Botrytis pseudocinerea is resistant to the fungicide fenhexamid due to detoxification by the cytochrome P450 monooxygenase Cyp684

The Botrytis species complex responsible for the grey mould disease found on grapevines is composed of two species: Botrytis cinerea, to a large extent (roughly 90%), and Botrytis pseudocinera. Despite their genetic polymorphism, these species cannot be morphologically distinguished. However, they do differ in their response to several fungicides, especially to the sterol biosynthesis inhibitor fenhexamid. While B. cinerea is sensitive to this hydroxyanilide fungicide, B. pseudocinerea is naturally resistant. Because a strong synergism was found on B. pseudocinerea between fenhexamid and sterol 14α-demethylation inhibitors (DMIs) known to inhibit Cyp51, a cytochrome P450 monooxygenase, it was hypothetized that the detoxification of fenhexamid by a cytochrome P450 monooxygenase similar to Cyp51 is involved in the resistance B. pseudocinerea. To test this, we sought the gene overexpressed in the presence of fenhexamid with the highest similarity to cyp51. Taking into account the Cyp P450 classification based on homology and phylogenetic criteria, this gene, whose function remains unknown, belongs to the Cyp684 family. It was then deleted in a B. pseudocinerea strain. cyp684 knock out mutants exhibit a loss of fenhexamid resistance and synergism between DMIs and fenhexamid, showing that the Cyp684 cytochrome P450 protein is responsible for B. pseudocinerea’s natural resistance to fenhexamid and is involved in fenhexamid detoxification. Although cyp684 is also present in B. cinerea, which is sensitive to fenhexamid, polymorphism was observed between the two species: in B. pseudocinerea the cyp684 promoter shows a deletion of 25 bp; an insertion of 10 bp is observed in the 3’ UTR of the gene. In addition, cyp684 expression is induced after exposure to fenhexamid in B. pseudocinerea suggesting that one of these gene modifications might be responsible for cyp684 overexpression. We are currently conducting experiments to correlate gene structure and resistance in different Botrytis species. The Cyp684 protein will be expressed in a heterologous host to easily identify the produced metabolites.