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Keywords:

  • CYP51;
  • epitasis;
  • fungicide resistance;
  • Zymoseptoria tritici

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cost and trade-offs associated with azole resistance mechanisms
  5. Conclusions and perspectives
  6. Acknowledgement
  7. Conflicts of interest
  8. References

The durability of azole fungicides in controlling agriculturally important pathogenic fungi is unique amongst modern single site fungicides. Today, azoles are still relied on to control pathogens of many crops including cereals, fruits and vegetables, canola and soybeans. Significantly, this widespread use continues despite many reports of azole-resistant fungal strains. In this review, recent reports of azole resistance and the mechanisms associated with resistant phenotypes are discussed. The example of the complex evolution of the azole target sterol 14α-demethylase (CYP51) enzyme in modern European populations of the wheat pathogen Mycosphaerella graminicola is used to describe the quantitative and epistatic effects on fungicide sensitivity and enzyme function of target site mutations, and to explore the hypothesis that constraints on CYP51 evolution have ensured the longevity of azoles. In addition, the threats posed by alternative resistance mechanisms causing cross-resistance to all azoles or even unrelated fungicides are discussed, and postulations are made on how using new genomic technologies to gain a greater understanding of azole resistance evolution should enhance the ability to control azole-resistant strains of plant pathogenic fungi in the future.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cost and trade-offs associated with azole resistance mechanisms
  5. Conclusions and perspectives
  6. Acknowledgement
  7. Conflicts of interest
  8. References

Azoles (imidazoles and triazoles) are the largest group of sterol 14α-demethylation inhibiting (DMI) fungicides and the most widely used class of antifungal agents for the control of pathogenic fungi of humans and plants, dominating the agricultural fungicide market since their introduction in the 1970s. In contrast to other single site fungicides, and despite their widespread long-term use, control failures with azoles are rare. When resistance occurs, resistance levels are often low and cross-resistance between members of the azole class incomplete. Therefore, disease control can be maintained by the use of more active compounds of the same class. Recently, more studies of azole resistance in plant pathogenic fungi have started to define the molecular mechanisms underlying less sensitive or resistant (here defined as reductions in sensitivity that may cause control failures in the field) phenotypes. Together with studies of human pathogens, these investigations have defined three primary mechanisms of azole resistance. These are (i) mutations in the target-encoding CYP51 gene resulting in decreased affinity of the protein for inhibitors, (ii) over-expression of the target CYP51 gene most frequently caused by insertions in the predicted promoter regions, and (iii) increased efflux caused by the over-expression of genes encoding membrane transporters. These mechanisms can combine, and resistance levels are often determined by combinations of CYP51 amino acid alterations, CYP51 gene over-expression and/or increased efflux (Cools & Fraaije, 2013). Furthermore, increasing genome sequence information has revealed that many filamentous fungi, particularly filamentous Ascomycetes (subphylum Pezizomycotina), possess two or more paralogous CYP51 genes (Becher et al., 2011). Species with multiple CYP51s are intrinsically less sensitive to some azoles, and mutations conferring acquired resistance to effective azoles are usually restricted to one paralogue, most often CYP51A (Becher & Wirsel, 2012).

The most recent studies describing the impact of genetic changes conferring azole resistance on fungicide sensitivity and the efficacy of azole sprays in the field will be discussed. Based on these data, the proposition that the longevity of azole fungicides in agriculture is a product of the costs and trade-offs associated with the genetic alterations conferring a less sensitive phenotype will be explored, with the hypothesis that these preclude the development of widespread resistance to all azoles, and should ensure this group of fungicides, if they are available to growers, remain an important class of antifungal agent in the future.

Cost and trade-offs associated with azole resistance mechanisms

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cost and trade-offs associated with azole resistance mechanisms
  5. Conclusions and perspectives
  6. Acknowledgement
  7. Conflicts of interest
  8. References

CYP51 alteration

Azoles inhibit sterol 14α-demethylase (CYP51). This P450 enzyme is essential for the biosynthesis of sterols, critical components of cell membranes that are considered prerequisite for the evolution of eukaryotes. Therefore, CYP51 activity is widely viewed as the ancestral P450 activity (Kelly & Kelly, 2013). Many CYP51 mutations associated with altered azole fungicide sensitivity phenotypes have been reported in fungi, some at the equivalent positions in both human and plant pathogens, and others unique to one species or genus (Becher & Wirsel, 2012). Unlike target site mutations causing resistance to other single site fungicides, CYP51 mutations often specifically affect individual, or a subset of azole compounds, with cross-resistance across the whole class generally incomplete. For example, substitutions at the equivalent residue to Y136 are the most frequently reported CYP51 alterations in azole-resistant isolates of human and plant pathogenic fungi (Becher & Wirsel, 2012). In agriculture, Y136F was first reported in grape and cereal powdery mildews resistant to triadimenol and propiconazole (Table 1; Délye et al., 1997, 1998; Wyand & Brown, 2005). However, isolates of other fungi carrying substitutions at the equivalent residue, for example the septoria leaf blotch pathogen Mycosphaerella graminicola (Y137F; Leroux et al., 2007; Cools et al., 2011) and Puccinia triticina (Y134F; Stammler et al., 2009), the wheat brown rust pathogen, although resistant to triadimenol, can be controlled by newer azoles such as epoxiconazole. Modelling of the M. graminicola CYP51 confirms this differential effect, as the exchange to F137 pushes this residue into an obstructive position specifically prohibiting the binding of triadimenol (Mullins et al., 2011). In Europe, cereal powdery mildews are generally controlled by mildewicides and host resistance. However, in Australia, where azoles are the only systemic fungicide class registered for use on cereals, incomplete cross-resistance between azoles is now relied on for powdery mildew control (M. Tucker and R. Oliver, Curtin University, Australia, personal communication).

Table 1. Examples of azole-resistant field isolates of plant pathogenic fungi
OrganismCropAzole sensitivity phenotypeMechanism(s)CommentsaSelected reference(s)
  1. a

    Current control strategies.

Blumeria graminis f.sp. hordeiBarleyVarying levels of resistance to triadimenol and propiconazole. Positive cross-resistance to other azolesCYP51 alterations (Y136F, K147Q)CYP51 alterations accumulate to confer highest levels of resistance. In Europe B. graminis f.sp. hordei is currently controlled by mildewicides and host resistanceDélye et al. (1998); Wyand & Brown (2005)
Blumeria graminis f.sp. triticiWheatVarying levels of resistance to triadimenol and propiconazole. Positive cross-resistance to other azolesCYP51 alteration (Y136F)In Europe B. graminis f.sp. tritici is currently controlled by mildewicides and host resistanceKuck & Mehl (2004); Wyand & Brown (2005)
Botrytis cinerea Various fruit and vegetablesReduced azole sensitivity and cross-resistance to unrelated fungicides (multiple drug resistance (MDR))Increased efflux (MfsM2 and Mrr1 alteration)Resistance levels low. Azoles not currently relied onKretschmer et al. (2009)
Cercospora beticola Sugar beetCross-resistant to epoxiconazole and flutriafol. Reduced tetraconazole, prothioconazole and difenoconazole sensitivityConstitutive CYP51 over-expression (CbCYP51)Azoles used to control C. beticolaNikou et al. (2009); Bolton et al. (2012)
Erysiphe necator GrapevineVarying levels of resistance to triadimenol. Restricted cross-resistance to mycobutanil and fenarimolCYP51 alteration (Y136F)Isolates carrying Y136F are least sensitive to triadimenol. Most azoles still effective although highly resistant isolates in the USA do not have a CYP51 mutationDélye et al. (1997); Gadoury et al. (2012)
Monilinia fructicola Stone fruitResistant to propiconazole. Reduced sensitivity to tebuconazole and fenbuconazoleConstitutive CYP51 over-expression (MfCYP51 promoter insert ‘Mona’)Isolates resistant to propiconazole can be controlled by higher doses or azoles with greater intrinsic activityHolb & Schnabel (2007); Chen et al. (2012)
Mycosphaerella fijiensis BananaVarying levels of resistance to propiconazole. Cross-resistance between propiconazole and cyproconazole, but not imazalilCYP51 alterations (Y136F, A313G, A381G, Y461D, G462A, Y463D, Y463H and Y463N). Inserts in the predicted MfCYP51 promoter also foundMost resistant isolates found in areas with highest numbers of sprays. Newer azoles (e.g. epoxiconazole) remain very effectiveCañas-Gutiérrez et al. (2009); Chong et al. (2011)
Mycosphaerella graminicola WheatVarying levels of resistance to all azoles registered for M. graminciola controlCYP51 alterations (>30 reported, e.g. D134G, V136A, Y137F, A379G, I381V, Y459D, Y461H, ΔY459/G460, S524T). Constitutive MgCYP51 over-expression. Increased efflux suggestedAlthough some recent isolates are less sensitive to epoxiconazole and prothioconazole, these compounds remain an important component of disease controlLeroux & Walker (2011); Cools & Fraaije (2013)
Oculimacula acuformis WheatIntrinsically resistant to some triazoles. Acquired resistance to the imidazole prochloraz. Sensitive to prothioconazoleMechanism on intrinsic or acquired resistance unknown, although CYP51 sequence variation suggestedProthioconazole remains effectiveLeroux et al. (2013)
Oculimacula yallundae WheatAcquired resistance to some azoles. Sensitive to prothioconazoleMechanism of acquired resistance unknown, although efflux pump activity suggestedProthioconazole remains effectiveLeroux et al. (2013)
Penicillium digitatum CitrusResistant to imazalil. Cross-resistant to prochloraz, myclobutanil and propiconazoleConstitutive CYP51 over-expression (PdCYP51A or PdCYP51B promoter insert, transposable element). ABC transporter PMR1 over-expressionImazalil widely used. High doses control resistant isolatesNakaune et al. (1998); Ghosoph et al. (2007); Sun et al. (2013)
Podosphaera fusca CucumberLower sensitivities to triadimenol and fenarimol. Cross-resistance between triadimenol and myclobutanil. No cross-resistance between fenarimol and triadimenol or myclobutanilNo CYP51 over-expression. CYP51 alteration suggestedAzole fungicides, in mixture with alternative modes of action, still recommended for P. fusca controlLópez-Ruiz et al. (2010, 2011)
Puccinia triticina WheatVarying sensitivity of European isolates to epoxiconazoleCYP51 alteration (Y134F) and CYP51 over-expression identified in sensitive and less sensitive isolatesDeceased sensitivity phenotypes are still rare. Azoles remain very effectiveStammler et al. (2009)
Rhynchosporium commune BarleyPositive cross-resistance between propiconazole, tebuconazole, epoxiconazole. Incomplete cross-resistance to prothioconazolePresence of a second CYP51 paralogue (RcCYP51A). Mechanism responsible for more recent shifts unknownProthioconazole remains very effectiveHawkins et al. (2011)
Sclerotinia homoeocarpa Turf grassReduced sensitivities to propiconazole. Sensitivities between azoles as well as to plant growth regulators highly correlatedInduced CYP51 over-expression. Constitutive and induced efflux pump (ShatrD) over-expressionAzoles still relied on for S. homoeocarpa control, resistance to unrelated fungicides a possibilityOk et al. (2011); Hulvey et al. (2012)
Venturia inaequalis AppleResistance to myclobutanil and reduced sensitivity to fenbuconazole and difenoconazole reported. Cross-resistance between azoles. Some local variationConstitutive CYP51 (CYP51A1) over-expression caused by a promoter insertAzoles still used although other modes of action are availableSchnabel & Jones (2001); Pfeufer & Ngugi (2012)

Other CYP51 mutations confer contrasting effects on azole sensitivity. For example, M. graminicola isolates carrying the substitution V136A are less sensitive to the imidazole prochloraz, but sensitive to tebuconazole, whereas isolates with I381V have lower sensitivities to most triazoles, particularly tebuconazole, but remain sensitive to prochloraz (Fraaije et al., 2007; Leroux et al., 2007). Furthermore, until recently V136A and I381V had not been found in combination. Consequently, it was proposed this trade-off could be exploited to prevent the further evolution of azole resistance by using mixtures or alternations of azoles that are differentially affected by V136A and I381V (Cools & Fraaije, 2008). However, in modern M. graminicola populations the sequential accumulation of CYP51 mutations has generated CYP51 variants with both V136A and I381V, often combined with the more recently emerged substitutions D134G and/or S524T (Leroux & Walker, 2011; Cools & Fraaije, 2013). Isolates carrying these CYP51 variants are becoming more common as they are less sensitive to the most widely used azoles epoxiconazole and prothioconazole, and also prochloraz, although the presence of V136A seems to maintain isolate sensitivity to tebuconazole.

In fact, the accumulation of CYP51 mutations seems to be critical for the stepwise evolution of new resistant phenotypes of M. graminicola in response to the introduction of progressively more active azoles. However, the sequence and frequency by which CYP51 mutations emerge, driven by azole selection, is constrained by effects on enzyme activity. For example, western European populations of M. graminicola are dominated by isolates with CYP51 variants carrying V136A and/or I381V, combined with changes at residues Y459–Y461 (Stammler et al., 2008), and different combinations of these mutations can confer decreased sensitivity to all azoles currently registered for septoria leaf blotch control (Cools et al., 2011; Cools & Fraaije, 2013). Heterologous expression in Saccharomyces cerevisiae showed that when introduced as single amino acid substitutions, V136A and I381V destroy the capacity of the M. graminicola CYP51 protein to complement yeast CYP51, indicating a loss of sterol 14α-demethylase activity. However, this impairment of function can be restored by combining V136A and I381V with changes at Y459–Y461 (Cools et al., 2010). Therefore, the rise of mutations encoding alterations at residues Y459–Y461 in the mid- to late 1990s (Cools & Fraaije, 2013) was a precondition to the diversity of CYP51 variants and azole resistance phenotypes observed in modern M. graminicola populations. The most recent M. graminicola CYP51 variants, for example, have up to eight amino acid alterations compared to the wild type. The identification of the equivalent mutations in isolates of the banana pathogen Mycosphaerella fijiensis (Y461–Y463, Table 1; Cañas-Gutiérrez et al., 2009) may suggest a propensity for alterations in this region in fungi related to M. graminicola. However, although rare in plant pathogenic fungi, amino acid changes in this region have been found in azole-resistant isolates of human pathogenic fungi not closely related to M. graminicola, including Candida albicans and Aspergillus fumigatus.

CYP51 over-expression

As a mechanism of acquired resistance to fungicides, increased expression of the target-encoding gene is unique to the azoles. In plant pathogens it is quite common, with CYP51 over-expression contributing to azole-resistant phenotypes in Venturia inaequalis (Table 1; Schnabel & Jones, 2001), Penicillium digitatum (Sun et al., 2013), Cercospora beticola (Bolton et al., 2012), Monilinia fructicola (Luo & Schnabel, 2008), Blumeriella jaapii (Ma et al., 2006), P. triticina (Stammler et al., 2009) and M. graminicola (Cools et al., 2012). Increases in expression are most often constitutive and are frequently caused by alterations in the predicted regulatory regions. For example, higher CYP51 expression was associated with insertions in the predicted promoter regions of V. inaequalis (Schnabel & Jones, 2001), M. fructicola (Luo & Schnabel, 2008), M. graminicola (Cools et al., 2012) and P. digitatum (Sun et al., 2013). These inserts vary in size, often have the signatures of transposable elements, and contain powerful promoter sequences (Sun et al., 2013). For some fungi, including C. beticola (Bolton et al., 2012) and P. triticina (Stammler et al., 2009), the genetic changes responsible for over-expression are currently unknown.

CYP51 over-expression may offer some selective advantages in comparison to other resistance mechanisms. Unlike alterations of CYP51 primary sequence, changes in sensitivity of individuals over-expressing CYP51 are not compound-specific, with complete cross-resistance between the azoles common (Table 1). However, resistance levels are generally lower than those caused by target site alteration, and therefore higher doses or more active compounds can be effective against isolates over-expressing CYP51 (Table 1). Recently, M. graminicola isolates over-expressing azole-resistant forms of the CYP51 gene have been identified. This combination of target site mutation and over-expression causes a pan-azole-resistant phenotype, with high levels of resistance to compounds specifically affected by mutations (Cools et al., 2012), leading to the concern that over-expression of the most resistant CYP51 variants could confer a phenotype that may affect the field performance of all azoles in the future.

Enhanced fungicide efflux

Enhanced fungicide efflux, leading to resistance to multiple unrelated drugs, a so-called multidrug-resistant (MDR) phenotype, is viewed as a major threat to the control of fungal pathogens of humans (Gulshan & Moye-Rowley, 2007). Indeed in pathogenic yeasts, for example Candida glabrata, Candida krusei and C. albicans, the impact of over-expression of ATP binding cassette (ABC) or major facilitator superfamily (MFS) transporter genes on intrinsic and acquired resistance to multiple antifungals is well established (Thakur et al., 2008; Morschhäuser, 2010). However, in filamentous fungi, particularly pathogens of plants, the importance of efflux pump activity is less clear. For example, genes encoding ABC or MFS transporters have been identified in plant pathogenic fungi, and their capacity to export azoles by heterologous expression and targeted knockout studies has been demonstrated (de Waard et al., 2006). Genome-wide transcriptional studies have identified ABC transporter genes responsive to azole treatment (Becher et al., 2011), and the use of putative chemical efflux antagonists has shifted resistant phenotypes, as well as sensitizing wildtype isolates (Roohparvar et al., 2002; Leroux & Walker, 2011). Yet, although MDR phenotypes of plant pathogens, including M. graminicola and Oculimacula yallundae, have been proposed to impact on fungicide performance (Table 1; Leroux & Walker, 2011; Leroux et al., 2013), to date, only in Botrytis cinerea has a genetic mechanism conferring enhanced efflux to multiple fungicides been characterized and shown to impact on the performance of fungicides in the field (Table 1; Kretschmer et al., 2009). The factors suggested as responsible for the limited occurrence of MDR in the field, including large population size, the restricted number of sprays per season and the occurrence of refugia, rely on fitness costs in the absence of fungicide selection (de Waard et al., 2006). However, as shown in experimentally evolved populations of C. albicans (Cowen et al., 2001), the biological potential to compensate for fitness costs associated with an MDR phenotype exists.

Multiple CYP51s

As more genomes of plant pathogenic fungi become available, the influence of multiple target site-encoding genes on intrinsic and acquired fungicide resistance is becoming more apparent (Cools & Hammond-Kosack, 2013). Multiple CYP51 paralogues have been identified in a number of plant pathogens including species of Aspergillus, Fusarium, Penicillium and Rhynchosporium (Becher et al., 2011; Hawkins et al., 2011; Liu et al., 2011). In Fusarium graminearum, heterologous expression and gene knockout studies have shown two of the three CYP51 paralogues, CYP51A and CYP51B, to be functionally redundant, both encoding sterol 14α-demethylases, but CYP51A is rapidly induced upon ergosterol depletion caused by azole treatment, and is thus responsible for the intrinsically low sensitivity of F. graminearum to some azole fungicides (Fan et al., 2013). Although tebuconazole resistance in four isolates of F. graminearum and Fusarium asiaticum was not associated with CYP51 changes (Yin et al., 2009), numerous studies in A. fumigatus (Becher & Wirsel, 2012) and P. digitatum (Sun et al., 2013) have shown acquired resistance to effective azoles is caused largely by mutations and/or over-expression of the CYP51A paralogue. This implies that carrying additional CYP51s may offer an advantage under selection by azoles, as any costs or trade-offs associated with changes in protein structure or gene over-expression of one paralogue are circumvented by the presence of an unchanged enzyme with paralogous wildtype activity.

Conclusions and perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cost and trade-offs associated with azole resistance mechanisms
  5. Conclusions and perspectives
  6. Acknowledgement
  7. Conflicts of interest
  8. References

Early studies seeking to predict the risk of resistance to azoles using experimentally evolved fungal populations of, for example, Cladosporium cucumerinum (Fuchs & Drandarevski, 1976), concluded that resistance under practical conditions was unlikely as spore formation, mycelial growth rate and germination were all retarded in resistant isolates. Although the conclusions ultimately proved incorrect, these studies identified a possible cost to azole resistance. With the molecular tools now available, it is known that target site changes contributing to an azole-resistant phenotype have interacting effects, and mutations that alone may be deleterious can be maintained in populations in the presence of compensatory mutations. The evolution of CYP51 in European Mgraminicola populations under selection by azoles is an elegant demonstration of these epistatic effects, where the phenotypic consequences of individual mutations are dependent on the genetic background in which they occur (Poelwijk et al., 2007). In addition, as target site resistance is generally compound-specific, the occurrence of particular mutations reflects the history of azole use. Therefore, a requirement for multiple mutations to confer resistance, and the diversity of azoles available to growers, have extended the effective life of this chemistry and have ensured that newly developed compounds, for example the recently introduced prothioconazole, can still have a profitable share of the market, despite the existence of azole-resistant strains of target pathogens. A current example is the potential use of prothioconazole to control azole-resistant strains of Asian soybean rust, Phakopsora pachyrhizi (Koga et al., 2011; Schmitz et al., 2013).

In the future, it may be that mechanisms that can provide cross-resistance across the azole class, for example CYP51 over-expression and enhanced fungicide efflux activity, will become more common, although the infrequency of these mechanisms in azole-resistant field isolates in current populations of plant pathogens suggests fitness costs (Leroux et al., 2013). Whether additional mutations will alleviate these costs in the future is unknown. However, as genome-wide analysis of different plant pathogens becomes increasing feasible (Cools & Hammond-Kosack, 2013), and we are able to identify genetic changes in azole-resistant strains and gain a greater understanding of the interactions between them, it may be possible to potentiate the activity of azoles or alternative fungicides in the future by inhibiting compensatory mechanisms.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Cost and trade-offs associated with azole resistance mechanisms
  5. Conclusions and perspectives
  6. Acknowledgement
  7. Conflicts of interest
  8. References

Rothamsted Research receives grant-aided support from the UK Biotechnology and Biological Sciences Research Council (BBSRC).

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  2. Abstract
  3. Introduction
  4. Cost and trade-offs associated with azole resistance mechanisms
  5. Conclusions and perspectives
  6. Acknowledgement
  7. Conflicts of interest
  8. References
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