space like the processing and storage packing houses, many resistant strains for these fungicides of the main pathogens have been widely detected in the last decades.

The urgency to find alternative technologies of control against postharvest pathogens of horticultural commodities is clearly evidenced and sustained by the following arguments:


#### **2. The thermotherapy of horticultural commodities**

The use of the heat to control some postharvest citrus pathogens was experimented in the first decades of the past century (Mulas & Schirra, 2007). The first experiment cited in the literature was the postharvest use of hot water and hot sodium hydroxide solutions to control citrus moulds. Subsequently, the use of the thermotherapy was also widely applied against parasites of horticultural commodities (Barkai-Golan & Phillips, 1991; Lurie, 1998; Schirra et al., 2000a; Shellie & Robert, 2000; Fallik, 2004; Fallik & Lurie, 2007).

In the 70th years a wide availability and diffusion of fungicides made less rentable the use of heat in post-harvest treatments which always were linked to supplementary energy costs (Schirra, 2005). However, it was just in the years of widest diffusion of synthetic fungicides that the advantages to combine postharvest treatments with warming of active solutions were observed. This improved efficiency was associated to a better distribution, penetration and cuticle diffusion of fungicides in the commodity tissues (Cabras et al., 1999; Schirra et al., 2002a). Usually the cuticle showed a relatively low permeability to most agricultural chemicals (Baur & Schönherr, 1995; Schreiber & Schönherr, 2009) but we have to consider the presence of cracks or other breaks in the cuticle surface that increases with fruits maturation (Faust & Shear, 1972; Freeman et al., 1979; Barthlott, 1990; Bianchi, 1995). These cracks are important for the fungicide uptake in plant tissues but, when the application is combined with thermotherapy, there is also a partial fusion and distribution of cuticular waxes that cover these cracks and reduce the ways of fungal infection (Gleen et al., 1985; El-Otmani et al., 1989; Roy et al., 1994; 1999; Bally, 1999). Many studies have demonstrated this evidence in different commodities, including citrus (Schirra & D'hallewin, 1997; Schirra et al., 1998a; Porat et al., 2000), cactus pear (D'hallewin et al., 1999; Schirra et al., 1999), sweet pepper (Fallik et al., 1999), and melon (Fallik et al., 2000).

Evaluations and experiments of thermotherapy combination have been reported on the application of a widely used fungicide to control moulds in citrus fruit (Eckert & Ogawa, 1985; Eckert & Eaks, 1988). Treatments with Sodium ortho-phenylphenate (SOPP) were performed after warming at 40 °C to control citrus moulds on 'Pineapple' and 'Valencia' oranges (Hayward & Grieson, 1960). The same fungicide was used at a concentration of 2%, with 1% hexamine and 0.2% sodium hydroxide in water at 22.8 and 37.8 °C. In this case the cold solution was more effective to control mould decay than the heated solution because of the phytotoxic effect of the SOPP (McCornack & Hayward, 1968).

Heated and unheated fungicides were tested on apples to control blue mould development (Spalding et al., 1969).

With the aim of controlling *Monilinia fructicola,* a solution of 2,6-diclhoro-4-nitroaniline was positively used at 51.5 °C for one and a half minute dip on peaches, plums, and nectarines (Wells & Harvey, 1970). These results were reconsidered and the research on thermotherapy was newly promoted in the 80th years (Carter, 1981a) due in part to the new cultural approach to the environment problems, the higher care for the human health and the difficulties to accept the use of fungicides and other chemical controlling pests largely used in the food industry (Schirra, 2005).

Actually we have to consider that few postharvest fungicides (Tomlin, 1997; Adaskaveg & Förster, 2010) are admitted to postharvest treatments of horticultural commodities and that their effectiveness is largely compromised by the natural selection of resistant strains of the main postharvest pathogens, as a consequence of their repeated use (Schmidt et al., 2006). For this reason, the research of alternatives to chemical pesticides for the control of postharvest disorders is considered as a prioritary objective for many researcher involved in the test of many methods: thermotherapy, use of radiations, gas and vapour, use of microbiological resources (like antagonist or toxic strains), use of chemical compounds of not toxic salts and compounds generically recognized as safe (GRAS).

#### **2.1 Applications of thermotherapy**

The thermotherapy is possible thank to the transfer of thermal energy to the horticultural commodity by means of a fluid mean which can be liquid, water vapour or air saturated of humidity. Hot dip are made in water with temperatures ranging from 43 to 53 °C and time of dipping comprised between 1-3 minutes and 2 hours (Ben-Yehoshua et al., 2000; Fallik, 2004). A different kind of dip treatment is the brushing of horticultural commodities under a hot water flow. Water temperature in this case are higher than in the case of hot dip, ranging between 48 and 60-63 °C during shorter periods of 15-30 seconds until 1 minute.

Treatments with air or hot vapour showed some technical difficulties and need more time but often their effectiveness is more prolonged with respect to hot dip (Mulas & Schirra, 2007).

As a further possibility among others, applications of radiofrequency waves may be useful for thermotherapy of horticultural commodities as actually in the wood industry or for the treatment of cereals (Johnson et al., 2003).

#### **2.2 Control of physiological disorders**

Fruits and vegetables of tropical and subtropical origin like citrus are particularly sensitive to chilling injury. Also this physiological disorder may be reduced or avoided by means of the thermotherapy, like the exposition of the commodities to hot air at 37 °C during 3 days under saturated humidity (Lafuente & Zacarias, 2006), or by mean of applications of hot dip (Wild & Hood, 1989; McDonald et al., 1991; Wang, 1993; Gonzales-Aguilar et al., 1997; Mulas, 2006). More indications are available in order to control chilling injury of horticultural commodities of the temperate regions, like apples, by means of the thermotherapy (Lurie, 1998; Mulas & Schirra, 2009).

The effectiveness of thermotherapy to control oxidative stress in chilled fruits seems also linked directly to the enhanced activity of enzymes controlling antioxidant responses (Sala, 1998; Sala & Lafuente, 1999; 2000).

#### **2.3 Control of pests**

136 Fungicides – Beneficial and Harmful Aspects

1985; Eckert & Eaks, 1988). Treatments with Sodium ortho-phenylphenate (SOPP) were performed after warming at 40 °C to control citrus moulds on 'Pineapple' and 'Valencia' oranges (Hayward & Grieson, 1960). The same fungicide was used at a concentration of 2%, with 1% hexamine and 0.2% sodium hydroxide in water at 22.8 and 37.8 °C. In this case the cold solution was more effective to control mould decay than the heated solution because of

Heated and unheated fungicides were tested on apples to control blue mould development

With the aim of controlling *Monilinia fructicola,* a solution of 2,6-diclhoro-4-nitroaniline was positively used at 51.5 °C for one and a half minute dip on peaches, plums, and nectarines (Wells & Harvey, 1970). These results were reconsidered and the research on thermotherapy was newly promoted in the 80th years (Carter, 1981a) due in part to the new cultural approach to the environment problems, the higher care for the human health and the difficulties to accept the use of fungicides and other chemical controlling pests largely used

Actually we have to consider that few postharvest fungicides (Tomlin, 1997; Adaskaveg & Förster, 2010) are admitted to postharvest treatments of horticultural commodities and that their effectiveness is largely compromised by the natural selection of resistant strains of the main postharvest pathogens, as a consequence of their repeated use (Schmidt et al., 2006). For this reason, the research of alternatives to chemical pesticides for the control of postharvest disorders is considered as a prioritary objective for many researcher involved in the test of many methods: thermotherapy, use of radiations, gas and vapour, use of microbiological resources (like antagonist or toxic strains), use of chemical compounds of

The thermotherapy is possible thank to the transfer of thermal energy to the horticultural commodity by means of a fluid mean which can be liquid, water vapour or air saturated of humidity. Hot dip are made in water with temperatures ranging from 43 to 53 °C and time of dipping comprised between 1-3 minutes and 2 hours (Ben-Yehoshua et al., 2000; Fallik, 2004). A different kind of dip treatment is the brushing of horticultural commodities under a hot water flow. Water temperature in this case are higher than in the case of hot dip, ranging

Treatments with air or hot vapour showed some technical difficulties and need more time but often their effectiveness is more prolonged with respect to hot dip (Mulas & Schirra, 2007).

As a further possibility among others, applications of radiofrequency waves may be useful for thermotherapy of horticultural commodities as actually in the wood industry or for the

Fruits and vegetables of tropical and subtropical origin like citrus are particularly sensitive to chilling injury. Also this physiological disorder may be reduced or avoided by means of the thermotherapy, like the exposition of the commodities to hot air at 37 °C during 3 days under saturated humidity (Lafuente & Zacarias, 2006), or by mean of applications of hot dip

between 48 and 60-63 °C during shorter periods of 15-30 seconds until 1 minute.

the phytotoxic effect of the SOPP (McCornack & Hayward, 1968).

not toxic salts and compounds generically recognized as safe (GRAS).

(Spalding et al., 1969).

in the food industry (Schirra, 2005).

**2.1 Applications of thermotherapy** 

treatment of cereals (Johnson et al., 2003).

**2.2 Control of physiological disorders** 

Many markets require horticultural commodities absolutely free of pesticide residues but at the same time also free from eggs, larvae and adults of insects. Also in this direction, alternative methods to the use of chemical fumigants, like methyl-bromide (forbidden in many countries), have been developed. Optimal times and heating temperatures for disinfestations depend on species and cultivars. For citrus, temperature of 44 °C for 90 minutes or 46 °C for 50 minutes are indicated as effective, but it is true that not all the citrus cultivars withstand these treatments. Thus, symptoms of senescence have been observed after this kind of treatments in bloody and 'Valencia late' oranges (Mulas et al., 2001; Schirra et al., 2004; 2005a).

Good results have been obtained against some coleoptera infesting dates by heating them at 55 °C for 2 hours and 30 minutes (Rafaeli et al., 2006).

#### **2.4 Direct control of postharvest pathogens**

Hot dip is effective controlling main postharvest pathogens of horticultural commodities both as a consequence of the fruit surface washing leading to reduced inoculums, as well as because of the thermal inactivation of the microorganisms. Hot dips and high temperature conditioning were effective to improve shelf quality of late crop cactus pear fruit (Schirra et al., 1996a).

Furthermore, we have to consider the possibility that thermotherapy may stimulate the biosynthesis or elicitation of endogenous compounds having fungicide effects (Lurie, 1998; Schirra et al., 2000a).

Evidence of these effects on fruit surface have been showed in a research carried out on 'Montenergrina' mandarins (Montero et al., 2010), where hot dip treatments at 60 °C with brushing significantly reduced the number of tangerines affected by decay.

In a recent study, hot water dip at 48 °C for 12 or 6 minutes showed good control of postharvest brown rot on 'Roig' peach and 'Venus' nectarine, without visual symptoms of heat damage and significant loss of fruit quality (Jemric et al., 2011). The elicitation of compounds involved in the plant defence as a consequence of hot air treatments at 38 °C during 24-72 hours was further demonstrated on tomato cherry fruit by means of higher lignin deposition, and higher activation of phenylalanine ammonia-lyase and β-1,3 glucanase (Zhao et al., 2009; 2010).

#### **3. Combination of thermotherapy with fungicides**

In spite of the beneficial effects of thermotherapy to the control of microbial postharvest disorders of fresh horticultural commodities, this treatment is not able to have the same effectiveness of fungicides, particularly when the marketing is made after a period more or less long of storage (Schirra, 2005; Mulas, 2008). In some countries, thermotherapy is applied to control mould decay in commodities from organic cultivation or when the postharvest fungicide treatment is completely forbidden (Fallik, 2004). In other cases, the chemical defence is always of fundamental importance against mould decay and there is not a convenience to use other methods (Dezman et al., 1986; Papadopoulou-Mourkidou, 1991).

#### **3.1 Old fungicides**

After the first experiences in the 60th years, many investigations have been promoted in the 70th and 80th years, but a new start of researches on old fungicides was observed in the 90th.

Mango decay was controlled by combination of hot dips with Benomyl (Spalding & Reeder, 1972; 1978). Brown rot and rhyzopus of peaches, nectarines and stone fruits were controlled by different combination of Benomyl or other fungicides and thermotherapy (Smith, 1971; Wells & Gerdts, 1971; Wells, 1972; Jones & Burton, 1973; Smith & Anderson, 1975; Wang & Anderson, 1985).

The control of rots of guavas was tested by application of heated Benomyl and Guazatine dips (Wills et al., 1982). Rotting and browning of lytchee fruit were controlled by hot benomyl and plastic film (Scott et al., 1982; Wong et al., 1991).

New experiments were carried out to demonstrate the synergistic effect of heat and sodium ortho-phenylpenate to inactivate *Penicillium* spores and suppress decay in citrus fruits (Barkai-Golan & Appelbaum, 1991). The efficacy of hot water and Carbendazim treatments was tested against the brown rot of apple (Sharma & Kaul, 1990).

#### **3.2 Thiabendazole and Imazalil**

The most used fungicides in postharvest of horticultural commodities are Imazalil and Thiabendazole. Both are synthetic compounds with different mode of action that can be applied in waxes or water (Brown et al., 1983; Brown, 1984). Imazalil is very effective against the green mould (*Penicillium digitatum*), including benzimidazole-resistant strains, while Thiabendazole is effective to a wide range of pathogens and often may have beneficial effects also against chilling injury (Schiffman-Nadel et al., 1972; 1975; Brown & Dezman, 1990; McDonald et al., 1991; Schirra et al., 2000b).

Many studies demonstrate that the efficacy of Imazalil and/or Prochloraz increases when applied in hot water in a number of fruits such as mango (Spalding & Reeder, 1986; Johnson et al., 1990; Prusky et al., 1990; Dodd et al., 1991; Coates et al., 1993; McGuire & Campbell, 1993; Waskar, 2005); melons (Carter, 1981b; Mayberry & Kartz, 1992), taro corms (Quevedo et al., 1991), red tamarillos (Yearsley et a., 1988), and in citrus fruit (Ansari & Feridoon, 2008).

That is because of the best cuticle and tissue penetration by mean of active compounds (Schirra et al., 2002a; 2008a). Wide demonstration of these effects has been reported in the case of the Imazalil and Thiabendazole use against *Penicillium* mould of citrus (Mulas & Schirra, 2007; Dore et al., 2009). The two fungicides resulted effective in some cases with very low concentration, up to 50 mg·L-1, if the application was made in hot water (50 °C) (Schirra & Mulas, 1993; 1994; 1995a; b; c; Schirra et al., 1995; 1997a).

effectiveness of fungicides, particularly when the marketing is made after a period more or less long of storage (Schirra, 2005; Mulas, 2008). In some countries, thermotherapy is applied to control mould decay in commodities from organic cultivation or when the postharvest fungicide treatment is completely forbidden (Fallik, 2004). In other cases, the chemical defence is always of fundamental importance against mould decay and there is not a convenience to use other methods (Dezman et al., 1986; Papadopoulou-Mourkidou, 1991).

After the first experiences in the 60th years, many investigations have been promoted in the 70th and 80th years, but a new start of researches on old fungicides was observed in the 90th. Mango decay was controlled by combination of hot dips with Benomyl (Spalding & Reeder, 1972; 1978). Brown rot and rhyzopus of peaches, nectarines and stone fruits were controlled by different combination of Benomyl or other fungicides and thermotherapy (Smith, 1971; Wells & Gerdts, 1971; Wells, 1972; Jones & Burton, 1973; Smith & Anderson, 1975; Wang &

The control of rots of guavas was tested by application of heated Benomyl and Guazatine dips (Wills et al., 1982). Rotting and browning of lytchee fruit were controlled by hot

New experiments were carried out to demonstrate the synergistic effect of heat and sodium ortho-phenylpenate to inactivate *Penicillium* spores and suppress decay in citrus fruits (Barkai-Golan & Appelbaum, 1991). The efficacy of hot water and Carbendazim treatments

The most used fungicides in postharvest of horticultural commodities are Imazalil and Thiabendazole. Both are synthetic compounds with different mode of action that can be applied in waxes or water (Brown et al., 1983; Brown, 1984). Imazalil is very effective against the green mould (*Penicillium digitatum*), including benzimidazole-resistant strains, while Thiabendazole is effective to a wide range of pathogens and often may have beneficial effects also against chilling injury (Schiffman-Nadel et al., 1972; 1975; Brown & Dezman,

Many studies demonstrate that the efficacy of Imazalil and/or Prochloraz increases when applied in hot water in a number of fruits such as mango (Spalding & Reeder, 1986; Johnson et al., 1990; Prusky et al., 1990; Dodd et al., 1991; Coates et al., 1993; McGuire & Campbell, 1993; Waskar, 2005); melons (Carter, 1981b; Mayberry & Kartz, 1992), taro corms (Quevedo et al., 1991), red tamarillos (Yearsley et a., 1988), and in citrus fruit (Ansari & Feridoon, 2008).

That is because of the best cuticle and tissue penetration by mean of active compounds (Schirra et al., 2002a; 2008a). Wide demonstration of these effects has been reported in the case of the Imazalil and Thiabendazole use against *Penicillium* mould of citrus (Mulas & Schirra, 2007; Dore et al., 2009). The two fungicides resulted effective in some cases with very low concentration, up to 50 mg·L-1, if the application was made in hot water (50 °C)

benomyl and plastic film (Scott et al., 1982; Wong et al., 1991).

was tested against the brown rot of apple (Sharma & Kaul, 1990).

(Schirra & Mulas, 1993; 1994; 1995a; b; c; Schirra et al., 1995; 1997a).

**3.1 Old fungicides** 

Anderson, 1985).

**3.2 Thiabendazole and Imazalil** 

1990; McDonald et al., 1991; Schirra et al., 2000b).

Treatments of citrus with Imazalil at 490 mg·L-1, in water at 37.8 °C resulted more effective in mould control than in wax mixture at 4200 mg·L-1 at ambient temperature (Smilanick et al., 1997).

The residue control of Imazalil in lemons after applications at 50 °C of concentrations from 250 to 1500 mg·L-1 demonstrated that fruit submitted to hot dip contained about 4.5-fold higher fungicide concentration (Schirra et al., 1996b). Good results were obtained with hot dip in Imazalil mixtures of lemons (Schirra et al., 1997b) and 'Marsh' grapefruits (Schirra et al., 1998b).

Thiabendazole residues are dependent on pH of solutions (Wardowski et al., 1974), not influenced by duration of the treatment (Cabras et al., 1999), and correlated to the amount of fungicide applied (Schirra et al., 1998c)

Synergic effect of thermotherapy with fungicide application was not always so clear, and in the past there is evidence of some negative combination of the two treatments, like in the case of 'Tarocco' oranges when the preharvest treatment with Thiabendazole was effective in the postharvest control of *Penicillium* moulds and against chilling injury, but the combination with a curing treatment at 37 °C during 48 hours resulted favourable to the decay by *Phytophthora citrophtora* (Schirra et al., 2002a).

Positive effects of Thiabendazole in combination with hot dip have been confirmed by treatments on 'Eureka' lemons at 0, 25, 50 e 100 μg·mL-1 in water containing 200 μg·mL-1 of sodium hypochlorite and 0.2 ml·L-1 of Triton X-100, at temperature of 16, 27, 38, or 49 °C after artificial inoculation with a Thiabendazole resistant strain of *Penicillium digitatum* (M6R) (Smilanick et al., 2006a). In fact, mould control by only dip temperature was of 50% at 49 °C, while the addition of only 100 μg·mL-1 of Thiabendazole was effective for an almost total control of the pathogen.

A further confirmation of the synergic effect between postharvest low dose treatment with Thiabendazole and warming of the dip solution at 52 °C was observed with the control of pathogens and, partially, of the chilling injury in cactus fruit of the 'Gialla' cultivar (Schirra et al., 2002b).

In tamarillos, Imazalil was effective against *Collectotricum gleosporioides* or *C. acutatum* both in water and in wax if fungicide application followed a hot dip treatment in water at 50 °C for 10 minutes (Yearsley et al., 1987).

Good results were also recorded for the control of *Penicillium* mould and of chilling injury in citrus fruits by the fungicide Imazalil when this compound was used after molecular complexation with β-cyclodestrine at 100 mg·L-1 in water dip at 50 °C (Schirra et al., 2002c). Different works made with other fungicides demonstrate the effectiveness of β-cyclodestrine since it is a carrier of active molecules that prevents their degradation (Szejtli, 1988; Kenawy et al., 1992; Lezcano et al., 2002).

#### **3.3 New fungicides**

Most of the positive effects of synergy between thermotherapy and low dose applications of fungicides has been confirmed with the experimental use of a new generation of fungicides so called "natural mimetic"(Gullino et al., 2000; Ragsdale, 2000; Leroux, 2003). These compounds are also defined like generally recognized as safe (GRAS) with respect to the previously used fungicides. They showed higher effectiveness at low doses, a more favourable toxicological and eco-toxicological profile and different mechanisms of action with respect to the old generation of fungicides (Heye et al., 1994; Errampalli & Crnko, 2004). Therefore, these new fungicides may be very useful as an alternative to traditional fungicides which are ineffective against resistant strains of pathogens (Schirra, 2005).

Among others it is important to consider the experiences realized with the strobilurine-like compounds Azoxystrobine and Trifloxistrobine (Margot et al., 1998; Reuveni, 2000; 2001; Barlett et al., 2002; Wood & Hollomon, 2003; Schirra et al., 2006), which have their site of action in the fungal mitochondrion and are quickly destroyed in soil and groundwater (Sudisha et al., 2010). Preharvest application of Azoxystrobine were effective to control *Alternaria alternata* in mandarin cultivars 'Minneola' and 'Nova' (Oren et al., 1999) and citrus scab and melanose in seedlings of rough lemon and grapefruit (Bushong and Timmer, 2000).

Azoxystrobine in postharvest was applied to 'Star Ruby' grapefruits and oranges by 3 minutes dip at 50 °C and low concentration (50 mg·L-1) (Schirra et al., 2002d; 2010). The long ability of Azoxystrobine residues to remain constant during cold storage and in darkness was confirmed by other studies on apples (Ticha et al., 2008) and peppers (Garau et al., 2002), while rapid decline of residues was observed with preharvest treatments (Angioni et al., 2004). However, Azoxystrobine showed good natural decay control but was less effective against artificially inoculated *P. digitatum*, while Trifloxistrobine was highly effective against blue and green mould after inoculation and in association with hot dip at 50 °C for 3 minutes (Schirra et al., 2006).

Further experiences were made with Fludioxonil (synthetic analogous of pyrrolnitrine), which after fruit inoculation with *Penicillium* provided the same results as Imazalil at 100 mg·L-1 in water dip at 50 °C and 400 mg·L-1 at 20 °C (Schirra et al., 2005b), and with Pyrimethanil (anilinepyrimidine) that resulted effective at 400 mg·L-1 at 20 °C or 100 mg·L-1 at 50 °C against *P. digitatum* and *P. italicum* (D'Aquino et al., 2006). Pyrimethanil inhibits elongation of mycelium and the secretion of cell wall degradation enzymes (Daniels & Lucas, 1995; Milling & Richardson, 1995; Rosslenbroich & Stuebler, 2000; Sholberg et al., 2005; Kanetis et al., 2008a). Pyrimethanil increased notably their effectiveness when coupled with thermotherapy (D'Aquino et al., 2006; Smilanick et al., 2006b).

Cyprodinil was positively tested in combination with hot water dip against apple moulds (Errampalli & Brubacher, 2006) and inoculated *Penicillium digitatum* moulds on 'Valencia' oranges (Schirra et al., 2009a).

The possibility to control many postharvest pathogens has been investigated on a wide range of horticultural commodities (Smilanick et al., 2006b; Zhang, 2007; Kanetis et al., 2008a; Montesinos-Herrero & Palou, 2010) and recently Azoxystrobine, Fludioxonil and Pyrimethanil have been authorized for postharvest treatments in U.S.A. (Kanetis et al., 2007; Förster et al., 2007).

The mode of action of Fludioxonil is that of a mutagen-activated protein chinase pathway that stimulates glycerol synthesis (Kanetis et al., 2008b).

Recently, fludioxonil was also positively tested in association with thermotherapy on mango (Swart et al., 2009), pomegranates (Palou et al., 2007; D'Aquino et al., 2009), apple (Errampalli, 2004; Errampalli et al., 2005), citrus (Schirra et al., 2005), nectarines, apricots and peaches (D'Aquino et al., 2007), and 'Precoce di Fiorano', 'Coscia' and 'Spadona estiva' summer pears after inoculation with *Penicillium expansum* and *Botrytis cinerea* (Schirra et al., 2008b; 2009b).

favourable toxicological and eco-toxicological profile and different mechanisms of action with respect to the old generation of fungicides (Heye et al., 1994; Errampalli & Crnko, 2004). Therefore, these new fungicides may be very useful as an alternative to traditional

Among others it is important to consider the experiences realized with the strobilurine-like compounds Azoxystrobine and Trifloxistrobine (Margot et al., 1998; Reuveni, 2000; 2001; Barlett et al., 2002; Wood & Hollomon, 2003; Schirra et al., 2006), which have their site of action in the fungal mitochondrion and are quickly destroyed in soil and groundwater (Sudisha et al., 2010). Preharvest application of Azoxystrobine were effective to control *Alternaria alternata* in mandarin cultivars 'Minneola' and 'Nova' (Oren et al., 1999) and citrus scab and melanose in seedlings of rough lemon and grapefruit (Bushong and Timmer, 2000). Azoxystrobine in postharvest was applied to 'Star Ruby' grapefruits and oranges by 3 minutes dip at 50 °C and low concentration (50 mg·L-1) (Schirra et al., 2002d; 2010). The long ability of Azoxystrobine residues to remain constant during cold storage and in darkness was confirmed by other studies on apples (Ticha et al., 2008) and peppers (Garau et al., 2002), while rapid decline of residues was observed with preharvest treatments (Angioni et al., 2004). However, Azoxystrobine showed good natural decay control but was less effective against artificially inoculated *P. digitatum*, while Trifloxistrobine was highly effective against blue and green mould after inoculation and in association with hot dip at

Further experiences were made with Fludioxonil (synthetic analogous of pyrrolnitrine), which after fruit inoculation with *Penicillium* provided the same results as Imazalil at 100 mg·L-1 in water dip at 50 °C and 400 mg·L-1 at 20 °C (Schirra et al., 2005b), and with Pyrimethanil (anilinepyrimidine) that resulted effective at 400 mg·L-1 at 20 °C or 100 mg·L-1 at 50 °C against *P. digitatum* and *P. italicum* (D'Aquino et al., 2006). Pyrimethanil inhibits elongation of mycelium and the secretion of cell wall degradation enzymes (Daniels & Lucas, 1995; Milling & Richardson, 1995; Rosslenbroich & Stuebler, 2000; Sholberg et al., 2005; Kanetis et al., 2008a). Pyrimethanil increased notably their effectiveness when coupled

Cyprodinil was positively tested in combination with hot water dip against apple moulds (Errampalli & Brubacher, 2006) and inoculated *Penicillium digitatum* moulds on 'Valencia'

The possibility to control many postharvest pathogens has been investigated on a wide range of horticultural commodities (Smilanick et al., 2006b; Zhang, 2007; Kanetis et al., 2008a; Montesinos-Herrero & Palou, 2010) and recently Azoxystrobine, Fludioxonil and Pyrimethanil have been authorized for postharvest treatments in U.S.A. (Kanetis et al., 2007;

The mode of action of Fludioxonil is that of a mutagen-activated protein chinase pathway

Recently, fludioxonil was also positively tested in association with thermotherapy on mango (Swart et al., 2009), pomegranates (Palou et al., 2007; D'Aquino et al., 2009), apple (Errampalli, 2004; Errampalli et al., 2005), citrus (Schirra et al., 2005), nectarines, apricots and peaches (D'Aquino et al., 2007), and 'Precoce di Fiorano', 'Coscia' and 'Spadona estiva' summer pears after inoculation with *Penicillium expansum* and *Botrytis cinerea* (Schirra et al., 2008b; 2009b).

with thermotherapy (D'Aquino et al., 2006; Smilanick et al., 2006b).

that stimulates glycerol synthesis (Kanetis et al., 2008b).

fungicides which are ineffective against resistant strains of pathogens (Schirra, 2005).

50 °C for 3 minutes (Schirra et al., 2006).

oranges (Schirra et al., 2009a).

Förster et al., 2007).

Fig. 2. Stored mandarins treated with Imazalil at 100 mg·L-1 and 50 °C and 400 mg·L-1 at 20 °C against *P. italicum* (Schirra et al., 2005b; courtesy of Dr. Mario Schirra).

Fig. 3. Stored mandarins treatment with pyrimethanil at 100 mg·L-1 and 50 °C against *P. italicum* (D'Aquino et al., 2006; courtesy of Dr. Mario Schirra).

Fig. 4. Wounding of 'Coscia' pears before inoculation with *Penicillium expansum* (upper), apparatus for water dip at 50 °C (middle), and mould development after shelf life (above) (Schirra et a., 2008b; pictures of the author).

Fig. 4. Wounding of 'Coscia' pears before inoculation with *Penicillium expansum* (upper), apparatus for water dip at 50 °C (middle), and mould development after shelf life (above)

(Schirra et a., 2008b; pictures of the author).

Fig. 5. Stored 'Coscia' pears after inoculation with *Penicillium expansum* (upper), water dip at 50 °C (middle), and treatment with fludioxonil at 100 mg·L-1 and 50 °C (above) (Schirra et a., 2008b; pictures of the author).

#### **4. GRAS compounds**

Researchers have focused their interest on GRAS compounds in order to find alternatives to traditional fungicides or to enhance their effectiveness by means of synergistic effects when combined with thermotherapy. Among others, it is interesting to point out calcium chloride preharvest applications in combination with 2,4-D, hot dip and fungicides on 'Satsuma' mandarins (Yildix et al., 2005); the ethanol used for the control of *B. cinerea* after inoculation on table grape (Karabulut et al., 2004; Gabler et al., 2005); the acetic acid as an alternative to ethanol or water vapour to control *B. cinerea* on kiwi fruit (Lagopodi et al., 2009); the sodium carbonate and bicarbonate, which were effective against *Penicillium* mould in citrus fruits dipped for 150 seconds in water at 45 °C containing a 3% of the salt (Palou et al., 2001). This is also the case of the bicarbonate, which resulted useful on citrus fruit in order to increase the effectiveness of Imazalil to inhibit germination of *P. digitatum* (Smilanick et al., 2005), and of Thiabendazole against the resistant strain of *P. digitatum* M6R in combination with dip at 49 °C during 60 seconds and addition of sodium hypochlorite at 200 μg·mL-1 (Smilanick et al., 2006). Good results have been also obtained by application of Imazalil or Thiabendazole in combination with potassium sorbate (Smilanick et al., 2008).

Potassium phosphite (2 mg·mL-1) in combination with hot dip at 50 °C for 3 minutes induced a three-fold reduction in blue mould incidence and was as effective as Thiabendazole after six months of storage at 2 °C of 'Elstar' apples (Amiri & Bompeix, 2011).

The positive effect of carbonate and bicarbonate addition to solutions of Thiabendazole used to control *P. digitatum* was demonstrated also on clementine fruits, 'Nova' mandarins, 'Valencia late' oranges with a higher penetration of the fungicide in the fruit tissues (Schirra et al., 2008c). 'Montenegrina' tangerines were exposed to postharvest thermotherapy and sodium carbonate and bicarbonate treatments in combination with carnauba wax application, which resulted in fruit protection against mould decay (Montero et al., 2010).

On 'Satsuma' mandarins, the combination of thermotherapy and fungicides was effective against resistant strains of pathogens, and the combination of thermotherapy with antagonist microorganisms was effective against the *Rhizopus* decay on strawberry (Zhang et al., 2005). On the other hand, the synergic effect of thermotherapy with the use of antagonist strains of yeasts and sodium bicarbonate was demonstrated against *Colletotrichum acutatum* and *P. expansum* in stored apples (Mulas & Schirra, 2007), as well as of thermotherapy in combination with *Cryptococcus laurentii* against *P. italicum* and *Rhizopus stolonifer* on peach, and with *Rhodotorula glutinis* against *P. italicum* on pear storage (Zhang et al., 2007; 2008).

Prevention of spoilage caused by fungi in cherry tomato was provided by heat treatment at 38 °C (24-72 hours) followed by *Pichia guilliermondii* application (Zhao et al., 2009; 2010). Other results showed that the combined application of hot air at 38 °C for 36 hours and the same yeast antagonist *Pichia guilliermondii* was effective in the control of postharvest anthracnose rot of loquat fruit (Liu et al., 2010).

The residue determination of pesticides, or of other chemicals used in combination with thermotherapy, is an essential condition to guarantee treatment effectiveness avoiding to overcome levels of tolerance. This is the only way to maintain the safety of horticultural commodity, which is associated with the absence of chemical residues (Schirra, 2005).

#### **5. Main effects on quality of commodities**

In spite of the numerous applications of thermotherapy to many horticultural commodities, there are generalized recurrences of not complete control of mould decay if the treatments are limited to the physical means (Lurie, 2006). At the present state of knowledge, it seems more appropriate the use of thermotherapy as a synergic tool of the available fungicides and GRAS compounds (Schirra, 1995; Mulas & Schirra, 2007).

Among quality traits is important to point out that thermotherapy may build-up volatile compounds as shown in 'Tarocco' (Schirra et al., 2002b) and other bloody oranges (Mulas et al., 2001; Schirra et al., 2004; 2005a). Increases in endogenous ethanol and acetaldehyde production that change fruit taste have been reported in this fruits, but studies for a complete definition of gas exchange during and after thermotherapy are in a phase of development (Mulas et al., 2004; 2006; 2008a; 2008b).

#### **5.1 Contra-indications**

144 Fungicides – Beneficial and Harmful Aspects

Researchers have focused their interest on GRAS compounds in order to find alternatives to traditional fungicides or to enhance their effectiveness by means of synergistic effects when combined with thermotherapy. Among others, it is interesting to point out calcium chloride preharvest applications in combination with 2,4-D, hot dip and fungicides on 'Satsuma' mandarins (Yildix et al., 2005); the ethanol used for the control of *B. cinerea* after inoculation on table grape (Karabulut et al., 2004; Gabler et al., 2005); the acetic acid as an alternative to ethanol or water vapour to control *B. cinerea* on kiwi fruit (Lagopodi et al., 2009); the sodium carbonate and bicarbonate, which were effective against *Penicillium* mould in citrus fruits dipped for 150 seconds in water at 45 °C containing a 3% of the salt (Palou et al., 2001). This is also the case of the bicarbonate, which resulted useful on citrus fruit in order to increase the effectiveness of Imazalil to inhibit germination of *P. digitatum* (Smilanick et al., 2005), and of Thiabendazole against the resistant strain of *P. digitatum* M6R in combination with dip at 49 °C during 60 seconds and addition of sodium hypochlorite at 200 μg·mL-1 (Smilanick et al., 2006). Good results have been also obtained by application of Imazalil or

Thiabendazole in combination with potassium sorbate (Smilanick et al., 2008).

Potassium phosphite (2 mg·mL-1) in combination with hot dip at 50 °C for 3 minutes induced a three-fold reduction in blue mould incidence and was as effective as Thiabendazole after six months of storage at 2 °C of 'Elstar' apples (Amiri & Bompeix, 2011). The positive effect of carbonate and bicarbonate addition to solutions of Thiabendazole used to control *P. digitatum* was demonstrated also on clementine fruits, 'Nova' mandarins, 'Valencia late' oranges with a higher penetration of the fungicide in the fruit tissues (Schirra et al., 2008c). 'Montenegrina' tangerines were exposed to postharvest thermotherapy and sodium carbonate and bicarbonate treatments in combination with carnauba wax application, which resulted in fruit protection against mould decay (Montero et al., 2010).

On 'Satsuma' mandarins, the combination of thermotherapy and fungicides was effective against resistant strains of pathogens, and the combination of thermotherapy with antagonist microorganisms was effective against the *Rhizopus* decay on strawberry (Zhang et al., 2005). On the other hand, the synergic effect of thermotherapy with the use of antagonist strains of yeasts and sodium bicarbonate was demonstrated against *Colletotrichum acutatum* and *P. expansum* in stored apples (Mulas & Schirra, 2007), as well as of thermotherapy in combination with *Cryptococcus laurentii* against *P. italicum* and *Rhizopus stolonifer* on peach, and with *Rhodotorula glutinis* against *P. italicum* on pear storage (Zhang et

Prevention of spoilage caused by fungi in cherry tomato was provided by heat treatment at 38 °C (24-72 hours) followed by *Pichia guilliermondii* application (Zhao et al., 2009; 2010). Other results showed that the combined application of hot air at 38 °C for 36 hours and the same yeast antagonist *Pichia guilliermondii* was effective in the control of postharvest

The residue determination of pesticides, or of other chemicals used in combination with thermotherapy, is an essential condition to guarantee treatment effectiveness avoiding to overcome levels of tolerance. This is the only way to maintain the safety of horticultural commodity, which is associated with the absence of chemical residues (Schirra, 2005).

**4. GRAS compounds** 

al., 2007; 2008).

anthracnose rot of loquat fruit (Liu et al., 2010).

Many experiments of thermotherapy were designed to optimize the protocol to avoid decay or physiological disorders, but damages derived from this kind of treatments have been observed (Lurie, 2006). Negative effects have been described on apple fruit after brushing with water at 60 or 65 °C for 15 seconds (Fallik et al., 2001). Heat-damages manifested as increased electrolyte leakage from biological membranes and surface browning may occur in cactus pear fruit (*Opuntia ficus-indica* Miller L.) after brushing with water at 65-70 °C for 10-30 seconds (Dimitris et al., 2005). Negative effects of thermotherapy were also observed on strawberry (Wszelaki & Mitcham, 2003). Short hot-water rinsing and brushing treatment for 20 seconds at 55, 60, and 65 °C significantly reduced the epiphytic microbial population on fruit surface of strawberry cultivar 'Feng xiang', as well as decay development and weight loss (Jing et al., 2010). However, about 60% of the fruit treated at 65 °C showed heat damages.

The effects of treatment temperatures within the range from 20 to 75 °C were studied on 'Navelate' oranges after dip during 150 seconds (Palou et al., 2001). Any negative effect was recorded until 45 °C, while 17 and 28% of fruit treated at 53 or 55 °C showed slight or medium symptoms of heat damages on 100% of fruit surface.

The contemporary evolution of chilling injury and heat damage has been described in 'Satsuma' mandarins, as well as the involvement of antioxidant enzymes, vacuolar ATPase, and pyrophosphatase (Ghasemnezhad et al., 2007).

Heat damages may produce symptoms in the internal tissues of different commodities even in total absence of any external alteration. This is the case of the internal browning, which was observed in avocados, citrus, peaches, nectarines, and lytchees (Zhou et al., 2002; Follet & Sanxter, 2003; Lurie, 2006). Other symptoms, like low pulp colour evolution, anomalous softening, lack of starch hydrolysis, and development of internal cavities have been observed on mangoes and papaya (Jacobi et al., 2001; Lurie, 2006). Lytchee fruits of the cultivar 'McLean's Red' after water dip at 50 °C for 2 minutes or water dip at 55 °C for 1 minute showed superficial scald, while the treatment at 60 °C for 1 second was less harmful (Sivakumar & Korsten, 2006). Dragon fruits (*Hylocereus undatus*) were tolerant to hot air disinfestations treatments until a temperature of 46.5-48.5 °C for 20 minutes as measured in the fruit centre. Because the lack of significant differences for bracteas and peduncle turgidity, fruit general appearance and presence of mould decay, peel colour and pulp firmness, total soluble solids concentration, acidity, taste and pulp brightness, fruit quality was kept immediately after the treatment, after 4 weeks storage at 5 °C in propylene bags, and after the shelf life period at 20 °C (Hoa et al., 2006).

Some field variables, like seasonal temperatures and rainfall may influence the thermotherapy effectiveness, particularly in those commodity very sensitive to chilling injury or to heat scald like citrus fruit. Some studies demonstrate thermotherapy effectiveness in the control of chilling injury, but also that the same treatment may be harmful depending on the harvest date (Schirra et al., 1997; Lafuente et al., 2005; Lafuente & Zacarias, 2006).

#### **5.2 Effects on maturation and senescence**

Postharvest thermotherapy treatments slow down maturation of climacteric fruits (Fallik, 2004; Lurie, 2006). Ethylene biosynthesis inhibition by heat treatments slows pulp softening and favours low colour and aromatic compound development in apples and kiwis treated at 38 °C, while the treatment at 39 °C for 90 minutes slow down colour development in tomatoes (Ali et al., 2004).

Investigations on 'Caldesi 2000' nectarines and 'Royal Glory' peaches showed that water dip at 46 °C reduced pulp softening of fruits sealed in thin polyethylene bags and stored at 0 °C for one or two weeks (Malakou & Nanos, 2005). This effect is the result of the combination of hot water treatment, modified atmosphere application and package, particularly in white pulp nectarines, which maintain functional cell membranes because cell wall hydrolytic enzyme inactivation, mainly polygalacturonase. Hot air treatment may also change the organoleptic characteristics of peaches 'Dixiland' by decreasing total acidity and increasing red pigments in pulp and peel (Budde et al., 2006).

Usually the effects of postharvest heat treatments are reversible, if the application is not too long, and then the physiological damage is avoided. This is the case of the tomatoes treated with water at 42 °C that showed regular biosynthesis of aromatic compounds and of lycopene (Mulas & Schirra, 2007)

High temperature may induce temporary inhibition of polygalacturonase in mango and tomato, as well as low activity of other enzymes involved in softening. Changes in other characters linked to maturation may be reduced in non-climacteric fruits such as strawberries with slow colour development and pulp softening (Lurie, 2006), which may be associated with low acidity (Vicente et al., 2002).

Water dip at 45 °C improves strawberry resistance to pathogens but determined external damages and reduction of the solubility of cell wall polysaccharides (Lara et al., 2006).

Heated vapour treatment at 52.5 °C or 55 °C during 18-27 minutes of table grape of the 'Sultanina' cultivar did not affect weight loss, berry firmness, colour, and total soluble solid and acid content modification. Nevertheless, treatment at higher temperature (58 °C) or for a more prolonged period (55 °C, 30 minutes) reduced fruit quality since it increased weight loss and berry browning (Lydakis & Aked, 2003).

Among effects of heat treatments on tissue senescence of horticultural commodities, it has been shown that the natural yellowish of broccoli is delayed both after exposing it to water dip at 45 °C for 10 minutes or after air conditioning at 50 °C for 2 hours (Funamoto et al., 2003).

Other symptoms of senescence, like geotropism deviation of spears of asparagus, and sprouting of onion, garlic and potato have been controlled by dip treatments in water at 50- 55 °C for 2-4 minutes (Cantwell et al., 2003; Lurie, 2006). Pineapple fruit treatment at 38-60 °C for 60 minutes was effective to control internal browning during cold storage (Weerahewa & Adikaram, 2005). Sapote fruit [*Pouteria sapota* (Jacq.) H.E. Moore & Stearn], exposed to disinfestations with dip treatment at 60 °C for 60 minutes showed less pulp browning respect to untreated control (Diaz Perez et al., 2001).

Postharvest treatments of early ripening pears of 'Camusina' and 'Precoce di Fiorano' cultivars with hot dip at 50 or 60 °C for 3 minutes or 1-Methylcyclopropene at 20 °C were useful to delay senescence and internal browning, while soy lecithin and calcium chloride resulted less effective (Mulas et al., 2008a). However, dip treatment at 60 °C determined heat damages in the peel of the two cultivars.

Water dip treatment for 3 minutes a 50 °C and with hot air at 37 °C for 48 hours were effective reducing chilling injury on bloody oranges of the cultivars 'Tarocco', 'Moro', 'Doppio sanguigno', and 'Sanguinello'. Any treatment caused visible damages during quarantine storage of 16 days at 1 °C, subsequent storage of 3 weeks at 8 °C and a further week of simulated shelf life at 20 °C. However, while dip treatment was not influent on fruit firmness, taste, aroma, juice content and composition (total soluble solids, titratable acidity, ascorbic acid and ethanol content), hot air treatment negatively influenced fruit firmness, taste and chemical composition (Schirra et al., 2004).

Postharvest treatments of disinfestations against pests with humid air at 44 or 46 °C, measured inside the fruit during 100 and 50 minutes respectively, do not produce negative effects on 'Olinda' and 'Campbel' oranges (clones of 'Valencia late' cultivar) both for the external appearance and the internal composition (Schirra et al., 2005a). However, these protocols cannot be recommended to the bloody oranges because of the negative influence on fruit quality (off flavours development, fruit softening, high weight loss), and of the reduced resistance to moulds (Mulas et al., 2001).

#### **5.3 Nutritional value**

146 Fungicides – Beneficial and Harmful Aspects

the fruit centre. Because the lack of significant differences for bracteas and peduncle turgidity, fruit general appearance and presence of mould decay, peel colour and pulp firmness, total soluble solids concentration, acidity, taste and pulp brightness, fruit quality was kept immediately after the treatment, after 4 weeks storage at 5 °C in propylene bags,

Some field variables, like seasonal temperatures and rainfall may influence the thermotherapy effectiveness, particularly in those commodity very sensitive to chilling injury or to heat scald like citrus fruit. Some studies demonstrate thermotherapy effectiveness in the control of chilling injury, but also that the same treatment may be harmful depending on the harvest date (Schirra et al., 1997; Lafuente et al., 2005; Lafuente &

Postharvest thermotherapy treatments slow down maturation of climacteric fruits (Fallik, 2004; Lurie, 2006). Ethylene biosynthesis inhibition by heat treatments slows pulp softening and favours low colour and aromatic compound development in apples and kiwis treated at 38 °C, while the treatment at 39 °C for 90 minutes slow down colour development in

Investigations on 'Caldesi 2000' nectarines and 'Royal Glory' peaches showed that water dip at 46 °C reduced pulp softening of fruits sealed in thin polyethylene bags and stored at 0 °C for one or two weeks (Malakou & Nanos, 2005). This effect is the result of the combination of hot water treatment, modified atmosphere application and package, particularly in white pulp nectarines, which maintain functional cell membranes because cell wall hydrolytic enzyme inactivation, mainly polygalacturonase. Hot air treatment may also change the organoleptic characteristics of peaches 'Dixiland' by decreasing total acidity and increasing

Usually the effects of postharvest heat treatments are reversible, if the application is not too long, and then the physiological damage is avoided. This is the case of the tomatoes treated with water at 42 °C that showed regular biosynthesis of aromatic compounds and of

High temperature may induce temporary inhibition of polygalacturonase in mango and tomato, as well as low activity of other enzymes involved in softening. Changes in other characters linked to maturation may be reduced in non-climacteric fruits such as strawberries with slow colour development and pulp softening (Lurie, 2006), which may be

Water dip at 45 °C improves strawberry resistance to pathogens but determined external damages and reduction of the solubility of cell wall polysaccharides (Lara et al., 2006).

Heated vapour treatment at 52.5 °C or 55 °C during 18-27 minutes of table grape of the 'Sultanina' cultivar did not affect weight loss, berry firmness, colour, and total soluble solid and acid content modification. Nevertheless, treatment at higher temperature (58 °C) or for a more prolonged period (55 °C, 30 minutes) reduced fruit quality since it increased weight

and after the shelf life period at 20 °C (Hoa et al., 2006).

**5.2 Effects on maturation and senescence** 

red pigments in pulp and peel (Budde et al., 2006).

associated with low acidity (Vicente et al., 2002).

loss and berry browning (Lydakis & Aked, 2003).

Zacarias, 2006).

tomatoes (Ali et al., 2004).

lycopene (Mulas & Schirra, 2007)

Many studies demonstrated that thermotherapy may influence the biosynthesis of antioxidant or nutraceutical compounds (Schreiner & Huyskens-Keil, 2006) and researches in this direction are increasing. In papaya (*Carica papaya*) fruit, for example, thermotherapy reduces chilling injury, slow down superoxide-dismutase and catalase activities and stops the increase of peroxidase activity (Huajaikaew et al., 2005). Hot air treatment at 34 °C and 50% R.H. during 24 hours of the tomato 'Rhapsody' did not affect to the antioxidant properties of the fruit that developed a normal colour during storage at 10 °C (Soto-Zamora et al., 2005). Otherwise, the fruit exposition to 38 °C during 24 hours in air o in an atmosphere containing a 5% of oxygen determined some negative effects, like loss of antioxidant properties and lack of colour evolution.

Dipping of mango fruit at 50 °C for 60 hours to kill pests may enhance carotene biosynthesis, reduce fruit shelf life, while the thermotherapy associated with cold storage slows carotene development (Talcott et al., 2005). More studies on the cultivar 'Kensington Pride' showed high effectiveness in maintaining fruit quality of thermotherapy in air at 40 °C for 8 hours, or with water dip at 52 °C for 10 minutes (Dang et al., 2008).

Broccoli (*Brassica oleracea* L.) treated at 48 °C for 3 hours with hot air showed slow senescence at 20 °C, better quality and a significant higher contents of chlorophyll, sugars, proteins and antioxidants (Costa et al., 2005). A slow degradation of chlorophyll and an increase in antioxidant properties has been also reported in spinach after water dip treatment at 40 °C for 3 minutes (Gomez et al., 2008).

Dip treatment at 45 °C for 4 minutes of pomegranate fruits (*Punica granatum* L. cultivar 'Mollar de Elche') also produced higher antioxidant activity, total phenols, ascorbic acid, anthocyans, sugars, and organic acids (Mirdehghan et al., 2006).

Studies on different horticultural commodities showed that hot dip at 35 °C for 12 hours (tomato), at 55 °C for 5 minutes (melon), and at 42 °C for 24 hours (mango) inhibited polyphenol-oxidase and peroxidase activities and reduced slow biosynthesis of antocyans, but maintain good nutraceutical properties in the fruits (Cisneros-Zevallos, 2003; Brovelli, 2006; Mulas & Schirra, 2007).

Good results have been obtained with water dip at 50 °C for 2 minutes in order to maintain nutritional and functional properties of kumquat fruit (Schirra et al., 2008d), and the quality of blueberry was also satisfactory after thermotherapy application (Fan et al., 2008).

#### **6. Conclusions and future perspectives**

In spite of the beneficial effects, thermotherapy is not sufficient alone to provide protection against postharvest disorders during long term storage (Tab. 1-4). This is because some of the induced mechanisms are only transient. However, it is very clear their synergic effect with the old fungicides and the need of maintaining also this possible supplement of effectiveness for the applications of the new fungicides. In fact there is yet some signal of pathogen resistance for the recently admitted postharvest fungicides.

Further investigations are necessary to optimize protocols for different horticultural commodities, cultivars and zone of production. A good direction to develop more studies is the possibility to combine the thermotherapy with other physical and chemical treatments. In the Tables 1-4 a synthesis of the main effects and indication of the termotherapy is proposed to stimulate new ideas and investigations.

The heat effect on temporary inhibition or enhancement of the enzyme activity, as well as on slowing maturation and senescence of commodities, are some of the evidences more critical to get a general insight of the complex physiological consequences of thermotherapy. The influence of this practice on the biosynthesis of ethylene and of phytochemicals of nutraceutical value, and on the development of aromatic and off volatiles are also some directions for future researches (Cisneros-Zevallos, 2003; Mulas et al., 2006; 2008b; Lafuente et al., 2011).

Dipping of mango fruit at 50 °C for 60 hours to kill pests may enhance carotene biosynthesis, reduce fruit shelf life, while the thermotherapy associated with cold storage slows carotene development (Talcott et al., 2005). More studies on the cultivar 'Kensington Pride' showed high effectiveness in maintaining fruit quality of thermotherapy in air at

Broccoli (*Brassica oleracea* L.) treated at 48 °C for 3 hours with hot air showed slow senescence at 20 °C, better quality and a significant higher contents of chlorophyll, sugars, proteins and antioxidants (Costa et al., 2005). A slow degradation of chlorophyll and an increase in antioxidant properties has been also reported in spinach after water dip

Dip treatment at 45 °C for 4 minutes of pomegranate fruits (*Punica granatum* L. cultivar 'Mollar de Elche') also produced higher antioxidant activity, total phenols, ascorbic acid,

Studies on different horticultural commodities showed that hot dip at 35 °C for 12 hours (tomato), at 55 °C for 5 minutes (melon), and at 42 °C for 24 hours (mango) inhibited polyphenol-oxidase and peroxidase activities and reduced slow biosynthesis of antocyans, but maintain good nutraceutical properties in the fruits (Cisneros-Zevallos, 2003; Brovelli,

Good results have been obtained with water dip at 50 °C for 2 minutes in order to maintain nutritional and functional properties of kumquat fruit (Schirra et al., 2008d), and the quality

In spite of the beneficial effects, thermotherapy is not sufficient alone to provide protection against postharvest disorders during long term storage (Tab. 1-4). This is because some of the induced mechanisms are only transient. However, it is very clear their synergic effect with the old fungicides and the need of maintaining also this possible supplement of effectiveness for the applications of the new fungicides. In fact there is yet some signal of

Further investigations are necessary to optimize protocols for different horticultural commodities, cultivars and zone of production. A good direction to develop more studies is the possibility to combine the thermotherapy with other physical and chemical treatments. In the Tables 1-4 a synthesis of the main effects and indication of the termotherapy is

The heat effect on temporary inhibition or enhancement of the enzyme activity, as well as on slowing maturation and senescence of commodities, are some of the evidences more critical to get a general insight of the complex physiological consequences of thermotherapy. The influence of this practice on the biosynthesis of ethylene and of phytochemicals of nutraceutical value, and on the development of aromatic and off volatiles are also some directions for future researches (Cisneros-Zevallos, 2003; Mulas et al., 2006; 2008b; Lafuente

of blueberry was also satisfactory after thermotherapy application (Fan et al., 2008).

pathogen resistance for the recently admitted postharvest fungicides.

40 °C for 8 hours, or with water dip at 52 °C for 10 minutes (Dang et al., 2008).

treatment at 40 °C for 3 minutes (Gomez et al., 2008).

**6. Conclusions and future perspectives** 

proposed to stimulate new ideas and investigations.

2006; Mulas & Schirra, 2007).

et al., 2011).

anthocyans, sugars, and organic acids (Mirdehghan et al., 2006).


Table 1. Thermotherapy treatments and their different effects.



minutes)

hours)

50-60 °C (2 minutes -1 second)

minutes)

51,5 °C (125 minutes) 46-48 °C (3-5 hours) 42-49 °C (7-120 minutes)

52 °C (10 minutes)

Water dip 46 °C (25 minutes) Slow pulp firmness

Mangoes Water dip 50 °C (60 minutes) Quarantine.

Lemons Water dip 50 °C (2 minutes) Synergic to Imazalil Dore et al., 2009

Lytchee Water dip 49 °C (20 minutes) Heat damages. Follet & Sanxter,

and other citrus Hot humid air. 37 °C (3 days) Chilling injury control. Lafuente & Zacarias,

Lemons Water dip 49 °C (60 seconds) Synergic to

Loquat Hot humid air 38 °C (36 hours) Synergic to antagonist

**(time) Effect References** 

30 minutes) Quarantine. Rafaeli et al., 2006

minutes) Avoid spruce Lurie, 2006

Synergic to ethanol Lagopodi et al., 2009

Thiabendazole Smilanick et al., 2006

strains. Liu et al., 2010

Heat damages. Sivakumar &

Heat damages. Jacobi et al., 2001

Quick maturation. Talcott et al., 2005

2003

Korsten, 2006

2006

Ghasemnezhad et al., 2007

Lafuente et al., 2011

Mulas & Schirra, 2007

Swart et al., 2009

Mulas & Schirra, 2007

Malakou & Nanos, 2005

colour and aroma. Lurie, 2006

Mould control.

Low development of

hours) Heat damages. Lurie, 2006

Heat damages. ATPase, pyrophosphatase and antioxidant enzymes.

Chilling injury control without loss of flavonoids and vitamin C.

40 °C (8 hours) Mould control. Dang et al., 2008

PPO and POD inhibition with antocyans storage.

Synergic to Fludioxonil and Prochloraz

PPO and POD inhibition with antocyan storage.

hours) Heat damages Lurie, 2006

loss.

minutes) Avoid sprouting. Lurie, 2006

Brushing. 60 °C (30 seconds) Synergic to Imazalil Montero et al., 2010

**Commodity Treatment Temperature** 

Dates Hot humid air. 55 °C (2 hours and

Garlic Water dip 50-55 °C (4-2

Kiwi Hot vapour. 47-53 °C (3-6

Kiwi Hot humid air. 38 °C (30-120

Lytchee ('McLean's

Mandarins ('Fortune')

Mandarins

Mandarins ('Montenegrina')

Mangoes

Mangoes ('Kensington Pride')

Nectarine ('Caldesi 2000' and 'Royal Glory')

Red') Water dip

Mandarins ('Dancy') Hot humid air. 45-48 °C (1-4

('Satsumas') Water dip 45-55 °C (2-5

Mandarins ('Fortune') Hot humid air. 37 °C (1-2 days)

Water dip.

Hot humid air. Hot vapour. Water dip

Water dip. Hot humid air.

Mangoes Water dip 42 °C (24 hours)

Mangoes Water dip 50 °C (30 seconds)

Melons Water dip 55 °C (5 minutes)

Nectarine Hot humid air. 41-46 °C (24-48

Onions Water dip 50-55 °C(4-2

Table 2. Thermotherapy treatments and their different effects.




Table 4. Thermotherapy treatments and their different effects.

#### **7. Acknowledgements**

The author want to thank Dr. Mario Schirra for the permission to print the pictures of the Figs. 2 and 3, as well as the Dr. Maria Teresa Lafuente for the critical contribution to the manuscript.

#### **8. References**


Strawberry Water dip 63 °C (12 seconds) Heat damages. Wszelaki e Mitcham,

40-50 °C (30-75 minutes.) 45 °C (15 minutes).

seconds)

38 °C (24 hours.)

The author want to thank Dr. Mario Schirra for the permission to print the pictures of the Figs. 2 and 3, as well as the Dr. Maria Teresa Lafuente for the critical contribution to the

Adaskaveg, J. E. & Förster, H. (2010). New developments in postharvest fungicide

Amiri, A. & Bompeix, G. (2011). Control of *Penicillium expansum* with potassium phosphite

Angioni, A., Schirra, M., Garau, V.L., Melis, M., Tuberoso, C.I.G. & Cabras, P. (2004).

registrations for edible horticultural crops and use strategies in the United States. *In:* Prusky, D. & Gullino, M.L. (eds.). *Postharvest pathology.* Springer, pp. 107-116. Ali, M.S., Nakano, K. & Maezawa, S. (2004). Combination effect of heat treatment and

modified atmosphere packaging on the colour development of cherry tomato.

Residues of azoxystrobin, fenhexamid and pyrimethanil in strawberry following field treatments and the effect of domestic washing. *Food Additives and* 

Tomato Hot humid air. 39 °C (90 minutes) Delayed colour

Tomato Hot humid air. 38 °C (24 hours) Synergic to

Mould control. Synergic to antagonist strains.

Slow pulp firmness loss and colour development. Low acidity.

Heat colour. Low hydrolysis of polysaccharides

Mould control. Synergic to ethanol.

Quarantine. Loss of antioxidants and anomalous colour.

PPO and POD inhibition with storage of antocyans.

antagonist strains.

minutes) Heat damages Lydakis & Aked,

development. Ali et al., 2004

and brushing 60 °C (20 seconds) Mould control. Jing et al., 2008

Zhang et al., 2005

2003

Vicente et al., 2002;

Lara et al., 2006

Karabulut et al., 2004; Gabler et al., 2005

2003

Soto-Zamora et al., 2005

Mulas & Schirra, 2007

Zhao et al., 2009; 2010

Strawberry Water dip 55 °C (30 seconds)

Strawberry Hot humid air. 45 °C (3 hours)

Water dip.

Hit water rinsing

Table grape Water dip 50-60 °C (30-60

Table grape Heated vapour 52,5-58 °C (18-30

('Rhapsody') Hot air with 50% RH 34 °C (24 hours).

Tomato Water dip 35 °C (12 hours)

Table 4. Thermotherapy treatments and their different effects.

*Postharvest Biology and Technology*, 34, pp. 113-116.

and heat treatment. *Crop Protection*, 30, pp. 222-227.

*Contaminants,* 21, pp. 1065–1070.

Strawberry ('Pàjaro') Hot humid air.

Strawberry ('Feng xiang')

Tomato

**7. Acknowledgements** 

manuscript.

**8. References** 


Carter, W.W. (1981a). Reevaluation of heated water dip as a postharvest treatment for

Carter, W.W. (1981b). Postharvest treatments for control of stem-scar, rind, and decay fungi

Cisneros-Zevallos, L. (2003). The use of controlled postharvest abiotic stresses as a tool for

Coates, L.M., Johnson, G.I. & Cooke A. W. (1993). Postharvest disease control in mangoes

Costa, M.L., Civello, P.M., Chaves, A.R. & Martinez, G.A. (2005). Effect of hot air treatment

Dang, K.T.H., Singh, Z. & Swinny, E.E. (2008). Impact of postharvest disease control

Daniels, A. & Lucas, J. A. (1995). Mode of action of anilino-pyrimidine fungicide

D'Aquino, S., Schirra, M., Palma, A., Angioni, A., Cabras, P. & Migheli, Q. (2006). Residue

D'Aquino, S., Schirra, M., Palma, A., Tedde, M., Angioni, A., Garau, A. & Cabras, P. (2007).

D'Aquino, S., Schirra, M., Palma, A., Angioni, A., Cabras, P., Gentile, A. & Tribulato, E.

Dezman, D. J., Nagy, S. & Brown, G. E. (1986). Postharvest fungal decay control chemicals: treatments and residues in citrus fruits. *Residue Reviews*, 97, pp. 37-92. D'hallewin, G., Schirra, M. & Manueddu. E. (1999). Effect of heat on epicuticular wax of

Diaz Perez, J.C., Mejia, A., Bautista, S., Zaveleta, R., Villanueva, R. & Gomez, R.L. (2001).

Dodd, J.C., Bugante, R., Koomen, I., Jeffries, P. & Jeger, M.J. (1991). Pre- and postharvest control of mango anthracnose in the Philippines. *Plant Pathology*, 40, pp. 576-583.

water treatments. *Postharvest Biology and Technology*, 22, pp. 156-167. Dimitris, L., Pompodakis, N., Markellou, E. & Lionakis, S.M. (2005). Storage response of

Italica). *Journal of Science and Food Agriculture*, 85, pp. 1154-1160.

on cantaloup. *Plant Disease*, 65, pp. 815-816.

leaves. *Pesticide Science*, 45, pp. 33-41.

*and Food Chemistry*, 54, pp. 4721-4726.

*Acta Horticulturae,* 818, pp. 313-318.

cactus pear fruit. *Tropical Science*, 39, pp. 244-247.

*Chemistry*, 55, pp. 825-831.

38, pp. 145-151.

vegetables. *Journal of Food Science*, 68, pp. 1560-1565.

335.

123, pp. 441-448.

10667-10674.

controlling surface and decay fungi of muskmelon fruits. *HortScience*, 16, pp. 334-

enhancing the nutraceutical content and adding value of fresh fruits and

using high humidity, hot air and fungicide treatments. *Annals of Applied Biology*,

on senescence and quality parameters of harvested broccoli (*Brassica oleracea* L. var.

methods and cold storage on volatiles, color development and fruit quality in ripe 'Kensington Pride' mangoes. *Journal of Agricultural and Food Chemistry*, 56, pp.

pyrimethanil. 1. *In vivo* activity against *Botrytis fabae* on broad bean (*Vicia faba*)

levels and effectiveness of pyrimethanil vs imazalil when using heated postharvest dip treatment for control of *Penicillium* decay on citrus fruit. *Journal of Agricultural* 

Residue levels and storage responses of nectarines, apricots and peaches after dip treatments with fludioxonil fungicide mixtures. *Journal of Agricultural and Food* 

(2009). Effectiveness of fludioxonil in control storage decay on pomegranate fruit.

Response of sapote mamey [*Pouteria sapota* (Jacq.) H.E. Moore & Stearn] fruit to hot

cactus pear fruit following hot water brushing. *Postharvest Biology and Technology*,


Förster, H., Driever, G.F., Thompson, D.C. & Adaskaveg, J.E. (2007). Postharvest decay

Freeman, B., Albrigo, L.G. & Biggs, R.H. (1979). Ultrastructure and chemistry of cuticular

Funamoto, Y., Yamauchi, N., Shigenaga, T. & Shigyo, M. (2003). Involvement of peroxidase

activity by heat treatments. *Postharvest Biology and Technology*, 28, pp. 39-46. Gabler, F.M., Smilanick, J.L., Ghosoph, J.M. & Margosan, D.A. (2005). Impact of postharvest

Garau, V. L., Angioni, A., Aguilera Del Real, A., Russo, M. T. & Cabras, P. (2002).

Gleen, G.M., Poovaiah, B.W. & Rasmussen, H.P. (1985). Pathways of calcium penetration

Gomez, F., Fernandez, L., Gergoff, G., Guiamet, J.J., Chaves, A. & Bartoli, C.G. (2008). Heat

Gonzales-Aguilar, G.A., Zacarias, L., Mulas, M. & Lafuente, M.T. (1997). Temperature and

Hayward, F.W. & Grieson, W. (1960). Effects of treatment conditions on o-Phenilphenol residues in oranges. *Journal of Agricultural and Food Chemistry*, 8, pp. 308-310. Heye, U.J., Speich, J., Siegle, H., Steinemann, A., Förster, B., Knauf Beiter, G., Herzog, J. &

Hoa, T.T., Clark, C.J., Waddell, B.C. & Woolf, A.B. (2006). Postharvest quality of Dragon

Huajaikaew, L., Uthairatankij, A., Kanlayanarat, S. & Gemma, H. (2005). Effect of heat

Jacobi, K.K., Macrae, E.A. & Hetherington, S.E. (2001). Postharvest heat disinfestation

Jemric, T., Ivic, D., Fruk, G., Matijas, H.S., Cvjetkovic, B., Bupic, M. & Pavkovic, B. (2011).

using hot water dipping. *Food and Bioprocess Technology*, 4, pp. 149-154.

treatments of mango fruit. *Scientia Horticulturae*, 89, pp. 171-193.

spinach leaves. *Postharvest Biology and Technology*, 49, pp. 229-234.

'Fortune' mandarins. *Postharvest Biology and Technology*, 12, pp. 61-69. Gullino, M.L., Leroux, P. & Smith, C.M. (2000). Uses and challenges of novel compounds for

greenhouse. *Journal of Agricultural and Food Chemistry*, 50, pp. 1929–1932. Ghasemnezhad, M., Marsh, K., Shilton, R., Babalar, M. & Woolf, A. (2007). Effect of hot

fludioxonil and fenhexamid. *Plant Disease*, 91, pp. 209-215.

ethanol content. *Plant Disease*, 89(3), pp. 309-316.

*Society for Horticultural Science*, 110, pp. 166-171.

plant disease control. *Crop Protection*, 19, pp. 1-11.

temperatures. *Acta Horticulturae*, 682, 1063-1068

*Biology and Technology*, 41, pp. 62-69.

13, pp. 541–549.

*Biology and Technology*, 48, pp. 364-371.

*Horticultural Science*, 104, 801-808.

management for stone fruit crops in California using the "reduced-risk" fungicides

waxes of developing *Citrus* leaves and fruits. *Journal of American Society for* 

in chlorophyll degradation in stored broccoli (*Brassica oleracea* L.) and inhibition of

hot water or ethanol treatment of table grapes on gray mold incidence, quality, and

Disappearance of azoxystrobin, cyprodinil, and fludioxonil on tomatoes in a

water treatments on chilling injury and heat damage in 'Satsuma' mandarins: antioxidant enzymes and vacuolar ATPase, and pyrophosphatase. *Postharvest* 

through isolated cuticles of 'Golden Delicious' apple fruit. *Journal of American* 

shock increases mitochondrial H2O2 production and extends postharvest life of

duration of water dips influence chilling injury, decay and polyamine content in

Hubele, A. (1994). CGA 219417–a novel broad-spectrum fungicide. *Crop Protection*,

fruit (*Hylocereus hundatus*) following disinfesting hot air treatments. *Postharvest* 

treatment on antioxidants and quality changes in papaya fruit stored at low

Reduction of postharvest decay of peach and nectarine caused by *Monilinia laxa*


Lezcano, M., Al-Soufi, W., Novo, M., Rodrìguez-Nuñez, E. & Tato, J. V. (2002).

Lurie, S. (1998). Postharvest heat treatments of horticultural crops. *Horticultural Review,* 22,

Lurie, S. (2006). The effect of high temperature treatment on quality of fruits and vegetables.

Lydakis, D., & Aked, J. (2003). Vapour heat treatment of Sultanina table grapes. II: effects on postharvest quality. *Postharvest Biology and Technology*, 27, pp. 117-126. Malakou, A. & Nanos, G.D. (2005). A combination of hot water treatment and modified

Margot, P., Huggenberger, F., Amrein, J. & Weiss, B. (1998). CGA 279202: A new broad

Mayberry, K.S. & Hartz, T.K. (1992). Extension muskmelon storage life through the use of hot water treatment and polyethylene wraps. *HortScience*, 27, pp. 324-326. McCornack, A.A. & Hayward, F.W. (1968). Factors affecting decay control of Dowcide A-

McDonald, R.E., Miller, W.R., McCollum, T.G. & Brown, G.E. (1991). Thiabendazole and

McGuire, R.G. & Campbell, C.A. (1993). Imazalil for postharvest control of anthracnose on

Milling, R. J. & Richardson, C. J. (1995). Mode of action of the anilino-pyrimidine fungicide

Mirdehghan, S.H., Rahemi, M., Serrano, M., Guillen, F., Martinez-Romero, D. & Valero, D.

Montero, C.R.S., Antes, R.B., Schwarz, L.L., Cunha dos Santos, L., Pires dos Santos, R. &

Montesinos-Herrero, C. & Palou, L. (2010). Combination of physical and low-toxicity

Mulas, M. (2006). Tratamientos postcosecha de los citricos con calor para el control del daño

of Montenegrina tangerines. *Crop Protection*, 29, pp. 1076-1083.

fruit: a review. *Stewart Postharvest Review*, *1* (*1*), pp. 1-11.

cyclodextrins*. Journal of Agricultural and Food Chemistry, 50,* pp. 108-112. Liu, F., Tu, K., Shao, X., Zhao, Y., Tu, S., Su, J., Hou, Y. & Zou, X. (2010). Effect of hot air

of loquat fruit. *Postharvest Biology and Technology*, 58, pp. 65-71.

pp. 91-121.

106-114.

290-293.

26, pp. 397-399.

pp. 43- 48.

21.

*Chemistry*, 54, pp. 8495-8500.

*Acta Horticulturae*, 712, pp. 165-174.

*Conference, Pests and Diseases,* pp. 375-382.

mango fruit. *Acta Horticulturae*, 341, pp. 371-376.

Complexation of several benzimidazole-type fungicides with α- and β-

treatment in combination with *Pichia guilliermondii* on postharvest anthracnose rot

atmosphere packaging maintains quality of advanced maturity 'Caldesi 2000' nectarines and 'Royal Glory' peaches. *Postharvest Biology and Technology*, 38, pp.

spectrum strobilurin fungicide. *In: Proceedings of the Brighton Crop Protection*

hexamine treated citrus fruit. *Proceeding of Florida State Horticultural Society*, 81, pp.

imazalil applied at 53C reduce chilling injury and decay of grapefruit. *HortScience*,

pyrimethanil. 2. Effects on enzyme secretion in *Botrytis cinerea*. *Pesticide Science*, 45,

(2006). Prestorage heat treatment to maintain nutritive and functional properties during postharvest cold storage of pomegranate. *Journal of Agricultural and Food* 

Bender, R.J. (2010). Complementary physical and chemical treatments as an alternative to fungicide use to control postharvest decay incidence and fruit quality

chemicals postharvest treatments for integrating disease management of citrus

por frio y la mejor efficacia de los tratamientos fungicidas. *Todo Citrus*, 8(35), pp. 13-


Effect of hot water brushing, prochloraz treatment and waxing on the incidence of black spot decay caused by *Alternaria alternata* in mango fruits. *Postharvest Biology and Technology*, 15, pp. 165-174.


Quevedo, M.A., Sanico, R.T. & Baliad, M.E. (1991). The control of postharvest diseases of

Rafaeli, A., Kostukovshy, A. & Carmeli, D. (2006). Successful disinfestation of sap-beetle

Ragsdale, N.N. (2000). The impact of food quality protection act on the future of plant disease management. *Annual Review of Phytopathology,* 38, pp. 577-596. Reuveni, M. (2000). Efficacy of trifloxystrobin (Flint), a new strobilurin fungicide, in

Reuveni, M. (2001). Activity of trifloxystrobin against powdery and downy mildew diseases

Rosslenbroich, H. J. & Stuebler, D. (2000). *Botrytis cinerea* – history of chemical control and novel fungicides for its management. *Crop Protection*, 19, pp. 557-561. Roy, S., Conway, W.S., Watada, A.E., Sams, C.E., Erbe, E.F. & Wergin, W.P. (1994). Heat

Roy, S., Conway, W S., Watada, A.E., Sams, C.E., Erbe, E.F.m & Wergin, W.P. (1999).

Sala, J. M. (1998). Involvement of oxidative stress in chilling injury 885 in cold-stored

Sala, J.M. & Lafuente, M.T. (1999). Catalase in the heat induced chilling injury of cold-stored

Sala, J.M. & Lafuente, M.T. (2000). Catalase enzyme activity is related to tolerance of mandarin fruits to chilling. *Postharvest Biology and Technology*, 20, pp. 81-89. Schiffman-Nadel, M., Chalutz, E., Waks, J. & Lattar, F.S. (1972). Reduction of pitting of

Schiffman-Nadel, M., Chalutz, E., Waks, J. & Dagan, M. (1975). Reduction of chilling injury

Schirra, M. (2005). Postharvest pest management of horticultural crops by combined heat therapy and fungicide treatment. *Acta Horticulturae*, 682, pp. 2127-2132. Schirra, M. & D'hallewin, G. (1997). Storage performance of Fortune mandarins following

Schirra, M. & Mulas, M. (1993). Keeping quality of 'Oroblanco' grapefruit-type as affected by

Schirra, M. & Mulas, M. (1994). Storage of 'Monreal' clementines as affected by CaCl2 and TBZ postharvest treatments. *Agricoltura Mediterranea*, 124, pp. 238-248. Schirra, M. & Mulas, M. (1995a). Improving storability of 'Tarocco' oranges by postharvest hot-dip fungicide treatments. *Postharvest Biology and Technology*, 6, pp. 129-138.

mandarin fruit. *Postharvest Biology and Technology*, 13, pp. 255-161.

of grapevines. *Canadian Journal of Plant Pathology*, 23, pp. 52-59.

'Golden Delicious' apples. *HortScience*, 29, pp. 1056-1058.

*American Society for Horticultural Science*, 100, pp. 270-272.

hot water dips. *Postharvest Biology and Technology*, 10, pp. 229-238.

hot dip treatments. *Advances in Horticultural Science,* 7, pp. 73-76.

*and Technology*, 15, pp. 165-174.

*Phytoparasitica*, 34(2), pp. 204-212.

trees. *Crop Protection*, 19, pp. 335-341.

in apples. *HortScience*, 34, 121-124.

2410-2414.

395.

taro corms. *Tropical Science*, 31, pp. 359-364.

Effect of hot water brushing, prochloraz treatment and waxing on the incidence of black spot decay caused by *Alternaria alternata* in mango fruits. *Postharvest Biology* 

contaminations from organically grown dates using heat treatment. A case study.

controlling powdery mildews on apple, mango and nectarine, and rust on prune

treatment affects epicuticular wax structure and postharvest calcium uptake in

Changes in ultrastructure of the epicuticular wax and postharvest calcium uptake

hybrid Fortune mandarin fruits. *Journal of Agricultural and Food Chemistry*, 47, pp.

grapefruit by thiabendazole during long-term cold storage. *HortScience*, 7, pp. 394-

in grapefruit by thiabendazole and benomyl during long-term storage*. Journal of* 


Gialla) fruit following postharvest treatments with reduced doses of fungicide at 52 °C. *Journal of Agricultural and Food Chemistry*, 50, pp. 739-743.


Schirra, M., Delogu, G., Cabras, P., Angioni, A., D'hallewin, G., Veyrat, A., Marcos, J.F. &

Schirra, M., Cabras, P., Angioni, A. & Brandolini, V. (2002d). Residue levels and storage

Schirra, M., Mulas, M., Fadda, A. & Cauli, E. (2004). Cold quarantine responses of blood

Schirra, M., Mulas, M., Fadda, A., Mignani, I. & Lurie, S. (2005a). Chemical and quality traits

Schirra, M., D'Aquino, S., Palma, A., Angioni, A., Cabras, P. & Migheli, Q. (2006). Residues

Schirra, M., D'Aquino, S., Palma, A., Angioni, A. & Cabras, P. (2008a). Factors affecting the

Schirra, M.; D'Aquino, S.; Mulas, M.; Melis, R.A.M.; Giobbe, S.; Migheli, Q.; Garau, A.;

Schirra, M., Palma, A., D'Aquino, S., Angioni, A., Minello, E.V., Melis, M. & Cabras, P.

Schirra, M., D'Aquino, S., Cabras, P. & Angioni, A. (2009a). Influence of post-harvest

Schirra, M., D'Aquino, S., Migheli, Q., Pirisi, F.M. & Angioni, A. (2009b). Influence of

fludioxonil residues in fruit. *Journal of Food Protection*, 71(5): 967-972. Schirra, M., D'Aquino, S., Palma, A., Angioni, A. & Cabras, P. (2008c). Factors affecting the

*Agricultural and Food Chemistry*, 56, pp. 455-460.

*Contaminants*, 26 (7), pp. 1033-1037.

°C. *Journal of Agricultural and Food Chemistry*, 50, pp. 739-743.

*Journal of Agricultural and Food Chemistry, 50,* pp. 1461-1465.

6797.

6718-6724.

pp. 1646-1652.

10798.

10798.

*Technology*, 31, pp. 191-200.

Gialla) fruit following postharvest treatments with reduced doses of fungicide at 52

Candelas, L.G. (2002c). Complexation of Imazalil with β-cyclodestrin, residue uptake, persistence, and activity against *Penicillium* decay in Citrus fruit following postharvest dip treatment. *Journal of Agricultural and Food Chemistry*, 50, pp. 6790-

decay control in 'Star Ruby' grapefruit after dip treatments with azoxystrobin.

oranges to postharvest hot water and hot air treatments. *Postharvest Biology and* 

of 'Olinda' and 'Campbell' oranges after heat treatment at 44 or 46 °C for fruit fly disinfestation. *LWT - Lebensmittel-Wissenshaft und-Technologie*, 38, pp. 519-527. Schirra, M., D'Aquino, S., Palma, A., Marceddu, S., Angioni, A., Cabras, P., Scherm, B. &

Migheli, Q. (2005b). Residue level, persistence, and storage performance of Citrus fruit treated with fludioxonil. *Journal of Agricultural and Food Chemistry*, 53, pp.

of the quinone outside inhibitor fungicide trifloxystrobin after postharvest dip treatments to control *Penicillium* spp. on citrus fruit. *Journal of Food Protection*, 69,

sinergy of thiabendazole, sodium bicarbonate, and heat to control postharvest green mold of citrus fruit. *Journal of Agricultural and Food Chemistry*, 56, pp. 10793-

Angioni, A. & Cabras, P. (2008b). Efficacy of heat treatments with water and fludioxonil for postharvest control of blue and gray molds on inoculated pears and

synergy of thiabendazole, sodium bicarbonate, and heat to control postharvest green mould of citrus fruit. *Journal of Agricultural and Food Chemistry*, 56,. 10793-

(2008d). Influence of postharvest hot water treatment on nutritional and functional properties of kumquat (*Fortunella japonica* Lour. Swingle cv. Ovale) fruit. *Journal of* 

application rates of cyprodinil, treatment time and temperature on residue levels and efficacy in controlling green mould on 'Valencia' oranges. *Food Additives and* 

postharvest treatments with fludioxonil and soy lecithin co-application in

controlling blue and gray mold and fludioxonil residues in Coscia pears. *Food Additives and Contaminants. Part A*, 26, pp. 68-72.


Smilanick, J.L., Mansour, M.F., Mlikota Gabler, F. & Sorenson, D. (2008). Control of citrus

Smith, W.L.Jr. (1971). Control of brown rot and rhyzopus of inoculated peaches with hot water and hot chemical suspensions. *Plant Disease Reporter*, 55, pp. 228-230. Smith, W.L. & Anderson, R.E. (1975). Decay control of peaches and nectarines during and

Soto-Zamora, G., Yahia, E., Brecht, J.K. & Gardea, A. (2005). Effect of postharvest hot air

Spalding, D.H., Vaught, H.C., Day, R.H. & Brown, G. A. (1969). Control of blue mold rot

Spalding, D.H. & Reeder, W.F. (1972). Postharvest disorders of mangos as affected by fungicides and heat treatments. *Plant Disease. Reporter*, 56, pp. 751-753. Spalding, D.H. & Reeder, W.F. (1978). Controlling market diseases of mangos with heated benomyl. *Proceedings of Florida State Horticultural Society*, 91, pp. 186-187. Spalding, D.H. & Reeder, W.F. (1986). Decay and acceptability of mangos treated with

Sudisha, J., Niranjana, S.R., Sukanya, S.L., Girijamba, R., Lakshmi Devi, N. & Shekar Shetty,

Swart, S., H., Serfontein, J.J., Swart, C. & Labuschagne, C. (2009). Chemical control of

and low-temperature storage. *Journal of Food Science*, 70(5), pp. 337-341. Ticha, J., Hajslova, J., Jech, M., Honzicek, J., Lacina, O., Kohoutkova, J, Kocourek, V., Lansky,

Tomlin, C. (1997). *The pesticide manual: a world compendium.* British Crop Protection Council,

Vicente, A.R., Martinez, G.A., Civello, P.M. & Chaves, A.R. (2002). Quality of heat-treated

Wang, C.Y. (1993). Approaches to reduce chilling injury of fruits and vegetables.

Wang, C.Y. & Anderson, R.E. (1985) Progress on controlled atmosphere storage and

mildew of sunflower. *Journal of Pest Science*, 83, pp. 461-470.

during cold storage. *Food Control*, 19, pp. 247-256.

University, School of Agriculture. Vol. 1, pp. 221–228.

fungicides. *Postharvest Biology and Technology*, 47, pp. 226–238.

*Horticultural Science,* 100, pp. 84-86.

*Reporter,* 53, pp. 738-742.

1151.

Farnham (U.K.).

*Horticultural Review*, 15. pp. 63-95.

pp. 59-71.

*Wissenshaft und Technologie*, 38, pp. 657-663.

postharvest green mold and sour rot by potassium sorbate combined with heat and

after controlled atmosphere and air storage. *Journal of American Society for* 

treatment on the quality and antioxidant level in tomato fruit. *Lebensmittel-*

development in apples treated with heated and unheated fungicides. *Plant Disease* 

combinations of hot water, imazalil, and γ-radiation. *Plant Disease*, 70, pp. 1149-

H. (2010). Relative efficacy of strobilurin formulations in the control of downy

postharvest disease of mango: the effect of fludioxonil and prochloraz on soft brown rot, stem end rot and antrachnose. *Acta Horticulturae*, 820, pp. 503-509. Szejtli, J. (1988). Cyclodextrin Technology. *In:* Davies, J.E.D. (ed.). *Topics in Inclusion Science.*  Kluwer Academic Publishers, Dordrecht (The Netherlands), Vol. 8, pp. 1-450. Talcott, S.T., Moore, J.P., Lounds-Singleton, A.J. & Percival, S.S. (2005). Ripening associated

phytochemicals changes in mangos (*Mangifera indica*) following thermal quarantine

M., Kloutvorova, J. & Falta, V. (2008). Changes of pesticide residues in apples

strawberry fruit during refrigerated storage. *Postharvest Biology and Technology*, 25,

intermittent warming of peaches and nectarines. In: *Symposium series*, Oregon State


### **Role of MAP Kinase Signaling in Secondary Metabolism and Adaptation to Abiotic/Fungicide Stress in** *Fusarium*

Emese D. Nagygyörgy, László Hornok and Attila L. Ádám *Agricultural Biotechnology Center and Department of Plant Protection, Szent István University, Mycology Group of the Hungarian Academy of Sciences, Gödöllő, Hungary* 

#### **1. Introduction**

166 Fungicides – Beneficial and Harmful Aspects

Zhang, H., Zheng, X., Wang, L., Li, S. & Liu, R. (2005). Effect of yeast antagonist in

Zhang, H., Wang, L., Zheng, X. & Dong, Y. (2007). Effect of yeast antagonist in combination

Zhang, H., Wang, L., Huang, X., Dong, Y. & Zheng, X. (2008). Integrated control of

Zhao, Y., Tu, K., Su, J., Tu, S., Hou, Y., Liu, F. & Zou, X. (2009). Heat treatment in

Zhao, Y., Tu, K., Tu, S., Liu, M., Su, J. & Hou, Y. (2010). A combination of heat treatment and

Zhou, T., Xu, S.Y., Sun, D.W. & Wang, Z. (2002). Effects of heat treatment on postharvest

peaches. *International Journal of Food Microbiology*, 115, pp. 53-58.

*glutinis*. *Postharvest Biology and Technology*, 49, pp. 308-313.

quality of peaches. *Journal of Food Engineering*, 34, pp. 17-22.

*Journal of Food Engineering*, 78, pp. 281-287.

*Chemistry*, 57, pp. 7565-7570.

*of Food Microbiology*, 137, pp. 106-110.

combination with hot water dips on postharvest *Rhizopus* rot of strawberries.

with heat treatment on postharvest blue mold decay and *Rhizopus* decay of

postharvest blue mold decay of pears with hot water treatment and *Rhodotorula* 

combination with antagonistic yeast reduces diseases and elicits the active defense responses in harvested cherry tomato fruit. *Journal of Agricultural and Food* 

*Pichia guilliermondii* prevents cherry tomato spoilage by fungi. *International Journal* 

Phosphorylation by protein kinases including mitogen activated protein kinases (MAPKs) is a major signal transduction mechanism used by eukaryotic cells to regulate different functions, virtually almost all activities that define their phenotypic behavior. Considering their diverse cellular roles, it was not surprising to find that significant portions of eukaryotic genes are devoted to code for protein kinases. For example, the genome of *Saccharomyces cerevisiae,* the budding yeast contains 130 distinct protein kinase encoding genes, representing approximately 2% of the entire yeast genome (Hunter and Plowman, 1997). The human genome contains 518 protein kinase genes comprising 1.7% of the genome (Manning *et al.,* 2002).

The MAPK signal transduction pathways constitute a cascade of phosphorylation events that transmit extracellular signals from membrane-bound receptors to the nucleus. MAPKs are highly selective for phosphorylation of serine/threonine residues lying immediately Nterminal to a proline residue within a peptide substrate (Hanks and Hunter, 1995; Brábek and Hanks, 2004). MAP kinase cascades control almost all aspects of fungal growth, development, sexual and asexual reproduction, metabolism, proliferation and stress tolerance.

Two of the three MAP kinase pathways of filamentous fungi, the HOG1 (*h*igh *o*smolarity *g*lycerol according to yeast nomenclature) MAPK and the CWI (*c*ell *w*all *i*ntegrity) MAPK (homologous to Slt2/Mpk1 in yeast) pathways take part in abiotic stress tolerance including tolerance to salt, osmotic, oxidative, heat, cold, arsenite and citric acid stressors. Recently the HOG MAPK pathway has also been indicated in tunicamycin induced endoplasmic reticulum stress in *S. cerevisiae* (Torres-Quiroz *et al.,* 2010). The third MAPK route, the socalled *p*athogenicity *M*AP *k*inase (PMK) pathway is homologous to the mating/ filamentation Fus3/Kss1 MAPK pathway of the yeast. PMK is required for the infection process including penetration into the host cells and invasive growth. This pathway is also involved in the yeast-to-hyphal transition in dimorphic species. As far as studies on *Fusarium* species are concerned, the Fmk1 and the Gpmk1 MAP kinases of *Fusarium oxysporum* (Di Pietro *et al.,* 2001) and *Fusarium graminearum* (Jenczmionka *et al.,* 2003), respectively were also found to be essential for pathogenicity. These PMK-type MAP kinases regulates the expression of several genes encoding cell-wall degrading hydrolytic enzymes (Jenczmionka and Schäfer , 2005).

Although PMK-type MAP kinases were generally regarded to have not much role in stress adaptation, recent studies demonstrated that the pathogenicity MAPK pathway controls the oxidative stress response in *Cochliobolus heterostrophus* (Izumitsu *et al.,* 2009). These observations highlight a more complex nature of stress signaling in filamentous fungi as it was anticipated previously (Aguirre *et al.,* 2006).

We present here a functional analysis of ∆*Fvhog1* and ∆*Fvmk2* CWI MAPK mutants of *F. verticillioides* by comparing their sensitivity to different oxidative stressors. *Fusarium verticillioides* (teleomorph: *Gibberella moniliformis*) is a world-wide occurring pathogen of maize that synthesizes a range of secondary metabolites, including fumonisins and carotenoids. To the best of our knowledge this is the first report on the comparison of oxidative stress tolerance of different MAPK mutants of the same filamentous fungus species. We also found that both ∆*Fvhog1* and ∆*Fvmk2* CWI MAPK gene-disruption mutants of *F. verticillioides* resulted in increased sensitivity to methylglyoxal, a toxic glycolytic byproduct suggesting a double MAPK regulation of the cellular response to this compound. Secondary metabolite production is also regulated by different MAPK pathways in fungi: we provide here additional information on the recent findings available for fusaria. As the highly conserved fungicide signaling by fludioxonil is not dependent on the histidine kinase-HOG1 MAPK route in all filamentous fungi (Liu *et al.,* 2008) we compared the fludioxonil and hydrogen peroxide sensitivity of three *Fusarium* species. The extreme sensitivity *of F. graminearum* to fludioxonil and hydrogen peroxide was not associated with substantial changes in HOG MAPK mediated osmotic stress tolerance. We also found that ∆*hog1* mutants of two other *Fusarium* species showed fludioxonil tolerant and hydrogenperoxide sensitive phenotypes, similarly to other filamentous species.

#### **2. The role of HOG1 MAPK signaling in stress and fungicide tolerance of**  *Fusarium* **species**

Orthologues of the yeast HOG1 pathway genes have been identified either by functional or *in silico* analysis in several *Fusarium* species, including *F. graminearum, Fusarium proliferatum, F. oxysporum,* and *F. verticillioides* (Di Pietro *et al.,* 2001; Ochiai *et al.,* 2007; Ádám *et al.,* 2008a, b; Rispail *et al.,* 2009; Rispail and Di Pietro, 2010). In *F. proliferatum,* the HOG1 MAPK pathway plays a pivotal role in stress tolerance: this route takes part in salt, osmotic, heat, UV and oxidative (hydrogen peroxide) stress responses, but it is not required for invasive growth, sexual and asexual sporulation (Ádám *et al.,* 2008a, b). Osmotic stress caused a considerably higher rate of cell death in the ∆*Fphog1* MAPK gene disruption mutants as compared to the wild type strain. More importantly, when the fungi were subjected to osmotic (4% NaCl) stress, levels of reactive oxygen species (ROS), mitochondrial membrane permeability transition, nuclear disintegration and DNA fragmentation, four independent markers of programmed cell death (PCD) all showed significant increases in the ∆*Fphog1* mutants in comparison to the wild type strain suggesting that an important role of the functional Hog1 MAPK gene is attenuating apoptotic phenotypes under stress conditions (Ádám *et al.,* 2008a). Fig. 1. shows intense cell death symptoms and accumulation of ROS indicated by blue stained morphologically abnormal cells and green fluorescent cells (indicated by arrow), respectively in a ∆*Fphog1* gene-disruption mutant subjected to salt stress after adding 4%

respectively were also found to be essential for pathogenicity. These PMK-type MAP kinases regulates the expression of several genes encoding cell-wall degrading hydrolytic

Although PMK-type MAP kinases were generally regarded to have not much role in stress adaptation, recent studies demonstrated that the pathogenicity MAPK pathway controls the oxidative stress response in *Cochliobolus heterostrophus* (Izumitsu *et al.,* 2009). These observations highlight a more complex nature of stress signaling in filamentous fungi as it

We present here a functional analysis of ∆*Fvhog1* and ∆*Fvmk2* CWI MAPK mutants of *F. verticillioides* by comparing their sensitivity to different oxidative stressors. *Fusarium verticillioides* (teleomorph: *Gibberella moniliformis*) is a world-wide occurring pathogen of maize that synthesizes a range of secondary metabolites, including fumonisins and carotenoids. To the best of our knowledge this is the first report on the comparison of oxidative stress tolerance of different MAPK mutants of the same filamentous fungus species. We also found that both ∆*Fvhog1* and ∆*Fvmk2* CWI MAPK gene-disruption mutants of *F. verticillioides* resulted in increased sensitivity to methylglyoxal, a toxic glycolytic byproduct suggesting a double MAPK regulation of the cellular response to this compound. Secondary metabolite production is also regulated by different MAPK pathways in fungi: we provide here additional information on the recent findings available for fusaria. As the highly conserved fungicide signaling by fludioxonil is not dependent on the histidine kinase-HOG1 MAPK route in all filamentous fungi (Liu *et al.,* 2008) we compared the fludioxonil and hydrogen peroxide sensitivity of three *Fusarium* species. The extreme sensitivity *of F. graminearum* to fludioxonil and hydrogen peroxide was not associated with substantial changes in HOG MAPK mediated osmotic stress tolerance. We also found that ∆*hog1* mutants of two other *Fusarium* species showed fludioxonil tolerant and hydrogen-

enzymes (Jenczmionka and Schäfer , 2005).

was anticipated previously (Aguirre *et al.,* 2006).

*Fusarium* **species** 

peroxide sensitive phenotypes, similarly to other filamentous species.

**2. The role of HOG1 MAPK signaling in stress and fungicide tolerance of** 

Orthologues of the yeast HOG1 pathway genes have been identified either by functional or *in silico* analysis in several *Fusarium* species, including *F. graminearum, Fusarium proliferatum, F. oxysporum,* and *F. verticillioides* (Di Pietro *et al.,* 2001; Ochiai *et al.,* 2007; Ádám *et al.,* 2008a, b; Rispail *et al.,* 2009; Rispail and Di Pietro, 2010). In *F. proliferatum,* the HOG1 MAPK pathway plays a pivotal role in stress tolerance: this route takes part in salt, osmotic, heat, UV and oxidative (hydrogen peroxide) stress responses, but it is not required for invasive growth, sexual and asexual sporulation (Ádám *et al.,* 2008a, b). Osmotic stress caused a considerably higher rate of cell death in the ∆*Fphog1* MAPK gene disruption mutants as compared to the wild type strain. More importantly, when the fungi were subjected to osmotic (4% NaCl) stress, levels of reactive oxygen species (ROS), mitochondrial membrane permeability transition, nuclear disintegration and DNA fragmentation, four independent markers of programmed cell death (PCD) all showed significant increases in the ∆*Fphog1* mutants in comparison to the wild type strain suggesting that an important role of the functional Hog1 MAPK gene is attenuating apoptotic phenotypes under stress conditions (Ádám *et al.,* 2008a). Fig. 1. shows intense cell death symptoms and accumulation of ROS indicated by blue stained morphologically abnormal cells and green fluorescent cells (indicated by arrow), respectively in a ∆*Fphog1* gene-disruption mutant subjected to salt stress after adding 4% (w/v) NaCl to the culture medium. Similarly to ∆*hog1* mutants of other fungi, like *Neurospora crassa*, *Aspergillus nidulans*, *C. heterostrophus* and *Colletotrichum lagenarium* the ∆*Fphog1* MAPK mutants of *F. proliferatum* became tolerant to phenylpyrrole and dicarboximide fungicides (Zhang *et al.,* 2002; Noguchi *et al.,* 2004; Yoshimi *et al.,* 2005; Ádám *et al.,* 2008b; Hagiwara *et al.,* 2009). Although the exact mode of action of these compounds is still unclear, the finding that heterologous expression of Hik1, the histidine kinase (HK) gene of *Magnaporthe oryzae* in the yeast, *S. cerevisiae* that contain only one HK gene, Sln1 confers susceptibility in this otherwise fludioxonil-resistant organism, suggests that class III HKs, located upstream of the HOG1 MAPK cascade are possible targets of this fungicide (Motoyama *et al.,* 2005). The class III HKs responsible for elevated osmo-tolerance and increased fludioxonil sensitivity in filamentous fungi are not the orthologues of Sln1 of the yeast (Catlett *et al.,* 2003). Inactivation of Fhk1, a class III HK in *F. oxysporum* resulted in osmo-sensitivity and resistance to phenylpyrrole and dicarboximide fungicides (Rispail and Di Pietro, 2010). The increased tolerance of the ∆*Fphog1* mutants of *F. proliferatum* to fludioxonil and vinclozoline (Ádám *et al.,* 2008b) suggests that functional HK-HOG1 MAPK pathway is required for sensitive response to these fungicides in *Fusarium* species. *In silico* analysis of HKs by reciprocal BLASTP searches in *Fusarium* genome sequences led to the identification of Fhk1 (FOXG\_01684) orthologues both in *F. verticillioides* (hypothetical protein FVEG\_08048) and *F*. *graminearum* (hypothetical protein FGSG\_07118) (Nagygyörgy and Ádám, unpublished).

Fig. 1. Double staining of *Fusarium proliferatum* ∆*Fphog1*-*24* gene-disruption MAP kinase mutant with 2,7-dichlorodihydrofluorescein diacetate (DCHFDA) and Evans blue after NaCl (4% w/v) exposition. Intensive green fluorescence (indicated by arrow) and dark blue discoloration of the cells indicate accumulation of reactive oxygen species (ROS) and cell death, respectively.

HKs have five HAMP (*h*istidine kinase, *a*denylate cyclase, *m*ethyl binding protein and *p*hosphatase) repeats: mutations in these sequences are responsible for the increased osmosensitivity and fungicide resistance of *N. crassa*, *C. heterostrophus*, *Alternaria brassicicola* and *Botrytis cinerea* (Ochiai *et al.,* 2001; Yoshimi *et al.,* 2004; Motoyama *et al.,* 2005; Viaud *et al.,* 2006). Recent microarray analyses have further shown that the transcriptional response to fludioxonil depends on a Hog1 orthologue in *A. nidulans*. This response overlaps, in part with the transcriptional response to hyperosmotic stress but depends on factors other than the AtfA transcription factor responsible for conidial stress tolerance (Hagiwara *et al.,* 2009). Thus the identification of transcription factor(s), that are located downstream of Hog1 MAPK and influence gene expression response to fludioxonil requires further studies.

Although fungicide signaling by fludioxonil is highly conserved in filamentous fungi, response to this compound is still not entirely dependent on the HK-HOG1 MAPK route in all species. For example, the ∆*sak1* (Hog1 orthologue) knockout mutants of *B. cinerea* maintained their sensitive phenotype to fludioxionil (Liu *et al.,* 2008) indicating the complex nature of this signaling pathway. On-going research of our laboratory on fludioxonil sensitivity of three *Fusarium* species with available genome sequences (*F. graminearum* PH-1/NRRL 31084, *F. oxysporum* 4287 and *F. verticillioides* FGSC 7600; http: //www.broadinstitute. org/annotation/genome/Fusariumgroup/MultiHome.html) led to somewhat surprising results (Fig. 2).

Fig. 2. Growth sensitivity of three *Fusarium* species: *Fusarium oxysporum* 4287, *Fusarium verticillioides* FGSC 7600 and *Fusarium graminearum* PH-1/NRRL 31084 against different stressors (a-d) and sensitivity of ∆*hog1* mutants of *F. verticillioides* FGSC 7603 and *F. proliferatum* FGSC 2287 (∆*Fvhog1-14* and ∆*Fphog1-24,* respectively*)* against fludioxonil (e-f). Five-five μl of indicated concentrations of conidia (cells/ml) was spotted on (a, e) complex medium (CM) agar plates and CM agar plates supplemented with (b, f) 10 μg/ml fludioxonil, (c) 4% (w/v) NaCl and (d) 2 M sorbitol. Incubation time was 3 days for all plates at 24 oC.

HKs have five HAMP (*h*istidine kinase, *a*denylate cyclase, *m*ethyl binding protein and *p*hosphatase) repeats: mutations in these sequences are responsible for the increased osmosensitivity and fungicide resistance of *N. crassa*, *C. heterostrophus*, *Alternaria brassicicola* and *Botrytis cinerea* (Ochiai *et al.,* 2001; Yoshimi *et al.,* 2004; Motoyama *et al.,* 2005; Viaud *et al.,* 2006). Recent microarray analyses have further shown that the transcriptional response to fludioxonil depends on a Hog1 orthologue in *A. nidulans*. This response overlaps, in part with the transcriptional response to hyperosmotic stress but depends on factors other than the AtfA transcription factor responsible for conidial stress tolerance (Hagiwara *et al.,* 2009). Thus the identification of transcription factor(s), that are located downstream of Hog1 MAPK and influence gene expression response to fludioxonil requires further studies.

Although fungicide signaling by fludioxonil is highly conserved in filamentous fungi, response to this compound is still not entirely dependent on the HK-HOG1 MAPK route in all species. For example, the ∆*sak1* (Hog1 orthologue) knockout mutants of *B. cinerea* maintained their sensitive phenotype to fludioxionil (Liu *et al.,* 2008) indicating the complex nature of this signaling pathway. On-going research of our laboratory on fludioxonil sensitivity of three *Fusarium* species with available genome sequences (*F. graminearum* PH-1/NRRL 31084, *F. oxysporum* 4287 and *F. verticillioides* FGSC 7600; http: //www.broadinstitute. org/annotation/genome/Fusariumgroup/MultiHome.html) led to somewhat surprising results (Fig. 2).

**107 106 105 104**

Fig. 2. Growth sensitivity of three *Fusarium* species: *Fusarium oxysporum* 4287, *Fusarium verticillioides* FGSC 7600 and *Fusarium graminearum* PH-1/NRRL 31084 against different stressors (a-d) and sensitivity of ∆*hog1* mutants of *F. verticillioides* FGSC 7603 and *F. proliferatum* FGSC 2287 (∆*Fvhog1-14* and ∆*Fphog1-24,* respectively*)* against fludioxonil (e-f). Five-five μl of indicated concentrations of conidia (cells/ml) was spotted on (a, e) complex medium (CM) agar plates and CM agar plates supplemented with (b, f) 10 μg/ml fludioxonil, (c) 4% (w/v)

NaCl and (d) 2 M sorbitol. Incubation time was 3 days for all plates at 24 oC.

**bc d**

**f** 

**107 106 105 104**

**107 106 105 104 107 106 105 104**

**a**

**107 106 105 104**

**e**

**107 106 105 104**

*F.v*. 7600

*F.o*. 4287

*F.g.* 31084

*F.v.* 7603

∆*Fvhog1*

*F.p.* 2287

∆*Fphog1*

*F. graminearum* showed increased sensitivity to this fungicide as compared to the other two species (Fig. 2a and b), but fludioxonil sensitivity was not accompanied with substantial changes in osmo-sensitivity of this fungus (Fig. 2c and d). On the other hand, ∆*hog1* mutants of *F. proliferatum* and *F. verticillioides* showed fludioxonil tolerance (Fig. 2e and f), paralleled with elevated osmo-sensitivity. Although these results do not exclude the involvement of the HK-HOG1 MAPK pathway in these phenotypes, further studies are needed to a better understanding of fungicide stress responses in *Fusarium* species.

#### **3. The role of MAPK pathways in secondary metabolism of** *Fusarium* **species**

In addition to its role in stress and fungicide tolerance, the HOG1 MAPK pathway plays also an important role in the regulation of secondary metabolism in different *Fusarium* species (Ochiai *et al.,* 2007; Kohut *et al.,* 2009). Disruption of either Fgos2 (a HOG-type MAPK orthologue) or Fgos4 (encoding a MAPK kinase) or Fgos5 (encoding a MAPK kinase kinase) blocked trichotecene production in *F. graminearum* and substantially reduced expression of the trichotecene gene cluster. On the other hand, amounts of aurofusarin were increased in all three types of mutants (Ochiai *et al.,* 2007). Deoxynivalenol production is controlled by Mgv1 CWI MAPK in *F. graminearum* (Hou *et al.,* 2002). Nitrogen depletion induced the production of fumonisin B1, a polyketide derivative mycotoxin and increased the expression of fuminisin biosynthesis genes in *F. proliferatum*. Under nitrogen starvation (absence of any N-source) conditions deletion of Fphog1, a HOG-type MAP kinase gene resulted in further increases in FUM1 and FUM8 gene expression, as well as fumonisin B1 production suggesting that this response is mediated via the HOG-type MAPK pathway in *F. proliferatum* (Kohut *et al.,* 2009). In a more recent study Fvmk1, a PMK-type MAPK gene was identified as a positive regulator of fumonisin B1 production in *F. verticillioides* (Zhang *et al.,*  2011). On the contrary, fumonisin B1 production was not regulated by cAMP signaling either in *F. proliferatum* (Kohut *et al.,* 2010) or *F. verticillioides* (Choi and Xu, 2010). This signaling route regulates, however negatively regulates the production of bikaverin, another polyketide metabolite in *Fusarium* species (Kohut *et al.,* 2010; Choi and Xu, 2010; García-Martínez *et al.,* 2011). Moreover, the production of another secondary metabolite such as carotenoids is upregulated in *Fusarium* species not only by cAMP signaling (García-Martínez *et al*., 2011) but other regulatory elements related to sexual reproduction (Ádám *et al.,* 2011).

#### **4. Complexity of oxidative stress signaling in fungi: Role of the HOG1 and the CWI MAPK pathways**

Previous research with different species indicated that, besides the HOG MAPK pathway (Aguirre *et al.,* 2006; Du *et al.,* 2006; Ádám *et al.,* 2008a), the CWI MAPK pathway (Krasley *et al.,* 2006; Valiante *et al.,* 2007) also has a role in oxidative stress tolerance of fungi. To compare the particularities of the two pathways we generated both ∆*hog1* (∆*Fvhog1*) and CWI MAPK (∆*Fvmk2*) gene-disruption mutants in a single fungus species, *F. verticillioides* as we have described earlier (Ádám *et al.,* 2008a). These mutants were tested for oxidative stress tolerance in conidial dilution assay using hydrogen peroxide, menadione, diamide and methylglyoxal as stressors. Although all of these compounds elicit finally oxidative stress, the mechanisms they do this are different. Hydrogen peroxide induces lipid peroxidation, protein and DNA damage directly or indirectly and contributes to the formation of hydroxyl radicals (OH**. )** via the Fenton reaction (Thön *et al.,* 2007). Menadione is a redox cycling reagent that acts by generating superoxide radicals (O2 **.-)** using NADPH as a cofactor. Diamide causes depletion of the reduced glutathione pool and perturbation of the redox balance of cells; this compound also reacts with sulfhydryl groups of proteins in a reversible way (Pócsi *et al.,* 2005; Thön *et al.,* 2007). Methylglyoxal is a highly toxic natural glycolytic by-product interacting with proteins in a reversible way and, at higher concentrations (8-10 mM) it can deplete the glutathione pool in yeast cells (Aguilera *et al.,* 2005).

Fig. 3*.* Differential sensitivity of ∆*Fvhog1-14* and ∆*Fvmk2-16* CWI MAPK mutants of *Fusarium verticillioides* FGSC 7603 against oxidative stressors. Five-five μl of indicated concentrations of conidia (cells/ml) was spotted on (a) complex medium (CM) agar plate and CM agar plates supplemented with (b) 4% (w/v) NaCl, (c) 2 M sorbitol (S), (d) 2 mM hydrogen peroxide (H2O2), (e) 5 mM methylglyoxal (MG), (f) 10 mM methylglyoxal (MG), (g) 0,5 mM diamide (D) and (h) 0,03 mM menadione (M). Incubation time was 3 days for all plates at 24 oC.

The ∆*Fvhog1* mutant of *F. verticillioides* was highly sensitive to the osmotic stressors, sodium chloride and sorbitol (Fig 3a, b and c), similar to our former results with *F. proliferatum* ∆*hog1*  mutants (Ádám *et al.,* 2008a). On the contrary, the ∆*Fvmk2* mutant showed no elevated osmo-sensitivity (Fig 3a, b and c). The ∆*Fvhog1* mutant was sensitive not only to osmotic stressors but, as well as to hydrogen peroxide and methylglyoxal. However, this mutation caused no change in menadione and diamide sensitivity (Fig. 3a, d, e, f g and h). This is a first report on the involvement of HOG MAPK pathway in methylglyoxal tolerance of a filamentous species. Formerly Aguilera *et al.* (2005) reported on methylglyoxal sensitivity of ∆*hog1* mutants of *S. cerevisiae*. ∆*Hog1* mutants of filamentous species showed different oxidative stress sensitivity. Mutants of *A. fumigatus* lacking the MAP kinase ∆*sakA/*∆*hog1* were more sensitive to H2O2 and menadione compared to wild type strain (Du *et al.,* 2006).

the Fenton reaction (Thön *et al.,* 2007). Menadione is a redox cycling reagent that acts by

the reduced glutathione pool and perturbation of the redox balance of cells; this compound also reacts with sulfhydryl groups of proteins in a reversible way (Pócsi *et al.,* 2005; Thön *et al.,* 2007). Methylglyoxal is a highly toxic natural glycolytic by-product interacting with proteins in a reversible way and, at higher concentrations (8-10 mM) it can deplete the glutathione pool in

**10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup> 10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup> 10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup> 10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup>**

**10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup> 10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup> 10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup>** 10<sup>7</sup> 10<sup>6</sup> 10<sup>5</sup> 10<sup>4</sup>

**e fg** h

CM + MG CM + MG CM + D CM + M

Fig. 3*.* Differential sensitivity of ∆*Fvhog1-14* and ∆*Fvmk2-16* CWI MAPK mutants of *Fusarium verticillioides* FGSC 7603 against oxidative stressors. Five-five μl of indicated concentrations of conidia (cells/ml) was spotted on (a) complex medium (CM) agar plate and CM agar plates supplemented with (b) 4% (w/v) NaCl, (c) 2 M sorbitol (S), (d) 2 mM hydrogen peroxide (H2O2), (e) 5 mM methylglyoxal (MG), (f) 10 mM methylglyoxal (MG), (g) 0,5 mM diamide (D)

The ∆*Fvhog1* mutant of *F. verticillioides* was highly sensitive to the osmotic stressors, sodium chloride and sorbitol (Fig 3a, b and c), similar to our former results with *F. proliferatum* ∆*hog1*  mutants (Ádám *et al.,* 2008a). On the contrary, the ∆*Fvmk2* mutant showed no elevated osmo-sensitivity (Fig 3a, b and c). The ∆*Fvhog1* mutant was sensitive not only to osmotic stressors but, as well as to hydrogen peroxide and methylglyoxal. However, this mutation caused no change in menadione and diamide sensitivity (Fig. 3a, d, e, f g and h). This is a first report on the involvement of HOG MAPK pathway in methylglyoxal tolerance of a filamentous species. Formerly Aguilera *et al.* (2005) reported on methylglyoxal sensitivity of ∆*hog1* mutants of *S. cerevisiae*. ∆*Hog1* mutants of filamentous species showed different oxidative stress sensitivity. Mutants of *A. fumigatus* lacking the MAP kinase ∆*sakA/*∆*hog1* were more sensitive to H2O2 and menadione compared to wild type strain (Du *et al.,* 2006).

and (h) 0,03 mM menadione (M). Incubation time was 3 days for all plates at 24 oC.

**a bc d**

CM + NaCl CM + S CM + H2O2

**.-)** using NADPH as a cofactor. Diamide causes depletion of

generating superoxide radicals (O2

yeast cells (Aguilera *et al.,* 2005).

*F. v.* 7603

∆*Fvmk2*

∆*Fvhog1*

*F. v.* 7603

∆*Fvhog1*

∆*Fvmk2*

CM

In *F. oxysporum* the *Fhk1* HK mutant, deficient in an upstream element of the HOG1 MAPK pathway is sensitive to menadione induced oxidative stress but not to H2O2 (Rispail and Di Pietro, 2010).

Results obtained with the ∆*Fvmk2* CWI MAPK mutants were completely different: they showed no elevated osmo-sensitivity, but their sensitivity to methylglyoxal and diamide increased as compared to the wild type strain (Figs. 3a, b, c, d, e, f, g and h). According to these results, the cellular response to methylglyoxal is regulated by both the HOG1 and CWI MAPK pathway, but the other stressors are signaled separately either by the HOG1 or CWI MAPK pathway. As both methylglyoxal and diamide interact mainly with glutathione metabolism, the possible role of CWI MAPK is to maintain glutathione pools under stress conditions. As regarding the oxidative stress sensitivity of other species, CWI MAPKs played a fluctuating but positive regulatory role in stress tolerance. An exceptional case is *A. fumigatus*: the deletion of MpkA CWI MAPK gene resulted in increased H2O2 tolerance and sensitivity to menadione and diamide (Valiante *et al.,* 2007). In the case of *S. cerevisiae* ∆*slt2/*∆*mpk1* mutants were sensitive to H2O2 (Krasley *et al.,* 2006). But in *C. albicans* and *S. pombe*, deletion mutants of ∆*mkc1* and ∆*pmk1*, respectively were sensitive to diamide but not to H2O2 and menadione (Navarro-Garcia *et al.,* 2005; Madrid *et al.,* 2006).

#### **5. Sensitivity of different** *Fusarium* **species to hydrogen peroxide**

We compared the hydrogen peroxide sensitivity of three *Fusarium* species, *F. graminearum* PH-1/NRRL 31084, *F. oxysporum* 4287 and *F. verticillioides* FGSC 7600 with available genome sequences (http://www.broadinstitute.org/annotation/genome/Fusariumgroup/ Multi Home.html). *F. gaminearum*, a causal agent of head blight of wheat and stalk/cob rot of maize was the most sensitive to this oxidative stressor both in a decimal conidium dilution assay (Valiante *et al.,* 2007) and in radial growth test (Ádám *et al.,* 2008a). Mycelial growth and conidial germination of this fungus was more strongly inhibited by 5-50 mM and 2 mM H2O2 concentrations as compared to *F. oxysporum* and *F. verticillioides* (Fig 4A, 4B), other two plant pathogenic species causing vascular wilt of a wide range of plants and maize cob rot, respectively. In a previous study (Nicolaou *et al.,* 2009), oxidative stress tolerance of 18 fungal species originating from different ecological niches and phylogenetic positions were compared and plant pathogenic species, like *Ustilago maydis*, *F. graminearum* and *M. grisea* were found to be relatively sensitive to oxidative stressors, including hydrogen peroxide. This result was somewhat surprising as both plant and animal pathogens that are exposed to massive oxidative and/or nitrosative stress by the host cells in many host-pathogen interactions (Brown *et al.,* 2009) would have been expected to acquire improved levels of oxidative stress tolerance during their evolution. When the hydrogen peroxide sensitivity tests were extended to other two *Fusarium* species, *F. fujikuroi* MP-C and *F. proliferatum*  FGSC 2287, causing the bakane disease of rice and crown and root rot of a wide range of plants, respectively they also showed higher levels of hydrogen peroxide tolerance as compared to that of *F. graminearum.* The increased H2O2 sensitivity of *F. graminearum* can be putatively explained by the long saprophytic phase in the life cycle of this species. During the saprophytic phase, *F. graminearum* lives and propagates on dead tissues and in this niche, the fungus is much less exposed to oxidative stress influence than the other species, that spend much of their life cycle inside living plant tissues either as endophytes or vascular colonizers.

Fig. 4. Growth sensitivity of three *Fusarium* species: *Fusarium verticillioides* FGSC 7600, *Fusarium oxysporum* 4287 and *Fusarium graminearum* PH-1/NRRL 31084 against hydrogen peroxide (H2O2) in radial growth test (A) and decimal conidial dilution assay (B). In (B) fivefive μl of indicated concentrations of conidia (cells/ml) was spotted on complex medium (CM) agar plate and CM agar plates supplemented with the indicated concentrations of hydrogen peroxide (H2O2). Incubation time was 5 days for plates in (A) and 3 days for plates in (B) at 24 oC.

In another approach we have studied the role of cAMP signaling in oxidative stress response of different *Fusarium* species. Previous studies in *N. crassa* demonstrated that cAMP signaling and HOG1 MAPK signaling play opposite role in respect to oxidative stress response: disturbances in the cAMP and the HOG1 pathways result in increased and decreased H2O2 tolerance, respectively. In *F. proliferatum* and *F*. *verticillioides,* disruption of Acy1, the adenylyl cyclase gene resulted in enhanced resistance to heat shock and oxidative stress (Kohut *et al.,* 2010; Choi and Xu, 2010). However, in contrast to these data, the *acyA*mutants of *F. fujikuroi* MP-C were more sensitive to H2O2 than the wild type (García-Martínez *et al.,* 2011). This finding suggests the high versatility of the cAMP signaling route even in closely related fungi. These differences are particularly important, if we consider that heat shock and oxidative stress pathways have at least partially overlapping signaling routes and regulated by the same transcription factors in yeast (Ikner and Shiozaki, 2005).

#### **6. Conclusion**

174 Fungicides – Beneficial and Harmful Aspects

CM 5 10 25 50 M H 0

CM 5 10 25 50 mM H202

**107 106 105 104 107 106 105 104 107 106 105 104**

CM+ 1 mM H202 CM+2 mM H202

*F.v.* 7600

**A**

*F.o.* 4287

*F.g.* 31084

**B**

*F.v.* 7600

*F.o.* 4287

*F.g.* 31084

in (B) at 24 oC.

CM

Fig. 4. Growth sensitivity of three *Fusarium* species: *Fusarium verticillioides* FGSC 7600, *Fusarium oxysporum* 4287 and *Fusarium graminearum* PH-1/NRRL 31084 against hydrogen peroxide (H2O2) in radial growth test (A) and decimal conidial dilution assay (B). In (B) fivefive μl of indicated concentrations of conidia (cells/ml) was spotted on complex medium (CM) agar plate and CM agar plates supplemented with the indicated concentrations of hydrogen peroxide (H2O2). Incubation time was 5 days for plates in (A) and 3 days for plates

In another approach we have studied the role of cAMP signaling in oxidative stress response of different *Fusarium* species. Previous studies in *N. crassa* demonstrated that cAMP signaling and HOG1 MAPK signaling play opposite role in respect to oxidative stress response: disturbances in the cAMP and the HOG1 pathways result in increased and decreased H2O2 tolerance, respectively. In *F. proliferatum* and *F*. *verticillioides,* disruption of Acy1, the adenylyl cyclase gene resulted in enhanced resistance to heat shock and oxidative stress (Kohut *et al.,* 2010; Choi and Xu, 2010). However, in contrast to these data, the *acyA*mutants of *F. fujikuroi* MP-C were more sensitive to H2O2 than the wild type (García-Martínez *et al.,* 2011). This finding suggests the high versatility of the cAMP signaling route even in closely related fungi. These differences are particularly important, if we consider that heat shock and oxidative stress pathways have at least partially overlapping signaling routes and regulated by the same transcription factors in yeast (Ikner and Shiozaki, 2005).

Functional analysis of orthologous signal transduction genes in different filamentous fungal species highlighted the complex nature of stress signal transduction. This is especially true for oxidative stress signaling, where all fungal MAPK cascades, the HOG1, CWI and PMK MAPK pathways participate and interact in this regulatory network depending on the fungal species. One of the oxidative agents, mehylglyoxal, a toxic by-product of glycolysis is signaled either by the HOG1 MAPK or CWI MAPK pathway in *F. verticillioides*. All these MAPK cascades are also involved either in positive or negative regulation of secondary metabolite production including mycotoxins in different *Fusarium* species. The high versatility of oxidative stress and secondary metabolite signaling by the above-mentioned MAPK pathways and the cAMP-PKA pathway in different *Fusarium* species denotes that stress signaling is exposed to rapid evolution to tune stress responses in a niche-specific manner, independently of the phylogenetic position of a given species.

#### **7. Acknowledgement**

This research was supported by an OTKA grant (National Scientific Research Council of Hungary, K 76067). We are indebted for support from the Office for Subsidized Research Units of the Hungarian Academy of Sciences. A.L.Á. is also grateful to the Hungarian-Spanish Bilateral Inter-Governmental S & T Project (OMFB 00666/2009).

#### **8. References**


Choi, Y.E. & Xu, J.R. (2010). The cAMP signaling pathway in *Fusarium verticillioides* is

Di Pietro, A., Garcia-Maceira, F. I., Meglecz, E., & Roncero, M. I. (2001). A MAP kinase of the

Du, C., Sarfati, J., Latgé, J.P. & Calderone, R. (2006). The role of the sakA (Hog1) and tcsB

García-Martínez, J., Ádám, A.L. & Avalos, J. (2011). Adenylyl cyclase plays a regulatory role

Hagiwara, D., Asano, Y., Marui, J., Yoshimi, A., Mizuno, T. & Abe, K. (2009). Transcriptional

Hou, Z., Xue, C., Peng, Y., Katan, T., Kistler, H. C. & Xu, J.R. (2002): A mitogen-activated

Hunter, T. & Plowman, G. (1997). The protein kinases of budding yeast: six score and more.

Ikner, A. & Shiozaki, K. (2005). Yeast signaling pathways in the oxidative stress response. *Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis* 6: 13-27. Izumitsu, K., Yoshimi, A., Kubo, D., Morita, A., Saitoh, Y. & Tanaka, C. (2009). The MAPKK

Jenczmionka, N. J, Maier, F. J., Lösch, A.P. & Schäfer, W. (2003). Mating, conidiation and

Jenczmionka, N.J. & Schäfer, W. (2005). The Gpmk1 kinase of *Fusarium graminearum* regulates the induction of specific secreted enzymes. *Current Genetics* 47: 29–36. Kohut, G., Ádám, A.L., Fazekas, B. & Hornok, L. (2009). N-starvation stress induced FUM

Kohut, G., Oláh, B., Ádám, A.L., García-Martínez, J. & Hornok, L. (2010). Adenylyl cyclase

colonization in *Fusarium proliferatum*. *Journal of Basic Microbiology* 50: 59-71.

fludioxonil and osmotic stress. *Fungal Genetics and Biology* 46: 868–878. Hanks, S.K. & Hunter, T. (1995). Protein kinases 6. The eukaryotic protein kinase

production. *Molecular Plant-Microbe Interactions* 23: 522-533.

pathogenesis. *Molecular Microbiology* 39: 1140-1152.

4765, Asilomar, California, USA, March, 15-20, 2011

211–218.

576-596.

95.

69.

*Interactions* 15: 1119–1127.

*Current Genetics* 55: 439–448.

*Trends in Biochemical Sciences* 22: 18–22.

important for conidiation, plant infection, and stress responses but not fumonisin

vascular wilt fungus *Fusarium oxysporum* is essential for root penetration and

(sln1) genes in the oxidant adaptation of *Aspergillus fumigatus*. *Medical Mycology* 44:

in development, stress and secondary metabolism in *Fusarium fujikuroi*. *26th Fungal Genetics Conference at Asilomar,* Fungal Genetics Reports, S58, p. 205, ISBN 1941-

profiling of *Aspergillus nidulans* HogA MAPK signaling pathway in response to

superfamily: kinase (catalytic) domain structure and classification. *FASEB Journal* 8:

protein kinase gene (MGV1) in *Fusarium graminearum* is required for female fertility, heterokaryon formation, and plant infection. *Molecular Plant-Microbe*

kinase ChSte11 regulates sexual/asexual development, melanization, pathogenicity, and adaptation to oxidative stress in *Cochliobolus heterostrophus*.

pathogenicity of *Fusarium graminearum*, the main causal agent of the head-blight disease of wheat, are regulated by the MAP kinase Gpmk1. *Current Genetics* 43: 87–

gene expression and fumonisin production is mediated via the HOG-type MAPK pathway in *Fusarium proliferatum*. *International Journal of Food Microbiology* 130: 65-

regulates heavy metal sensitivity, bikaverin production and plant tissue


## **Fungicides as Endocrine Disrupters in Non-Target Organisms**

Marco F. L. Lemos1,2, Ana C. Esteves2 and João L. T. Pestana2 *1ESTM and GIRM, Polytechnic Institute of Leiria 2Department of Biology and CESAM, University of Aveiro Portugal* 

#### **1. Introduction**

178 Fungicides – Beneficial and Harmful Aspects

Thön, M., Al-Abdallah, Q., Hortschansky, P. & Brakhage, A.A. (2007). The thioredoxin

oxidative stress response. *Journal of Biologycal Chemistry* 282: 27259–27269. Torres-Quiroz, F., García-Marqués, S., Coria, R., Randez-Gil, F. & Prieto, J.A. (2010). The

system of the filamentous fungus *Aspergillus nidulans:* impact on development and

activity of yeast Hog1 MAPK is required during endoplasmic reticulum stress induced by tunicamycin exposure. *Journal of Biological Chemistry* 285: 20088-20096. Yoshimi, A., Tsuda, M. & Tanaka, C. (2004). Cloning and characterization of the histidine

kinase gene Dic1 from *Cochliobolus heterostrophus* that confers dicarboximide

positive regulator of Hog1-type mitogen-activated protein kinase in filamentous

activated protein kinase MpkA of *Aspergillus fumigatus* regulates cell wall signaling

Legendre, L. (2006). A class III histidine kinase acts as a novel virulence factor in

kinase gene regulates conidiation, pathogenesis, and fumonisin production in

resistance: the *Neurospora crassa* os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue. *Applied and Environmental Microbiology* 68: 532-538.

resistance and osmotic adaptation. *Molecular Genetics Genomics* 271: 228–236. Yoshimi, A., Kojima, K., Takano, Y. & Tanaka, C. (2005). Group III histidine kinase is a

Valiante, V., Heinekamp, T., Jain, R., Härtl, A. & Brakhage, A.A. (2007). The mitogen-

Zhang, Y., Choi, Y.E., Zou, X. & Xu, J.R. (2011). The FvMK1 mitogen-activated protein

Zhang, Y. , Lamm, R. , Pillonel, C., Lam, S. & Xu, J.R. (2002). Osmoregulation and fungicide

and oxidative stress response. *Fungal Genetics and Biology* 45: 618-627. Viaud, M., Fillinger, S., Liu, W., Polepalli, J.S., Le Pêcheur, P., Kunduru, A.R., Leroux, P. &

*Botrytis cinerea*. *Molecular Plant-Microbe Interactions* 19: 1042-1050.

*Fusarium verticillioides. Fungal Genetics and Biology* 48: 71-9.

fungi. *Eukaryotic Cell 4:* 1820-1828.

In the past few decades concern has been growing about the possible consequences of environmental exposure to a group of chemicals (natural, synthetic, industrial chemicals or by-products) suspected to alter the functions of the endocrine system and consequently causing adverse health effects in an intact organism, its offspring, or (sub) population (European Commission, 2007), the Endocrine Disruptor Compounds (EDCs). Today, this concern is focused both on human health and on the impacts on wildlife and the environment, being already a priority in research and legislation within the European Union (European Commission, 1999, 2001, 2004, 2007), the US Environmental Protection Agency (Kavlock et al., 1996; U.S. EPA, 1998; Harding et al., 2006) and the World Health Organization (Damstra et al., 2002).

All vertebrate and invertebrate taxa use chemical signalling molecules (hormones). Changes of the endocrine function can occur when EDCs interfere with the synthesis, secretion, transport, action or elimination of natural hormones, which are responsible for homeostasis mechanisms, reproduction, growth and/or behaviour. These interferences can be caused by the direct binding of EDCs to hormone receptors – acting as hormone mimics (agonists) or as "anti-hormones" (antagonists) – or indirectly through modulation of endogenous hormone levels by interfering with biochemical processes associated with the production, availability, or metabolism of hormones or also by the modulation of their receptors (Rodriguez et al., 2007).

#### **2. Endocrine active compounds and invertebrates**

Although invertebrates dominate over 95% of the known animal species and represent more than 30 different phyla within the animal kingdom (Ruppert et al., 2003), potential effects of suspected EDCs on the various invertebrate endocrine systems have not been studied with comparable intensity as in vertebrates, especially in fish (e.g. Baker et al., 2009), reptiles (e.g. De Falco et al., 2007), amphibians (e.g. Kaneko et al., 2008), birds (e.g. Halldin et al., 2001), and mammals (e.g.Tabuchi et al., 2006).

Even though the issue of Endocrine Disruption (ED) in invertebrates received some scientific interest in the past, only a limited number of confirmed cases were reported (deFur et al., 1999). These are largely dominated by investigations on insect growth regulators (IGRs), which are designed to act as EDCs for insect pest control, and by studies on the antifouling biocide tributyltin (TBT) that has been shown to induce imposex and intersex in prosobranch snails (Matthiessen & Gibbs, 1998; Sousa, 2009b). Imposex has been associated with skewed sex ratios, reduced fecundity, population declines, and local extinctions of affected gastropod populations (Gibbs & Bryan, 1986). These are perhaps the most complete examples of ED studies in wildlife populations. Further examples for ED in invertebrates are scarce and limited to laboratory studies, where effects on endocrine regulated processes in marine and freshwater invertebrates (Porte et al., 2006), and in the soil compartment have been demonstrated for some compounds (Lemos et al., 2009, 2010a, 2010b, 2010c). Endocrine changes following exposure to certain compounds may therefore be missed or simply be immeasurable at present, even though there is increasing evidence indicating that invertebrates are susceptible to ED (Porte et al., 2006).

Consequently, there is no reason to suppose that far-reaching changes as demonstrated by TBT and its effects on prosobranch populations are in any sense unique within invertebrates (Matthiessen & Gibbs, 1998).

Additionally, since many chemicals have been considered as endocrine disrupters in vertebrates and chemical signalling systems and their basic mechanisms in the animal kingdom exhibit some degree of conservatism (McLachlan, 2001) we can presume that endocrine systems in invertebrates can be subject to modulation by identical or similar compounds as in vertebrates (Pinder and Pottinger, 1998). But, and as stated before, despite their abundance and variety, relatively little is known about their endocrine systems, making data obtained by studies on endocrine disruption rather difficult to interpret.

The "Endocrine Disruption in Invertebrates: Endocrinology, Testing, and Assessment" report (deFur et al., 1999) summarizes 56 studies where ED may have occurred in invertebrates, although non-endocrine mechanisms are also possible scenarios for the observed effects. Effects like reduced molting frequency, reduced fecundity, elevated ecdysteroid levels, delayed reproduction, reduced size of neonates, increased brood size, mortality, molting impairment, delayed maturation, impairment of reproduction, reduced egg production, delayed brood release, reduced elimination of testosterone metabolites, retardation of regenerative limb growth, suppression of ovarian growth, differential sex ratio and super-female induction, have been reviewed in this report. This includes several studies which comprise many compounds suspected of being hormonally active on aquatic crustaceans.

With the exception of TBT, effects in molluscs, that have been associated to a locally severe impact on community levels (e.g. Blaber, 1970), and IGRs in target terrestrial insects, there are only a few field examples of ED in invertebrates. Nevertheless, much more examples for ED affecting invertebrate populations and communities can be expected.

In fact, numerous studies provide strong evidence of effects on development, fecundity and reproductive output of invertebrates that can be attributed to substances acting as EDCs (Gibbs & Bryan, 1986; Matthiessen and Gibbs, 1998; Pinder and Pottinger, 1998; Oehlmann & Schulte-Oehlmann, 2003). So, carefully targeted monitoring programs are needed because effects in invertebrates are probably widespread but undetected (Fent, 2004).

et al., 1999). These are largely dominated by investigations on insect growth regulators (IGRs), which are designed to act as EDCs for insect pest control, and by studies on the antifouling biocide tributyltin (TBT) that has been shown to induce imposex and intersex in prosobranch snails (Matthiessen & Gibbs, 1998; Sousa, 2009b). Imposex has been associated with skewed sex ratios, reduced fecundity, population declines, and local extinctions of affected gastropod populations (Gibbs & Bryan, 1986). These are perhaps the most complete examples of ED studies in wildlife populations. Further examples for ED in invertebrates are scarce and limited to laboratory studies, where effects on endocrine regulated processes in marine and freshwater invertebrates (Porte et al., 2006), and in the soil compartment have been demonstrated for some compounds (Lemos et al., 2009, 2010a, 2010b, 2010c). Endocrine changes following exposure to certain compounds may therefore be missed or simply be immeasurable at present, even though there is increasing evidence indicating that

Consequently, there is no reason to suppose that far-reaching changes as demonstrated by TBT and its effects on prosobranch populations are in any sense unique within invertebrates

Additionally, since many chemicals have been considered as endocrine disrupters in vertebrates and chemical signalling systems and their basic mechanisms in the animal kingdom exhibit some degree of conservatism (McLachlan, 2001) we can presume that endocrine systems in invertebrates can be subject to modulation by identical or similar compounds as in vertebrates (Pinder and Pottinger, 1998). But, and as stated before, despite their abundance and variety, relatively little is known about their endocrine systems, making data obtained by studies on endocrine disruption rather difficult to

The "Endocrine Disruption in Invertebrates: Endocrinology, Testing, and Assessment" report (deFur et al., 1999) summarizes 56 studies where ED may have occurred in invertebrates, although non-endocrine mechanisms are also possible scenarios for the observed effects. Effects like reduced molting frequency, reduced fecundity, elevated ecdysteroid levels, delayed reproduction, reduced size of neonates, increased brood size, mortality, molting impairment, delayed maturation, impairment of reproduction, reduced egg production, delayed brood release, reduced elimination of testosterone metabolites, retardation of regenerative limb growth, suppression of ovarian growth, differential sex ratio and super-female induction, have been reviewed in this report. This includes several studies which comprise many compounds suspected of being hormonally active on aquatic

With the exception of TBT, effects in molluscs, that have been associated to a locally severe impact on community levels (e.g. Blaber, 1970), and IGRs in target terrestrial insects, there are only a few field examples of ED in invertebrates. Nevertheless, much more examples for

In fact, numerous studies provide strong evidence of effects on development, fecundity and reproductive output of invertebrates that can be attributed to substances acting as EDCs (Gibbs & Bryan, 1986; Matthiessen and Gibbs, 1998; Pinder and Pottinger, 1998; Oehlmann & Schulte-Oehlmann, 2003). So, carefully targeted monitoring programs are needed because

ED affecting invertebrate populations and communities can be expected.

effects in invertebrates are probably widespread but undetected (Fent, 2004).

invertebrates are susceptible to ED (Porte et al., 2006).

(Matthiessen & Gibbs, 1998).

interpret.

crustaceans.

#### **3. Pesticides as Endocrine Disruptor Compounds**

In June 2000 a list of 564 potential EDCs was published in two reports of BKH Consulting Engineers, Delft, and TNO Nutrition and Food Research, Zeist, both from The Netherlands (European Commission, 2000). This list of substances was compiled having in mind the compound persistence in the environment, its production volume, the scientific evidence of endocrine disruption and wildlife and human exposure. These criteria were used to categorise the candidate substances. From these, a group of 60 compounds considered to have endocrine disrupting activity (i.e. compounds for which endocrine activity has been shown in at least one *in vivo* study) and for which a high level of concern existed with regard to exposure, deserved a special attention. These 60 compounds were included in a high priority list of EDCs proposed by the EU Commission (European Commission DG ENV, 2002). This list includes industrial chemicals such as plasticizers (e.g. benzyl-nbutylphthalate, di-n-butylphthalate, bisphenol A) or flame retardants (e.g. PBBS) but also agrochemicals or crop protection products (e.g. lindane, vinclozolin, linuron, diuron, the common metabolite of linuron and diuron, 3,4-dichloroaniline, as well as triphenyltin compounds), and biocides with antifouling properties (tributyltin compounds).

#### **4. Fungicides as endocrine-disrupters**

The increased need for pest control has made pesticide use a major issue in environmental risk assessment. Within these compounds, despite some ecotoxicology studies reporting their low toxicity to non-target species (Jansch et al., 2005; Haeba et al., 2008), many fungicides have already been reported as affecting organisms and their molecular targets and effects are now well documented.

It is not surprising that several examples of endocrine disruption have been reported for terrestrial arthropods, as several pesticides have been specially tailored to affect insects endocrine systems (IGRs), eventually co-affecting several non-target invertebrates (deFur et al., 1999). In aquatic environments the examples are scarce and reduced to the effects of TBT in molluscs that have been associated with a locally severe impact at the marine community levels (Matthiessen & Gibbs, 1998).

For this review two compounds from the EU highest priority list were selected based on their environmental occurrence as well as the existing studies confirming ED effects: the fungicides tributyltin (androgen) and vinclozolin (anti-androgen). Focusing on these compounds and their effects on non-target invertebrates, an overview of terrestrial and aquatic compartments is here addressed.

#### **5. Case studies**

#### **5.1 Tributyltin**

Tributyltin (TBT) compounds are a subgroup of the trialkyl organotin family of compounds. Of all known organotins, some of the most toxic are tributyltin compounds like tributyltin oxide (TBTO; Fig.1A) and tributyltin chloride (TBTCl; Fig.1B) (Carfi 2008). TBT compounds are organic derivatives of tin (Sn4+) characterized by the presence of covalent bonds between three carbon atoms and a tin atom (Antizar-Ladislao, 2008). While inorganic forms of tin are regarded as non-toxic, these more lipid-soluble organotins can be highly toxic (Gadd, 2000).

Fig. 1. Molecular structures of tributyltin chloride (A) and tributyltin oxide (B).

They are the main active ingredients in pesticides used to control a broad spectrum of organisms as they act as biocides for fungi, bacteria and insects (Mimura et al., 2008; Fent, 2006). Nevertheless, research undertaken since the early 1970s has shown that TBT is highly toxic to a larger number of non target aquatic organisms (Antizar-Ladislao, 2008). TBTs' properties were recognized in the 1950s and since then this compound has been extensively used for various industrial purposes such as slime control in paper, as a wood preservative, as a polyvinyl chloride (PVC) stabilizer, and as fungicide in agriculture (Mimura et al., 2008). In the 1970s, TBT paints widely replaced copper-based paints due to their superior performance in terms of efficacy and duration (Sonak, 2009). Since then, TBT has been used mostly as an antifouling agent in marine paint formulations to prevent the attachment of barnacles and slime on boat hulls and aquaculture nets (Kannan et al., 1998).

Due to its widespread use as an antifouling agent in boat paints, TBT is a common contaminant of marine and freshwater ecosystems. Its damaging consequences to marine ecosystems were recognized in early 1980s as the cause for the decline of some marine molluscs (Smith, 1981; Waldock & Thain, 1983; Bryan et al., 1986). As a result of field evidences of negative ecological impact of organotins, the European Union published a Directive (89/677/CEE) banning TBT application on ships smaller than 25 m. On the assumption that TBT concentrations in the open sea were too low to cause effects, there were not many restrictions on the use of organotins in larger ships. However, a similar impact in the open sea has been shown for TBT with incidence being correlated with shipping density (Santos et al., 2002). Thus, the International Maritime Organization banned the application of TBT-based paints in 2003 and called for a global agreement for total prohibition of the presence of organotins on ship hulls in 2008 (International Maritime Organization, 2001).

TBT from hulls and nets can be adsorbed onto suspended particles in the water, sediment and biota (Gadd, 2000). Subsequently it is readily incorporated into the tissues of filterfeeding zooplankton, invertebrates and eventually higher organisms such as fish and mammals where it accumulates (Antizar-Ladislao, 2008). Despite the present restrictions, TBT and its degradation products will not disappear immediately from the marine environment, and it can be expected that TBT will remain in waters and sediments for long periods of time because of the moderate to high persistency in anoxic sediments and its' widespread presence, as was confirmed by the work of Sousa and co-workers along the Portuguese coast (Sousa et al., 2009). Together with its lipophilicity, it tends to accumulate in oysters, mussels, crustaceans, molluscs, fish, and algae favouring the bioconcentration up the marine predators' food chain (Santos et al., 2009; Cruz et al., 2010).

Sn

O

Sn

Sn

**A. B.** 

Cl

Fig. 1. Molecular structures of tributyltin chloride (A) and tributyltin oxide (B).

barnacles and slime on boat hulls and aquaculture nets (Kannan et al., 1998).

of organotins on ship hulls in 2008 (International Maritime Organization, 2001).

the marine predators' food chain (Santos et al., 2009; Cruz et al., 2010).

They are the main active ingredients in pesticides used to control a broad spectrum of organisms as they act as biocides for fungi, bacteria and insects (Mimura et al., 2008; Fent, 2006). Nevertheless, research undertaken since the early 1970s has shown that TBT is highly toxic to a larger number of non target aquatic organisms (Antizar-Ladislao, 2008). TBTs' properties were recognized in the 1950s and since then this compound has been extensively used for various industrial purposes such as slime control in paper, as a wood preservative, as a polyvinyl chloride (PVC) stabilizer, and as fungicide in agriculture (Mimura et al., 2008). In the 1970s, TBT paints widely replaced copper-based paints due to their superior performance in terms of efficacy and duration (Sonak, 2009). Since then, TBT has been used mostly as an antifouling agent in marine paint formulations to prevent the attachment of

Due to its widespread use as an antifouling agent in boat paints, TBT is a common contaminant of marine and freshwater ecosystems. Its damaging consequences to marine ecosystems were recognized in early 1980s as the cause for the decline of some marine molluscs (Smith, 1981; Waldock & Thain, 1983; Bryan et al., 1986). As a result of field evidences of negative ecological impact of organotins, the European Union published a Directive (89/677/CEE) banning TBT application on ships smaller than 25 m. On the assumption that TBT concentrations in the open sea were too low to cause effects, there were not many restrictions on the use of organotins in larger ships. However, a similar impact in the open sea has been shown for TBT with incidence being correlated with shipping density (Santos et al., 2002). Thus, the International Maritime Organization banned the application of TBT-based paints in 2003 and called for a global agreement for total prohibition of the presence

TBT from hulls and nets can be adsorbed onto suspended particles in the water, sediment and biota (Gadd, 2000). Subsequently it is readily incorporated into the tissues of filterfeeding zooplankton, invertebrates and eventually higher organisms such as fish and mammals where it accumulates (Antizar-Ladislao, 2008). Despite the present restrictions, TBT and its degradation products will not disappear immediately from the marine environment, and it can be expected that TBT will remain in waters and sediments for long periods of time because of the moderate to high persistency in anoxic sediments and its' widespread presence, as was confirmed by the work of Sousa and co-workers along the Portuguese coast (Sousa et al., 2009). Together with its lipophilicity, it tends to accumulate in oysters, mussels, crustaceans, molluscs, fish, and algae favouring the bioconcentration up Because of all the above mentioned, recently organotins have been considered as the most toxic substance ever introduced into the marine environment so far (Fent, 2006; Guo et al., 2010; Antizar-Ladislao, 2008; Sonak, 2009), and due to their use in a variety of industrial processes, their environmental fate and ecotoxicity as well as human exposure are topics of current concern.

In a study conducted by Guo et al (2010) on western clawed frog embryos (*Xenopus tropicalis*) the authors suggested that TBT might be the cause of several malformations. These include the loss of eye pigmentation, enlarged trunks and bent tails, in the presence of 50 ng/L of TBTCl after 24 hours of exposure. This is particularly relevant since the concentrations of TBT in open water, bays, estuaries, lakes and freshwater harbors commonly exceed this concentration with the highest TBT values found near marinas and seaports (Fent, 2006). At higher concentrations TBT may be lethal to several marine and freshwater species. Short and Thrower (1987) reported that the 96 hour LC50 for juvenile Chinook salmon (*Oncorhynchus tshawytscha*) is 1.5 μg/L.

TBT is known as an endocrine disruptor promoting adverse effects in organisms on diverse levels of biological organization (Guo et al., 2010). Matthiessen & Gibbs (1998) reported an interference with hormone metabolism, increasing the androgen levels of snails exposed to TBT. One of the best-documented and iconic adverse impacts of TBT in non-target organisms is imposex in molluscs. This pathology occurs when male sex characteristics and organs, such as penis, *vas deferens*, and seminiferous tubules are superimposed on normal female resulting in female sterilization, and even with spermatogenesis occurrence. Such pathology consequently has obvious impacts population dynamics (Gibbs et al. 1991). The first evidences linking TBT to imposex were reported in 1970 for the dog-whelk, *Nucella lapillus*, in the UK (Blaber, 1970). Since then, several studies have related TBT to the worldwide decline of marine mollusks (e.g. *Nassarius*, *Ilyanassa*, *Ocenebra* and *Urosalpinx)* in costal areas due to imposex (Gibbs et al., 1991). Administrating testosterone to the snail *Euchadra peliomphala* resulted in a stimulation of the development of male sex characters in female and castrated male gastropods (Takeda, 1980; Spooner et al., 1991). These authors also report increased testosterone titres in *N. lapillus* exposed to this fungicide. These findings have led to the hypothesis that the increased levels of testosterone in TBT-exposed organisms are responsible for the imposex development. The precise mechanism by which increased levels of testosterone are produced has not been fully described, but the weight of evidence suggests that TBT acts as a competitive inhibitor of cytochrome P450-mediated aromatase (Bettin et al., 1996). In laboratory tests, imposex was also reported from concentrations below 1 ng of TBT/L in the mud snail, *Ilyanassa obsoleta* (*Gooding* et al., 2003), and the dog-whelk, *N. lapillus* (Gibbs et al., 1988). There are still uncertainties regarding the sensitivity of the endocrine function in different genders and developmental stages in invertebrates exposed to EDCs (Rodriguez et al., 2007). Nevertheless, some authors agree that the hormonal impacts of TBT and EDCs in general may differ according to the specific life stage at which exposure occurs (e.g., embryolarval stages, gonadal development, etc.) (deFur et al., 1999; Lemos et al., 2009). Generally, the larvae/neonates of any tested species are more hormonally sensitive to tributyltin exposure than are the adults. For example, the adult female dog-whelk, *N. lapillus*, revealed imposex signs at concentrations as low as 5 ng of TBT/L while young and sexually immature females were more sensitive than adults with concentrations of 1 ng of TBT/L inducing the growth of penis and *vas deferens* tissue (Gibbs et al., 1987).

Fig. 2. Female gastropods exhibiting imposex: *Nucella lapillus* (A), and *Nassarius reticulatus*  (B). CG - capsule gland; Ft - foot; P – penis. Photographs are courtesy of Prof. T. Horiguchi and Dr. A. Sousa.

Deviations from normal limb regeneration in the prawn, *Caridina rajadhari* (Reddy et al. 1991), and in the fiddler crab *Uca pugilator* (Weis et al., 1987), have been observed in laboratory experiments involving TBT exposure. Nevertheless, despite the findings and the known hormonal regulation of the molting process, it has been argued whether these effects are due to endocrine disruption rather than regular systemic toxicity. Unless specific parameters such as hormone levels are assessed no accurate conclusion can be drawn (Barata et al., 2004; Lemos et al 2009).

Levels in harbour and port waters prior to restrictions on TBT use in antifouling paints have shown levels higher than 500 ng/L in North American and European marinas. For example, one year before the UK ban (1986), TBT concentrations in Wroxham Broad and at the nearby River Bure boatyard were 898 ng/L and 1540 ng/L, respectively (Waite et al., 1989). They were significantly higher than in open surface waters, bays and estuaries where commonly values of up to 50 ng/L were observed (Fent, 2006). Albeit this regulation for the use of TBT and consequent general decrease in environmental TBT levels (Antizar-Ladislao, 2008), recent surveys still account for levels higher than those reported to elicit effects at a global scale: for instance, 32 ng TBT/L in South Korea (Sidharthan et al., 2002), 2–160 ng/L in Japanese coastal waters (Takeuchi et al., 2004), in the UK 10–78 ng/L were detected in marinas and harbors in 1998 (Thomas, 2001), and 200 ng/L in ferry ports in Corsica, Italy (Michel et al., 2001).

The impact of TBT in freshwater systems has been studied at a much lesser extent compared to estuarine and coastal environments. As still, endocrine effects have also been shown for freshwater molluscs. For example, after a 3 month exposure to a concentration of 50 ng of TBT/L, the giant ramshorn snail, *Marisa cornuarietis*, showed recognizable morphological characteristics of imposex development (Shulte-Oehlmann et al., 1995). Despite the scarce information (possibly reflecting a reduced concern about the impacts in this ecossystem), Schulte-Oehlmann (1997) reported concentrations in European lakes up to 930 ng of TBT /L in water and reaching 340 µg of TBT/g wet weight in sediments of River Elbe in Central Europe.

For the soil compartment, to our knowledge, there are no reported data on the effects of TBT to edaphic organisms.

#### **5.2 Vinclozolin**

184 Fungicides – Beneficial and Harmful Aspects

Photo by T. Horiguchi

Fig. 2. Female gastropods exhibiting imposex: *Nucella lapillus* (A), and *Nassarius reticulatus*  (B). CG - capsule gland; Ft - foot; P – penis. Photographs are courtesy of Prof. T. Horiguchi

Deviations from normal limb regeneration in the prawn, *Caridina rajadhari* (Reddy et al. 1991), and in the fiddler crab *Uca pugilator* (Weis et al., 1987), have been observed in laboratory experiments involving TBT exposure. Nevertheless, despite the findings and the known hormonal regulation of the molting process, it has been argued whether these effects are due to endocrine disruption rather than regular systemic toxicity. Unless specific parameters such as hormone levels are assessed no accurate conclusion can be drawn

Levels in harbour and port waters prior to restrictions on TBT use in antifouling paints have shown levels higher than 500 ng/L in North American and European marinas. For example, one year before the UK ban (1986), TBT concentrations in Wroxham Broad and at the nearby River Bure boatyard were 898 ng/L and 1540 ng/L, respectively (Waite et al., 1989). They were significantly higher than in open surface waters, bays and estuaries where commonly values of up to 50 ng/L were observed (Fent, 2006). Albeit this regulation for the use of TBT and consequent general decrease in environmental TBT levels (Antizar-Ladislao, 2008), recent surveys still account for levels higher than those reported to elicit effects at a global scale: for instance, 32 ng TBT/L in South Korea (Sidharthan et al., 2002), 2–160 ng/L in Japanese coastal waters (Takeuchi et al., 2004), in the UK 10–78 ng/L were detected in marinas and harbors in 1998 (Thomas, 2001), and 200 ng/L in ferry ports in Corsica, Italy

The impact of TBT in freshwater systems has been studied at a much lesser extent compared to estuarine and coastal environments. As still, endocrine effects have also been shown for freshwater molluscs. For example, after a 3 month exposure to a concentration of 50 ng of TBT/L, the giant ramshorn snail, *Marisa cornuarietis*, showed recognizable morphological characteristics of imposex development (Shulte-Oehlmann et al., 1995). Despite the scarce information (possibly reflecting a reduced concern about the impacts in this ecossystem), Schulte-Oehlmann (1997) reported concentrations in European lakes up to 930 ng of TBT /L in water and reaching 340 µg of TBT/g wet weight in sediments of River Elbe in Central Europe. For the soil compartment, to our knowledge, there are no reported data on the effects of TBT

**CG**

**P**

**A B**

**Ft** Photo by A. Sousa

**P**

**Ft**

(Barata et al., 2004; Lemos et al 2009).

**CG**

and Dr. A. Sousa.

(Michel et al., 2001).

to edaphic organisms.

Vinclozolin [Vz, 3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione; Fig.3] is a non-systemic dicarboxymide fungicide, manufactured by BASF and commercially sold under the names Ronilan®, Curalan®, and Ornilan®. It is efficient in controlling plants and fruit diseases caused by *Botrytis* spp., *Monilia* spp., and *Sclerotinia* spp. (Bursztyka et al., 2008) that affect crops such as lettuce, raspberries, beans and onions (Price et al., 2007). This fungicide is widely used in the United States of America and throughout Europe. In Britain, as well as in Germany, up to 50 tonnes of Vz are used each year and it was estimated that in 2002, in the USA, 2,330,738 US dollars were spent on this compound for crop protection (Gianessi & Reigner, 2005).

When sprayed as Ronilan®, at the maximum recommended application rate, the concentration of Vz in the soil is 1 mg active ingredient (a.i.)/kg (assuming that 70 % of the fungicide will reach the surface and is homogeneously distributed over the top 5 cm soil layer and the soil bulk density is 1.4 kg/dm3) (Lemos et al., 2009). Vinclozolin has a low to moderate persistence in soil, with reported half-lives from 28-43 days in the laboratory up to 34-94 days in the field and 6-12% of the original compound is present after 1 year (U.S. EPA, 1991; IUPAC, 2006). Despite this low-persistency of Vz, a reported increase of mortality with exposure time (Lemos et al., 2009) may be due to the increased concentration of its' three major metabolites (out from 15): 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3 butenoic acid (M1), 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2), and 3,5 dichloroaniline (DCA), which are reported to be more active than the parent compound (Kelce et al., 1997; Anway et al., 2005; Kavlock and Cummings, 2005) and have half-lives ranging from 179 to >1000 days (U.S. EPA, 2000). It has been reported that these metabolites may be produced both spontaneously in the presence of aqueous buffers and by biotransformation of Vz (Bursztyka et al., 2008). M1 and M2 are able to bind to the androgen receptor (Vinggaard et al., 1999) and competitively inhibit the binding of androgens to the human androgen receptor (Kelce et al., 1997; Kavlock and Cummings, 2005). Concerning the parent compound, it is known that it inhibits vertebrate testosterone-induced growth of androgen-dependent tissues (Kang et al., 2004).

Fig. 3. Molecular structure of vinclozolin.

On plant leaves, Vz is detectable as the parent compound but does not wash off, since it is more soluble in oil than in water. This implies that Vz residues are commonly found on food (Szeto et al., 1989). Metabolites have also been found in human food (Gonzalez-Rodriguez et al., 2008).

Vinclozolin endocrine disruptor effects include induction of Leydig cell tumors, reduction of ejaculated sperm counts and prostate weight, and delayed puberty in exposed rats. One major concern is that Vz causes transgenerational effects. F1 to F4 generations of male rats exposed to Vz at the time of gonadal sex determination developed prostate disease, kidney disease, immune system abnormalities, spermatogenesis abnormalities, breast tumour development, and blood abnormalities as hypercholesterolemia, which have been associated with an alteration in the epigenetic programming of the male germ line (Anway et al., 2006; Anway & Skinner, 2008). Similar effects have been shown for pregnant rat exposed to Vz, where a transgenerational increase in pregnancy abnormalities and female adult onset disease states are promoted (Nilsson et al., 2008).

The existing information supports the hypothesis that Vz steroid-mediated actions in vertebrates have similar sub-lethal effects in invertebrates. In *Daphnia magna* it induces a decrease in the number of newborn males (Haeba et al., 2008). In molluscs Vz was shown to cause female virilisation (imposex development) and reduction of accessory sex organ expression in the fresh water snail *Marisa cornuarietis* and two marine prosobranchs *N. lapillus* and *Nassarius reticulatus* (Tillmann et al., 2001). Snails were exposed to nominal concentrations of Vz ranging from 0.03 to 1 µg/L for up to 5 months. In exposed juvenile *Marisa*, males had a slight decrease in the male accessory sex organ, particularly the penis and penis sheath. This response was only detected during the first 2-3 months of exposure for the lowest concentrations (0.03 and 0.1 µg of Vz/L), and was reversible once they attained puberty. Adult male *N. lapillus* exposed to the fungicide developed shorter penis, smaller prostate gland, and there were less males with ripe sperm stored in the seminal vesicle. Compared to the reported effects of estrogens and androgens on these two same species, these anti-androgenic responses seem to be less drastic, and might not have any biological effect at the population level (Tillmann et al., 2001). The immobilising effect (EC50, 48h) of Vz on the American oyster (*Crassostrea virginica*) and *D. magna* was reported to be 3.2 mg/L and 3.65 mg/L, respectively. For the opossum shrimp (*American bahia*) the LC50 (96h) was 1.5 -2.1 mg Vz/L (U.S. EPA, 2000).

Studies of Vz effects in the soil compartment are scarce. Vinclozolin has been reported as non-toxic to earthworms (Tomlin, 2003), but the most complete and extensive research was performed in a terrestrial isopod (Lemos et al. 2009, 2010a, 2010b, 2010c). In *Porcellio scaber*, Vz exhibited ecdysteroidal activity in ecdysone endogenous levels in a concentrationdependent way (Lemos et al., 2009). The results from this study demonstrated that the fungicide caused endocrine disruption in the isopod with an ecdysteroid up-regulation resulting in molting disturbances being further related to developmental and reproductive toxicity which enabled to suggest a causal link to ED in this class of organisms.

Since a sharp rise of ecdysteroid followed by a decrease of the hormone level triggers the ecdysis process (Bodar et al., 1990), when basal concentration of 20-hydroxyecdysone (20E) is maintained at high levels (hyperecdysonism) the shedding of the old cuticle is impaired and mortality due to incomplete ecdysis occurs (Fig. 4). Therefore, molting behaviour was associated with hyperecdysonism, delaying molt and in many cases impairing the molting process, and death surmounting at higher toxicant concentrations (1 g of a.i. Vz/kg of soil) (Lemos et al., 2009).

On plant leaves, Vz is detectable as the parent compound but does not wash off, since it is more soluble in oil than in water. This implies that Vz residues are commonly found on food (Szeto et al., 1989). Metabolites have also been found in human food (Gonzalez-Rodriguez et al., 2008). Vinclozolin endocrine disruptor effects include induction of Leydig cell tumors, reduction of ejaculated sperm counts and prostate weight, and delayed puberty in exposed rats. One major concern is that Vz causes transgenerational effects. F1 to F4 generations of male rats exposed to Vz at the time of gonadal sex determination developed prostate disease, kidney disease, immune system abnormalities, spermatogenesis abnormalities, breast tumour development, and blood abnormalities as hypercholesterolemia, which have been associated with an alteration in the epigenetic programming of the male germ line (Anway et al., 2006; Anway & Skinner, 2008). Similar effects have been shown for pregnant rat exposed to Vz, where a transgenerational increase in pregnancy abnormalities and female adult onset

The existing information supports the hypothesis that Vz steroid-mediated actions in vertebrates have similar sub-lethal effects in invertebrates. In *Daphnia magna* it induces a decrease in the number of newborn males (Haeba et al., 2008). In molluscs Vz was shown to cause female virilisation (imposex development) and reduction of accessory sex organ expression in the fresh water snail *Marisa cornuarietis* and two marine prosobranchs *N. lapillus* and *Nassarius reticulatus* (Tillmann et al., 2001). Snails were exposed to nominal concentrations of Vz ranging from 0.03 to 1 µg/L for up to 5 months. In exposed juvenile *Marisa*, males had a slight decrease in the male accessory sex organ, particularly the penis and penis sheath. This response was only detected during the first 2-3 months of exposure for the lowest concentrations (0.03 and 0.1 µg of Vz/L), and was reversible once they attained puberty. Adult male *N. lapillus* exposed to the fungicide developed shorter penis, smaller prostate gland, and there were less males with ripe sperm stored in the seminal vesicle. Compared to the reported effects of estrogens and androgens on these two same species, these anti-androgenic responses seem to be less drastic, and might not have any biological effect at the population level (Tillmann et al., 2001). The immobilising effect (EC50, 48h) of Vz on the American oyster (*Crassostrea virginica*) and *D. magna* was reported to be 3.2 mg/L and 3.65 mg/L, respectively. For the opossum shrimp (*American bahia*) the

Studies of Vz effects in the soil compartment are scarce. Vinclozolin has been reported as non-toxic to earthworms (Tomlin, 2003), but the most complete and extensive research was performed in a terrestrial isopod (Lemos et al. 2009, 2010a, 2010b, 2010c). In *Porcellio scaber*, Vz exhibited ecdysteroidal activity in ecdysone endogenous levels in a concentrationdependent way (Lemos et al., 2009). The results from this study demonstrated that the fungicide caused endocrine disruption in the isopod with an ecdysteroid up-regulation resulting in molting disturbances being further related to developmental and reproductive

Since a sharp rise of ecdysteroid followed by a decrease of the hormone level triggers the ecdysis process (Bodar et al., 1990), when basal concentration of 20-hydroxyecdysone (20E) is maintained at high levels (hyperecdysonism) the shedding of the old cuticle is impaired and mortality due to incomplete ecdysis occurs (Fig. 4). Therefore, molting behaviour was associated with hyperecdysonism, delaying molt and in many cases impairing the molting process, and death surmounting at higher toxicant concentrations (1 g of a.i. Vz/kg of soil)

toxicity which enabled to suggest a causal link to ED in this class of organisms.

disease states are promoted (Nilsson et al., 2008).

LC50 (96h) was 1.5 -2.1 mg Vz/L (U.S. EPA, 2000).

(Lemos et al., 2009).

Fig. 4. Isopod *Porcellio scaber* and shedded cuticle after posterior half molt (A), and dead animal within the unshedded old cuticle after failed posterior half molt, after exposure to vinclozolin contaminated soil (B).

The same authors (Lemos et al. 2010a) also reported that young isopods respond with reduced growth at lower concentrations (LOEC of 100 mg a.i. Vz/kg soil) compared to adults (LOECs 300 mg a.i. Vz/kg soil). The increased sensitivity of the juvenile life stage may be either due to easier absorption of the toxicant through their relatively larger body surface/volume ratio and thin cuticle, or due to their lower capacity to metabolize the contaminants (Fischer et al., 1997; Lemos et al., 20010a) but another possibility pointed was the differential life-stage endocrinology.

Vinclozolin also elicited overall reproductive toxicity to *P. scaber* (Lemos et al., 2010b) decreasing the reproductive allocation for exposed females, the number of pregnancies, and the number of juveniles, while increasing the percentage of abortions. It induced a decrease of the brood period, with the isopods releasing juveniles almost 43 hours earlier at 100 mg a.i./kg dry soil and five days earlier at 300 mg a.i./kg dry soil. These two factors together considerably reduced the total juvenile output.

One of the reasons attributed to this impairment was the increased 20E titres (Lemos et al., 2009) that have been previously correlated with increased vitellogenin - a key protein of extreme importance in crustacean reproduction - synthesis and uptake in developing oocytes (Gohar &d Souty, 1984).

The molecular effect of Vz was assessed by differential protein expression of Vz exposed *P. scaber* in the gut, hepatopancreas, and gonads (Lemos et al., 2010c). In this study it was possible to detected up-regulated proteins at concentrations as low as 10 mg a.i./kg soil in the testes while for hepatopancreas this was only possible at concentrations equal to 1000 mg a.i./kg soil. Proteins from the heat shock protein family, Hsp70 (known as ubiquitous stress response proteins, anti-apoptotic, and protects cells from cytotoxicity and inhibiting cell death induced by several agents) were over-expressed at the lower concentration of Vz in the testes of organisms exposed to Vz (around 160% increase after exposure to 10 mg a.i./kg and around 130% at 30 mg a.i./kg).

Vinclozolin up-regulated arginine kinase (around 150% at 1000 mg a.i./kg for Vz) in the isopods' hepatopancreas. This enzyme is involved in the cellular energy metabolism, suggesting an increase of resources allocated to the activation of metabolic processes related to detoxification and the metabolisation of energy reserves to provide for these processes (Lemos et al., 2010c).

The fact that the gonads showed increased protein expression at concentrations higher than 10 mg a.i./kg soil (Lemos et al., 2010c), suggests that testes are more susceptible to these compounds than other organs. Thus, male isopod reproductive traits may therefore be especially susceptible and sensitive to this fungicide. Previous studies have also stressed the gonads and reproductive traits as preferential targets by EDCs in vertebrates (Navas and Segner, 2006).

### **6. Conclusions**

Although TBT levels have been decreasing in the last decades, mainly due to restrictions in its use, it is still present at ng/L levels in the environment (Fent, 2006). Additionally, marine prosobranch gastropods and other invertebrates are extremely sensitive to TBT contamination, and imposex can be elicited in some species at concentrations of < 1 ng TBT/L (Gibbs et al. 1988). Moreover, TBT is very persistent in sediments being is considered as the most toxic substance ever introduced into the marine environment and a major threat to the environment for many years (e.g. Fent, 2006) since its sediment concentrations (up to several mg/kg sediment) are still highly toxic to benthic fauna while organotins resuspension is possible through storms or dredging leading to an increase of organotin levels in the water column (Unger et al., 1988). Additionally one should not forget that organotins residues such as TBT can also be transferred to humans via dietary uptake. Sousa and co-workers predict that in traditional seafood-consuming countries the estimated Sn daily intake is high enough to cause damage to humans (Sousa et al., 2009b), making TBT contamination more than "just" an environmental issue of concern.

Vinclozolin is classified as very toxic (EC50 < 1 mg/L), and it has been shown to have adverse effects in the laboratory on aquatic snail at concentrations below 1 µg/L. Nevertheless, Vz is non-persistent in the environment, and degrades rapidly, particularly under alkaline conditions (Tomlin, 2003; Ueoka *et al*., 1997). Moreover, a survey of German ground and surface waters found only 1% of water samples to have detectable levels (>0.1 g/L) of Vz (Funari *et al*., 1995). Mediterranean estuarine waters have also been investigated for fungicide contamination, and from the sites examined only the River Po in Italy had detectable levels of vinclozolin (Readman *et al.*, 1997).

In the soil compartment, despite these reported severe effects in isopods, the concentrations that elicit effects are far from ecological relevant concentrations.

So far there is no clear evidence suggesting effects of Vz on the terrestrial or aquatic ecosystems. Nevertheless, in Europe, the use of this substance is no longer authorized according to EC Directive 91/414.

Despite the examples showing that invertebrates are susceptible to ED, endocrine changes following exposure to certain compounds may be missed due to scarce knowledge of invertebrate endocrinology or due to very low concentrations of certain compounds (below detection limits) eliciting ED effects. In fact there is no reason to suppose that the array of endocrine changes such as the ones demonstrated for TBT and Vz are in any sense unique and most certainly similar effects are plausible and to be expected in most invertebrate species.

Through small biochemical and molecular changes, these contaminants may however interfere with different systems (e.g., reproductive, endocrine, immunological, and nervous) in different life stages of non-target species, causing medium and long term effects at the population level. Due to the nature of the ED mode of action, the consequences of the exposure to such class of compounds in the communities' structure and function are thus not always immediate and can be extremely hard to predict.

As said before many fungicides can behave like EDC, and because ED effects at the population and community levels might be only detected after several generations exposed to sub-lethal levels of pollutants, there is a need to develop and validate quantifiable tools for the identification of ED effects of fungicides and pesticides in general. It is our conviction that this has to be achieved with a mechanistic approach where alterations of hormonal vital processes in particular species are assessed. For that it is vital to conduct long term chronic assays where biochemical, organismal and reproductive parameters are measured and compared with changes in hormone levels of invertebrate species. It is not an easy task but due to the expected increase in fungicide production and utilization in the next decades, it is critical to adopt this kind of integrative approaches to better understand the mechanisms that link endocrine level responses to population- and community-level processes and to improve environmental risk assessment of these compounds

#### **7. Acknowledgements**

The authors are grateful to Professor Toshihiro Horiguchi and Dr. Ana Sousa for kindly providing the photos of imposex-exhibiting female gastropods used in this book chapter. AC Esteves and JLT Pestana wish to acknowledge the financial support given by Fundação para a Ciência e Tecnologia (SFRH/BPD/38008/2007 and SFRH/BPD/45342/2008).

#### **8. References**

188 Fungicides – Beneficial and Harmful Aspects

suggesting an increase of resources allocated to the activation of metabolic processes related to detoxification and the metabolisation of energy reserves to provide for these processes

The fact that the gonads showed increased protein expression at concentrations higher than 10 mg a.i./kg soil (Lemos et al., 2010c), suggests that testes are more susceptible to these compounds than other organs. Thus, male isopod reproductive traits may therefore be especially susceptible and sensitive to this fungicide. Previous studies have also stressed the gonads and reproductive traits as preferential targets by EDCs in vertebrates (Navas and

Although TBT levels have been decreasing in the last decades, mainly due to restrictions in its use, it is still present at ng/L levels in the environment (Fent, 2006). Additionally, marine prosobranch gastropods and other invertebrates are extremely sensitive to TBT contamination, and imposex can be elicited in some species at concentrations of < 1 ng TBT/L (Gibbs et al. 1988). Moreover, TBT is very persistent in sediments being is considered as the most toxic substance ever introduced into the marine environment and a major threat to the environment for many years (e.g. Fent, 2006) since its sediment concentrations (up to several mg/kg sediment) are still highly toxic to benthic fauna while organotins resuspension is possible through storms or dredging leading to an increase of organotin levels in the water column (Unger et al., 1988). Additionally one should not forget that organotins residues such as TBT can also be transferred to humans via dietary uptake. Sousa and co-workers predict that in traditional seafood-consuming countries the estimated Sn daily intake is high enough to cause damage to humans (Sousa et al., 2009b), making TBT

Vinclozolin is classified as very toxic (EC50 < 1 mg/L), and it has been shown to have adverse effects in the laboratory on aquatic snail at concentrations below 1 µg/L. Nevertheless, Vz is non-persistent in the environment, and degrades rapidly, particularly under alkaline conditions (Tomlin, 2003; Ueoka *et al*., 1997). Moreover, a survey of German ground and surface waters found only 1% of water samples to have detectable levels (>0.1 g/L) of Vz (Funari *et al*., 1995). Mediterranean estuarine waters have also been investigated for fungicide contamination, and from the sites examined only the River Po in

In the soil compartment, despite these reported severe effects in isopods, the concentrations

So far there is no clear evidence suggesting effects of Vz on the terrestrial or aquatic ecosystems. Nevertheless, in Europe, the use of this substance is no longer authorized

Despite the examples showing that invertebrates are susceptible to ED, endocrine changes following exposure to certain compounds may be missed due to scarce knowledge of invertebrate endocrinology or due to very low concentrations of certain compounds (below detection limits) eliciting ED effects. In fact there is no reason to suppose that the array of endocrine changes such as the ones demonstrated for TBT and Vz are in any sense unique

contamination more than "just" an environmental issue of concern.

Italy had detectable levels of vinclozolin (Readman *et al.*, 1997).

that elicit effects are far from ecological relevant concentrations.

according to EC Directive 91/414.

(Lemos et al., 2010c).

Segner, 2006).

**6. Conclusions** 


Barata, C., Porte, C., & Baird, D.J. (2004). Experimental designs to assess endocrine disrupting effects in invertebrates - A review. *Ecotoxicology*, 13, pp. 511-517. Bettin, C., Oehlmann, J., & Stroben, E. (1996). Induced imposex in marine neogastropods is

Blaber, S.J.M. (1970). The occurrence of a penis-like outgrowth behind the right tentacle in spent females of *Nucella lapillus* (L.). *Journal of Molluscan Studies*, 39, pp. 231-233. Bodar, C.W.M., Voogt, P.A., & Zandee, D.I. (1990). Ecdysteroids in *Dapnhia magna* - Their

Bryan, G.W., Gibbs, P.E., Hummerstone, L.G., & Burt, G.R. (1986). The decline of the

Bursztyka, J., Debrauwer, L., Perdu, E., Jouanin, I., Jaeg, J.P., & Cravedi, J.P. (2008).

Carfi, M., Croera, C., Ferrario, D., Campi, V., Bowe, G., Pieters, R., & Gribaldo, L. (2008).

Cruz, A., Caetano, T., Suzuki, S., & Mendo, S. (2007). *Aeromonas veronii*, a tributyltin (TBT)-

Damstra, T., Barlow, S., Bergman, A., Kavlock, R., & Van Der Kraak, G. (2002). Global

De Falco, M., Sciarrillo, R., Capaldo, A., Russo, T., Gay, F., Valiante, S., Varano, L., &

deFur, P.L., Crane, M., Ingersoll, C., & Tattersfield, L. (1999). Endocrine disruption in

European Commission (1999). Communication from the Commission to the Council and the

European Commission (2001). Communication to the Council and the European Parliament

Portugal. *Marine Environmental Research*, 64, pp. 639-50.

Chemical Safety, Geneva, Switzerland.

*Contamination and Toxicology*, 53, pp. 241-248.

Netherlands. SETAC Press, Pensacola, U.S.

317

4839.

P. 29.

*Aquatic Toxicology*, 17, pp. 339-350.

*United Kingdom*, 66, pp. 611-640.

*Toxicology*, 249, pp. 11-18.

mediated by an increasing androgen level. Helgoländer Meeresunters 50, pp. 299-

role in molting and reproduction and their levels upon exposure to cadmium.

gastropod *Nucella lapillus* around south-west England: evidence for the effects of tributyltin from antifouling paints. *Journal of the Marine Biological Association of the* 

Biotransformation of vinclozolin in rat precision-cut liver vices: Comparison with in vivo metabolic pattern. *Journal of Agricultural and Food Chemistry*, 56, pp. 4832-

TBTC induces adipocyte differentiation in human bone marrow long term culture.

degrading bacterium isolated from an estuarine environment, Ria de Aveiro in

Assessment of the State-of-the Science of Endocrine Disruptors. WHO/PCS/EDC/02.2. World Health Organisation, International Programme on

Laforgia, V. (2007). The effects of the fungicide methyl thiophanate on adrenal gland morphophysiology of the lizard, *Podarcis sicula*. *Archives of Environmental* 

invertebrates: endocrinology, testing, and assessment. *Proceeding of the Workshops on Endocrine Disruption in Invertebrates*, 12-15 December 1998, Noordwijkerhout, The

European Parliament - Community Strategy for Endocrine Disrupters. COM(1999)706. Commission of the European Communities, Brussels, Belgium. European Commission (2000). Towards the establishment of a priority list of substances for

further evaluation of their role in endocrine disruption - preparation of a candidate list of substances as a basis for priority setting. BKH-RPS Group, The Netherlands.

on the implementation of the Community Strategy for Endocrine Disrupters - a

range of substances suspected of interfering with the hormone systems of humans and wildlife. COM(2001)262. Commission of the European Communities, Brussels, Belgium.


Gonzalez-Rodriguez, R.M., Rial-Otero, R., Cancho-Grande, B., & Simal-Gandara, J. (2008).

Gooding, M.P., Wilson, V.S., Folmar, L.C., Marcovich, D.T., & LeBlanc, G.A. (2003)*.* The

Guo, S.Z., Qian, L.J., Shi, H.H., Barry, T., Cao, Q.Z., & Liu, J.K. (2010). Effects of tributyltin

Haeba, M.H., Hilscherova, K., Mazurova, E., & Blaha, L. (2008). Selected endocrine

Halldin, K., Berg, C., Bergman, A., Brandt, I., & Brunstrom, B. (2001). Distribution of

Harding, A.K., Daston, G.P., Boyd, G.R., Lucier, G.W., Safe, S.H., Stewart, J., Tillitt, D.E., &

International Maritime Organization (2001). Final Act. International Conference on the

IUPAC, 2006. *Global availability of information on agrochemicals: vinclozolin* (Ref: BAS 352F),

Jansch, S., Garcia, M., & Rombke, J. (2005). Acute and chronic isopod testing using tropical

Kaneko, M., Okada, R., Yamamoto, K., Nakamura, M., Moscom, G., Polzonetti-Magni, A.M.,

Kang, I.H., Kim, H.S., Shin, J.H., Kim, T.S., Moon, H.J., Kim, I.Y., Choi, K.S., Kil, K.S., Park,

Kannan, K., Guruge, K.S., Thomas, N.J., Tanabe, S., & Giesy, J.P. (1998). Butyltin residues in

Kavlock, R.J., Daston, G.P., DeRosa, C., FennerCrisp, P., Gray, L.E., Kaattari, S., Lucier, G.,

waters. *Environmental Science and Technology*, 32, pp. 1169-1175.

AFS/CONF/25. London: International Maritime Organization.

*magna*. *Environmental Science and Pollution Research*, 15, pp. 222-227.

*Environmental Health Perspectives*, 114, pp. 1276-1282.

http://sitem.herts.ac.uk/aeru/iupac/680.htm

*General and Comparative Endocrinology*, 155, pp. 574-580.

Hershberger assay. *Toxicology*, 199, pp. 145-159.

100-109.

*Chemosphere*, 79, pp. 529-533.

*of Toxicology*, 75, pp. 597-603.

41, pp. 143-152.

Determination of 23 pesticide residues in leafy vegetables using gas chromatography-ion trap mass spectrometry and analyte protectants. *10th International Symposium on Advances in Extraction Techniques*, Bruges, Belgium, pp.

biocide tributyltin reduces the accumulation of testosterone as fatty acid esters in the mud snail (*Ilyanassa obsoleta*). *Environmental Health Perspectives*, 111, pp. 426-430.

(TBT) on *Xenopus tropicalis* embryos at environmentally relevant concentrations.

disrupting compounds (vinclozolin, flutamide, ketoconazole and dicofol): Effects on survival, occurrence of males, growth, molting and reproduction of *Daphnia* 

bisphenol A and tetrabromobisphenol A in quail eggs, embryos and laying birds and studies on reproduction variables in adults following in ovo exposure. *Archives* 

Van der Kraak, G. (2006). Endocrine disrupting chemicals research program of the US Environmental Protection Agency: Summary of a peer-review report.

Control of Harmful Anti-Fouling Systems for Ships, 1–5 October 2001. Report No.

accessed in 20 of July 2011, available from:

*Porcellionides pruinosus* and three model pesticides. *European Journal of Soil Biology*,

& Kikuyama, S. (2008). Bisphenol A acts differently from and independently of thyroid hormone in suppressing thyrotropin release from the bullfrog pituitary.

Y.I., Dong, M.S., & Han, S.Y. (2004). Comparison of anti-androgenic activity of flutamide, vinclozolin, procymidone, linuron, and p,p '-DDE in rodent 10-day

southern sea otters (*Enhydra lutris nereis*) found dead along California coastal

Luster, M., Mac, M.J., Maczka, C., Miller, R., Moore, J., Rolland, R., Scott, G.,

Sheehan, D.M., Sinks, T., & Tilson, H.A. (1996). Research needs for the risk assessment of health and environmental effects of endocrine disruptors: A report of the US EPA-sponsored workshop. *Environmental Health Perspectives*, 104, pp. 715- 740.


Kingdom Environmental Agency and the CEFIC Endocrine Modulator Steering Group, 178p.


Porte, C., Janer, G., Lorusso, L.C., Ortiz-Zarragoitia, M., Cajaraville, M.P., Fossi, M.C., &

Price, T.M., Murphy, S.K., & Younglai, E.V. (2007). Perspectives: The possible influence of

environmental endocrine disruptors. *Toxicological Sciences*, 96, pp. 218-226. Readman, J.W., Albanis, T.A., Barcelo, D., Galassi, S., Tronczynski, J., & Gabrielides, G.P.

Reddy, P.S., Sarojini, R., & Nagabhushanam, R. (1991). Impact of tributyltin oxide (TBTO) on

Rodriguez, E.M., Medesani, D.A., & Fingerman, M. (2007). Endocrine disruption in

Ruppert, E.E., Fox, R.S., & Barnes, R.D. (Eds.). (2003). *Invertebrate Zoology: A Functional* 

Santos, M.M., Ten Hallers-Tjabbes, C.C., Santos, A.M., & Vieira, N. (2002). Imposex in

Santos, M.M., Enes, P., Reis-Henriques, M., Kuballa, J., Castro, L.F.C., & Vieira, M.N. (2009).

Short, J.W., Thrower, F.P. (1987). Toxicity of tri-n-butyl-tin to chinook salmon, *Oncorhynchu* 

Shulte-Oehlmann, U., Bettin, C., Fioroni, P., Oehlmann J., & Stroben, E. (1995). *Marisa* 

Shulte-Oehlmann, U. (1997). *Fortpflanzungsstörungen bei süß- und brackwasserschnecken –* 

Sidharthan, M., Young, K.S., Woul, L.H., Soon, P.K., & Shin, H.W. (2002). TBT toxicity on the

Smith, B.S. (1981). Tribultyltin compounds induce male characteristics on female mud snails *Nassarius obsoletus* = *Ilyanassa obsolete*. *Journal of Applied Toxicology*, 1, pp. 141-144. Sonak, S. (2009). Implications of organotins in the marine environment and their prohibition.

Sousa, A., Ikemoto, T., Takahashi, S., Barroso, C., & Tanabe, S. (2009a). Distribution of

MED POL pilot survey. *Marine Pollution Bulletin,* 34, pp. 259-263

intervals of amputation. *Journal of Tissue Research*, 1, pp. 35-39.

*Evolutionary Approach*. Brooks Cole Thomson. Belmont, CA, U.S..

*tshawytscha*, adapted to seawater. *Aquaculture*, 61, pp. 193-200.

freshwater environments. *Ecotoxicology*, 4, pp. 372-384.

*Journal of Environmental Management*, 90, pp. 1-3.

Portuguese coast. *Marine Pollution Bulletin*, 58, pp. 1130-1136.

*Molecular and Integrative Physiology*, 146, pp. 661-671.

Group, 178p.

143, pp. 303-315.

*Research,* 48, pp. 217– 223.

*Chemosphere,* 75, pp- 661-666.

Germany.

180

Kingdom Environmental Agency and the CEFIC Endocrine Modulator Steering

Canesi, L. (2006). Endocrine disruptors in marine organisms: Approaches and perspectives. *Comparative Biochemistry and Physiology C-Toxicology & Pharmacology*,

assisted reproductive technologies on transgenerational reproductive effects of

(1997). Fungicide contamination of Mediterranean estuarine waters: Results from a

limb regeneration of the prawn, *Caridina rajadhari*, after exposure to different time

crustaceans due to pollutants: A review. *Comparative Biochemistry and Physiology A -* 

*Nucella lapillus*, a bioindicator for TBT contamination: re-survey along the Portuguese coast to monitor the effectiveness of EU regulation. *Journal of Sea* 

Organotin levels in seafood from Portuguese markets and the risk for consumers.

*cornuarietis* (Gastropoda, Prosobranchia): a potential TBT bioindicator for

*Einfluß der umweltchemikalie tributylzinn.* Wissenschaft und Technik Verlag. Berlin,

marine microalga Nannochloropsis oculata. *Marine Pollution Bulletin*, 45, pp. 177-

synthetic organotins and total tin levels in *Mytilus galloprovincialis* along the


### **Molecular Characterization of Carbendazim Resistance of Plant Pathogen (***Bipolaris oryzae***)**

S. Gomathinayagam1, N. Balasubramanian2,

V. Shanmugaiah3, M. Rekha4, P. T. Manoharan5 and D. Lalithakumari6 *1Faculty of Agriculture and Forestry, University of Guyana, Berbice Campus, Tain, 2CIRN and Department of Biology, University of Azores, Ponta Delgada, Azores,* 

*3Department of Microbial Technology, School of Biological Sciences,* 

*Madurai Kamaraj University, Madurai, Tamil Nadu,* 

*4Department of Biotechnology, Kalasalingam University, Krishnankovil, Tirunelveli, Tamil Nadu, 5Department of Botany, Vivekananda College, Thiruvedakam, Madurai, Tamil Nadu,* 

 *6Research and Development, Bio-Soil Enhancers, Hattiesburg, MS,* 

*1Guyana 2Portugal 3,4,5India 6USA* 

#### **1. Introduction**

196 Fungicides – Beneficial and Harmful Aspects

Waldock, M.J., Thain, J.E. (1983). Shell thickening in *Crassostrea gigas*: Organotin antifouling

Weis, J.S, Gottlieb, J., & Kwiatkowski, J. (1987). Tributyltin retards regeneration and

produces deformities in the limbs in the fiddler crab, Uca pugilator. *Archives of* 

or sediment induced? *Marine Pollution Bulletin*, 14, pp. 411-415

*Environmental Contamination and Toxicology*, 16, pp. 321-326

Agricultural practices are often portrayed as significantly contributing to environmental problems. When organic pesticides were discovered they were hailed for their positive contributions to increased food production. The challenges we are facing is that the changing environmental ethics coincide with rapid population growth. It is expected that within the next 50 years there will be approximately 50% more people on earth to feed, house and cloth. The world is not only increasing in population, but also in affluence. Invasive pests are a significant threat to the statuesque of our natural ecosystems, our health and agriculture. Many of the most serious plant diseases have already been spread throughout the world, but there is still good reason to be very diligent in monitoring and excluding those diseases that are still endemic to limited geographic areas. Emergence of new diseases and disease epidemics must be expected. One of the most important contributions of plant pathology research to science, is the recognition that pest evolve at a rapid rate and that single gene changes can turn an obscure microbe into the cause of an epidemic. The challenge for plant pathology is to be able to predict when such genetic changes will happen. The ability to genetically modified plants, animals and microbes in precise ways using molecular biology and biotechnology provides the present century best hope for meeting the food, fiber and nutritional needs of the growing population of the world without further compromising the quality of our environment. This technology is so important for the future that care has to be taken that no compromise is taken on long-term value by meeting short-term goals.

With the modern biotechnology on hand, research area calls for more attention in basic and applied research programmes in product, mode of action, mechanism of action and application of fungicides for safety and economic use. Development of fungicide resistance is a threatening topic to farmers who practice with potential systemic fungicides. When a fungicide fails because of the development of resistance by the target organism, in practice it is very important to know whether the effectiveness of other fungicides has been affected as this will lead to many more problems.

The developing research on biocontrol practice needs supportive control of some chemicals in the control of diseases that cannot be controlled by biocontrol. It is high time to go further with in-depth research on fungicides, to combat and overcome fungicide resistance in practice. Systemic chemicals are best alternatives for any failure practices of plant protection, provided they are carefully monitored for their mode of action, method of application and understood more on the mechanism of development of fungicide resistance.

The present research is case study on Carbendazim fungicide resistance in many important pathogens. *In vivo* and *in vitro* fungicide resistant mutants have been isolated and characterized for growth, sporulation and pathogenicity. The complete fitness and competition tests of the resistant strains we carefully carried out in the presence and absence of fungicide. Level of resistance, using petri plate method was compared to spot diagnosis techniques using allele specific oligonucleotides. The test fungicide chosen was Carbendazim, a Benzimidazole compound whose mode of action is inhibition of microtubules assembly. Benzimidazole compounds are increasingly important and most widely known, owing to the excellent systemic control of much important plant diseases. Development of fungicide resistance is now one of the major problems in plant disease control, but could be easily delayed or prevented through careful practices. This requires stable information's on genetics of resistance, level of resistance and mechanisms of resistance. The Present research give an insight on to the above factors to know more information's on fungicide resistance. New molecular techniques have been assessed for effective evaluation of the level of fungicide resistance at field level.

#### **2. Materials and methods**

#### **2.1 General methods**

Glassware was first soaked in chromic acid cleaning solutions (10% potassium dichromate solutions in 25% sulphuric acid) for few hours and washed thoroughly in tap water. After a second wash in detergent solution, they were again washed thoroughly in tap water, rinsed in distilled water and air dried. Glassware, media and buffers were sterilized at 120°C, 15 Ib pressure / inch2 for 20 min.

#### **2.2 Chemicals**

Chemicals and solvents used were of analar grade. Ethyl Methane Sulfate (EMS), calf thymus DNA, cesium chloride, agarose, DNA, restriction enzymes and polyethylene glycol (PEG) were purchased from Sigma chemicals Co., St. Louis, USA. Novozym –234 was purchased from Novo Industry, Denmark.

#### **2.3 Fungicides**

BASF (India) Ltd., Chennai generously supplied Carbendazim. Other fungicide used in the investigation was purchased from various sources.

#### **2.4 Preparation of stock solution of fungicide**

Stock solutions of fungicide were prepared by dissolving the fungicide in 1 mL acetone or ethanol and made up to a known volume with distilled water. Required concentrations were prepared by diluting the stock solution. Fungicide solutions were prepared just before use and the final concentration of the solvent did not exceed 0.5% in the medium. Sterilization of stock solution was made by filtering through sterilized millipore filter system (0.22µ).

#### **2.5 Isolation of fungi**

198 Fungicides – Beneficial and Harmful Aspects

is a threatening topic to farmers who practice with potential systemic fungicides. When a fungicide fails because of the development of resistance by the target organism, in practice it is very important to know whether the effectiveness of other fungicides has been affected as

The developing research on biocontrol practice needs supportive control of some chemicals in the control of diseases that cannot be controlled by biocontrol. It is high time to go further with in-depth research on fungicides, to combat and overcome fungicide resistance in practice. Systemic chemicals are best alternatives for any failure practices of plant protection, provided they are carefully monitored for their mode of action, method of application and understood more on the mechanism of development of fungicide resistance. The present research is case study on Carbendazim fungicide resistance in many important pathogens. *In vivo* and *in vitro* fungicide resistant mutants have been isolated and characterized for growth, sporulation and pathogenicity. The complete fitness and competition tests of the resistant strains we carefully carried out in the presence and absence of fungicide. Level of resistance, using petri plate method was compared to spot diagnosis techniques using allele specific oligonucleotides. The test fungicide chosen was Carbendazim, a Benzimidazole compound whose mode of action is inhibition of microtubules assembly. Benzimidazole compounds are increasingly important and most widely known, owing to the excellent systemic control of much important plant diseases. Development of fungicide resistance is now one of the major problems in plant disease control, but could be easily delayed or prevented through careful practices. This requires stable information's on genetics of resistance, level of resistance and mechanisms of resistance. The Present research give an insight on to the above factors to know more information's on fungicide resistance. New molecular techniques have been assessed for

Glassware was first soaked in chromic acid cleaning solutions (10% potassium dichromate solutions in 25% sulphuric acid) for few hours and washed thoroughly in tap water. After a second wash in detergent solution, they were again washed thoroughly in tap water, rinsed in distilled water and air dried. Glassware, media and buffers were sterilized at 120°C,

Chemicals and solvents used were of analar grade. Ethyl Methane Sulfate (EMS), calf thymus DNA, cesium chloride, agarose, DNA, restriction enzymes and polyethylene glycol (PEG) were purchased from Sigma chemicals Co., St. Louis, USA. Novozym –234 was

BASF (India) Ltd., Chennai generously supplied Carbendazim. Other fungicide used in the

effective evaluation of the level of fungicide resistance at field level.

this will lead to many more problems.

**2. Materials and methods** 

15 Ib pressure / inch2 for 20 min.

purchased from Novo Industry, Denmark.

investigation was purchased from various sources.

**2.1 General methods** 

**2.2 Chemicals** 

**2.3 Fungicides** 

#### **2.5.1 Test pathogen**

The test pathogen *Bipolaris oryzae* causing brown leaf spot were chosen as test pathogen. The phytopathogenic strains of *Bipolaris oryzae* were collected from infected paddy leaves in paddy filed of Siddi Vinayaga farm, Bandikavnoor, Chennai. Infected leaves were sterilized in mercuric chloride (0.01%) and placed onto PDA, to which streptomycin (50 g / mL) was added to suppress bacterial growth and incubated at 20oC for 3 days leaf bits were removed. Hundred agar blocks containing germlings of single conidia were picked up with a sterile needle under microscopic observation, transferred individually to PDA slants and incubated until they form sporulating colonies.

#### **2.5.2 Culture conditions of the fungus**

Fungus stock culture was maintained on PDA slants at 28oC and transferred to new media at regular intervals. Petri plate cultures were maintained as follows. To 3 days old culture, 5 mL of sterile distilled water was added and scrapped with an inoculation needle. The conidial suspension was transferred to a 250 mL conical flask containing 100 mL molten PDA (40oC), mixed thoroughly and poured into sterile Petri dishes. After 3 days of incubation, using a sterile cork borer, mycelial discs (8 mm dia) were cut at random of periphery region and were used for further experiments throughout the investigation.

#### **2.5.3 Preparation of conidial suspension**

Conidial suspension of phytopathogen of *Bipolaris oryzae* were prepared by washing the well sporulated slant cultures in sterile water containing one drop of Tween 20 and filtered through two layers of cheese cloth to remove hyphal fragments. The spore suspension was washed twice with sterile water and resuspended in sterile water. The concentration of conidia was determined using a Haemocytomer.

#### **2.5.4 Pathogenicity test on rice plants (Chevalier et at., 1991)**

Pathogenicity of rice pathogens was tested on rice plants under green house conditions. Forty five days old Ponni and IR 50 paddy plants were first sprayed with mycelial suspension of the sensitive strains of pathogen (*B. oryzae*) means of automizer. The plants were covered with individual's polythene bags to provide adequate humidity and kept at room temperature. The inoculated plants were observed after 7 days for characteristic symptoms.

#### **2.6 Morphological and physiological characterization**

#### **2.6.1 Growth of test pathogen (sensitive strain) on solid media**

Before the assessment of growth on fungicide amended medium, the maximum growth and sporulation rate inducing medium was screened. Growth rate of pathogens was assessed on five different solid media viz. PDA, PDYEA, CDA, MEA and OMA. Mycelial disc (8 mm dia) was placed at the center of the Petri plate in an inverted position containing media and kept for incubation. Quadruplicates were maintained for each treatment. At every 48 h, the diameter of the mycelial growth was measured. After incubation, 10 mycelial discs (8 mm dia) were cut at random from the periphery and transferred to 10 mL sterile water in 100 mL flasks. The flasks were kept on an orbital shaker for 30 min. The number of conidia /mL in different media was counted using Haemocytometer.

#### **2.6.2 Growth rate of test pathogen (sensitive strains) in PD broth**

The growth rates of test pathogen were studied by inoculating 4 mycelial discs (8 mm dia) into 250 mL conical flask containing 100 mL PD broth. One set of flasks were incubated on an orbital shaker (120 rpm) while another's sets of flasks were incubated under static conditions for 8 days. Every day, 4 flasks were removed from each set, the mycelial mat was filtered and dried separately in preweighted Whatman No. 1 filter paper at 80oC and the dry weights estimated.

#### **2.6.3 Sensitivity of test pathogen (wild strain) to Carbendazim on PDA**

Sensitivity of phytopathogen to Carbendazim was tested using poison food technique (Carpenter, 1942) by inoculating 8 mm mycelial discs at the centre of the Petri plates containing different concentration of the fungicide (i.e. 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500 and 1000M) amended with PDA. Four plates were maintained for each treatment. After incubation for 10 days, the diameter of mycelial growth was measured in all treatments and control. The percentange inhibition over control was calculated by plotting probit values of the percent inhibition of the growth against log concentration of the fungicide (Nageswara Rao, 1965) and ED50 dose was estimated.

#### **2.6.4 Sensitivity of test pathogen to Carbendazim in PD broth**

In PD broth, the sensitivity of phytopathogens to Carbendazim was carried out by inoculating 4 mycelial discs (8mm dia) in 250 mL flasks containing 100 mL of PD broth amended with different concentrations of fungicides. The flasks were kept on an orbital shaker (120 rpm) for 4 days. Quadruplicates were maintained for each treatment. Then the mycelial mat was than collected and dried on a reweighed, Whatman No. 1 filter paper and the dry weight estimated. The ED50 dosage was calculated by plotting probit values of the percent inhibition of the growth against log concentration of the fungicide.

#### **2.7 Development of** *in vitro* **resistant mutant of test pathogen against Carbendazim**

#### **2.7.1 Collection of conidia**

The fungus was grown in PD broth at 27oC on a reciprocal shaker for 5 days. The conidia were separated from mycelia by filtering through double layers of cheese cloth and the filtrate was centrifuged at 1000Xg for 10 min. The conidia were suspended in a known

Before the assessment of growth on fungicide amended medium, the maximum growth and sporulation rate inducing medium was screened. Growth rate of pathogens was assessed on five different solid media viz. PDA, PDYEA, CDA, MEA and OMA. Mycelial disc (8 mm dia) was placed at the center of the Petri plate in an inverted position containing media and kept for incubation. Quadruplicates were maintained for each treatment. At every 48 h, the diameter of the mycelial growth was measured. After incubation, 10 mycelial discs (8 mm dia) were cut at random from the periphery and transferred to 10 mL sterile water in 100 mL flasks. The flasks were kept on an orbital shaker for 30 min.

The number of conidia /mL in different media was counted using Haemocytometer.

The growth rates of test pathogen were studied by inoculating 4 mycelial discs (8 mm dia) into 250 mL conical flask containing 100 mL PD broth. One set of flasks were incubated on an orbital shaker (120 rpm) while another's sets of flasks were incubated under static conditions for 8 days. Every day, 4 flasks were removed from each set, the mycelial mat was filtered and dried separately in preweighted Whatman No. 1 filter paper at 80oC and the dry weights estimated.

Sensitivity of phytopathogen to Carbendazim was tested using poison food technique (Carpenter, 1942) by inoculating 8 mm mycelial discs at the centre of the Petri plates containing different concentration of the fungicide (i.e. 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500 and 1000M) amended with PDA. Four plates were maintained for each treatment. After incubation for 10 days, the diameter of mycelial growth was measured in all treatments and control. The percentange inhibition over control was calculated by plotting probit values of the percent inhibition of the growth against log concentration of the fungicide (Nageswara

In PD broth, the sensitivity of phytopathogens to Carbendazim was carried out by inoculating 4 mycelial discs (8mm dia) in 250 mL flasks containing 100 mL of PD broth amended with different concentrations of fungicides. The flasks were kept on an orbital shaker (120 rpm) for 4 days. Quadruplicates were maintained for each treatment. Then the mycelial mat was than collected and dried on a reweighed, Whatman No. 1 filter paper and the dry weight estimated. The ED50 dosage was calculated by plotting probit values of the

**2.7 Development of** *in vitro* **resistant mutant of test pathogen against Carbendazim** 

The fungus was grown in PD broth at 27oC on a reciprocal shaker for 5 days. The conidia were separated from mycelia by filtering through double layers of cheese cloth and the filtrate was centrifuged at 1000Xg for 10 min. The conidia were suspended in a known

**2.6 Morphological and physiological characterization** 

**2.6.1 Growth of test pathogen (sensitive strain) on solid media** 

**2.6.2 Growth rate of test pathogen (sensitive strains) in PD broth** 

**2.6.3 Sensitivity of test pathogen (wild strain) to Carbendazim on PDA** 

**2.6.4 Sensitivity of test pathogen to Carbendazim in PD broth** 

percent inhibition of the growth against log concentration of the fungicide.

Rao, 1965) and ED50 dose was estimated.

**2.7.1 Collection of conidia** 

volume of sterile water and the final concentration was adjusted to 1 X 106 conidia /mL by using Haemocytometer.

#### **2.7.2 Development of Carbendazim resistant mutants of test pathogen by UV – irradiation (Tanaka et al., 1988)**

The in vitro mutants resistant to Carbendazim were developed by following the procedure of Tanaka et al., (1988). Aliquots of conidial suspension (1 X 103 conidia / mL ) containing Tween 20 (0.02% V/V) was poured into a Petri plate and exposed to UV irradiation (254 nm Phillips TUV 51 W G15T8) at a distance of 30 cm for 15, 30, 45 and 60 min. After which, the conidia were incubated in the dark for 2 h to prevent photo reactivation.

Conidia treated with mutagens as described above, were plated separately on PDA amended with Carbendazim 5 times ED50 concentration of the sensitive strain. Medium devoid of fungicide served as control. The Petri plates were incubated at 20oC for 8 days. survival rates was calculated from the ratio of colonies which grew on the fungicide unamended medium and the number of conidia inoculated. Mutation rate was determined from the ratio of colonies which grew on fungicide amended media and number of conidia inoculated.

#### **2.7.3 Induction and isolation of Carbendazim resistant mutants of phytopathogen by Ethyl Methyl Sulfate (EMS) treatment (Tanaka et al., 1988)**

Conidia (1 X 106 /mL) of the sensitive strain of test pathogens were suspended in 100 mL conical flask containing 10 mL freshly prepared EMS solution at a concentration of 5, 10, 25, 50, 100, 250, 500, 1000,2000 and 5000m in 0.1 M sterile phosphate buffer (pH 7.0). All the flasks were kept on a shaker for 6 h at 27oC. Treated conidia were washed twice with sterile distilled water by centrifugation at 1000 X g for 10 min to remove traces on EMS. Subsequently, the conidial concentration was adjusted to 1 X 106 mL and plated on PDA amended with fungicide (five times the concentration of ED50 value of sensitive strain). The seeded plates were incubated for 5 d at 20oC. The colonies (mutants) that survived were counted and isolated for further studies and percent survival was also calculated.

#### **2.7.4 Development of resistant strains of test pathogen by adaptation technique**

Adaptability of phytopathogen to Carbendazim was carried out by the method of Rana and Sengupta (1977). Conidia (1 X 103 / mL) of the sensitive strain were placed on PDA medium amended with 5 times higher the ED50 value of fungicide and the plates were incubated for 5 d at 23oC. From the developed colonies, mycelial discs (8 mm dia) were cut from periphery using a sterile cork borer and then transferred to stepwise increasing concentration of the fungicide. All the laboratory mutants resistant to test fungicide Carbendazim were numbered and maintained.

#### **2.7.5 Collection of field resistant strains**

Field resistant mutants of *B. oryzae* were collected from diseased parts of plants treated continuously with Carbendazim.

#### **2.7.6 Stability test for fungicide resistance in the mutants of test pathogen**

Carbendazim resistant mutants were subcultured on fungicide free PDA medium for 10 generations. After 10 generations, the mutants were transferred to the respective fungicide (5 X the ED50 concentration) amended PDA medium. Stability rate was determined from the ratio of colonies which grew on the fungicide amended medium and number of colonies inoculated. The Carbendazim resistant mutant strains of test pathogens obtained by various mutagenesis techniques were designated.

#### **2.8 Level of fungicide resistance laboratory mutants and field mutants resistant to Carbendazim**

#### **2.8.1 Level of resistance based on the mycelial growth on PDA medium**

Young mycelial discs (8 mm dia) were aseptically transferred to the centre of the Petri plates containing PDA medium amended with different concentrations (always above 5 times the concentration of ED50 value of sensitive strain) of Carbendazim (5M to 1000M) and incubated at 20oC for 25days. Medium devoid of fungicide served as control. Four replicates were maintained for each treatment. Percent inhibition of growth was calculated and the ED50 values were derived by probit analysis.

#### **2.8.2 Level of resistance based on the mycelial growth and conidial production in PD broth**

One mL conidial suspension (1 X 103 conidia / mL) of the resistant mutants was inoculated in 100 mL PD broth amended with different concentrations of Carbendazim (5 to 1000M) and incubated orbital shaker (120 rpm) at 20oC for 12 days. PD broth devoid of fungicide served as control. Four replicates were maintained for each treatment. After the incubation period, mycelia were filtered and dry weight was determined. Numbers of conidia were counted in each flask per replicate per treatment. Percent inhibition of mycelial dry weight and conidial production was determined and ED50 values derived by probit analysis.

#### **2.8.3 Level of resistance based on germination and primary hyphal elongation in PD broth**

One ml conidial suspension (1 X 103 conidia / mL) of the resistant mutants was inoculated in 25 mL PD broth amended with higher concentration of Carbendazim (100 to 1000M) and incubated on an orbital shaker (120 rpm) at 20oC. Flasks inoculated with Cassava Root meal (CRM) were incubated for 30 h. PD broth devoid of fungicide served as control. Four replicates were maintained for each treatment. Percent inhibition of germination and primary hyphal elongation were calculated and ED50 values of the fungicide were derived by probit analysis.

#### **2.8.4 Morphological characterization of the resistant mutants of test fungi**

Colony morphology, pigmentation and conidial production of the resistant mutants in fungicide unamended and amended medium were compared with that of the sensitive strain. The conidia was measured using a calibrated microscope (Carl Zeiss, Germany)

#### **2.9.1 Measurement of oxygen uptake by sensitive strain and resistant mutants of test pathogen**

Oxygen uptake was measured (Johnson, 1972) polar graphically in clark type oxygen probe fitted to a YSI (Yellow Springs Instruments Ltd., Ohio., USA) model oxygen monitor. The probe was standardized with distilled water. The probe was inserted into the chamber and 100% saturation of air was set. For the measurements of oxygen, 1 g fresh mycelium suspended in 5 mL Potato Dextrose Yeast Extract (PDYE) broth was transferred into the chamber. The decrease in saturation percent was measured at 1 min interval for 10 min (Arditti and Dunn, 1969). Percent saturation of air measured was converted to oxygen concentration using Rawso's monogram (Welch, 1948).

#### **2.9.2 Determination of electrolytes**

202 Fungicides – Beneficial and Harmful Aspects

Carbendazim resistant mutants were subcultured on fungicide free PDA medium for 10 generations. After 10 generations, the mutants were transferred to the respective fungicide (5 X the ED50 concentration) amended PDA medium. Stability rate was determined from the ratio of colonies which grew on the fungicide amended medium and number of colonies inoculated. The Carbendazim resistant mutant strains of test pathogens obtained by various

**2.8 Level of fungicide resistance laboratory mutants and field mutants resistant to** 

Young mycelial discs (8 mm dia) were aseptically transferred to the centre of the Petri plates containing PDA medium amended with different concentrations (always above 5 times the concentration of ED50 value of sensitive strain) of Carbendazim (5M to 1000M) and incubated at 20oC for 25days. Medium devoid of fungicide served as control. Four replicates were maintained for each treatment. Percent inhibition of growth was calculated and the

**2.8.2 Level of resistance based on the mycelial growth and conidial production in PD** 

One mL conidial suspension (1 X 103 conidia / mL) of the resistant mutants was inoculated in 100 mL PD broth amended with different concentrations of Carbendazim (5 to 1000M) and incubated orbital shaker (120 rpm) at 20oC for 12 days. PD broth devoid of fungicide served as control. Four replicates were maintained for each treatment. After the incubation period, mycelia were filtered and dry weight was determined. Numbers of conidia were counted in each flask per replicate per treatment. Percent inhibition of mycelial dry weight and conidial production was determined and ED50 values derived by

**2.8.3 Level of resistance based on germination and primary hyphal elongation in PD** 

One ml conidial suspension (1 X 103 conidia / mL) of the resistant mutants was inoculated in 25 mL PD broth amended with higher concentration of Carbendazim (100 to 1000M) and incubated on an orbital shaker (120 rpm) at 20oC. Flasks inoculated with Cassava Root meal (CRM) were incubated for 30 h. PD broth devoid of fungicide served as control. Four replicates were maintained for each treatment. Percent inhibition of germination and primary hyphal elongation were calculated and ED50 values of the fungicide were derived

Colony morphology, pigmentation and conidial production of the resistant mutants in fungicide unamended and amended medium were compared with that of the sensitive strain. The conidia was measured using a calibrated microscope (Carl Zeiss, Germany)

**2.8.4 Morphological characterization of the resistant mutants of test fungi** 

**2.8.1 Level of resistance based on the mycelial growth on PDA medium** 

**2.7.6 Stability test for fungicide resistance in the mutants of test pathogen** 

mutagenesis techniques were designated.

ED50 values were derived by probit analysis.

**Carbendazim** 

**broth** 

**broth** 

probit analysis.

by probit analysis.

One gram fresh mycelium was washed with sterile double distilled water and incubated in 100 mL PD broth. One set of flasks was amended with ED50 concentration of the carbendazim and incubated on an orbital shaker (150rpm). Mycelium incubated in PD broth devoid of fungicide served as control. Four replicates were maintained for each treatment. Mycelium was harvested at every 4 h interval for 24 and washed with excess of distilled water. Washed mycelium was transferred to 25 mL of sterile double distilled water and incubated on an orbital shaker (150rpm) for 1 h. After incubation, myceliums were filtered and the conductance of the ambient water was measured using CM 82T Conductivity Bridge with a dip electrolytic cell. Dry weight of mycelium was determined.

#### **2.9.3 Extraction of protein**

Wet mycelium (500 mg) was ground with equal amount of acid washed sand and 0.1 M sodium phosphate buffer (pH 7.0) in a pre-chilled mortar and pestle for 20 min at 4oC. The ground material was centrifuged at 15, 000 x g for 15 min and the supernatant was made up to a known volume with the same buffer and dialyzed overnight against large volume of glass distilled water. The dialyzed extract was used for protein estimation.

#### **2.9.4 Protein estimation**

Protein was estimated by the method of Lowry et al., (1951) using Bovin Serum Albumin (BSA) as standard and the amount of protein was expressed as mg protein/g dry wt. of mycelium.

#### **2.10 Electrophoretic protein pattern of sensitive strain an resistant mutant**

Electrophoretic pattern of total protein of sensitive and resistant mutants was analysed by the method of Ornstein (1964).

#### **2.10.1 Analysis of protein by SDS –PAGE (Laemmli, 1970)**

The discontinuous buffer system of Laemmli (1970), which is a modification of Ornstein (1964), was used in the present study for the separation of proteins.

#### **2.11 Molecular characterization of sensitive strain and resistant mutants of test pathogen**

#### **2.11.1 Extraction of degraded DNA and RNA**

For quantification, DNA and RNA were extracted by the modified method of Scheneider (Munro and Fleck, 1966).

One gram fresh weight of the mycelium was treated with 5% trichloroacetic acid (TCA) for 60 min at 4oC. The supernatant containing the cold acid soluble compounds was discarded after centrifugation at 15,000 X g for 15 min at 4oC. The mycelium was their treated with 1 N perchloric acid (PCA) at 70oC in water bath for 20 min to hydrolyse the nucleic acid. The suspension was again centrifuged at 15, 000 X g for 30 min at 4oC and the supernatant was collected. The pellet was re extracted with 1 N PCA under the same conditions and the supernatants were pooled and used for DNA and RNA estimation.

#### **2.11.2 Calorimetric determination of DNA**

DNA was estimated by the improved method of Giles and Myers (1965) further modified by Lalithakumari et al (1975). The reaction mixture consisted of 2 mL of hydrolysate obtained after 1 N PCA treatment and 2 mL 1% freshly prepared diphenylamine in glacial acetic acid and was incubated at 30oC for 16 h in dark. The blue colour developed was measured at 595 nm and 700 nm in a Hitachi 150 – 20 spectrophotometer and the difference in optical densities were calculated. The solution without hydrolysate mixture was used for blank. The quantity of DNA was estimated using the standard curve prepared with calf thymus DNA.

#### **2.11.3 Determination of rate of uptake of Carbendazim**

One gram fresh mycelium grown in PDYE broth was harvested by filtration on a Buchner filter and washed thrice with distilled water. The mycelium was washed twice with incubation medium (25mM KH2PO4, 12.5 mM K2HPO4.3H2O buffer, pH 7.0, with 0.1 mM CaCl2. 2H2O and 1% glucose) (De Waard and Nistelrooy, 1980) and resuspended in 50 mL of the same at 37oC for 30 min. After 30 min, ED50 of fungicide (25m carbendazim) was amended in the mycelial suspension. Uninoculated medium served as control and four replicates were maintained for each treatment. At 10 min interval, 5 mL sample was filtered over Whatman No.1 filter paper. The mycelial residues were washed thrice with the incubation medium without fungicide and finally the incubation media were pooled and made up to a known volume. Similarly, 5 mL incubation medium was drawn from the control flask (incubation medium without fungicide) and made up to same volume as that of the test sample. Percent uptake of fungicide by each strain was determined from the difference in the amount of residual fungicide in the test and control medium. Sodium azide was added to the mycelial suspension 15 min prior to the addition of fungicide.

#### **2.12 Polymerase Chain Reaction (PCR)**

The polymerize chain reaction and then sequence analysis of the genomic DNA were used to rapidly characterize the sequence of β–tubulin DNA from the pathogens. Genomic DNA was prepared from each strain of test pathogens and subjected to PCR by using two generic

For quantification, DNA and RNA were extracted by the modified method of Scheneider

One gram fresh weight of the mycelium was treated with 5% trichloroacetic acid (TCA) for 60 min at 4oC. The supernatant containing the cold acid soluble compounds was discarded after centrifugation at 15,000 X g for 15 min at 4oC. The mycelium was their treated with 1 N perchloric acid (PCA) at 70oC in water bath for 20 min to hydrolyse the nucleic acid. The suspension was again centrifuged at 15, 000 X g for 30 min at 4oC and the supernatant was collected. The pellet was re extracted with 1 N PCA under the same conditions and the

DNA was estimated by the improved method of Giles and Myers (1965) further modified by Lalithakumari et al (1975). The reaction mixture consisted of 2 mL of hydrolysate obtained after 1 N PCA treatment and 2 mL 1% freshly prepared diphenylamine in glacial acetic acid and was incubated at 30oC for 16 h in dark. The blue colour developed was measured at 595 nm and 700 nm in a Hitachi 150 – 20 spectrophotometer and the difference in optical densities were calculated. The solution without hydrolysate mixture was used for blank. The quantity of DNA was estimated using the standard curve

One gram fresh mycelium grown in PDYE broth was harvested by filtration on a Buchner filter and washed thrice with distilled water. The mycelium was washed twice with incubation medium (25mM KH2PO4, 12.5 mM K2HPO4.3H2O buffer, pH 7.0, with 0.1 mM CaCl2. 2H2O and 1% glucose) (De Waard and Nistelrooy, 1980) and resuspended in 50 mL of the same at 37oC for 30 min. After 30 min, ED50 of fungicide (25m carbendazim) was amended in the mycelial suspension. Uninoculated medium served as control and four replicates were maintained for each treatment. At 10 min interval, 5 mL sample was filtered over Whatman No.1 filter paper. The mycelial residues were washed thrice with the incubation medium without fungicide and finally the incubation media were pooled and made up to a known volume. Similarly, 5 mL incubation medium was drawn from the control flask (incubation medium without fungicide) and made up to same volume as that of the test sample. Percent uptake of fungicide by each strain was determined from the difference in the amount of residual fungicide in the test and control medium. Sodium azide

was added to the mycelial suspension 15 min prior to the addition of fungicide.

The polymerize chain reaction and then sequence analysis of the genomic DNA were used to rapidly characterize the sequence of β–tubulin DNA from the pathogens. Genomic DNA was prepared from each strain of test pathogens and subjected to PCR by using two generic

**2.11 Molecular characterization of sensitive strain and resistant mutants of test** 

**pathogen** 

(Munro and Fleck, 1966).

**2.11.1 Extraction of degraded DNA and RNA** 

**2.11.2 Calorimetric determination of DNA** 

prepared with calf thymus DNA.

**2.12 Polymerase Chain Reaction (PCR)** 

supernatants were pooled and used for DNA and RNA estimation.

**2.11.3 Determination of rate of uptake of Carbendazim** 

β–tubulin primers. Constraints on primer design were that the amplified DNA had to contain codons 167 and 241, in which mutations were associated with resistance to benomly and that the primers had to anneal to a conserved region with minimal variation in the sequence. The 22-mer oligonucleotide A (5'- CAAACCATCTCTGGCGAACACG) and 22 mer oligonucleotide B (5' – TGGAGGACATCTTAAGACCACG) were used as primers. Primer A was identical in sequence to codons 22-28, and primer B was complementary in sequence to codons 359-365 of β–tubulin genes of *V.inaequalis* With these primers, a 1,191 bp fragment of the beta-tubulin gene was amplified. The primer was synthesized in Bangalore Gennie Pvt.Ltd, Bangalore.

The reactions were performed in a thermal cycler (35 cycles) with the repliprime DNA amplification system (Du Pont) according to the manufactures procedures. Each PCR reaction was performed in 25 l (final volume) of reaction mixture. It consisted of 1 l of DNA, 4 l (1.02l) 200M dNTPs, 5.5l (2.5 mM MgCl2), 5.5 l (50 mM KCl), 5.5 l (10 mM Tris HCl), Triton –100 X 0.05l, 0.5 M of each primer and 1 unit of Tag DNA polymerize (approximately 0.66l) 1.79l double distilled water. Negative controls were run in all the amplification reactions to detect contamination. In reactions involving primers A and B, 35 cycles were performed by pre –heating the sample for 5 min at 94oC for each reaction as follows. 94oC, 1 min; 55oC, 1min; and 72o C, 2 min. Amplification products were analyzed for the expected 1,191-bp or 436-bp fragments by 1.0% agarose gel electrophoresis in 1X TBE buffer (0.1 M Tris-HCl, 0.1 M boric acid, 0.02 mM EDTA, pH 8.3. following electrophoresis, the DNA was visualized after staining with ethidium bromide.

#### **2.12.1 Allele –Specific Oligonucleotide (ASO) analysis**

The single spore isolate of test pathogen used this study was from a large collection of field strains and laboratory strains previously characterized in studies on the inheritance to benomly resistance negatively correlated cross-resistance to diethofencarb (Jones, et al., 1987). The PCR amplified β–tubluin DNA (25ng per sample) was denatured in 0.25 N NaOH for 10 min and then applied to a nylon membrane (Gen Screen-Plus, Du Pont, Boston, MA) in a dot blot manifold. The dot blots were incubated in prehybridization solution (1 M NaCl, 50 mM Tris –Hcl, pH 7.5, 10% dextran sulfate, 1% SDS, 0.2% Ficoll (MW 400,000), 0.2% polyvinylprrolidone (MW 40,000), 0.2% bovine serum albumin, 0.1% sodium pyrophosphate, and 0.25 mg/mL denatured salmon sperm DNA) for 2 h at 5oC according to the manufacture's producer. An end-labeled ASO probe was then added to the prehybridization solution and incubated at 37ºC for at least 4 h. The blots were washed three times for 15 min each in 2 X SSC buffer (Saline – Sodium Citrate) (1 X SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 6.8) at room temperature. A high –stringency wash, three times for 2 min each in 2X SSC buffer, was then used to remove ASO probe with a single base pair mismatch from the blots. The optimum temperature for the high-stringency wash with each probe was determined empirically. The dot blots were exposed to X-ray film for 0.5-2 days at –70oC.

Four ASO probes for detecting allelic mutations in the β-tubulin gene of *V. inaequalis* were synthesized in the macromolecular structure facility, Department of Biochemistry, Michigan State University, East Lansing, USA. The 18-mer oligo-nucleotides were designated as ASOS-LR, ASOMR, ASOHR and ASOVHR probes according to the specificity of each probe.


#### **3. Results**

In recent years awareness of the importance of fungicide resistance in crop protection has been growing. Hence, in the present studies *B. oryzae* (Table 1) highly sensitive to benzimidazole of fungicide have been chosen to study the probability and level of resistance of Carbendazim in these pathogens and measured to overcome resistance by understanding the molecular mechanisms of resistance. All the test pathogen was tested for their virulence through pathogenicity test.


Table 1.Test pathogen used in the present study

#### **3.1 Growth kinetics of sensitive strains**

#### **3.1.1 Radial growth of sensitive strains of test pathogens on PDA**

Growth kinetics of the test pathogen *B. oryzae*, (Table 2) shows that *B. oryzae* takes 12 day for complete growth


\* Mean of 4 replicates

Table 2. Radial growth of sensitive strain of *B. oryzae* on PDA

#### **3.1.2 Growth rate of sensitive strains of test pathogens in PD broth**

Dry weight estimation in PD broth of test pathogen (Table 3) under static and shaken conditions always showed that under shaken condition the growth rate was fast with enhanced mycelial dry weight in all the test pathogen.

#### **3.1.3 Effect of Carbendazim on the growth of test pathogens**

Before understanding, the mechanism of resistance in different mutants, the base line sensitivity of the parent strains to test fungicide, Carbendazim was analyzed (Table 4). The ED50 value was calculated for all the test pathogen. The test pathogen the most sensitive to Carbendazim *B. oryzae* with ED50 values of 40 M.


\* Mean of four replications

206 Fungicides – Beneficial and Harmful Aspects

In recent years awareness of the importance of fungicide resistance in crop protection has been growing. Hence, in the present studies *B. oryzae* (Table 1) highly sensitive to benzimidazole of fungicide have been chosen to study the probability and level of resistance of Carbendazim in these pathogens and measured to overcome resistance by understanding the molecular mechanisms of resistance. All the test pathogen was tested for their virulence

**S. No Organism Place of collection Disease caused** 

Growth kinetics of the test pathogen *B. oryzae*, (Table 2) shows that *B. oryzae* takes 12 day for

2 2.7 4 3.3 6 4.6 8 6.9 10 8.8

Dry weight estimation in PD broth of test pathogen (Table 3) under static and shaken conditions always showed that under shaken condition the growth rate was fast with

Day Radial growth\* (cm)

12 Plate fully covered

Agriculture field Brown spot disease

1 *Bipolaris oryzae* Bandikkavanoor, Tamil Nadu, India.

**3.1.1 Radial growth of sensitive strains of test pathogens on PDA** 

Table 2. Radial growth of sensitive strain of *B. oryzae* on PDA

enhanced mycelial dry weight in all the test pathogen.

**3.1.2 Growth rate of sensitive strains of test pathogens in PD broth** 

Table 1.Test pathogen used in the present study

**3.1 Growth kinetics of sensitive strains** 

**3. Results** 

through pathogenicity test.

complete growth

\* Mean of 4 replicates

Table 3. Growth rate of sensitive strain of *B. oryzae* in PD broth


ED50 value = 40 M

Table 4. Effect of Carbendazim on the growth of *B. oryzae*

#### **3.1.4 Induction of mutation of test pathogen**

To understand the mechanisms of resistance EMS, UV and adaptation methods have been used to produce number of laboratory mutants (Table 5, Table 6, and Table 7). In the case of all the test pathogen nearly 200 mutants were produced and screened on PDA amended with five times the ED50 concentration of respective sensitive strains (according to test pathogen). Resistant colonies were picked up and stored in test tube slants amended with 5 times concentration of ED50 dose. The results in tables 7-9 clearly show that the percent survival rate depends on the test pathogen and the mutagenesis.


Spore concentration : 0.5 X 103 Incubation : 6 h

Table 5. Induction of mutation of *B. oryzae* by EMS


Spore concentration : 0.5 X 103 Incubation : 6

Table 6. Induction of mutation of *B. oryzae* by UV-irradiation


Incubation time : 6 d Medium : PDA

Table 7. Development of resistant strains of *B. oryzae* by adaptation

#### **3.1.5 Stability of test pathogen**

208 Fungicides – Beneficial and Harmful Aspects

all the test pathogen nearly 200 mutants were produced and screened on PDA amended with five times the ED50 concentration of respective sensitive strains (according to test pathogen). Resistant colonies were picked up and stored in test tube slants amended with 5 times concentration of ED50 dose. The results in tables 7-9 clearly show that the percent

**Concentration of EMS (M) No. of colonies survived % of survival** 

50 450 90 100 400 80 250 375 75 500 175 35 1000 75 15 2500 10 2

**Exposure time No. of colonies survived % of survival**  15 475 95 30 400 80 45 200 40 60 150 30 75 35 7

**Carbendazim (M)** 

I 5 75 II 10 60 III 25 58 IV 50 56 V 100 48 VI 250 45 VII 500 38 VIII 1000 20 IX 2,000 15 X 5,000 10

**Mycelial growth in diameter(mm)** 

survival rate depends on the test pathogen and the mutagenesis.

Spore concentration : 0.5 X 103 Incubation : 6 h

Spore concentration : 0.5 X 103 Incubation : 6

Incubation time : 6 d Medium : PDA

Table 5. Induction of mutation of *B. oryzae* by EMS

Table 6. Induction of mutation of *B. oryzae* by UV-irradiation

**Treatment Concentration of** 

Table 7. Development of resistant strains of *B. oryzae* by adaptation

The laboratory developed resistant mutants were subsequently tested for their stability for 10 generation in fungicide free medium (Table 8). All the tested fungicides which retained resistance after 10 generation only were taken for further studies. In *B. oryzae* the EMS mutants were more stable.


EMS : Carbendazim EMS resistant mutant

UV : Carbendazim UV resistant mutant

AD : Carbendazim Adapted resistant mutant

Table 8. Stability of fungicide resistance in *B. oryzae* 

#### **3.1.6 Pathogenicity test**

All the stable mutants of the test pathogen were subsequently tested for pathogenicity. From all the tested mutants only those that developed symptoms of the diseases were chosen for further studies.

#### **3.1.7 Categorization of level of fungicide resistance**

The stable resistant mutants were screened to evaluate the level of resistance and then to categorize them as LR, MR, HR and VHR based on to their level of resistance. To group the selected mutants, they were grown on 5-10 times (LR), 10-15 times (MR), 15-20 (HR), 20-25 times and above as VHR based on ED50 value of the sensitive strain of test pathogen. The ED50 value of *B. oryzae* was 40 m. accordingly various concentrations were prepared and 60 mutants in pathogen were screened (Table 9) and categorized.



ED50 of sensitive strain 12μM


Table 9. Categorization of level resistance based on mycelial growth of mutants of *B. oryzae*

#### **3.1.8 Grouping of mutants of test pathogen**

210 Fungicides – Beneficial and Harmful Aspects

ED50 of sensitive strain 12μM

1 –10 time 15 mutants LR 10 - 15 time 20 mutants MR 15 –20 time 10 mutants HR

Table 9. Categorization of level resistance based on mycelial growth of mutants of *B. oryzae*

In *B. oryzae* 15 mutants were classified under group of LR, 19 under MR, 14 under HR and 12 as VHR were obtained (Table 10).


Table 10. Grouping of mutants of test pathogen

#### **3.1.9 Sporulation of resistant strains of test pathogen**

After grouping the resistant mutants as LR, MR, HR, and VHR, morphological characterization studies were carried out. Sporulation of the resistant mutants were evaluated for all the test pathogen and compared with the sensitive strain (Table 11). The sporulation was always more in sensitive strain than the resistant mutants. The rate of sporulation decreased according to level of resistance.


Table 11. Sporulation of resistant strains of test pathogen

#### **3.1.10 Rate of conidial germination and primary hyphal elongation of test pathogen**

Following sporulation, the spore germination and primary hyphal length were measured and presented in Table 12 for the test pathogen. Interestingly the results show a distinct reduction in spore germination and primary hyphal elongation as the level of resistant increase. The time taken for spore germination and hyphal elongation was more in resistant mutants over the control. The test pathogen, *B. oryzae* showed similar results.

#### **3.1.11 Oxygen uptake by the sensitive and resistant mutant of test pathogen**

Physiological characterization was initiated with oxygen uptake (Table 13). The oxygen uptake in n mole/min/mg dry wt of mycelium showed a distinct reduction in all the mutants irrespective of the test pathogen.


Shaken conditions

Incubation : 10 3 conidia/ mL

Temperature : 20 oC

\* Values are mean of 5 replicates.

Table 12. Rate of conidial germination and primary hyphal elongation of *B. oryzae* under shaken conditions (without fungicide)


Table 13. Oxygen uptake by the sensitive and resistant mutant of test pathogen

#### **3.1.12 Rate of efflux of electrolytes of sensitive and resistant mutants of test pathogen**

Electrolytic leakage studies on various levels of mutants of test pathogens (Table 14) also showed decrease in efflux when compared to the sensitive strains. Though there was reduction in electrolytic leakage, it was increasing with incubation time.

#### **3.1.13 Amino acid composition of sensitive and resistant mutants of test pathogens**

Amino acid composition of sensitive and resistant mutants of test pathogen (Table 15) showed distinct variations in the quantity of amino acids. Trypsin, Cystine and Glycine were totally absent in sensitive and LR mutants of *B. oryzae*. In all the mutants of test pathogens, interestingly the Proline content was more and this was absent in sensitive strain.


+ mean of 10 replication

212 Fungicides – Beneficial and Harmful Aspects

**Strain SEN MR HR VHR SEN MR HR VHR**  3 0 0 0 0 0 0 0 0 6 20 0 0 0 35 0 0 0 12 40 30 38 45 50 45 48 47 15 45 38 46 59 69 55 50 49 18 78 49 67 69 68 60 55 51 21 89 53 71 79 80 70 69 70 24 90 69 65 60 150 145 140 125

Table 12. Rate of conidial germination and primary hyphal elongation of *B. oryzae* under

SEN 27.18 MR 22.12 HR 24.10 VHR 24.00 Table 13. Oxygen uptake by the sensitive and resistant mutant of test pathogen

**3.1.12 Rate of efflux of electrolytes of sensitive and resistant mutants of test** 

reduction in electrolytic leakage, it was increasing with incubation time.

**3.1.13 Amino acid composition of sensitive and resistant mutants of test** 

Electrolytic leakage studies on various levels of mutants of test pathogens (Table 14) also showed decrease in efflux when compared to the sensitive strains. Though there was

Amino acid composition of sensitive and resistant mutants of test pathogen (Table 15) showed distinct variations in the quantity of amino acids. Trypsin, Cystine and Glycine were totally absent in sensitive and LR mutants of *B. oryzae*. In all the mutants of test pathogens, interestingly the Proline content was more and this was absent in sensitive

*B. oryzae*  **Oxygen uptake n moles/min/mg/ dry wet. Mycelium** 

**Incubation % of germination\* Primary hyphal length (M)** 

Shaken conditions

**pathogen** 

**pathogens** 

strain.

Temperature : 20 oC \* Values are mean of 5 replicates.

Incubation : 10 3 conidia/ mL

shaken conditions (without fungicide)

**Name of strains** 

\* Figure in parenthesis indicate per cell (+) increase (or) (-) decrease over sensitive strain.

Table 14. Rate of efflux of electrolytes of sensitive and resistant mutants of *B. oryzae* 


\*Mean of four replicates

Table 15. Amino acid composition of sensitive and resistant strains of *B. oryzae*

#### **3.1.14 SDS gel electrophoresis of sensitive and resistant strains of test pathogen**

Agarose gel electrophoresis of genomic DNA of all the levels of mutants of test pathogen did not show any variations among the levels of mutants and also between sensitive and resistant mutants.

#### **3.1.15 GC% and Tm of resistant mutants of test pathogen**

Results on the GC% (Table 16) and Tm showed an increase in GC% and as a result increase in melting temperature. Always the GC% was less in sensitive strains while all levels of mutants of test pathogens showed an increase in GC%.


Figure in parenthesis indicate percent over sensitive strain # Mean of strain

Table 16. Base composition of DNA of sensitive and resistant strains of *B. oryzae* 


Figure in parenthesis indicate percent over sensitive strain # Mean of strain

Table 17. Base composition of (GC%) of mitochondrial DNA of sensitive strain and resistant mutants of *B. oryzae* 

Agrose gel electrophoresis of mitochondrial DNA of various levels of mutants of all the test pathogens when compared to control did not show variation at all but the mitochondrial DNA was subjected to Hind III restriction enzyme and the restriction enzyme digested profile of various levels of mutants showed distinct variation in the number of discrete bands. The profile pattern varied between the levels of mutants and test pathogens. The number of discrete bands developed varied among the levels of mutants.

In mitochondrial DNA GC% was assessed (Table 17) and always in all levels of mutants the GC% was more and consequently the melting temperature was always more than the control.


#### **3.1.16 Cross resistance of resistant mutants of test pathogen**

\* Negatively correlated

Table 18. Cross resistance in resistant mutants of *B. oryzae*

### **4. Discussion**

214 Fungicides – Beneficial and Harmful Aspects

Agarose gel electrophoresis of genomic DNA of all the levels of mutants of test pathogen did not show any variations among the levels of mutants and also between sensitive and

Results on the GC% (Table 16) and Tm showed an increase in GC% and as a result increase in melting temperature. Always the GC% was less in sensitive strains while all levels of

**GC% of undegraded DNA** 

(6.64) <sup>88</sup>

(6.66) <sup>90</sup>

(9.00) <sup>91</sup>

(8.63) <sup>93</sup>

(10.52) <sup>94</sup>

(14.66) <sup>95</sup>

**Melting temperature (oC)** 

> **Melting temperature (oC)**

**Thermal Denaturation** 

64.18

64.19

65.60

**GC% of mt. DNA** 

**Denaturation** 

65.83

66.98

68.80

**3.1.14 SDS gel electrophoresis of sensitive and resistant strains of test pathogen** 

**3.1.15 GC% and Tm of resistant mutants of test pathogen** 

**UV absorbance** 

> 65.14 (8.34)

> 63.80 (6.12)

> 62.16 (3.40)

Table 16. Base composition of DNA of sensitive and resistant strains of *B. oryzae* 

**UV absorbance Thermal** 

Table 17. Base composition of (GC%) of mitochondrial DNA of sensitive strain and resistant

1 Sensitive 50.00 60.60 89

55.60 (11.20)

54.65 (9.30)

56.80 (13.60)

1 Sensitive 60.12 60.18 80

mutants of test pathogens showed an increase in GC%.

resistant mutants.

**S. No. Name of** 

<sup>2</sup>MR

<sup>3</sup>HR

<sup>4</sup>VHR

**S.No. Strains** 

<sup>2</sup>MR

<sup>3</sup>HR

<sup>4</sup>VHR
