The evolution of fungicide resistance.

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The Evolution of Fungicide Resistance John A. Lucas1, Nichola J. Hawkins and Bart A. Fraaije Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, UK 1 Corresponding author: E-mail: [email protected]

Contents 1. 2. 3. 4. 5.

Introduction Fungicide Resistance: The Evolutionary Context Fungicide Use on Cereals in Europe Mechanisms of Resistance to Single-Site Inhibitors Case Histories 5.1 Eyespot of Cereals

2 3 8 10 11 11

5.1.1 Changes in Field Populations of the Cereal Eyespot Pathogens in Response to Fungicide Use

5.2 Septoria tritici Blotch of Wheat

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5.2.1 Changes in CYP51 5.2.2 Additional Resistance Mechanisms to Azoles 5.2.3 SDHI Fungicides and Z. tritici

24 26 27

5.3 Powdery Mildew of Cereals, B. graminis 5.4 Fusarium Ear Blight 6. Predictability of Resistance Evolution 6.1 Mutagenesis and in vitro Selection 6.2 Fitness Costs 6.3 Parallel Evolution 6.4 Functional Constraints and Epistasis 7. Estimating Resistance Risk 8. Implications for Resistance Management 8.1 Resistance Diagnostics 8.2 Evaluating Management Strategies 8.3 The Impact of Genomics 9. Conclusions Acknowledgments References

28 31 33 33 35 36 38 41 43 43 44 46 47 48 48

Abstract Fungicides are widely used in developed agricultural systems to control disease and safeguard crop yield and quality. Over time, however, resistance to many of the Advances in Applied Microbiology, Volume 90 ISSN 0065-2164 http://dx.doi.org/10.1016/bs.aambs.2014.09.001

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most effective fungicides has emerged and spread in pathogen populations, compromising disease control. This review describes the development of resistance using case histories based on four important diseases of temperate cereal crops: eyespot (Oculimacula yallundae and Oculimacula acuformis), Septoria tritici blotch (Zymoseptoria tritici), powdery mildew (Blumeria graminis), and Fusarium ear blight (a complex of Fusarium and Microdochium spp). The sequential emergence of variant genotypes of these pathogens with reduced sensitivity to the most active single-site fungicides, methyl benzimidazole carbamates, demethylation inhibitors, quinone outside inhibitors, and succinate dehydrogenase inhibitors illustrates an ongoing evolutionary process in response to the introduction and use of different chemical classes. Analysis of the molecular mechanisms and genetic basis of resistance has provided more rapid and precise methods for detecting and monitoring the incidence of resistance in field populations, but when or where resistance will occur remains difficult to predict. The extent to which the predictability of resistance evolution can be improved by laboratory mutagenesis studies and fitness measurements, comparison between pathogens, and reconstruction of evolutionary pathways is discussed. Risk models based on fungal life cycles, fungicide properties, and exposure to the fungicide are now being refined to take account of additional traits associated with the rate of pathogen evolution. Experimental data on the selection of specific mutations or resistant genotypes in pathogen populations in response to fungicide treatments can be used in models evaluating the most effective strategies for reducing or preventing resistance. Resistance management based on robust scientific evidence is vital to prolong the effective life of fungicides and safeguard their future use in crop protection.

1. INTRODUCTION The routine use of fungicides to control crop diseases has been an important element in the intensification of modern agriculture and has helped to boost crop yields, improve quality, and ensure stability of production. Farmers and growers have had access to a range of effective chemicals that are active at low doses and provide a high level of disease control (Russell, 2005). This scenario is now changing. The cost and difficulty of discovery and registration of new actives has led to a declining product pipeline; an increasingly adverse regulatory environment, especially in Europe, has resulted in the withdrawal of many current actives; and the emergence of resistance to some of the most important classes of fungicides in many target pathogens is now compromising control. With some plant pathogens there is a concern that chemical options for their control are becoming limited or even unavailable, analogous to the situation with antibiotics in the management of human diseases (D’Costa et al., 2011; Livermore, 2009; Spellberg et al., 2008).

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Fungicides have been used in agriculture for well over a century, and initially there were no reports of losses of efficacy in the field. A comprehensive early text on fungicides and their action (Horsfall, 1945) does not include any reference to resistance. The earliest documented instances date from the 1960s and include reduced sensitivity to aromatic hydrocarbons in Penicillium species causing citrus storage rots and dodine in the apple scab fungus Venturia inaequalis (Brent, 2012, Table 1). A surprising case was adaptation to organomercurial fungicides in some strains of the seed-borne pathogen of oats, Pyrenophora avenae (Noble, Macgarvi, Hams, & Leafe, 1966). But overall, instances of confirmed resistance to fungicides remained rare until the 1970s, when novel classes of antifungal chemicals with specific modes of action were introduced and became widely used (Brent, 2012). Since then there has been an ever-increasing incidence of reported cases in a wide range of plant pathogenic fungi (Table 1). Resistance has become a fact of life for the crop protection industry and impacts directly on product stewardship and use in practice (Urech, Staub, & Voss, 1997). There is a large and expanding literature on fungicide resistance, concerning different fungal pathogens and crops, contrasting fungicide modes of action, and strategies for resistance management. A recent volume on fungicide resistance in crop protection covers several of these aspects with case histories from different countries (Thind, 2012). In this review, we focus primarily on pathogens of cereal crops in Europe (Table 2) and experiences with the major classes of fungicides used for their control. We explore the underlying mechanisms of resistance and their genetic control, and how knowledge of these factors might explain the emergence and impact of resistance. We consider the evolutionary pathways of resistance development, and whether such analysis can improve our ability to predict future problems and inform risk assessment. Finally, we discuss the implications of such mechanistic and evolutionary knowledge for practical resistance management.

2. FUNGICIDE RESISTANCE: THE EVOLUTIONARY CONTEXT Modern selective fungicides disrupt particular cellular processes and bind to specific protein targets (Table 3); they are therefore described as single-site (site-specific) in contrast to earlier fungicide classes that act on a range of cellular processes and are considered to be multisite inhibitors. Insensitivity to single-site fungicides can occur as a result of a change in a

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Table 1 Timeline of fungicide resistance in crop diseases: some examples Time to resistance Date Fungicide class (approx. yrs) Disease example

Aromatic hydrocarbons Organomercury Dodine (Guanidine)

20 40 10

Citrus storage rots Penicillium spp Cereal leaf spot Pyrenophora spp Apple scab Venturia inaequalis

1970 1971 1980

Benzimidazoles (MBCs) 2-Aminopyrimidines Phenylamides

2 2 2

1982

Demethylation inhibitors (DMIs) Quinone outside inhibitors (QoIs) Succinate dehydrogenase inhibitors

7

Many pathogens Powdery mildews Potato late blight, grape downy mildew Cereal powdery mildew and other diseases Cereal powdery mildew

1998 2007

4e5

Alternaria alternata (nuts), early blight of potato (Alternaria solani)

De Waard et al. (1994) Chin et al. (2001) Avenot and Michailides (2007) and Miles, Miles, Fairchild, and Wharton (2014)


Adapted from Hewitt (1998) and Brent (2012).

2

Eckert (1982) Noble et al. (1966) Szkolnik and Gilpatri (1973) Dekker (1976) Brent (1982) Staub (1994)

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1960 1964 1969

References

Puccinia striiformis

Brown rust Powdery mildew

Puccinia triticina Blumeria graminis

Septoria tritici blotch

Zymoseptoria tritici (formerly Mycosphaerella graminicola) Pyrenophora tritici-repentis Pyrenophora teres Rhynchosporium commune Ramularia collo-cygni Fusarium spp (especially Fusarium culmorum and Fusarium graminearum) and Microdochium nivale Oculimacula yallundae and Oculimacula acuformis (formerly Tapesia spp) Gaeumannomyces graminis

Tan spot Net blotch Rhynchosporium Ramularia Fusarium ear blight

Eyespot

Take-all

Tissues affected

Primarily wheat (form species tritici) Wheat Wheat (f. sp. tritici) Barley (f. sp. hordei) Wheat

Leaves and glumes

Wheat Barley Barley Barley Wheat, barley, oats, triticale

Leaves Leaves Leaves Leaves Ears, can also cause foot rot (stem base) and seedling blight

Wheat, barley, rye (O. acuformis)

Stem base

Wheat, barley, rye

Roots

Leaves Leaves Leaves

Examples in bold are the main focus of this review.

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Yellow rust

Host(s)

The Evolution of Fungicide Resistance

Table 2 Major fungal pathogens of cereal crops in Europe Disease Pathogen(s)

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Table 3 Single-site inhibitors used in cereal fungicide programs and their targets Cellular function Fungicide class affected Target protein

Methyl benzimidazoles (MBCs) Demethylation inhibitors (DMIs) e.g., azoles Quinone outside inhibitors (QoIs) e.g., strobilurins Succinate dehydrogenase inhibitors (SDHIs)

Cytoskeleton

b-tubulin

Membrane biosynthesis Respiration

Sterol 14a-demethylase (CYP51) Mitochondrial cytochrome b Succinate dehydrogenase

Respiration

Multisite inhibitors used in cereal programs: Chlorothalonil, folpet, dithiocarbamates.

single-target protein, whereas for multisite fungicides it is assumed that multiple changes are required. Single-site fungicides are also highly active and often systemic (taken up and distributed in plant tissues), giving good disease control at very low dose rates. Hence, following fungicide application, the majority of individuals in the pathogen population are either removed or inhibited from completing their life cycle, resulting in strong selection for any resistant individuals. Many plant pathogenic fungi have short generation times and rapid reproductive rates, producing large numbers of propagules (usually spores) that can be dispersed over long distances. This combination of high fungicide efficacy and large pathogen population size means that even rare mutations altering sensitivity to the fungicide will be selected, and survive and propagate, provided there is no major fitness cost associated with the change. The vulnerability of single-site fungicides to resistance development is a consequence of these different factors: the fungicide mode of action and curative use, high efficacy, and pathogen biology and epidemiology. The first requirement for resistance to occur is for heritable variability in sensitivity to the fungicide to be present in the pathogen population (Georgopoulos & Skylakakis, 1986). The nature of the fungicide and the genetic determinant(s) of resistance influence the rate and pattern of emergence of resistance. With single-site inhibitors, where a single mutation in the target protein can confer a high level of resistance, a qualitative change takes place, resulting in two distinct populations with a bimodal sensitivity distribution (Figure 1). With multisite inhibitors, or some single-site compounds where more than one gene or allele contributes to resistance, a unimodal distribution is observed. In both cases, there is directional selection

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Figure 1 Development of fungicide resistance in a pathogen population, shown as frequency against resistance level. (a) and (b) Hypothetical frequency distributions for discrete versus continuous sensitivity shifts. (a) Separation into sensitive and resistant subpopulations, typical of resistance due to a single genetic change of major effect. (b) Unimodal sensitivity distribution shifting toward resistance over time, due to multiple genetic changes of smaller effect. (c) and (d) Sensitivity distribution of isolates of Zymoseptoria tritici obtained from the field at Rothamsted in successive seasons (2003–07). (c) Response to the quinone outside inhibitor (QoI) fungicide azoxystrobin, showing increasing proportion of isolates highly resistant to the QoI. (d) Response to the azole fungicide epoxiconazole, showing a progressive shift toward resistance over the sampling period. See Section 5.2 for discussion of the genetic basis of these distribution patterns. (a) and (b) based on Georgopoulos and Skylakakis (1986).

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for reduced sensitivity, but in the first example it is acting on discrete variation, as opposed to the continuous distribution observed in the second, characterized by gradual shifts toward resistance over time. A further distinction should be drawn between the intrinsic resistance of some fungal species to particular classes of fungicide and acquired resistance that develops in response to selection by exposure to the fungicide. Until recently, comparatively little was known about the basis of intrinsic resistance, whereby some fungal taxa are naturally insensitive to a specific chemical group, and fungicides therefore have a particular spectrum of activity. Molecular variation in the target site is one likely explanation that has been confirmed for the strobilurin (quinone outside inhibitors, QoI) fungicides, which are themselves natural products of certain basidiomycete fungi. Alternatively, there may be redundancy in the protein target due to the presence of additional copies of the encoding gene (see Section 6.3). Comparative genomics of the target sites of single-site fungicides should shed further light on these types of intrinsic resistance.

3. FUNGICIDE USE ON CEREALS IN EUROPE Prior to the mid-1970s, there was little systematic use of fungicides on cereal crops, predominantly comprising wheat and barley, in Europe. The introduction of aminopyrimidines such as ethirimol for control of powdery mildew Blumeria graminis and the methyl benzimidazole carbamates (MBCs) for control of eyespot, Oculimacula species (Brent, 2012), marked the start of more intensive programs of foliar fungicide use, which coincided with progressive increases in crop yields over the next 30 years (Figure 2). Once the benefits of fungicide applications were appreciated, the proportion of cereal crops in the UK receiving fungicides rose steadily to its current level, close to 100%. In addition to seed treatments, on average wheat crops receive two or three sprays during the growing season, and in years of high disease pressure additional sprays may be applied. Since 2000 the average number of fungicide applications on wheat crops in England has risen from around 2.5 to 3.1, with a peak of 3.7 in 2012, a season of exceptional disease pressure (data from the Defra annual survey of winter wheat pests and diseases). The increase in fungicide use has been accompanied by sequential introductions of new classes of single-site inhibitors, starting with the MBCs, then followed by the demethylation inhibitors (DMIs), exemplified by the azoles, in the late 1970s, the QoIs in the 1990s, and the succinate dehydrogenase inhibitors (SDHIs) from 2002 onward (Figure 2; Table 3). While advice is

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Figure 2 Wheat yields and fungicide use in the UK, 1960–2013. Wheat yields (source: Cereal Production Surveys, Defra); percentage of crops sprayed with fungicides; average number of sprays per season (Defra annual survey of winter wheat pests and diseases); introduction of main fungicide groups; updated from Lucas (2006).

now issued to limit the number of sprays containing a specific mode of action in fungicide programs within a season, and wherever possible to mix different modes of action, the overall trend in the UK has been toward increased fungicide use, with extensive use of single-site inhibitors. Perhaps inevitably, this has increased the selection pressure for resistance. In some other European countries and regions, especially Scandinavia, there have been concerted efforts to reduce fungicide use. In Denmark, for instance, farmers have had a tradition of minimizing and optimizing fungicide inputs in cereals based on integrated pest management principles (Jorgensen, Nielsen, Orum, Jensen, & Pinnschmidt, 2008), with an emphasis on use of resistant cultivars, disease monitoring, and use of thresholds informing fungicide application. However, scope for further reductions is limited and influenced by market forces such as the price of grain. In the United States, while overall fungicide use in agriculture rose between the 1960s and 1990s, it has since declined (Osteen & Fernandez-Cornejo, 2013). Fruit and vegetable crops, including potatoes, account for over 90% of fungicide quantity applied. Foliar applications to cereal crops such as wheat are less intensively used, due to a less favorable climate for foliar

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pathogens and a low economic return on fungicide treatment on cereal crops with a low yield potential.

4. MECHANISMS OF RESISTANCE TO SINGLE-SITE INHIBITORS To understand the emergence of resistance to fungicides in field populations of pathogens, it is necessary to characterize the mechanisms leading to reduced sensitivity to the compound and the genetic basis of the resistance trait. Studies with several classes of single-site inhibitors, and a range of plant pathogenic fungi, have implicated several mechanisms underlying reduced sensitivity. Additional evidence has been gained from work on drug resistance in model species such as yeast (Fisher & Meunier, 2005), and fungi of clinical importance, such as Candida spp and Aspergillus (Camps et al., 2012; Cowen et al., 2000; Snelders, Karawajczyk, Schaftenaar, Verweij, & Melchers, 2010). Four main mechanisms have been implicated in the development of acquired resistance to fungicides (Figure 3). Alteration of the target protein due to mutations in the encoding gene has been confirmed for many single-site fungicides, including the MBCs, azoles, QoIs, and SDHIs. Efflux of the fungicide due to the action of ABC or other transporters has been reported in several plant pathogens and is a common mechanism in clinically

Figure 3 Mechanisms of resistance to single-site fungicides. 1. Alteration of the target protein prevents fungicide binding (target-site resistance). 2. Overexpression of target protein increases concentration of fungicide necessary for inhibition. 3. Efflux pumps expel fungicide from cell. 4. Degradation of fungicide by metabolic enzymes.

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important fungi such as Candida (Hiller, Sanglard, & Morschhauser, 2006; Rajendran et al., 2011; Sanglard et al., 1995). Overexpression of the target due to upregulation of the encoding gene has been confirmed in some cases (Cools, Bayon, Atkins, Lucas, & Fraaije, 2012; Ma & Michailides, 2005) but does not appear to be a widespread mechanism in plant pathogens. Degradation of the pesticide due to detoxification by metabolic enzymes such as cytochrome P450s or glutathione transferase is a commonly reported scenario in weeds and insects developing resistance to herbicides (Cummins et al., 2013; Powles & Yu, 2010) and insecticides (Puinean et al., 2010), respectively, but does not appear to be common in cases of resistance to fungicides, with only one report on the degradation of the QoI fungicide kresoxim-methyl through esterase activity (Jabs, Cronshaw, & Freund, 2001). This is surprising given the well-known propensity of fungal P450 monooxygenases to detoxify a range of xenobiotics (Lah et al., 2011) and the activity of some soil fungi in degrading fungicides (e.g., Bailey & Coffey, 1985; Rosario Martins, Pereira, Lima, & Cruz-Morais, 2013).

5. CASE HISTORIES 5.1 Eyespot of Cereals Eyespot disease of wheat and other cereals, caused by the closely related fungi Oculimacula yallundae and Oculimacula acuformis, formerly Tapesia spp, and prior to that W- and R-pathotypes of Pseudocercosporella herpotrichoides (Crous, Groenewald, & Gams, 2003), occurs in many temperate cereal-growing regions of the world (Lucas, Dyer, & Murray, 2000). The fungi infect the stem base of the host plant leading to reduced water and nutrient transport, premature ripening, and eventually lodging of the crop. In NW Europe, the disease is usually controlled by fungicide application between the end of tillering (growth stage (GS) 29) and the second node stage (GS32). The first fungicides used against the eyespot fungi were the MBCs introduced in the mid-1970s (Leroux, Gredt, Remuson, Micoud, & Walker, 2013). The eyespot pathogens were regarded as at relatively low risk of developing resistance, as the disease is monocyclic (one generation per season) and was only known to produce asexual spores, which are splash-dispersed over short distances. Early work in Germany (Fehrmann, Horsten, & Siebrasse, 1982; Horsten & Fehrmann, 1980) showed that MBC treatments in the field increased the frequency of resistant spores in fungal populations, but overall the proportions of resistant spores were very low, and one or two applications of these compounds per season

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were therefore recommended as acceptable practice. Nonetheless, control failures after MBC treatment were reported at two sites in the UK in 1981, and isolates obtained from infected plants at each site were subsequently shown to be highly resistant to carbendazim and related MBC compounds, with resistance factors (RF ¼ difference in EC50 (concentration of fungicide required to inhibit growth by 50%) between sensitive and resistant strains) greater than 1000 (Brown, Taylor, & Epton, 1984). The resistant isolates had growth rates in culture similar to sensitive isolates and were equally pathogenic in infection tests, suggesting that the change(s) conferring resistance did not have a significant fitness cost. The frequency of resistance to MBC fungicides in the field populations of the eyespot pathogens increased rapidly; a survey of winter wheat and barley crops in the UK in 1983 (King & Griffin, 1985) showed that resistance was now widespread with the proportion of resistant isolates in some fields exceeding 50%. Significantly, resistant isolates were also common in fields where MBC fungicides had not been used, indicating that invasion of resistant strains had occurred more quickly and on a wider scale than might have been anticipated from the known epidemiology of the disease. A similar increase in the incidence of highly resistant strains was also reported in France and other European countries (Leroux et al., 2013). More detailed characterization of field isolates showed that the majority fell into either the sensitive or highly resistant category, with the latter exhibiting cross-resistance to different fungicides in the MBC class (Hocart, Lucas, & Peberdy, 1990). The apparent preponderance of highly resistant isolates in samples from the field might have been a consequence of the screening procedure, whereby a single discriminatory concentration of fungicide was used to select them. Interestingly, many highly resistant isolates had increased sensitivity to N-phenylcarbamates such as diethofencarb, a phenomenon described as negatively correlated cross-resistance (Kato, Suzuki, Takahashi, & Kamoshita, 1984). Laboratory studies with initially sensitive isolates of both species (at that time classified as W- and R-pathotypes) showed that spontaneous mutants insensitive to MBC compounds could be selected on fungicide-amended agar at low frequencies (1 106) (Hocart et al., 1990). The laboratory-selected and induced mutants, unlike most field isolates, showed a range of resistance phenotypes, from high to intermediate to low, with some variation in the degree of crossresistance to MBCs and negatively correlated cross-resistance to phenylcarbamates, suggesting the involvement of different mutations or more than

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one resistance mechanism. Spontaneous revertants to phenylcarbamate insensitivity from some isolates highly resistant to MBCs were also recovered. The ease with which such doubly resistant strains could be selected suggested that the potential use of phenylcarbamates to manage MBC resistance in the field was unlikely to prove a durable strategy. Subsequent analysis of French field populations of both Oculimacula species identified at least seven benzimidazole-resistant phenotypes (Albertini, Gredt, & Leroux, 1999) with different levels of resistance and patterns of cross-resistance to MBCs and phenylcarbamates. PCR amplification of partial sequences of the b-tubulin target-encoding gene of benzimidazoles revealed identical amino acid sequences for wild-type MBC-sensitive strains of both O. yallundae and O. acuformis, and mutations leading to single amino acid substitutions at positions 198, 200, or 240 in MBC-resistant field isolates. Each of the seven phenotypes had a change at a particular position or a substitution with a different amino acid that correlated with the different resistance patterns. For instance, substitution of glutamic acid by alanine at position 198 (E198A) was found in strains highly resistant to MBCs but with increased sensitivity to phenylcarbamates, whereas substitution of phenylalanine by tyrosine at position 200 (F200Y) was correlated with resistance to both chemical classes. Overall, changes at position 198 were most common in resistant field isolates, suggesting that such alterations, while conferring resistance to MBCs, do not impact significantly on the functioning of the b-tubulin protein. Analysis of UV-induced lab mutants revealed more complex resistance patterns as well as changes at additional positions in the b-tubulin sequence. Hence, the emergence of resistance to MBC fungicides in Oculimacula species can be accounted for by the presence of specific mutations in the gene encoding the b-tubulin protein target that were selected by exposure to these compounds. But why did resistance, once it had emerged, predominate so quickly in a fungus that had been widely regarded as low risk? Were the relatively low rates of spontaneous mutation to resistance observed in the above studies sufficient, even in a monocyclic pathogen with only one spore generation per season, to quickly establish a resistant subpopulation for rapid selection by a highly active fungicide, or was some other factor(s) involved? Not long after the first reports of MBC resistance in the eyespot fungi, a sexual stage (teleomorph) was discovered in Australia for the W-pathotype (Wallwork, 1987) that was subsequently renamed Tapesia yallundae (Wallwork & Spooner, 1988). The sexual reproductive structures (apothecia) produce forcibly ejected ascospores that are dispersed by wind, potentially over

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much longer distances than the rain-splashed asexual conidiospores. Once the sexual stage had been described, it was found in the field in other cereal-growing regions, including New Zealand, South Africa, and many European countries, confirming that it might be a regular feature of the pathogen life cycle (Lucas et al., 2000). There was a single report of a similar sexual stage for the R-pathotype from Germany (King, 1990). Attempts to induce the formation of a sexual stage in the R-pathotype by crossing isolates on straw in sterile culture eventually succeeded with production of apothecia and ascospores and confirmation as the separate species Tapesia acuformis (Dyer, Nicholson, Lucas, & Peberdy, 1996; Moreau & Maraite, 1996). To date, however, the overall incidence of a sexual stage in this species in the field is not known, although surveys have also found apothecia in NW United States (Douhan, Murray, & Dyer, 2002). While the epidemiological significance of a sexual cycle in Oculimacula (previously Tapesia) species has not yet been conclusively resolved, its potential importance for adaptation to fungicides is clear, as a mechanism for both generating variation through recombination (Dyer et al., 1996; Moreau & Maraite, 1996) and dispersing any resistant isolates over a wider range than previously understood. This case history illustrates the need for more fundamental knowledge of pathogen biology to inform estimates of resistance risk. In practical terms, the emergence of resistance to MBC fungicides led to withdrawal of these compounds for cereal eyespot control in France (Leroux & Gredt, 1997) and other European countries, and the use of alternative chemistry for management of the disease. Among the available sterol 14ademethlylase Inhibitor (DMI) fungicides then available, none of the triazoles showed good activity against the eyespot pathogens, especially O. acuformis, but the imidazole prochloraz was effective against both species. Prochloraz was consequently widely used in cereal fungicide programs in Europe, mainly for eyespot control. However, isolates of O. acuformis with reduced sensitivity to prochloraz were reported from Northern France in 1991 (Leroux & Marchegay, 1991) followed by detection of a highly resistant phenotype of O. yallundae the following season (Leroux & Gredt, 1997). The latter was at low frequency in the population, but resistant strains were shown to be selected by treatment with prochloraz or the triazole flusilazole in field trials. Laboratory studies with isolates of both O. yallundae and O. acuformis showed that spontaneous variants with reduced sensitivity to prochloraz could be selected in vitro at frequencies between 106 and 107, and that this rate could be increased by UV mutagenesis (Julian, Hardy, & Lucas,

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1994). However, the majority of isolates recovered showed low levels of resistance, within the natural range of sensitivities found in field populations (Gallimore, Knights, & Barnes, 1987), and many of the UV-induced variants reverted to wild-type sensitivity after repeated subculture, indicating that the shift in sensitivity was not stable. A proportion of such isolates also showed reduced pathogenicity in infection tests. Subsequent rounds of UV mutagenesis, however, produced some isolates with higher levels of resistance, a proportion of which retained pathogenicity and maintained the same degree of resistance after 1 year of serial subculture in the absence of the fungicide. One conclusion of this study was that resistance to imidazoles in Oculimacula was most likely multifactorial, and an example of continuous directional selection, similar to that reported in other plant pathogens (Gullino & De Waard, 1984; Koller & Scheinpflug, 1987). The discovery of a sexual cycle in Oculimacula provided the opportunity to investigate the genetic basis of prochloraz resistance in O. yallundae through analysis of ascospore progeny arising from crosses between sensitive and resistant parental strains (Dyer, Hansen, Delaney, & Lucas, 2000). The resistant parents were two field isolates from Northern France and one from New Zealand that were classified as having low-, medium-, and high-level resistance phenotypes, respectively. The progeny from a cross between a sensitive parent and the low-resistance strain showed a bimodal segregation for EC50 (concentration of fungicide required to inhibit growth by 50%), suggesting segregation of a single major gene for resistance. Crosses between a sensitive parent and the medium- and high-resistance isolates yielded approximately equal proportions of sensitive and resistant progeny, but with a skewed distribution of the latter, with a range of EC50 values within the resistant class. The proposed segregation of one major gene associated with prochloraz resistance was further supported by analysis of backcrosses and crosses between F1 progeny, but the presence of additional quantitative genetic components contributing to the EC50 of the more resistant isolates was once again inferred. Overall the analysis indicated the presence of a single gene required for resistance to prochloraz, with additional genes having smaller additive effects on the resistance phenotype. A similar conclusion was reached in studies on the inheritance of resistance to the triazole fungicide triadimenol in another cereal pathogen, Pyrenophora teres (Peever & Milgroom, 1992). As mutations in the sterol 14a-demethylase (CYP51) target protein of azole fungicides were known to contribute to resistance to these compounds in both plant (Délye, Laigret, & Corio-Costet, 1997) and human (Sanglard,

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Ischer, Koymans, & Bille, 1998) pathogens, the CYP51 gene was subsequently cloned from prochloraz-sensitive and prochloraz-resistant isolates of O. yallundae (Wood, Dickinson, Lucas, & Dyer, 2001). Two sensitive isolates had identical CYP51 sequences, while some substitutions were detected in two resistant field isolates, that both possessed the same variant allele of the CYP51 gene. However, further analysis of another sensitive field isolate and progeny from sexual cross between sensitive and resistant parental strains showed no consistent correlation between CYP51 sequence and the prochloraz-resistant phenotype. In a separate study, cloning of the CYP51 gene from both O. yallundae and O. acuformis showed 98% identity in amino acid sequence, confirming the close relatedness of the two species, while sequencing of the gene from additional field isolates revealed several species-specific polymorphisms (Albertini, Gredt, & Leroux, 2003). One substitution (F180L) was postulated to contribute to the lower baseline sensitivity of O. acuformis than O. yallundae to triazole fungicides, but comparisons between isolates possessing a range of prochloraz-resistant phenotypes showed no consistent association between a particular polymorphism and dose response to this fungicide. These authors also concluded that reduced sensitivity to prochloraz was not correlated with target-site mutations and was likely to have a polygenic basis. Subsequently, newer compounds with activity against the eyespot fungi have been identified and used in cereal fungicide programs in Europe, notably the anilinopyrimidine cyprodinil (Babij, Zhu, Brain, & Hollomon, 2000), and the triazolinethione prothioconazole (Mauler-Machnik et al., 2002), which has a different mechanism of binding to the CYP51 target protein from related triazoles or imidazoles (Parker et al., 2011). With the availability of new chemistry, treatment with prochloraz has declined. 5.1.1 Changes in Field Populations of the Cereal Eyespot Pathogens in Response to Fungicide Use Further insights into the evolution of fungicide resistance in Oculimacula spp. have been gained due to the availability of data from field trials run over several seasons (Bierman et al., 2002) as well as isolate collections from field populations of the pathogens sampled over many years in France (Leroux et al., 2013) and more widely across N. Europe (Parnell, Gilligan, Lucas, Bock, & van den Bosch, 2008). These surveys monitored not only the incidence of different resistance phenotypes but also trends in the frequency of the two species under selection by different fungicide treatments. Hence, it is possible to address questions relating to the emergence, invasion, and

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persistence of resistant strains, as well as pathogen population dynamics and interspecific competition. Bierman et al. (2002) set up a long-term field experiment with winter wheat plots inoculated with mixtures of MBC-sensitive or MBC-resistant isolates of either O. yallundae or O. acuformis, in proportions of 5% sensitive:95% resistant, or vice versa. Over the first five seasons, the populations in both MBC sprayed and unsprayed plots changed toward a stable equilibrium between the two species, suggesting that coexistence occurs in continuous wheat crops, indicative of niche separation. Treatment with carbendazim, an MBC fungicide, or carbendazim plus prochloraz, resulted in populations that were almost 100% MBC resistant within one or two seasons, respectively, demonstrating very strong selection. In plots sprayed with prochloraz or the carbendazim plus prochloraz mixture, the proportion of O. acuformis isolates in the population shifted to >80% over five seasons. This might be accounted for by the intrinsic sensitivity of the two species to prochloraz, with O. acuformis able to tolerate higher concentrations in vitro. Interestingly, the proportion of MBC-resistant isolates initially declined over the first few seasons in plots treated with prochloraz, but subsequently increased. This might suggest some reduced competitive ability of MBC-resistant strains, followed by the emergence and immigration from MBC-treated plots of resistant strains of greater fitness. By the end of the 15-year experiment, MBC-resistant strains predominated in all plots, irrespective of treatment. The sampling of eyespot populations in NW France started in the mid1980s and has continued until the present, with results summarized by Leroux and Gredt (1997) and Leroux et al. (2013). The latter publication covers the period from 1997 until 2010. As in the UK, resistance to MBC fungicides emerged in the 1980s and quickly became common and widespread. Following the withdrawal of MBCs for eyespot control in France in 1991, resistance has remained widespread, occurring in more than 90% of isolates of both species. The predominant strains have high-level resistance conferred by the E198A substitution, while other MBC-resistant genotypes have declined. This confirms that a single mutation in the target b-tubulin protein associated with complete loss of efficacy of the fungicide emerged and spread in field populations of both Oculimacula species and has persisted for almost 20 years in the presence of alternative fungicide treatments and absence of selection by MBCs. Resistance to prochloraz was first identified in 1990 in O. acuformis. These strains belonged to a phenotypic class (ProR1) displaying relatively

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low RF. In subsequent seasons, a second more resistant phenotype was identified in O. acuformis (ProR2) as well as the first prochloraz-resistant strains of O. yallundae (classified as TriR2). Coincident with these developments, the relative abundance of O. acuformis increased to around 60%, most likely due to the use of DMI fungicides. The ProR1 phenotype became the most common form of azole resistance. Since the mid1990s, however, this trend has reversed, possibly due to differential effects of another fungicide, cyprodinil, on the two species, and O. yallundae now predominates in field populations. Consequently, there has been a steady increase in TriR2 strains that accounts for around 20% of unselected populations, rising to more than 60% following prochloraz treatment (Leroux et al., 2013). This resistance has persisted for several seasons at some sites in the absence of any prochloraz use, implying that there is little fitness cost in such strains. Following the introduction of cyprodinil for eyespot control in the mid1990s, field strains with varying degrees of resistance to this compound were detected in France. However, the incidence of such resistance has mainly remained low in eyespot populations, and in vitro studies showed that it often attenuates on media lacking fungicide (Leroux et al., 2013). Similar instability of cyprodinil-insensitive strains was also reported in studies on UK eyespot populations (Babij et al., 2000). To date, no control failures due to resistance to anilinopyrimidine fungicides have been confirmed in practice in commercial crops. Despite regular use of prothioconazole in cereal fungicide programs in Europe since its introduction in 2005–2006, there have been no reports of Oculimacula isolates with reduced sensitivity. There remains a concern, however, that the occurrence of some strains of O. yallundae, at low frequency, possessing a putative multidrug resistance (MDR) phenotype similar to that reported for Mycosphaerella graminicola (Zymoseptoria tritici) (Leroux & Walker, 2011), might pose a potential threat to future chemical control of the disease. The second long-term survey of fungicide resistance and the population dynamics of the two Oculimacula species is based on annual isolate collections made from the field in France, Germany, and the UK over a 17-year period (1984–2000). These collections were coordinated by Aventis Crop Science as part of their monitoring program, in which more than 16,000 isolates were characterized by species and by mycelial growth on agar amended with a discriminatory dose of either MBC (carbendazim or benomyl) or a DMI (prochloraz). Testing with MBC fungicides was discontinued in 1994. The data set was analyzed retrospectively to determine trends in the

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incidence of the two species and their resistance to MBC or prochloraz (Parnell et al., 2008). The annual frequency distributions of response to MBC fungicide showed a bimodal shape in all years for each country consistent with the monogenic nature of MBC resistance. There was some variation between countries in the proportion of sensitive and resistant isolates, but a general decline in sensitive strains over the survey period, especially in France and the UK. The frequency of MBC-resistant strains was on the whole higher in O. acuformis populations than in O. yallundae. At the start of the survey, populations of both species were almost universally sensitive to prochloraz, but from 1985 onward there was a progressive increase in less-sensitive strains, especially in O. acuformis, with emergence of a higher resistance group of isolates between 1990 and 1996. This was not seen in O. yallundae populations. The relative frequency of the two species also changed over time, with O. acuformis becoming more common in the middle period of the survey, coinciding with the increase in prochloraz-resistant strains, and then declining again in all three countries. This later increase in the proportion of O. yallundae isolates coincided with reduced use of prochloraz, and the introduction of cyprodinil, which may differentially affect the two species. These studies demonstrate how fungicide use can influence the dynamics and abundance of pathogen populations, with selection of resistant subpopulations, which may persist after removal of selection by the chemical, depending on the stability of the trait and the fitness of resistant strains. The broad trends observed in the two surveys are similar, with some variation possibly due to differences in sampling protocols and the bioassay methods used to determine sensitivity.

5.2 Septoria tritici Blotch of Wheat Septoria tritici blotch (Stb), caused by the fungus Z. tritici (formerly Septoria tritici, teleomorph M. graminicola), is an important disease of wheat crops worldwide (Eyal, 1999). In many European countries, including the UK, Stb has been considered the most damaging foliar pathogen of winter wheat (Cook, Polley, & Thomas, 1991; Hardwick, Jones, & Slough, 2001) since the 1980s (Bearchell, Fraaije, Shaw, & Fitt, 2005), and fungicide programs are primarily targeted against this disease. The MBC fungicides have been used as foliar sprays and seed treatments on cereal crops since the mid-1970s, initially for control of eyespot (see Section 5.1 above) and also showed good activity in controlling Stb. There

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is little published information on the development of resistance to MBCs in Z. tritici. The sensitivity of isolates from the field in 1984 was compared with isolates collected in 1973, prior to MBC use (Fisher & Griffin, 1984). Around half of the more recent isolates showed normal growth on high concentrations of benomyl. These isolates were also highly cross-resistant to other MBC compounds, carbendazim, thiabendazole, and thiophanatemethyl. During the 1984–1985 season, poor control of Stb was seen in field trials following five sprays of carbendazim, associated with the presence of a high proportion of MBC-resistant strains in the pathogen population (Metcalfe, Sanderson, & Griffin, 1985). It seems likely that selection for the mutation encoding the amino acid substitution most often associated with MBC resistance (E198A) had taken place at some time before the 1984 trial. However, analysis of stored crop samples from the long-term continuous wheat trial at Rothamsted (Broadbalk) using an allele-specific PCR assay able to detect both the wild-type b-tubulin and the variant (E198A) form showed that no change could be detected prior to 1984. Subsequently the variant allele almost completely replaced the wild type within a single season, coinciding with the first use of MBC fungicides in this trial (Lucas & Fraaije, 2008a). The most likely explanation for this sudden switch was strong selection operating on a pathogen population in which spores founding the epidemic, migrating from areas previously treated with MBCs, carried the mutation at low frequency. Samples from following years maintained the E198A substitution at high frequency, even beyond the period of use of this chemistry, confirming the stability of the trait and lack of fitness costs. This observation has been corroborated by survey data from France in which amid a collection of Z. tritici isolates from 1988 to 2005, 85% were shown to be highly resistant to carbendazim (Leroux, Albertini, Gautier, Gredt, & Walker, 2007). Hence, MBC fungicides would not be expected to provide significant control of Stb disease by this time. QoI fungicides were introduced in the UK from 1997 onward and initially showed excellent activity against Stb. This new chemical group was considered to be at moderate risk of resistance, due to the fungicide target (cytochrome b) being a mitochondrially rather than nuclearencoded protein (Table 2). However, resistance quickly developed in the field in the powdery mildew pathogens of wheat and barley (B. graminis) (Chin, Chavaillaz, Kaesbohrer, Staub, & Felsenstein, 2001), raising concerns that other cereal diseases might also be at risk. The emergence of resistance in powdery mildews (see Section 5.3) was shown to be due to a single amino

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acid substitution (G143A) in the mitochondrial cytochrome b, that could be detected at high sensitivity using an allele-specific quantitative real-time PCR assay (Fraaije, Butters, Coelho, Jones, & Hollomon, 2002). M. graminicola isolates with reduced sensitivity to QoIs were first detected in the UK in 2002, including from wheat crops that had not received any QoI treatment, suggesting that resistance had been present in ascospore populations founding the epidemic the previous autumn (Fraaije, Lucas, Clark, & Burnett, 2003). These QoI-insensitive UK isolates were all shown to have the G143A substitution conferring a high level of resistance to these fungicides. Retrospective analysis of stored diseased leaves from 2001 confirmed that the G143A mutation was already present in the M. graminicola population albeit at very low frequency (Fraaije, Burnett, Clark, Motteram & Lucas, 2005). Interestingly, a few isolates carrying F129L cytochrome b alleles were reported in Ireland (Kildea et al., 2010; Lucas & Fraaije, 2008b) among a majority of G143A isolates. Due to a much lower RF to QoIs, F129L isolates will be outcompeted by G143A isolates under selection by QoI fungicides, assuming no fitness penalties are associated with either mutation. Following the discovery of QoI resistance in Z. tritici, measures were introduced to try to limit the further development and spread of the problem. The use of QoI fungicides as single products was discontinued, and they were applied only in mixtures with an alternative mode of action compound. The number of QoI sprays in cereal fungicide programs was also limited to either one (in Ireland) or two (in the UK) to reduce selection for resistance. Despite these precautionary measures, the frequency of the G143A mutation in field populations of Z. tritici increased rapidly; in 2003 incidence in UK, wheat crops ranged from 12% to 87%, and plot experiments with QoI treatments demonstrated an increase from 41% to 81% within a single season (Fraaije, Burnett, Clark, Motteram, & Lucas, 2005). This trend was repeated over much of NW Europe (Figure 4) so that by 2004 QoI fungicides were no longer effective in the control of Stb in this region (Lucas & Fraaije, 2008b). Resistance emerged less quickly in southern and eastern regions of Europe; for instance, analysis of isolates of Z. tritici from France in 2005 showed a gradient of incidence from 70% in the north to 30% in central regions to none detected in the south (Siah, Deweer, Morand, Reignault, & Halama, 2010). Two recent surveys (Boukef, McDonald, Yahyaoui, Rezgui, & Brunner, 2012; Stammler et al., 2012) of 357 and 58 isolates from Tunisia found no evidence of QoI resistance, and the G143A mutation was not detected. While Z. tritici has a global distribution, resistance to QoIs appears to be rare outside Europe. However,

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Figure 4 The incidence of the G143A mutation conferring resistance to QoI fungicides in European populations of Zymoseptoria tritici in 2003. From Lucas and Fraaije (2008b). Original data provided by K. H. Kuck and the Fungicide Resistance Action Committee.

isolates with the G143A mutation have now been found in Oregon, USA (Estep et al., 2013), and New Zealand (Suvi Viljanen-Rollinson, personal communication), a significant number being from field plots that had not received a QoI treatment, suggesting that resistance is now established in pathogen populations founding disease outbreaks. The mutation resulting in a glycine-to-alanine substitution at position 143 in the mitochondrial cytochrome b is a notable example of a single change in a fungicide target site conferring a highly resistant phenotype emerging and rapidly coming to predominate in pathogen populations under fungicide selection. Analysis of mitochondrial haplotypes suggests that the G143A mutation emerged independently on at least four occasions in European populations of Z. tritici and was then spread from west to east

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by airborne ascospores (Torriani, Brunner, McDonald, & Sierotzki, 2009). Spore trapping, combined with a PCR assay diagnostic for the G143A mutation, has shown rapid selection of the resistant allele in ascospore populations within treated crops and dispersal into adjacent untreated areas (Fraaije, Cools et al., 2005b). Airborne inoculum, in the form of windborne ascospores, is known to be the initial source of infection in wheat crops in the late summer and autumn (Shaw & Royle, 1989). Other mechanisms of insensitivity to QoI fungicides, such as alternative respiration (Wood & Hollomon, 2003) and metabolization of kresoxim-methyl through fungal esterase activity (Jabs et al., 2001), have also been described, but these seem to have little impact on field resistance development. The demise of QoI fungicides for the control of Stb meant that alternative chemistry was now required to protect winter wheat crops in Europe. The multisite inhibitor chlorothalonil was more extensively used, especially for the early T0 and T1 sprays, but there was also increased reliance on the azole fungicides for cereal disease management, especially for Stb. The azoles had been in use in fungicide programs since the 1980s, but while there was variation in the sensitivity of Z. tritici to different compounds within the azole class, there was little evidence for several years for significant changes in sensitivity following exposure to these fungicides. A study of Z. tritici populations from the UK, France, Germany, and Switzerland from 1992 to 1996 (Gisi, Hermann, Ohl, & Steden, 1997) found a range of sensitivities among isolates, but no indications of decreased sensitivity over time, with the overall sensitivity profiles remaining unchanged. Mavroeidi and Shaw (2005) tested 73 isolates of Z. tritici collected in the UK from 1993 to 2005 and found that sensitivity to the triazole fluquinconazole and the imidazole prochloraz had decreased by factors of 10 and 2, respectively. Moreover, field observations on the efficacy of azoles in controlling Stb, along with results from monitoring programs in the UK (Turner, Elcock, & Hims, 1996), began to suggest that the performance of some triazole fungicides was declining. This trend was confirmed in subsequent annual efficacy trials in the UK, in which the shift in sensitivity of the fungal population was shown to differentially impact on different azoles, with the older compounds that had been in commercial use for longer most affected. A field experiment comparing the performance of 12 azole fungicides found wide variation in disease control, with compounds such as cyproconazole, propiconazole, and tebuconazole showing very poor activity, while other, more recently introduced azoles, such as metconazole, epoxiconazole, and prothioconazole, were still highly effective (Clark, 2006).

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Unlike the MBC and QoI fungicides, with which the emergence of resistance in Z. tritici led to a bimodal sensitivity distribution in the pathogen population, the development of resistance to azoles occurred gradually in a stepwise manner, with a continued unimodal sensitivity profile, but a higher incidence of less sensitive phenotypes emerging, year on year (Figure 1). A further contrast was the incomplete cross-resistance between different members of the azole class. This pattern of resistance development suggested that more than one mechanism might account for the observed shifts in sensitivity. Proposed mechanisms included alterations in the CYP51 gene encoding the azole target, overexpression of the CYP51 gene, and efflux of the fungicide by membrane drug transporters (Leroux & Walker, 2011). 5.2.1 Changes in CYP51 Sequencing of the CYP51 gene, or pyrosequencing of key regions of the gene implicated in target-site resistance, from numerous Z. tritici field isolates from different countries in Europe has revealed a complex series of changes over time in different locations. More than 30 different MgCYP51 mutations leading to amino acid substitutions or deletions have been reported (Cools & Fraaije, 2013), a dynamic process that continues to date. Field populations of the pathogen are highly heterogeneous and vary in the frequency of different CYP51 genotypes, both between different regions and between different seasons (Leroux et al., 2007; Stammler et al., 2008). Comparison of isolate collections and archived crop samples has allowed a temporal reconstruction of the recent evolution of the protein under selection by azole fungicides. Brunner, Stephanoto, and McDonald (2008) determined CYP51 haplotypes from non-European, “old” European, and “recent” European collections and concluded that mutations conferring azole resistance most likely arose in NW Europe and then spread eastward via airborne ascospores with continual recombination and haplotype replacement by more resistant invading strains. The overall pattern is the sequential emergence over time of increasingly complex CYP51 genotypes with multiple changes in the protein target (Cools & Fraaije, 2013). The fact that most of the reported mutations in MgCYP51 occur in combination rather than alone means that determining the impact of individual changes on both azole sensitivity and sterol 14a-demethylase function is difficult. Some but not all of the changes are located in predicted substrate recognition sites (Cools & Fraaije, 2008) and several, for instance, the amino acid substitution Y137F, are equivalent to those altered in azole-resistant isolates of Candida albicans (Sanglard et al., 1998). Others, including V136A/C,

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A379G, and I381V, appear to be unique to Z. tritici. Systematic analysis of resistance phenotypes and correlation with particular MgCYP51 genotypes (Fraaije et al., 2007; Leroux et al., 2007) has identified certain key mutations that have a measurable effect on azole sensitivity. For example, Leroux et al. (2007) reported that the substitution V136A has little effect on sensitivity to some azoles, such as propiconazole and metconazole, whereas it decreased sensitivity to prochloraz and increased sensitivity to tebuconazole. Isolates with the highest RF had the substitution I381V in combination with either a double amino acid deletion DY459/G460, or point mutations at positions 459 or 461. The I381V substitution, first detected in 2001, is now widespread and common in European Z. tritici populations, and its emergence has coincided with a significant shift in resistance to azoles, especially tebuconazole. It has been shown to be differentially selected by tebuconazole and confers an adaptive advantage in the presence of this compound (Fraaije et al., 2007). In contrast, Y137F, which has been shown to increase resistance to triadimenol (Leroux et al., 2007), is now very rare or absent from Z. tritici populations, coinciding with reduced use of triadimenol, as it has been superseded by more modern azoles. Recent increases in resistance to the two currently most effective and widely used azoles, epoxiconazole and prothioconazole, have coincided with the emergence of novel MgCYP51 variants, often including the substitution S524T (Cools et al., 2011). These temporal shifts suggest that the pathogen population is continually adapting to changing patterns of fungicide use. Sterol analysis of isolates carrying some combinations of CYP51 alterations has, however, shown quantitative differences in sterol pathway intermediates, especially the CYP51 substrate eburicol, suggesting some impact on enzyme function, and by inference, the potential fitness of such mutant strains (Bean et al., 2009). More detailed characterization of individual mutations and the most commonly occurring combinations have now been done by site-directed mutagenesis, heterologous expression in yeast, and molecular modeling. Wild-type and mutated MgCYP51 variants were expressed in a Saccharomyces strain with a regulatable promoter controlling native CYP51 expression (Cools et al., 2010). The wild-type MgCYP51 protein effectively complemented the function of the orthologous protein in yeast. Amino acid alterations at positions 50, 459, and 461, as well as DY459/G460, all commonly found in recent Z. tritici populations, had no discernable effect on the functionality of the protein. Yeast transformants containing MgCYP51 proteins with these alterations at codons 459–461 were less

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sensitive to azoles. In contrast, the I381V substitution destroyed the capacity of MgCYP51 to complement in yeast when introduced alone. When combined with changes between residues Y459 and Y461, however, functionality of the enzyme was partially restored. This provided the first demonstration of the impact of certain alterations on protein function, as well as a rationale for the temporal sequence of such alterations in the Z. tritici population, with, for instance, changes between 459 and 461 predating the emergence of I381V. This analysis was subsequently extended to more recently occurring combinations of changes such as D134G, V136A, Y461S, and S524T, in which transformants showed large reductions in sensitivity to the previously most effective azoles, epoxiconazole and prothioconazole (Cools et al., 2011). The lack of a eukaryotic CYP51 crystal structure initially constrained efforts to model the potential impact of these amino acid changes on azole binding by the protein. The recent publication of structures for Trypanosoma (Lepesheva et al., 2010) and human CYP51 (Strushkevich, Usanov, & Park, 2010) together with the increasing database information for other species provided the basis for multihomologue modeling of wild-type and mutant MgCYP51 structures (Mullins et al., 2011). The model confirmed the specific effects of Y137F on triadimenol, I381V on tebuconazole, and V136A on prochloraz binding, as well as confirming the importance of the Y459– Y461 region, specific for fungal CYP51s, in changing the binding pocket volume. Overall, this molecular modeling provides a robust structure–function rationale for the binding of different azole molecules, supporting the observations on resistance to these fungicides in field populations of the pathogen. 5.2.2 Additional Resistance Mechanisms to Azoles While target-site changes are now known to account for much of the loss of efficacy of azole fungicides in controlling Stb in the field, other mechanisms have also been implicated, and in some cases confirmed to contribute to resistance. Leroux and Walker (2011) report the isolation of Z. tritici strains from France and the UK that have reduced sensitivity not only to azole fungicides but also to some unrelated compounds such as tolnaftate, an antifungal thiocarbamate terbinafine, and some SDHI fungicides, including boscalid and bixafen. The suggested MDR phenotype is believed to be due to overexpression of plasma membrane efflux transporters. Proteins belonging to both the ABC and major facilitator families of transporters are known to contribute to protection of Z. tritici against natural xenobiotics

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and fungicides (Roohparvar, De Waard, Kema, & Zwiers, 2007; Zwiers, Stergiopoulos, Van Nistelrooy, & De Waard, 2002). Putative efflux pump inhibitors have been shown to alter azole sensitivity in laboratory bioassays, but the contribution of efflux to resistance in planta is not clear (Roohparvar, Huser, Zwiers, & De Waard, 2007). Variation in the EC50 in bioassays of some Z. tritici isolates with identical mutant CYP51 genotypes suggests that additional mechanisms might contribute to the enhanced resistance phenotype. Isolates showing on average a 10-fold reduction in sensitivity to azoles in vitro, and also growth on wheat seedlings at fungicide doses higher than those required to inhibit the original mutant genotypes, were found to constitutively overexpress CYP51 (Cools et al., 2012). Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp insertion in isolates overexpressing MgCYP51. In a field survey in France, Leroux and Walker (2011) found that around 10% of isolates analyzed contained a larger insertion upstream of the start codon, but overexpression of MgCYP51 was not confirmed. This larger insert was also reported earlier (Chassot, Hugelshofer, Sierotzki, & Gisi, 2008), but its impact on MgCYP51 expression seems negligible. However, recombination of the overexpression trait with the most resistant CYP51 variants now present in the field, or further evolution of resistance mutations in the overexpression background, would pose an increased threat to effective control of Stb by azole fungicides. 5.2.3 SDHI Fungicides and Z. tritici A class of compounds inhibiting complex II of fungal respiration was first introduced in the 1960s with the fungicide carboxin. This had a narrow spectrum of activity against basidiomycete pathogens (Sierotzki & Scalliet, 2013). More recently, a new series of complex II inhibitors acting on succinate dehydrogenase has been introduced. These next-generation SDHIs, which include boscalid, bixafen, fluopyram, fluxapyroxad, isopyrazam, and penthiopyrad, have a much broader spectrum of activity, including against Stb. They are already in use on potato, fruit, and vegetable crops in the USA, and cereal crops in Europe. As SDHIs are single-site inhibitors of high efficacy that have been quickly adopted by growers, they are regarded as of moderate to high risk of resistance development (www. frac.info/; Sierotzki & Scalliet, 2013), and intensive resistance monitoring programs are under way for several pathogens and crops. Succinate dehydrogenase is composed of four subunits, three of which (SdhB, SdhC, and SdhD) contribute to the active site. To date, mutations

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affecting sensitivity to SDHIs have been reported in at least 14 fungi (Sierotzki & Scalliet, 2013). Mutants of Z. tritici resistant to carboxin with a single amino acid substitution, H267L or H267Y in SdhB, were produced in the laboratory (Skinner et al., 1998) and subsequently shown to be crossresistant to boscalid. In a more recent study (Fraaije et al., 2012), UV mutagenesis of two SDHI-sensitive field isolates produced 124 mutants with reduced sensitivity to carboxin; all harbored amino acid substitutions in at least one subunit. The majority of variants were single changes in SdhB, but mutations also occurred in SdhC and SdhD. A few mutants had two or three alterations in two different subunits. Bioassays with carboxin, boscalid, bixafen, and isopyrazam revealed a range of different resistance phenotypes and degrees of cross-resistance between the four SDHIs tested. All had reduced sensitivity to boscalid, the compound used for selection, but the degree of resistance to the other SDHIs varied widely. Generally, boscalid was more affected than bixafen or isopyrazam; for instance, the most commonly recovered alteration (B-H267Y, found as a single change in 62 of the 124 mutants) conferred resistance to carboxin and boscalid, but retained sensitivity to bixafen and isopyrazam. However, two substitutions, B-H267L and C-N86K, conferred high levels of resistance to all four SDHIs tested. Europe-wide monitoring programs since 2003 of field isolates of Z. tritici by agrochemical companies have, to date, shown no significant shifts in sensitivity likely to affect field performance of SDHIs. Two isolates with reduced sensitivity were found in 2012, one in France, the other in the UK, with substitutions C-T79N or C-W80S, respectively. These changes were associated with low RF and considered unlikely to impact on Stb control. No such isolates were found in 2013 (www.frac.info/SDHI Working Group Minutes December 2013). However, the recovery of a range of resistant isolates of Z. tritici in the laboratory (Fraaije et al., 2012; Scalliet et al., 2012) along with the recent detection of five different target site changes in the related cereal pathogen P. teres on barley, conferring low or moderate RF, (www.frac.info/SDHI Working Group Minutes December 2013) indicates that the potential exists for resistance to emerge.

5.3 Powdery Mildew of Cereals, B. graminis Collectively, the powdery mildew fungi (Erysiphales) have shown a propensity to rapidly develop resistance to single-site inhibitor fungicides (Brent, 1982; McGrath, 2001). The reasons for this are not fully understood, although the epiphytic growth habit of these pathogens, ensuring high

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exposure to any foliar applied chemical, rapid reproductive cycles, and large population size are likely contributory factors. The 2-aminopyrimidine fungicide ethirimol was widely used as a seed treatment in the 1970s to control barley powdery mildew (B. graminis f. sp. hordei - Bgh) (Brent, 2012). Field monitoring showed variation between isolates in sensitivity to the fungicide, and some selection for less sensitive strains in treated crops (Wolfe, 1984). However, control of the disease by ethirimol continued to be effective, and there was no major shift in the sensitivity of the pathogen population. Experiments on mixtures of mildew strains differing in sensitivity (Hollomon, 1978) suggested that the more resistant strains were less competitive than strains of intermediate sensitivity, hence the population overall stabilized. One key factor in this scenario was maintenance of a sufficient area of untreated crop on which the less sensitive strains were most likely at a disadvantage compared to the wild type (Wolfe, 1984). Initially, resistance to 2-aminopyrimidine fungicides was believed to be quantitative in nature, but crosses between sensitive and insensitive isolates subsequently showed the difference in response to ethirimol to be associated with one major allele (Brown, Jessop, Thomas, & Rezanoor, 1992). Control of cereal powdery mildew with 2-aminopyrimidines was superseded by use of the morpholines and azoles. Shifts in sensitivity were recorded for both types of fungicides, but as the azoles were much more widely used, the shift in this case was greater (Wolfe, 1984), and wheat mildew (B. graminis f. sp. tritici- Bgt) was more affected than the barley form (Bgh) (Napier, Bayles, Stigwood, & Burnett, 2000). The degree of insensitivity to morpholines was relatively low, with isolates inhibited at concentrations of fungicide less than field rates, and hence considered unlikely to impact on practical control (Brown & Evans, 1992). A range of different azole-resistant phenotypes were reported in Bgh, and subsequently the CYP51 gene from both Bgh and Bgt isolates differing in sensitivity to azoles was sequenced (Wyand & Brown, 2005). Two amino acid substitutions, Y136F and K147Q, were detected, the latter a novel mutation found only in the most resistant isolates of Bgh. Genetic crosses confirmed that both mutations segregated with the resistant phenotype, confirming that changes in CYP51 account for most of the resistance to azoles. However, analysis of responses of progeny to the azole triadimenol in one cross showed a range of resistance phenotypes indicating that more than one mechanism might be operating. The overall conclusion is therefore similar to that observed in Z. tritici, with alterations in the fungicide target protein explaining most of the observed resistance, but other mechanisms influencing the final phenotype.

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The launch in 1996 of the QoI fungicides provided a new mode of action highly effective against a range of important cereal pathogens, including the powdery mildews. Within two seasons, isolates of Bgt highly resistant to QoIs were found in Germany, and subsequently in France, Belgium, Denmark, and the UK (Chin et al., 2001). QoI resistance in Bgt isolates was shown to be due to a single substitution (G143A) in the mitochondrial cytochrome b (Sierotzki, Wullschleger, & Gisi, 2000). Crosses between QoI-sensitive and QoI-resistant isolates confirmed that inheritance was mitochondrial, hermaphroditic, and isogamous, with either parent acting as the maternal parent (Robinson, Ridout, Sierotzki, Gisi, & Brown, 2002). An allele-specific PCR assay capable of detecting either G143 (sensitive) or A143 (resistant) forms at very low frequencies was used to follow the dynamics of resistance in field populations of Bgt before and after fungicide application (Fraaije et al., 2002). This demonstrated very rapid selection by QoI treatments, with an increase in frequency of the A143 allele from around 2–58% after application of three sprays of the QoI fungicide azoxystrobin. Shortly after, resistance to QoIs was also confirmed in Bgh, again associated with the same single nucleotide polymorphism in the cytochrome b gene (Baeumler, Felsenstein, & Schwarz, 2003). Once again, resistance emerged and spread very rapidly in Europe. In a field trial with Bgh, evaluating strategies to manage resistance, both fungicide dose and number of applications were shown to influence the strength of selection for the R-allele, while mixing with an alternative mode of action fungicide delayed selection (Burnett, Clark, Fraaije, & Lucas, 2006). Nonetheless, once resistance had emerged, it quickly reached levels in the pathogen population at which control of powdery mildew disease was no longer feasible with QoI chemistry. Fortuitously, during the same period several novel compounds with alternative modes of action effective against powdery mildews were reaching the market. These included cyflufenamid, quinoxyfen, proquinazid, spiroxamine, and metrafenone. The mode of action of cyflufenamid is unknown. Quinoxyfen and proquinazid both interfere with differentiation of the fungal infection structure, the appressorium, while spiroxamine inhibits sterol biosynthesis at the D14 reductase step, and metrafenone is believed to act by disruption of the actin cytoskeleton at the hyphal tip (Opalski et al., 2006). More detailed analyses of quinoxyfen mode of action have suggested disruption of early signaling events on the host surface (Hollomon, Wheeler, Dixon, Longhurst, & Skylakakis, 1997), possibly via interference with host signals derived from the cuticle (Lee, Gustafson, Skamnioti, Baloch, & Gurr,

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2008). Variation in the sensitivity of cereal powdery mildew to quinoxyfen and proquinazid has been observed, but to date this has not seriously impacted on disease control in the field. Quinoxyfen-resistant laboratory mutants and some field isolates of Bgh with reduced sensitivity were shown to be defective in sporulation and hence unlikely to compete effectively in the absence of the fungicide (Hollomon et al., 1997). Quinoxyfen-resistant isolates of Bgt showed a slight reduction in sensitivity to proquinazid (Genet & Jaworska, 2009), but were still inhibited at low doses. Cross-resistance in Bgt therefore seems to be incomplete between these compounds, although they have been placed in the same resistance risk category by FRAC (see www.frac.info/AZN working group). With the diversity of chemistry now available for controlling powdery mildew diseases, resistance management should be easily achievable.

5.4 Fusarium Ear Blight Ear blight (or scab) caused by a complex of Fusarium and Microdochium species is a threat to cereal production and food safety in many countries. The disease affects not only grain yield but also quality due to the production of trichothecene mycotoxins by some of the causal pathogens. Fusarium graminearum has a worldwide distribution (Backhouse, 2014) and is an emerging problem in many countries (McMullen, Jones, & Gallenberg, 1997), including the UK. Severity of disease caused by this toxigenic species is predicted to increase due to climate change and an increased area of susceptible maize crops grown in the UK (West et al., 2012). Climate and crop growth models also suggest that FEB will increase in other important wheat production areas, such as China (Zhang et al., 2014). Control of FEB is problematical due to the relative lack of good sources of genetic resistance to the disease in current wheat cultivars, and the difficulty of timing and targeting fungicides to the ear during the critical infection period at anthesis (Trail, 2009). There is also a relative lack of fungicides effective against Fusarium species. Intrinsic resistance of F. graminearum to the QoI fungicide trifloxystrobin has been implied from a survey of 55 strains from 6 countries, including some strains isolated prior to the market introduction of QoIs (Dubos, Pasquali, Pogoda, Hoffmann, & Beyer, 2011). F. graminearum strains from Europe and the USA were also found to be insensitive to the newgeneration SDHI fungicide isopyrazam (Dubos et al., 2013). Furthermore, Fusarium species have recently been shown to possess three paralogues of CYP51, the gene encoding the 14a-demethylase target for azole fungicides (Liu et al., 2011). The CYP51A paralogue is upregulated in response to

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ergosterol depletion, including following azole treatment, and results in reduced intrinsic sensitivity of Fusarium species to some azoles, including epoxiconazole (Fan et al., 2013). To date, control of FEB has relied on MBC fungicides and the few triazoles with good activity toward Fusarium species, notably tebuconazole, metconazole, and prothioconazole. However, acquired resistance to MBCs in F. graminearum is now widespread; control failures with the MBC carbendazim have been reported after extensive use of this fungicide in China (Chen, Wang, Luo, Yuan, & Zhou, 2007), with most field isolates showing intermediate or high levels of resistance, related to mutations in the b2-tubulin target (Chen et al., 2009). It has recently been found that F. graminearum has two b-tubulins (Liu, Duan, Ge, Chen, & Zhou, 2013), with MBC resistance being conferred by mutations in one of them (b2-tubulin). A survey of more than 9000 isolates from the 10 main wheat-producing regions found a high incidence of MBC resistance in two regions, but less in others, related to the duration and intensity of MBC use (Liu et al., 2014). Genotyping detected several substitutions in the b2-tubulin (F167Y, E198Q, E198L, E198K, and F200Y), with F167Y or E198K accounting for more than 95% of the resistant isolates. MBC-resistant strains showed increased production of trichothecene mycotoxins in shake cultures and in the field (Zhang et al., 2009). Variation between isolates in sensitivity to triazoles has also been shown. In a survey of more than 150 isolates from China (Yin, Liu, Li, & Ma, 2009), three showed an azole-resistant phenotype, but no changes in either the CYP51A- or CYP51B-deduced amino acid sequence were found. More recently, field isolates of F. graminearum with reduced sensitivity to tebuconazole and metconazole have been reported in Brazil (Spolti, de Jorge, & Del Ponte, 2012), and one strain with reduced azole sensitivity was found in the USA (Spolti, Del Ponte, Dong, Cummings, & Bergstrom, 2014). Control of FEB following application of commercial rates of tebuconazole was reduced in infection tests with an azole-resistant isolate. Metconazole was ineffective at either controlling FEB or reducing mycotoxin contamination (Spolti et al, 2014). The mechanistic basis of this azole resistance is not yet known. Becher, Hettwer, Karlovsky, Deising, and Wirsel (2010) studied adaptation to azoles in F. graminearum by growing the fungus in vitro on sublethal concentrations of tebuconazole. Two azole-resistant phenotypes were recovered, differing in morphology, virulence, and mycotoxin production. One of the adapted strains showed an apparent MDR phenotype with reduced sensitivity to unrelated fungicides. ABC transporter genes have

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been implicated in resistance of F. graminearum to azole fungicides as they are upregulated in response to azole treatment, and deletion of two ABC transporter genes considerably increased sensitivity to tebuconazole (Abou Ammar et al., 2013). These authors concluded that one gene (FgABC3) may encode a transporter protecting the fungus from antifungal metabolites produced by the host plant. Overall, there is a need to better understand the basis of adaptation to azoles in F. graminearum, given the limited options for effective disease management, and the critical role of azole fungicides in limiting the losses caused by FEB.

6. PREDICTABILITY OF RESISTANCE EVOLUTION Whether evolutionary processes are repeatable and therefore evolution is inherently predictable, or whether complexity, contingency, and chance mean evolutionary pathways can only be determined retrospectively, has long been debated by evolutionary theorists. However, in fungicide resistance, attempting to predict evolutionary outcomes is of great practical importance, and so attempts have been made to predict future steps in pathogen evolution.

6.1 Mutagenesis and in vitro Selection One approach to predicting future resistance in the field involves forced selection in the laboratory, generally using UV or chemical mutagenesis to increase the mutation frequency, and strong fungicide selection to ensure rapid emergence of resistant genotypes. In the case of the MBCs, initial mutagenesis work in Aspergillus nidulans (Jung & Oakley, 1990; Jung, Wilder, & Oakley, 1992; Koenraadt, Somerville, & Jones, 1992) and Neurospora crassa models (Fujimura, Kamakura, Inoue, & Yamaguchi, 1994; Fujimura, Kamakura, Inoue, Inoue, & Yamaguchi, 1992; Orbach, Porro, & Yanofsky, 1986) identified 15 mutations over 12 codons in benzimidazole-resistant laboratory mutants. Some of these mutations were subsequently identified in field isolates of plant pathogens, including Venturia and Penicillium spp. (Koenraadt et al., 1992) and Botrytis cinerea (Yarden & Katan, 1993). Reijo, Cooper, Beagle, and Huffaker (1994) carried out systematic sitedirected mutagenesis of b-tubulin in Saccharomyces cerevisiae, and 27 of the 55 mutations generated had an effect on benzimidazole sensitivity. These included E198A, the only charged-to-alanine substitution reported in field isolates of plant pathogens. Systematic mutagenesis enables the identification

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of deleterious mutations, which may not be selected after random mutagenesis, but the exponential number of possible mutations relative to protein length means only a limited subset can be considered; in this case, charged-to-alanine substitutions. Therefore, this approach is more suited to characterizing protein regions involved in binding, and those which are structurally conserved, than generating a range of mutations conferring resistance. When Davidse and Ishii (1995) reviewed b-tubulin mutations associated with benzimidazole resistance, of the 10 codons identified in laboratory mutations, only mutations at codons 198 and 200 had been reported in field isolates. Furthermore, Albertini et al. (1999) generated nine mutations in vitro in Tapesia spp., of which only two were found in field isolates, although four other mutations were found only in field isolates. A review of b-tubulin mutations in field isolates of plant pathogens (Ma & Michailides, 2005) listed mutations at six codons, five of which had previously been reported in laboratory mutants, although codon 198 changes remained by far the most frequent. Similarly, for the QoI fungicides, a review of cytochrome b mutations in yeast and other eukaryotes listed 22 mutations over 12 codons conferring resistance to QoIs (Brasseur, Saribas, & Daldal, 1996), of which 3 (G143A, F129L, and G137R) have been reported in field isolates of phytopathogenic fungi (Sierotzki et al., 2007). In Magnaporthe grisea, laboratory mutants had G143A or G143S substitutions in cytochrome b (Avila-Adame & K€ oller, 2003), but only G143A has been reported in the field (Ma & Uddin, 2009). In Cercospora beticola, UV mutants were generated with G143S or F129V (Malandrakis, Markoglou, Nikou, Vontas, & Ziogas, 2006), but again, only G143A has been reported in the field (Birla, Rivera-Varas, Secor, Khan, & Bolton, 2012). Spontaneous mutants of B. cinerea with resistance to the QoI trifloxystrobin were selected in the laboratory from wild-type sensitive isolates (Angelini et al., 2012), but the G143A mutation could not be detected, unlike resistant isolates from the field that all had the mutation. The laboratory mutants were unstable in the absence of fungicide selection, and these authors suggested that the heteroplasmic state of resistant mitochondria might account for the lack of detection of G143A and rapid loss of resistance. Recently, in vitro mutagenesis studies have been carried out to predict possible mechanisms of resistance to the new SDHI fungicides (Avenot & Michailides, 2010; Sierotzki & Scalliet, 2013). In B. cinerea, mutations in sdhB codon 225 and 272 have been generated in vitro and reported in the field (Walker, Gredt, & Leroux, 2011). The B-R54G substitution has

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been reported in both laboratory mutants and field isolates (Fraaije et al., 2012), but only in combination with different further changes. Eight mutations found in laboratory mutants of Z. tritici (Fraaije et al., 2012; Scalliet et al., 2012) and Aspergillus oryzae (Shima et al., 2009) have been reported in field isolates of other species, such as B. cinerea (Walker et al., 2011), P. teres (FRAC SDHI Working Group, 2014), Alternaria alternata (Avenot et al., 2014), and V. inaequalis (FRAC SDHI Working Group, 2014), but a further 30 mutations have so far only been reported in laboratory mutants of various species and 20 other mutations only in the field, although not all are associated with reduced fungicide sensitivity. In some cases, the difference between laboratory and field mutations could be attributed to mutational bias. The chemical mutagen EMS predominantly causes A:T transitions (Burns, Allen, & Glickman, 1986), and consequently chemical mutagenesis in Colletotrichum gloeosporioides produced E198K mutants (Buhr & Dickman, 1994), as opposed to the E198A subsequently found in the field (Maymon, Zveibil, Pivonia, Minz, & Freeman, 2006). However, benzimidazole-resistant UV mutants reported so far encompass three of four possible transitions and seven of eight possible transversions. Therefore, it appears that UV mutagenesis produces a range of possible mutants, of which a subset can be expected to occur in the field: it shows the range of possible variation in sensitivity to a fungicide, but not which of the possible mutations may be selected under field conditions.

6.2 Fitness Costs A frequently suggested reason for mutations generated in vitro not emerging in the field is that some mutations result in pleiotropic fitness costs (AvilaAdame & K€ oller, 2003; Davidse & Ishii, 1995). In typical in vitro selection experiments, fungicide-resistant mutants are able to grow on agar plates as single-spore colonies unless the mutations are actually lethal, whereas in the field, mutants must be pathogenic in planta and competitive against other isolates. Fusarium moniliforme Y50R mutants (Yan & Dickman, 1996), S. cerevisiae F167Y mutants (Li, Katiyar, & Edlind, 1996), and three of the benomyl-resistant S. cerevisiae mutants generated by Reijo et al. (1994) were cold sensitive. A. nidulans mutants carrying Y50S, Q134K, or to a lesser extent Y50N or M257L, were heat sensitive, due to effects on microtubule stability preventing normal microtubule disassembly and spindle formation during mitosis (Jung, May, & Oakley, 1998). In the case of QoI resistance, fitness costs were detected in the form of reduced conidial production in G143S laboratory mutants of Magnaporthe

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grisea, but not in G143A mutants, (Avila-Adame & K€ oller, 2003), and it was G143A that later emerged in the field (Ma & Uddin, 2009). However, Monilinia fructicola isolates with b-tubulin H6Y and cold sensitivity, or E198A and heat sensitivity, and Monilinia laxa isolates with L240F and heat sensitivity were found in the field (Ma, Yoshimura, & Michailides, 2003; Ma, Yoshimura, Holtz, & Michailides, 2005), and Hsiang and Chastagner (1990) report reduced growth or sporulation in planta of benzimidazole-resistant field isolates of Botrytis spp. This suggests that either fungicide selection in the field is sufficient to outweigh some fitness penalties, or some fitness penalties observed in the laboratory, such as decreased growth as temperature approaches 40 C, are not applicable in the field. Similarly, for the SDHIs, decreased succinate dehydrogenase activity has been measured in some mutants, but this does not always correlate with reduced overall growth rate or in planta pathogenicity (Laleve, Fillinger, & Walker, 2014; Scalliet et al., 2012). In the case of QoIs, of the 22 mutations listed from QoI-resistant mutants of yeast and other eukaryotes, 9 mutations resulted in respiration deficiency or detectable effects on protein stability or activity, whereas G143A, F129L, and G137R had no such detectable fitness costs, but neither did 10 other mutations (Brasseur et al., 1996), suggesting that if the prevalence of G143A and absence of those 10 other mutations is due to fitness costs, those fitness costs were not readily detectable. Differences in the level of resistance conferred by different mutations may also affect their selection in the field. In M. fructicola, E198A and E198K b-tubulin mutants are described as highly resistant to benzimidazoles, whereas H6Y mutants are low resistant (Ma et al., 2003). In C. beticola, E198A mutants, first reported in 2006, are highly resistant to benzimidazoles (Davidson, Hanson, Franc, & Panella, 2006), whereas F167Y, only found at low frequencies from 2008, confers only moderate, temperature-dependent resistance (Trkulja et al., 2013). In V. inaequalis (Quello, Chapman, & Beckerman, 2010) and Pyrenopeziza brassicae (Carter, Cools, West, Shaw, & Fraaije, 2013), E198A correlates with higher resistance than L240F. Therefore, it seems likely that mutations conferring higher levels of resistance, with lower fitness costs under field conditions, are more likely to be selected.

6.3 Parallel Evolution In addition to in vitro mutagenesis experiments, if intrinsic resistance is found in some fungi, a similar mechanism may be predicted in other species. In the

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case of the QoIs, fungi that synthesize natural strobilurin products are intrinsically resistant. Mycena gallopoda has an alanine at codon 143 (Kraiczy et al., 1996), as in the G143A mutation subsequently reported in many cases of acquired resistance. However, M. gallopoda also has A153S, whereas Strobilurus tenacellus has S/P254Q and N261D (Kraiczy et al., 1996), and so the predominance of G143A in acquired resistance appears more predictable after the event. Intrinsic MBC resistance reported in Cochliobolus heterostrophus (Gafur, Tanaka, Shimizu, Ouchi, & Tsuda, 1998) and related Stemphylium species (Huang, Shi, Xie, Wang, & Li, 2013) is attributable to F167Y, a mutation that has been reported in some cases of acquired resistance, but more rarely than codon 198 and 200 substitutions. Intrinsic insensitivity in Colletotrichum acutatum has been attributed to b-tubulin overexpression, which has not yet been reported as a mechanism of acquired resistance (Nakaune & Nakano, 2007). Presence of the inducibly upregulated CYP51A paralogue reduces intrinsic sensitivity to azoles (Mellado et al., 2005), whereas acquired resistance is generally due to point mutations or constitutive upregulation of CYP51 (Becher, Weihmann, Deising, & Wirsel, 2011). Intrinsic sequence differences in SDHI-insensitive F. graminearum include sdhB-D130N, sdhB-A275T, and an additional S at amino acid position 83–84 of sdhC (Dubos et al., 2013), of which sdhB-D130N is equivalent to sdhB-106N in the intrinsically insensitive Schizophyllum commune (Oita, Fushimi, Ookura, Ito, & Yanagi, 1997), but none correspond to any mutations associated with acquired resistance. While intrinsic QoI resistance is an adaptation to naturally occurring strobilurins, the selective pressure resulting in intrinsic resistance to MBCs, azoles, and SDHIs is not clear, and perhaps should not be expected to select the same mechanisms as those fungicides: an undiscovered natural inhibitor may not show full cross-resistance with current fungicides, overexpression may be due to increased demand for enzyme activity rather than inhibition, or point mutations may be coincidental. However, once acquired resistance emerges in one species, a similar mechanism may be expected in other fungi. As MBC resistance emerged in the first plant pathogens, it soon became apparent that of all the mutations generated in vitro, E198A/K and F200Y would be the predominant resistant mechanisms in the field (Davidse & Ishii, 1995). Resistance to QoIs due to G143A was first reported in Erysiphe graminis f. sp. tritici isolated in 1998 (Sierotzki, Wullschleger, & Gisi, 2000) and Mycosphaerella fijiensis isolated in 1997 (Sierotzki et al., 2000), and parallel evolution of G143A has since

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been reported in over 20 species (Fernandez-Ortuno, Tores, De Vicente, & Perez-Garcia, 2008). In Magnaporthe grisea, an alternative mutation, F129L, was been reported, conferring a lower level of resistance (Farman, 2001), and F129L has since been reported in multiple species, including Pythium aphanidermatum (Gisi, Sierotzki, Cook, & McCaffery, 2002), Alternaria solani (Pasche, Piche, & Gudmestad, 2005), and Pyrenophora spp. (Sierotzki et al., 2007). Of around 40 mutations reported in mutagenesis studies of SDHI resistance, so far the most common in the field are equivalent to sdhB-H277Y/R/L, sdhC-H134R, and sdhD-H132R in A. alternata (FRAC SDHI Working Group, 2012; Sierotzki & Scalliet, 2013). Further evidence for the repeatability of resistance evolution can be found in apparent multiple origins of the same resistance mutation within species. Population genetic studies have inferred at least two independent origins of G143A in Plasmopara viticola (Chen et al., 2007) and four in Z. tritici (Torriani et al., 2009). Furthermore, G143A was found in five mitochondrial genetic backgrounds in M. grisea isolates (Kim, Dixon, Vincelli, & Farman, 2003), and three different codons for leucine-129 were found in F129L field isolates of P. teres (Sierotzki et al., 2007). For the azole fungicides, the range of reported mutations is far greater, but there are still some cases of parallel evolution. The Y136F mutation was first reported in Erysiphe necator (Délye, Laigret, & Corio-Costet, 1997), and equivalent mutations have since been reported in five other plant pathogens and three human pathogens (Becher & Wirsel, 2012). However, in contrast to MBCs and QoIs, where a single mutation can effectively confer qualitative resistance across the whole fungicide class, CYP51 mutations have smaller effects with incomplete cross-resistance, and so evolution has proceeded beyond an initial mutation in many species: Z tritici is the most extreme case among plant pathogens, with over 30 mutations in various combinations (Cools & Fraaije, 2013). Some parallel mutations have occurred in Z. tritici and M. fijiensis (Canas-Gutierrez et al., 2009), especially at codons 459–461 (Z. tritici numbering), and between clinical C. albicans and other clinical and plant pathogens (Becher & Wirsel, 2012), but the mutations are found in different combinations, which may reflect functional constraints, epistatic interactions, or the use of different azoles.

6.4 Functional Constraints and Epistasis In most species, the G143A mutation results in increased fitness under selection by QoI fungicides. However, some fungal cytochrome b genes have an intron after codon 143, and in these fungi, the mutation encoding G143A

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has the pleiotropic effect of preventing intron splicing and is lethal (Grasso, Palermo, Sierotzki, Garibaldi, & Gisi, 2006). This is a simple example of epistasis, whereby the effect of a mutation differs depending on the genetic background, and the need to preserve the intron splice site is a simple example of a functional constraint. Understanding such epistatic interaction can make evolution more predictable: fungal lineages with the intron will not evolve G143A, and so are more likely to acquire alternative mutations such as F129L (Grasso et al., 2006). This prediction has so far been borne out in Pyrenophora spp., where in P. tritici-repentis the intron is absent and G143A has emerged, but in P. teres the intron is present and only F129L and G137R have emerged (Sierotzki et al., 2007); and in B. cinerea, where some isolates have the intron and some do not, but G143A is only found in isolates lacking the intron (Banno et al., 2009). However, functional constraints and pleiotropic and epistatic effects at the protein level are more complex. In the case of MBC resistance, functional constraints on b-tubulin evolution relate to tubulin stability, polymerization, and depolymerization (Jung et al., 1998), but despite such constraints, 10 different mutations have emerged in different plant pathogenic fungi in the field (Ma & Michailides, 2005). Pleiotropic fitness effects of mutations may vary depending on genetic background. F200Y mutants in Saccharomyces pombe had impaired microtubule dynamics resulting in meiotic failure, whereas tyrosine-200 is the wild-type sequence in many animals (Paluh, Killilea, Detrich, & Downing, 2004) and is a common mutation in fungal field isolates (Ma & Michailides, 2005). However, current understanding of the effects of mutations on b-tubulin structure and epistatic interactions with genetic background is not sufficient to predict which resistance mutations will arise in which genetic background. The situation is especially complex for the azole fungicides, as reviewed by Cools, Hawkins, and Fraaije (2013): not only does CYP51 have more reported resistance mutations than other target sites, but multiple mutations may be combined in a single haplotype (Becher & Wirsel, 2012), as well as other resistance mechanisms (Leroux & Walker, 2011). One major difference in CYP51 genetic background is the presence of multiple CYP51 paralogues in some filamentous ascomycetes (Deng, Carbone, & Dean, 2007; Mellado, Diaz-Guerra, Cuenca-Estrella, & Rodriguez-Tudela, 2001; Yan et al., 2011). The CYP51B paralogue is present in all filamentous ascomycetes, and in species with no other paralogues, such as Z. tritici, M. fijiensis, B. graminis, V. inaequalis and M. fructicola, target-site mutations and overexpression involve CYP51B (Canas-Gutierrez et al., 2009; Cools

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et al., 2012; Luo & Schnabel, 2008; Schnabel & Jones, 2001; Wyand & Brown, 2005). In species with CYP51A, such as Penicillium digitatum and the clinical pathogens Aspergillus fumigatus and Aspergillus parasiticus, mutations and overexpression mainly involve CYP51A (Alcazar-Fuoli, Mellado, Cuenca-Estrella, & Sanglard, 2011; Doukas, Markoglou, Vontas, & Ziogas, 2012; Hamamoto et al., 2000; Mellado et al., 2007). However, intraspecific variation in CYP51 paralogue number may provide an unexpected source of adaptive potential for resistance (Hawkins et al., 2014). The nature of the mutations likely to occur in either paralogue is not so clear. The Y136F substitution is common in CYP51B and also found in the single CYP51 orthologues of yeasts and basidiomycetes, but further mutations vary between species (Becher & Wirsel, 2012). In Z. tritici, with over 30 reported mutations, the order and combinations in which those mutations have and have not occurred may partly reflect functional constraints (Cools & Fraaije, 2013). Protein modeling can explain the structural basis for the individual effects and epistatic interactions of known mutations (Mullins et al., 2011), and site-directed mutagenesis and heterologous expression in yeast make is possible to dissect multimutation haplotypes and investigate the effect of mutations singly or in alternative combinations (Cools et al., 2010). For example, I381V emerged after mutations at codon 459–461, and yeast expression studies have shown that I381V alone destroys enzyme function, whereas the Y461H single mutant and I381V/Y461H double mutant are viable (Cools et al., 2010). Therefore, functional constraints may have prevented I381V emerging in a wild-type background due to its pleiotropic effects on enzyme function, but it was able to emerge later in other genetic backgrounds due to epistatic interactions with other mutations such as Y461H (Cools et al., 2013). However, S524T also emerged after other mutations, but the single mutant is able to complement yeast CYP51 and confers reduced sensitivity to prothioconazole. In this case, the later emergence may be due to incomplete cross-resistance between azoles, with S524T selected following the introduction of prothioconazole, when other mutations were already widespread in the population (Cools et al., 2011): such contingency on historical factors may make evolution less predictable. Alternative mechanisms such as overexpression (Cools et al., 2012) and efflux (Leroux & Walker, 2011) add to the range of possibilities, making it more difficult to predict which mechanisms will emerge in which species or population. Therefore, where functional constraints are simple, known, and applicable across a range of taxa and genetic backgrounds, they can make

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evolution more predictable. More subtle constraints, subject to epistatic interactions and variation between lineages, make evolution more difficult to predict, and so far more detailed knowledge of protein structure and genetic background is needed.

7. ESTIMATING RESISTANCE RISK The vulnerability of single-site fungicides to resistance development is a consequence of three different factors: the fungicide mode of action and use, high efficacy, and pathogen biology and epidemiology. These criteria have been used in combination to estimate the overall risk of resistance occurring in contrasting pathogens and production systems to different chemical classes (Brent & Hollomon, 2007; Kuck & Russell, 2006). While such risk models, based initially on practical experience with different fungicides and pathogens, provide a general guide, they cannot predict when or where resistance will occur, or how quickly it might spread to compromise disease control (Lucas, 2006). This would require precise measures of mutation rates and population size, proportions of sensitive and insensitive individuals in the pathogen population (van den Bosch & Gilligan, 2008), the selection coefficient (the difference in fitness between the resistant and the sensitive strain due to application of the fungicide (van den Bosch, Oliver, van den Berg, & Paveley, 2014)), as well as other factors influencing the survival and invasion of resistant strains (Gubbins & Gilligan, 1999). A recent analysis of such risk matrix fungicide x pathogen assessment schemes, in which the time from introduction of a fungicide to the first emergence of resistance was compared (Grimmer, van den Bosch, Powers, & Paveley, 2014a), concluded that while the system had useful predictive power for the broad risk categories (low, moderate, and high) for type of fungicide, pathogen, and agronomic system, it had limited predictive value within the now dominant single-site fungicide group. The same authors have now proposed a revised scheme based on comparison of 61 documented cases of fungicide resistance, and candidate traits potentially associated with the rate of evolution to resistance (Grimmer, van den Bosch, Powers, & Paveley, 2014b). This risk assessment identified some key traits that are important determinants of resistance risk, including pathogen latent periods per year (a measure of duration of the disease epidemic divided by the time from infection to pathogen reproduction), number of crop species infected by the pathogen (narrow versus wide host range, with less intensive fungicide selection acting on the latter), protected versus open field production

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system (with the confined environment allowing greater selection of resistant strains), and fungicide molecular complexity (with high-complexity molecules exhibiting greater binding specificity to their target site, with a higher probability of small changes compromising efficacy). A model combining such key traits explained 61% of the variation in time in years to emergence of resistance for single-site fungicides. It is also proposed that such trait-based resistance risk assessments could be used to predict the potential resistance risk status of fungicides with novel modes of action, when there is no prior knowledge of their behavior in practical use. Pathogen risk levels may be revised over time based on responses to new fungicide classes or changes in fungicide use. Overall, the rust fungi have been regarded as at relatively low risk of developing resistance to other fungicide classes, but recently this has been questioned (Oliver, 2014). This author argues that many rusts have until recently had limited exposure to fungicides at greatest risk of resistance, and that when selection pressure increases through heavy fungicide use, as has occurred with azoles and Asian Soybean rust in South America, shifts in sensitivity take place (Scherm, Christiano, Esker, Del Ponte, & Godoy, 2009), at least partly based on familiar mechanisms such as CYP51 mutations or overexpression (Schmitz, Medeiros, Craig, & Stammler, 2014). A further dimension to risk assessment with agricultural fungicides has recently emerged in Europe, concerning the possibility that widespread use of chemical classes such as the azoles that are also important drugs for the management of opportunistic fungal pathogens in humans, might be selecting for resistance in such pathogens, especially A. fumigatus. The evidence is mainly correlative and circumstantial, based on the presence and increased frequency of specific SNPs in the cyp51 gene found in both clinical and environmental A. fumigatus isolates (Snelders et al., 2008; Verweij, Snelders, Kema, Mellado, & Melchers, 2009). The origin of such azoleresistant isolates in the environment is not known, but should the pathway from fungicide selection in the field to antifungal drug resistance in the clinic be confirmed, it would have serious implications for how fungicides are used in agriculture, horticulture, and some other trades such as protecting building materials from biodeterioration (Bowyer & Denning, 2014). A risk assessment has now been published (Gisi, 2014) in which the primary exposure events for A. fumigatus and azoles were calculated on a case-by-case basis. This review concluded that the selection risk for resistance evolution is highest for medical and veterinary applications, and lower for agricultural and horticultural uses. However, it was acknowledged that certain fruit and

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seed treatments, along with applications in wood preservation, might pose some risk, while the much lower exposure levels in fungicide-sprayed crops would exert only limited selection on fungi in soil or crop residues.

8. IMPLICATIONS FOR RESISTANCE MANAGEMENT It is now well understood that modern, intensive agriculture exerts strong selection on other species in the agroecosystem, driving ecological and evolutionary processes (Hollomon & Brent, 2009; Thrall et al., 2011). The often rapid shifts in sensitivity to agrochemicals, or breakdown of initially resistant crop genotypes to new virulent biotypes of pathogens (Brown & Tellier, 2011), are all too familiar examples of such dynamic responses. How can the improved understanding of fungicide-resistance mechanisms, their genetic control, and development inform strategies for preventing resistance or minimize its impact once it has occurred? In the absence of clear experimental evidence on the merits or otherwise of particular resistance management strategies, initial guidance was empirical and based on a conceptual approach. The overall aim was twofold. Firstly to prevent emergence of variants that could survive fungicide treatment (the “emergence phase” of resistance development), and subsequently to reduce selection for such variants in the pathogen population (the “selection phase”; van den Bosch & Gilligan, 2008). In reality, resistance management strategies were focused exclusively on the second phase, reducing either the rate of increase of resistant strains or time span over which selection occurs (van den Bosch et al., 2014), as the circumstances leading to the initial occurrence of resistance at low frequencies in a usually very large pathogen population are difficult to measure. A similar question was faced with bacterial resistance to antibiotics, which has now been partly answered by the discovery of natural resistance mechanisms in ancient bacterial populations, confirming that the ability to resist these natural products long pre-dated their clinical use (D’Costa et al., 2011). As the majority of fungicides currently used are synthetic products, coevolution of fungal pathogens with such chemicals is presumed to be a recent phenomenon in most cases.

8.1 Resistance Diagnostics Initially, identification of a change in the properties of a fungal pathogen that might impact on its chemical control relied on time-consuming cultural tests and bioassays to determine resistance phenotypes. These were refined to permit higher throughput methods using multiwell plates and automated

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data capture, but such assays were still based on testing individual isolates of the fungus. In the case of nonculturable fungi, such as the powdery mildews, labor intensive tests with living plants or detached leaves sprayed with fungicides were required. An important breakthrough that has facilitated detection and more rapid quantification of resistance in field populations of pathogens is the development of specific molecular diagnostic tests for mutations or other genetic changes conferring resistance (Aoki, Hada, & Suzuki, 2013; Banno et al., 2008; Beckerman, 2013; Fontaine et al., 2009; Ma & Michailides, 2005; Yin, Kim, & Xiao, 2011). It is now possible to sample crops or air samples directly using bulk DNA extraction techniques and PCR-based assays to not only detect specific mutations conferring resistance but also determine the proportion of the pathogen population carrying the resistance trait. This has been of considerable practical value in determining R-allele frequencies and monitoring the development of resistance in space and time (Fraaije, Cools, et al., 2005; Stammler et al., 2008; Torriani et al., 2009). It is also possible, for the first time, to directly measure the outcomes of different resistance management regimes in terms of their effects on pathogen population structure (see below).

8.2 Evaluating Management Strategies The aim of all resistance management strategies is to reduce directional selection for resistance in the pathogen population. The challenge in practical terms is to achieve this without compromising disease control or the economic sustainability of the crop production system. Resistance management options include limiting the amount of fungicide applied, by either reducing the dose or the number of applications, or mixing or alternating treatments with a fungicide with an alternative mode of action. The general principle behind resistance management, defined in terms of the selection coefficient (the difference in fitness between resistant and sensitive strains in the presence of fungicide) has remained more or less unchanged for 25 years (Milgroom & Fry, 1988), for the most part supported only by empirical observations rather than experimental data. This has recently been revisited with a modeling approach and detailed analysis of the literature to validate or challenge assumptions about the most effective resistant management tactics (van den Bosch et al., 2014). There has been a long-standing debate about the effect of fungicide dose on selection for resistance. Intuitively, one would expect higher doses to exert stronger selection pressure. Closer consideration of the model presented in Figure 3 suggests, however, that this assumption might

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not apply in all cases. Where there is a discrete separation into a sensitive and a resistant subpopulation (Figure 3(a)), it is likely that high doses will strongly select for the latter. Where resistance occurs as a continuous series of slight shifts in sensitivity (Figure 3(b)), one could argue that a high dose might control all of the different resistance classes, together with the sensitive wild types, thereby reducing the selection coefficient, whereas a lower dose might allow the most resistant types to survive. Further complexity is added, however, by the observation that a fungicide is unlikely to be distributed equally within a crop, and the amount will decline through weathering or degradation, so not all individuals in the pathogen population will experience the same exposure. The only way to resolve these theoretical considerations is through experimentation. Mavroeidi and Shaw (2006) tested the effect of different doses of a triazole fungicide, with or without a QoI partner, on selection for azole resistance in Z. tritici. Selection for resistance was shown to increase in proportion to dose, while addition of the QoI at higher azole doses reduced selection. The effects of the mixture varied, however, depending on dose rate and the different levels of disease control in the different treatments. These results supported the idea that selection for resistance is positively related to fungicide dose, but that effects of mixtures on selection may be variable. One limitation with this and other earlier experiments on selection was a focus on phenotypes rather than specific genotypes in which selection of specific genes or alleles conferring resistance could be measured. Hobbelen, Paveley, Fraaije et al., (2011) developed a mathematical model to predict selection for fungicide resistance in foliar pathogens of cereals and then tested its predictions using data from field trials with powdery mildew (B. graminis f. sp. hordei) on barley in which the ratio of QoI-sensitive and QoI-resistant (G143A) alleles was quantified following treatments differing in the overall dose and number of sprays of the QoI fungicide azoxystrobin. Selection was shown to increase with increasing dose, and the model successfully predicted between 75 and 90% of the variation in mean selection ratio for most sites and seasons. This approach could be extended to evaluate resistance management in any pathosystem in which resistance is conferred by known genes or alleles of major effect. Further evidence relating to risk of fungicide resistance and dose rate was reviewed by van den Bosch, Paveley, Shaw, Hobbelen, and Oliver (2011), who concluded that almost all experimental studies and models published to date support the hypothesis that higher doses select for resistance.

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At present, a commonly adopted strategy intended to reduce selection for resistance is to mix a high-risk, single-site inhibitor with a low-risk, multisite fungicide. The expectation is that resistance to the low-risk fungicide is unlikely to emerge, and hence it will remain effective and help to reduce selection for resistance to the high-risk component of the mixture. It may also be possible to reduce the dose of the high-risk fungicide without compromising control. Hobbelen, Paveley, and van den Bosch (2011) tested this scenario using a modeling approach in which resistance to the high-risk fungicide carries no fitness cost and showed that maintaining a full dose of the low-risk partner while varying the dose of the high-risk fungicide delayed selection and extended the effective life of the latter. More recently, Mikaberidze, McDonald, and Bonhoeffer (2014) revisited this question, but included the potential effect of a fitness cost of resistance in their model. The outcome confirmed that mixing in the absence of a fitness cost can delay resistance, while in the presence of a fitness cost it might be possible to find an optimal proportion of the two fungicides at which de novo emergence of resistance is prevented. Models are now being extended to estimate time to emergence based on mutation probabilities, fitness costs of resistance, and sensitivity of the resistant strain (Hobbelen, Paveley, & van den Bosch, 2014). This theoretical framework might be tested experimentally in a pathosystem where mutations potentially causing resistance are known from laboratory studies or can be inferred from the emergence of resistance in related fungi. It is also important to obtain more detailed experimental measures of the fitness costs of specific genetic changes associated with resistance; molecular tools can provide more accurate assessment of their potential effects, albeit in the laboratory or glasshouse rather than in the crop environment. A recent study of resistance to SDHI fungicides in the gray mold pathogen B. cinerea (Laleve, Fillinger, & Walker, 2014) used recombinant strains containing different sdhB mutations in a fixed genetic background to assess the impact of these changes on a range of fitness parameters, including growth, reproduction, survival, sensitivity to stress, pathogenicity, and competitiveness, and showed that different mutations varied in their effects. Evidence was obtained for possible compensatory mechanisms modulating the impact of some mutations, hence allowing them to survive in field populations.

8.3 The Impact of Genomics Genome sequences are now available for three of the five fungal species featured in the case histories (Section 5)dM. graminicola (Z. tritici) (Goodwin

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et al., 2011), B. graminis (Spanu et al., 2010), and F. graminearum (Cuomo et al., 2007)dproviding a complete inventory of genes including those encoding known and potential fungicide targets. With the ever-increasing speed and declining cost of next-generation sequencing technologies one can anticipate, in the near future, parallel sequencing of numerous strains of the major crop pathogens, revealing detailed information on their natural diversity, and regions of the genome that are rapidly evolving in response to selection by fungicides and other agricultural practices. This should shed light on the different patterns of cross-resistance to new and existing chemicals, and help to improve risk assessment and stewardship of novel chemical classes prior to and after their market launch. There is also the opportunity to revisit existing target sites through modeling and design of potential “resistance-busting” chemistry (Cools & Hammond-Kosack, 2013).

9. CONCLUSIONS Fungicides have played a key role in crop protection and are likely to remain an important means of disease control for the foreseeable future (Lucas, 2011). But their continued use and effectiveness is under pressure from several sources. The specter of resistance is increasing worldwide, with limited options for effective management. The regulatory environment for new chemistry, along with soaring development costs, means that the pipeline of new products is running low. Recent EU legislation on pesticides is likely to further reduce the diversity of chemistry available for use, and thereby increase the risk of resistance occurring and reducing options for its management (Hillocks, 2012; Leadbeater, 2011). Stringent new hazard criteria may lead to removal of some existing products, limiting flexibility and potentially impacting on disease control. Removal of azole fungicides, for instance, would compromise control of already problematical diseases, such as fusarium ear blight, with an increased risk of mycotoxin contamination, and consequences for human and animal health. All this is taking place at a time when the threats posed by fungal pathogens are increasing rather than diminishing (Fisher et al., 2012). If we are to counter the global risks to plant health (Fears, Aro, Pais, & ter Meulen, 2014) and meet the challenge of food security in a changing environment, a more coordinated approach is required, integrating plant breeding and biotechnology, chemical discovery, and coherent policies on sustainable use of pesticides, as well as continuing innovation in alternative crop protection technologies. More effective management of fungicide resistance through

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improved knowledge, risk assessment, and monitoring will be vital to safeguard both existing and future chemistry.

ACKNOWLEDGMENTS Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK.

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Fungicide resistance risk assessment based on traits associated with the rate of pathogen evolution.

Mixtures as a fungicide resistance management tactic.

Identification of Genes Related to Fungicide Resistance in Fusarium fujikuroi.

Does farm fungicide use induce azole resistance in Aspergillus fumigatus?

Governing principles can guide fungicide-resistance management tactics.

The Evolution of Fungicide Resistance Resulting from Combinations of Foliar-Acting Systemic Seed Treatments and Foliar-Applied Fungicides: A Modeling Analysis.

Molecular evolution. The resistance movement.

Epistasis and the Evolution of Antimicrobial Resistance.

Mechanisms of Resistance to an Azole Fungicide in the Grapevine Powdery Mildew Fungus, Erysiphe necator.

Managing the evolution of herbicide resistance.

Resistance and resilience responses of a range of soil eukaryote and bacterial taxa to fungicide application.

Decomposition of the systemic fungicide 1991 (Benlate).

Suspension Array for Multiplex Detection of Eight Fungicide-Resistance Related Alleles in Botrytis cinerea.

Spatial distribution of single-nucleotide polymorphisms related to fungicide resistance and implications for sampling.

Thiol-reactivity of the fungicide maneb.

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata.

Occupational asthma induced by the fungicide tetrachloroisophthalonitrile.

Random recombination and evolution of drug resistance.

Microbial conversion of fungicide vinclozolin.

Evolution of resistance to thyroid cancer therapy.

Seeking Goldilocks During Evolution of Drug Resistance.

Molecular Evolution of Antifungal Drug Resistance.

Does fungicide application in vineyards induce resistance to medical azoles in Aspergillus species?

Enhanced fungicide resistance in Isaria fumosorosea following ionizing radiation-induced mutagenesis.