Field strains of Botrytis cinerea Pers ex Fr, the causal agent of grey mould diseases, were collected from French vineyards between 1993 and 2000. Several phenotypes have been characterized according to the inhibitory effects of fungicides towards germ-tube elongation and mycelial growth. Two types of benzimidazole-resistant strains (Ben R1 and Ben R2) could be detected; negative cross-resistance to phenylcarbamates (e.g. diethofencarb) was only found in Ben R1. Benzimidazole resistance was related to point mutations at codon 198 (Ben R1) or 200 (Ben R2) of the beta-tubulin gene. Most dicarboximide-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. dicloran) but remained sensitive to phenylpyrroles (e.g. fludioxonil). These resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Three anilinopyrimidine-resistant phenotypes have been identified. In the most resistant one (Ani R1), resistance was restricted to anilinopyrimidines, but no differences were observed in the amino-acid sequences of cystathionine beta-lyase (the potential target site of these fungicides) from Ani R1 or wild-type strains. In the two other phenotypes (Ani R2 and Ani R3), resistance extended to various other groups of fungicide, including dicarboximides, phenylpyrroles and sterol biosynthesis inhibitors. This multi-drug resistance was probably determined by over-production of ATP-binding cassette transporters. The hydroxyanilide fenhexamid is a novel botryticide whose primary target site is the 3-keto reductase involved in sterol C-4 demethylations. Apart from the multi-drug-resistant strain Ani R3, three other fenhexamid-resistant phenotypes have been recognized. For two of them (Hyd R1 and Hyd R2) fenhexamid-resistance seemed to result from P450-mediated detoxification. Reduced sensitivity of the target site could be the putative resistance mechanism operating in the third resistant phenotype (Hyd R3). Increased sensitivity to inhibitors of sterol 14 alpha-demethylase recorded in Hyd R1 strains was related to two amino-acid changes at positions 15 and 105 of this enzyme.

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Fenpiclonil [4-(2,3-dichlorophenyl)pyrrole-3-carbonitrile], a new agricultural fungicide, is a structural analogue of the antibiotic pyrrolnitrin used as an antimycotic in animals. Its spectrum of antifungal activity is similar to that of iprodione and tolclofos-methyl, which are representatives of dicarboximide and aromatic hydrocarbon fungicides. Laboratory mutants of Botryris cinerea and Fusarium nivale were simultaneously resistant to fenpiclonil, iprodione and tolclofos-methyl and they showed an increased susceptibility to high osmotic pressure (sodium chloride, glucose). Strains of B. cinerea, collected in Champagne vineyards, which were resistant to dicarboximides were not resistant to fenpiclonil and tolclofos-methyl. The antioxidant a-tocopheryl acetate, and the cytochrome P-450 inhibitors piperonyl butoxide and tebuconazole antagonized the fungitoxicity of fenpiclonil and iprodione in a wild-type strain of B. cinerea. The uncoupling activity of fenpiclonil, iprodione and tolclofos-methyl was detected in mitochondria from both a wild-type strain and a laboratory mutant of B. cinerea.The concentrations effective towards this process were greater than those inhibiting the mycelial growth of wild-type strains. These results suggest that fenpiclonil, iprodione, tolclofos-methyl and related fungicides have the same biochemical mode of action. However, a primary effect on respiration, previously considered for pyrrolnitrin, seems to be improbable.

: When mycelium of Botrytis cinerea was treated with low concentrations of the anilinopyrimidine fungicide pyrimethanil the total amount of free amino acids increased. Qualitative variations were also induced : alanine, glutamine, lysine, glycine, histidine, asparagine, arginine, threonine and moreover, aaminobutyrate and b-alanine were accumulated ; cyst(e)ine, valine, leucine and citrulline were reduced. When mycelium of B. cinerea was incubated with pyrimethanil at 1É5 kM induced a decrease of [35S]methionine and Na, simultaneously an increase of [35S]cystathionine. These data indicate that the anilinopyrimidine fungicide pyrimethanil inhibits the biosynthesis of methionine and suggest that the primary target could be the cystathionine b-lyase.

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