Pierre Leroux

Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea

The grey mould fungus Botrytis cinerea causes losses of commercially important fruits, vegetables and ornamentals worldwide. Fungicide treatments are effective for disease control, but bear the risk of resistance development. The major resistance mechanism in fungi is target protein modification resulting in reduced drug binding. Multiple drug resistance (MDR) caused by increased efflux activity is common in human pathogenic microbes, but rarely described for plant pathogens. Annual monitoring for fungicide resistance in field isolates from fungicide-treated vineyards in France and Germany revealed a rapidly increasing appearance of B. cinerea field populations with three distinct MDR phenotypes. All MDR strains showed increased fungicide efflux activity and overexpression of efflux transporter genes. Similar to clinical MDR isolates of Candida yeasts that are due to transcription factor mutations, all MDR1 strains were shown to harbor activating mutations in a transcription factor (Mrr1) that controls the gene encoding ABC transporter AtrB. MDR2 strains had undergone a unique rearrangement in the promoter region of the major facilitator superfamily transporter gene mfsM2, induced by insertion of a retrotransposon-derived sequence. MDR2 strains carrying the same rearranged mfsM2 allele have probably migrated from French to German wine-growing regions. The roles of atrB, mrr1 and mfsM2 were proven by the phenotypes of knock-out and overexpression mutants. As confirmed by sexual crosses, combinations of mrr1 and mfsM2 mutations lead to MDR3 strains with higher broad-spectrum resistance. An MDR3 strain was shown in field experiments to be selected against sensitive strains by fungicide treatments. Our data document for the first time the rising prevalence, spread and molecular basis of MDR populations in a major plant pathogen in agricultural environments. These populations will increase the risk of grey mould rot and hamper the effectiveness of current strategies for fungicide resistance management.

Exploring Mechanisms of Resistance to Respiratory Inhibitors in Field Strains of Botrytis cinerea, the Causal Agent of Gray Mold

ABSTRACT Respiratory inhibitors are among the fungicides most widely used for disease control on crops. Most are strobilurins and carboxamides, inhibiting the cytochrome b of mitochondrial complex III and the succinate dehydrogenase of mitochondrial complex II, respectively. A few years after the approval of these inhibitors for use on grapevines, field isolates of Botrytis cinerea, the causal agent of gray mold, resistant to one or both of these classes of fungicide were recovered in France and Germany. However, little was known about the mechanisms underlying this resistance in field populations of this fungus. Such knowledge could facilitate resistance risk assessment. The aim of this study was to investigate the mechanisms of resistance occurring in B. cinerea populations. Highly specific resistance to strobilurins was correlated with a single mutation of the cytb target gene. Changes in its intronic structure may also have occurred due to an evolutionary process controlling selection for resistance. Specific resistance to carboxamides was identified for six phenotypes, with various patterns of resistance levels and cross-resistance. Several mutations specific to B. cinerea were identified within the sdhB and sdhD genes encoding the iron-sulfur protein and an anchor protein of the succinate dehydrogenase complex. Another as-yet-uncharacterized mechanism of resistance was also recorded. In addition to target site resistance mechanisms, multidrug resistance, linked to the overexpression of membrane transporters, was identified in strains with low to moderate resistance to several respiratory inhibitors. This diversity of resistance mechanisms makes resistance management difficult and must be taken into account when developing strategies for Botrytis control.

A Class III Histidine Kinase Acts as a Novel Virulence Factor in Botrytis cinerea

Filamentous ascomycetes contain large numbers of histidine kinases (HK) that belong to eleven classes. Members of class III from different species were previously shown to be involved in osmoregulation and resistance to dicarboximide and phenylpyrrole fungicides. We have inactivated the gene encoding the single group III HK, BOS1, in the economically important plant pathogen Botrytis cinerea. BOS1 inactivation had pleiotropic effects on the fungus. Besides the expected osmosensitivity and resistance to fungicides, null mutants presented additional characteristics indicating that BOS1 is necessary for normal macroconidiation and full virulence. On standard culture media, null mutants very rarely formed conidiophores and those few conidiophores failed to produce conidia. This defect could be partially restored with 1 M sorbitol, suggesting that another BOS1-independent signal cascade may be involved in macroconidiation. The mutants were not found to be hypersensitive to various oxidative stresses but were more resistant to menadione. Finally, pathogenicity tests showed that bos1-null mutants were significantly reduced in the ability to infect host plants. Appressorium morphogenesis was not altered; however, in planta growth was severely reduced. To our knowledge, this is the first class III HK characterized as a pathogenicity factor in a plant-pathogenic ascomycete.

Genetic Analysis of Fenhexamid-Resistant Field Isolates of the Phytopathogenic Fungus Botrytis cinerea

ABSTRACT The hydroxyanilide fenhexamid, one of the latest antibotrytis fungicides, active especially against leotiomycete plant-pathogenic fungi, inhibits 3-ketoreductase of the C-4-demethylation enzyme complex during ergosterol biosynthesis. We isolated Botrytis cinerea strains resistant to various levels of fenhexamid from French and German vineyards. The sequence of the gene encoding 3-ketoreductase, erg27, varied according to levels of resistance. Highly resistant isolates, termed HydR3+, all presented a modification of the phenylalanine at the C terminus of the putative transmembrane domain at position 412, either to serine (85% of the isolates), to isoleucine (11.5% of the isolates), or to valine (3.5% of the isolates). The introduction of the batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (erg27^{HydR3^{{+}}}) end{document} allele into a fenhexamid-sensitive strain by means of a replicative plasmid conferred fenhexamid resistance on the resulting transformants, showing that the mutations at position 412 are responsible for fenhexamid resistance. Weakly to moderately resistant isolates, termed HydR3−, showed different point mutations between the strains in the sequenced regions of the erg27 gene, corresponding to amino acid changes between positions 195 and 400 of the protein. The batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (erg27^{HydR3^{{-}}}) end{document} alleles on the replicative vector introduced into a sensitive strain did not confer resistance to fenhexamid. Genetic crosses between HydR3− and sensitive strains showed strict correlation between the sequenced mutation in the erg27 gene and the resistance phenotypes, suggesting that these mutations are linked to fenhexamid resistance. The HydR3 mutations possibly modify the affinity of the 3-ketoreductase enzyme for its specific inhibitor, fenhexamid.

The Allele-Specific Probe and Primer Amplification Assay, a New Real-Time PCR Method for Fine Quantification of Single-Nucleotide Polymorphisms in Pooled DNA

ABSTRACT The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.

Sterol Biosynthesis Inhibitors: C-4 Demethylation

Two available fungicides block sterol C-4 demethylation in ergosterol biosynthesis: the hydroxyanilide fenhexamid and the amino-pyrazolinone fenpyrazamine, introduced in the early 2000s and 2010s, respectively. Both these fungicides specifically target 3-ketoreductase (Erg27), the third enzyme of the C-4 demethylation enzyme complex. Highly effective against Botrytis cinerea, causing gray mold, they are also effective against other pathogens, such as Monilinia spp., Sclerotinia spp., and Oculimacula spp. Up to now, most available data on resistance phenomena have been recorded with fenhexamid on gray mold. The mechanism of acquired resistance to fenhexamid is linked to target modification in most resistant strains. Amino-acid changes due to erg27 gene mutations possibly decrease the affinity of fenhexamid for 3-ketoreductase. Twenty-three individual mutations, sometimes, but rarely, occurring in pairs, have been identified in moderately resistant strains. The principal highly resistant strains display one out of four detected amino-acid substitutions at position 412. In fungal species naturally resistant to fenhexamid, this intrinsic resistance also appears to be related to a low affinity of the hydroxyanilide for the 3-ketoreductase. Gray mold has recently been shown to be caused by a complex of two cryptic species living in sympatry: B. cinerea, the predominant species, and Botrytis pseudocinerea. B. pseudocinerea is naturally resistant to fenhexamid, due to its cytochrome P450 protein Cyp684 rather than erg27 polymorphism. The function of Cyp684 is unknown, but it may be involved in fenhexamid detoxification. Moreover, multidrug-resistant strains exhibiting reduced susceptibility to fenhexamid have been detected in B. cinerea and Oculimacula yallundae.

Grapevine Gray Mold in France

Chemical control remains the main approach for decreasing the incidence of gray mold, a disease of many crops, including grapevine, caused principally by Botrytis cinerea. Fungicides with seven different modes of action are currently authorized in French vineyards, but specific resistance has developed against five of these modes of action. Multidrug resistance caused by fungicide efflux has been detected and affects all the classes of fungicide used. Here, we present the history and current status of resistance to the various botryticides in French vineyards. We also discuss resistance management options, based on decreases in the number of botryticide applications per season and the alternation of single products from different classes of molecules with different biochemical modes of action.