Exploring SDHI resistance in Botrytis cinerea : from mutagenesis to enzymatic assays.

Botrytis cinerea is a phytopathogenic ascomycete responsible for grey mould on many crops. Respiration inhibitors play an increasing role in the control of this disease. Succinate dehydrogenase inhibitors (SDHIs, including carboxamides) inhibit the fungal respiration by blocking the ubiquinonebinding site of the mitochondrial complex II. Old SDHIs (i.e. carboxin), essentially active against Basidiomycetes were replaced in the 2000s by a new generation of SDHIs with a broader spectrum including Ascomycetes. Boscalid is the only representative of this new generation in France up to now. A few years after its market introduction, field mutants of B. cinerea, resistant to boscalid were isolated in France and Germany. At least six different phenotypes, named CarR1 to CarR6, have been pinpointed by characterizing their resistance pattern towards 20 SDHIs. CarR1 to R4 phenotypes exhibit low to medium level of resistance, whereas CarR5 and R6 show high level of resistance to different SDHIs, including boscalid. CarR1 and CarR2 strains are currently the most frequent strains detected in German and French vineyards. The resistance mechanism was investigated for the different phenotypes by searching for putative alterations in the SDH proteins, which could be responsible for the observed resistance. Our findings show point mutations in the sdhB gene lead to a specific amino acid change in SdhB for each phenotype. Isogenic mutants have been generated through a gene replacement strategy to confirm the role of these mutations in the various SDHI resistance phenotypes. We have shown that each mutation of the sdhB gene confers resistance to at least one SDHI. Isogenic mutants show similar resistance factors compared to field mutants carrying the same mutation. SDH enzyme activity and inhibition by different SDHIs were measured in these mutants. Our results provide evidence for differential SDH inhibition profiles according to the tested mutations, correlating with the mutants’ resistance pattern. SDH enzyme affinity to different SDHIs will be measured in these mutants.