Phenotypic Analyses of Fenhexamid Resistant Botrytis cinerea Mutants

Grey mould caused by the fungus Botrytis cinerea Pers ex Fr. [anamorph of Botryotinia fuckeliana (de Bary) Whets] is a major disease of grapes (Elad et al. 2004; Keller et al. 2004). Although biological control of fungal growth is becoming popular on account of environmental concerns (Elmer & Reglinski 2006), generally B. cinerea infections are prevented by the application of fungicides. Fenhexamid, a hydroxanilide derivative, is one of the newly developed fungicides that exhibit strong inhibitory activity against B. cinerea, owing to its ability to inhibit 3-ketoreductase in the C4-demethylation enzyme complex during ergosterol biosynthesis (Rosslenbroich et al. 2000; Debieu et al. 2001). Fenhexamid is used in many European countries and is considered to be an effective fungicide against B. cinerea due to its unique mode of action (Rosslenbroich et al. 2000; Debieu et al. 2001; Baroffio et al. 2003). However, B. cinerea wild types exhibiting resistance to fenhexamid were detected shortly after its introduction (Leroux et al. 2002; Baroffio et al. 2003). In Japan, although fenhexamid was registered in 2000, it is not widely used and thus, the existence of fenhexamid resistance in B. cinerea has not been reported in Japan. Molecular-based techniques have been developed to rapidly detect the sensitivity of fungi to several fungicides by taking advantage of point mutations in target genes (Oshima et al. 2002; Paplomatas et al. 2004; Banno et al. 2008, Furuya et al. 2009; Saito et al. 2009). A rapid method for the detection of fungicide resistance is necessary for viticulturists to better understand the incidence of resistance in B. cinerea populations in their vineyards. However, it is unknown whether molecular-based techniques for fungicide resistance detection could be applied to the study of fenhexamid resistance in B. cinerea. In fenhexamid-resistant field isolates of B. cinerea, Fillinger et al. (2008) found various point mutations leading to amino acid substitutions between 195 and 412 in the target protein, 3-ketoreductase, and revealed a relationship between high resistance to fenhexamid and the amino acid substitution at codon 412 in the protein. In the present study, in order to evaluate whether the detection of fenhexamid resistance with molecular-based techniques is possible and whether there are other point mutations in the target gene that are related to the resistance to fenhexamid, we generated 18 mutants showing resistance to fenhexamid by chemical mutagenesis and determined putative point mutations in the erg27 gene that conferred resistance to fenhexamid.