J. A. Lucas

Role of Ascospores in Further Spread of QoI-Resistant Cytochrome b Alleles (G143A) in Field Populations of Mycosphaerella graminicola

ABSTRACT Strobilurin fungicides or quinone outside inhibitors (QoIs) have been used successfully to control Septoria leaf blotch in the United Kingdom since 1997. However, QoI-resistant isolates of Mycosphaerella graminicola were reported for the first time at Rothamsted during the summer of 2002. Sequence analysis of the cytochrome b gene revealed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Extensive monitoring using real-time polymerase chain reaction (PCR) testing revealed that fungicide treatments based on QoIs rapidly selected for isolates carrying resistant A143 (R) alleles within field populations. This selection is driven mainly by polycyclic dispersal of abundantly produced asexual conidia over short distances. In order to investigate the role of sexually produced airborne ascospores in the further spread of R alleles, a method integrating spore trapping with real-time PCR assays was developed. This method enabled us to both quantify the number of M. graminicola ascospores in air samples as well as estimate the frequency of R alleles in ascospore populations. As expected, most ascospores were produced at the end of the growing season during senescence of the wheat crop. However, a rapid increase in R-allele frequency, from 35 to 80%, was measured immediately in airborne ascospore populations sampled in a wheat plot after the first QoI application at growth stage 32. After the second QoI application, most R-allele frequencies measured for M. graminicola populations present in leaves and aerosols sampled from the treated plot exceeded 90%. Spatial sampling and testing of M. graminicola flag leaf populations derived from ascospores in the surrounding crop showed that ascospores carrying R alleles can spread readily within the crop at distances of up to 85 m. After harvest, fewer ascospores were detected in air samples and the R-allele frequencies measured were influenced by ascospores originating from nearby wheat fields.

Analysis of Two in Planta Expressed LysM Effector Homologs from the Fungus Mycosphaerella graminicola Reveals Novel Functional Properties and Varying Contributions to Virulence on Wheat

Secreted effector proteins enable plant pathogenic fungi to manipulate host defenses for successful infection. Mycosphaerella graminicola causes Septoria tritici blotch disease of wheat (Triticum aestivum) leaves. Leaf infection involves a long (approximately 7 d) period of symptomless intercellular colonization prior to the appearance of necrotic disease lesions. Therefore, M. graminicola is considered as a hemibiotrophic (or necrotrophic) pathogen. Here, we describe the molecular and functional characterization of M. graminicola homologs of Ecp6 (for extracellular protein 6), the Lysin (LysM) domain-containing effector from the biotrophic tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum, which interferes with chitin-triggered immunity in plants. Three LysM effector homologs are present in the M. graminicola genome, referred to as Mg3LysM, Mg1LysM, and MgxLysM. Mg3LysM and Mg1LysM genes were strongly transcriptionally up-regulated specifically during symptomless leaf infection. Both proteins bind chitin; however, only Mg3LysM blocked the elicitation of chitin-induced plant defenses. In contrast to C. fulvum Ecp6, both Mg1LysM and Mg3LysM also protected fungal hyphae against plant-derived hydrolytic enzymes, and both genes show significantly more nucleotide polymorphism giving rise to nonsynonymous amino acid changes. While Mg1LysM deletion mutant strains of M. graminicola were fully pathogenic toward wheat leaves, Mg3LysM mutant strains were severely impaired in leaf colonization, did not trigger lesion formation, and were unable to undergo asexual sporulation. This virulence defect correlated with more rapid and pronounced expression of wheat defense genes during the symptomless phase of leaf colonization. These data highlight different functions for MgLysM effector homologs during plant infection, including novel activities that distinguish these proteins from C. fulvum Ecp6.

A novel substitution I381V in the sterol 14α-demethylase (CYP51) of Mycosphaerella graminicola is differentially selected by azole fungicides

SUMMARY The recent reduction in the efficacy of azole fungicides in controlling Septoria leaf blotch of wheat, caused by Mycosphaerella graminicola, has prompted concerns over possible development of resistance, particularly in light of the recent emergence of widespread resistance to quinone outside inhibitors (QoIs). We have recently implicated alterations in the target-encoding sterol 14alpha-demethylase protein (CYP51), and over-expression of genes encoding efflux pumps, in reducing sensitivity to the azole class of sterol demethylation inhibitors (DMIs) in M. graminicola. Here we report on the prevalence and selection of two CYP51 alterations, substitution I381V and deletion of codons 459 and 460 (DeltaY459/G460), in populations of M. graminicola. Neither alteration has previously been identified in human or plant pathogenic fungi resistant to azoles. The presence of DeltaY459/G460 showed a continuous distribution of EC(50) values across isolates with either I381 or V381, and had no measurable effect on azole sensitivity. Data linking fungicide sensitivity with the presence of I381V in M. graminicola show for the first time that a particular CYP51 alteration is differentially selected by different azoles in field populations of a plant pathogen. Substitution I381V although not an absolute requirement for reduced azole sensitivity, is selected by tebuconazole and difenoconazole treatment, suggesting an adaptive advantage in the presence of these two compounds. Prochloraz treatments appeared to select negatively for I381V, whereas other azole treatments did not or only weakly impacted on the prevalence of this substitution. These findings suggest treatments with different members of the azole class of fungicides could offer a resistance management strategy.

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new-generation carboxamide fungicides with broad-spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single-site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin-resistant mutants. A range of SDHI resistance-conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B-S221P/T, B-H267F/L/N/Y, B-I269V and D-D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild-type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross-resistance studies will aid the implementation of anti-resistance strategies to prolong the cost-effectiveness and lifetime of SDHI fungicides.

Impact of Recently Emerged Sterol 14α-Demethylase (CYP51) Variants of Mycosphaerella graminicola on Azole Fungicide Sensitivity

ABSTRACT The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.

Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola.

A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.

Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function

ABSTRACT The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO7-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.

Overexpression of the sterol 14α-demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype

BACKGROUND The recent evolution towards resistance to azole fungicides in European populations of the wheat pathogen Mycosphaerella graminicola has been caused by the progressive accumulation of mutations in MgCYP51 gene, encoding the azole target sterol 14α-demethylase. Particular combinations of mutations have been shown specifically to affect the interaction of the MgCYP51 protein with different members of the azole class. Although additional mechanisms, including increased MgCYP51 expression and enhanced active efflux, have been proposed, the genetic changes underlying these mechanisms are unknown. RESULTS Analysis of the azole sensitivities of recent M. graminicola isolates identified a novel phenotype, seemingly independent of changes in MgCYP51 coding sequence. Characterised by a 7-16-fold reduction in in vitro sensitivity to all azoles tested and by growth on seedlings at higher doses of azoles in glasshouse tests compared with isolates carrying the same MgCYP51 variant (L50S, S188N, I381V, ΔY459/G460, N513K), isolates with this phenotype constitutively overexpress MgCYP51 by between 10- and 40-fold compared with the wild type. Analysis of sequences upstream of the predicted MgCYP51 translation start codon identified a novel 120 bp indel, considered to be an insertion, in isolates overexpressing MgCYP51. CONCLUSIONS The identification of an insertion in the predicted MgCYP51 promoter in azole-resistant isolates overexpressing MgCYP51 is the first report of a genetic mechanism, other than changes in target-site coding sequence, affecting sensitivity to multiple azoles in field isolates of M. graminicola. The identification of recent isolates overexpressing MgCYP51 confirms the ongoing evolution and diversification of resistance mechanisms in European populations of M. graminicola.

The evolution of fungicide resistance.

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 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.

Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals

ABSTRACT Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml−1 resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.