Danièle Debieu

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.

Strong resistance to the fungicide fenhexamid entails a fitness cost in Botrytis cinerea, as shown by comparisons of isogenic strains

BACKGROUND Fenhexamid, a sterol biosynthesis inhibitor effective against Botrytis, inhibits the 3-ketoreductase (Erg27) involved in C-4 demethylation. Several fenhexamid-resistant phenotypes have been detected in Botrytis cinerea populations from French vineyards. The field isolates with the highest resistance levels display amino acid changes in Erg27 (F412S, F412I or F412V). RESULTS Fenhexamid-resistant mutants were generated by site-directed mutagenesis of the erg27 gene in a sensitive recipient strain to overcome the impact of different genetic backgrounds. The wild-type erg27 allele was replaced by the three mutated alleles (erg27(F412S/I/V)) by homologous recombination. These isogenic strains were shown to be fenhexamid-resistant and were used to quantify the impact of F412 mutations on fungal fitness. Several parameters, including radial growth, the production of sclerotia and conidia, freezing resistance and aggressiveness, were quantified in laboratory conditions. Analysis of variance demonstrated significant differences between the mutant and parental strains for some characters. In particular, the mutants grew more slowly than the wild-type strain and displayed variations in the production of sclerotia and conidia with temperature and susceptibility to freezing. CONCLUSIONS The results highlight a moderate but significant impact of F412 mutations on the survival capacity of B. cinerea strains displaying high levels of resistance to fenhexamid in laboratory conditions, potentially limiting their dispersal and persistence, particularly in terms of overwintering, in field conditions.

Ergosterol biosynthesis and its inhibition by fenpropimorph in Fusarium species

Abstract In the mycelium of all the Fusarium species studied, the major sterol was ergosterol in the absence of fenpropimorph. In the presence of this fungicide fenpropimorph-sensitive strains, with the exception of F. nivale , accumulated mainly Δ 8,14 -sterols and tolerant ones accumulated either both Δ 8,14 - and Δ 8 -sterols or only Δ 8 -sterols. This seemed to indicate that fenpropimorph toxicity was related to sterol Δ 14 -reductase sensitivity. In F. nivale , which was highly sensitive to fenpropimorph, the accumulation of only Δ 8 -sterols indicated that the main target enzyme was sterol Δ 8 →Δ 7 -isomerase. In addition to Δ 8,14 -and Δ 8 -sterols, squalene was accumulated in very high amounts in several strains; the level of squalene accumulation did not seem to be correlated with fenpropimorph sensitivity.

Site-directed mutagenesis of the P225, N230 and H272 residues of succinate dehydrogenase subunit B from Botrytis cinerea highlights different roles in enzyme activity and inhibitor binding.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recent field monitoring studies have identified Botrytis cinerea isolates resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B subunit. We confirmed, by site-directed mutagenesis of the sdhB gene, that each of the mutations identified in field strains conferred resistance to boscalid in B.cinerea, and in some cases cross-resistance to other SDHIs (fluopyram, carboxin). Enzyme inhibition studies showed that the studied modifications (SdhB_P225T/L/F, N230I, H272Y/R/L) affected the inhibition of SDH activity by SDHIs, directly contributing to resistance. Our results confirm the importance of H272, P225 and N230 for carboxamide binding. Modifications of P225 and N230 conferred resistance to the four carboxamides tested (boscalid, fluopyram, carboxin, bixafen). Modifications of H272 had differential effects on the susceptibility of SDH to SDHIs. SdhB(H272L) , affected susceptibility to all SDHIs, SdhB(H272R) conferred resistance to all SDHIs tested except fluopyram, and SdhB(H272Y) conferred fluopyram hypersensitivity. Affinity-binding studies with radiolabelled fluopyram revealed strong correlations among the affinity of SDHIs for SDH, SDH inhibition and in vivo growth inhibition in the wild type. The sdhB(H272Y) mutation did not affect SDH and respiration activities, whereas all the other mutations affected respiration by decreasing SDH activity.

Sterol composition of the vine powdery mildew fungus, Uncinula necator: Comparison of triadimenol-sensitive and resistant strains

Ergosta-5,24(241)-dien-3β-ol was the major sterol of conidia of Uncinula necator, an obligate pathogenic fungus, which is the causal agent of vine powdery mildew. Other Δ5-sterols, such as cholesterol, ergost-5-en-3β-ol, stigmasta-5,22-dien-3β-ol and stigmast-5-en-3β-ol found in U. ecator host plant (Vitis vinifera) were detected as minor sterols in conidia of U. necator. The triadimenol-sensitive and resistant strains contained similar sterol compositions. Triadimenol treatment led to accumulation of 14α-methylsterols, such as eburicol and obtusifoliol. This suggests that sterol C-14 demethylase is the target for triadimenol in sterol biosynthesis in U. necator.

Effects of sterol biosynthesis inhibitor fungicides in the phytopathogenic fungus, Nectria haematococca: ergosterol depletion versus precursor or abnormal sterol accumulation as the mechanism of fungitoxicity

The relative importance of the depletion of ergosterol versus the accumulation of precursor or abnormal sterols in the mechanism of fungal growth inhibition by sterol biosynthesis inhibitor fungicides is incompletely understood. In order to investigate this problem further, the degree of inhibition of the growth of Nectria haematococca by fungicides with different enzymatic targets in the sterol biosynthetic pathway was determined and compared with their effects on the sterol profile. The sensitivity of N. haematococca was highest towards fenpropimorph, followed by tebuconazole, terbinafine, fenpropidin and tridemorph. Terbinafine, a squalene epoxidase inhibitor, induced a very large accumulation of squalene without very significant inhibition of ergosterol biosynthesis and growth. The fungus appeared able to tolerate large amounts of squalene. In the case of tebuconazole, a sterol 14α-demethylase inhibitor, it seemed that the accumulation of C4 mono- and dimethyl sterols was responsible for fungitoxicity. Fenpropimorph and fenpropidin seemed to be good inhibitors of both sterol Δ14-reductase and Δ8Δ7-isomerase, whereas tridemorph was a better inhibitor of Δ8Δ7-isomerase than of the Δ14-reductase. Large accumulations of Δ8,14- or Δ8-sterols and correspondingly large decreases in the ergosterol content are both implicated in the fungitoxicity of these compounds in N. haematococca.

Role of sterol 3-ketoreductase sensitivity in susceptibility to the fungicide fenhexamid in Botrytis cinerea and other phytopathogenic fungi

BACKGROUND The narrow-spectrum fungicide fenhexamid was introduced into French vineyards in 2000 to control grey mould caused by a complex of two cryptic species: Botrytis cinerea, the predominant species sensitive to fenhexamid, and Botrytis pseudocinerea, naturally resistant. Fenhexamid was suggested to inhibit the 3-ketoreductase involved at C-4 demethylation steps during ergosterol biosynthesis, as revealed by its effects on the B. cinerea sterol profile. Resistance monitoring studies have hitherto identified two B. cinerea fenhexamid-resistant phenotypes, both resulting from mutations in the erg27 gene encoding 3-ketoreductase. RESULTS The role of 3-ketoreductase sensitivity in fungal susceptibility to fenhexamid was investigated by studying sterol profiles and microsomal 3-ketoreductase in various fungal strains. Fenhexamid does inhibit B. cinerea 3-ketoreductase activity. Erg27 mutations causing amino acid substitutions in or near the transmembrane domain strongly decrease the affinity of fenhexamid for 3-ketoreductase. Fenhexamid has very low affinities for 3-ketoreductase in inherently resistant species, whether closely related to B. cinerea, like B. pseudocinerea, or more distantly related, like Nectria haematococca. CONCLUSION erg27 mutation and erg27 polymorphism may therefore contribute to the unfavourable binding of fenhexamid to its target, 3-ketoreductase, explaining the acquisition of fenhexamid resistance in B. cinerea and the narrow spectrum of this fungicide.