Genetic Analysis of Isolates of Botrytis cinerea Sensitive and Resistant to Benzimidazole and Dicarboximide Fungicides

Abstract

A total of 56 isolates of B. cinerea collected from ornamental crops from commercial greenhouses were examined by random amplified polymorphic DNA (RAPD) fingerprint analyses. Isolates were examined as two independent sets of 35 and 36 isolates, with 15 isolates common to both sets. The isolates had four phenotypes: 17 were sensitive to two commonly used fungicides, thiophanate-methyl (a benzimidazole) and vinclozolin (a dicarboximide) (STSV); 18 were resistant to both fungicides (RTRV); 16 were resistant to thiophanate-methyl but sensitive to vinclozolin (RTSV); and 5 were sensitive to thiophanate-methyl but resistant to vinclozolin (STRV). Relationships among the isolates were determined by cluster analyses of mean character differences using the unweighted pair group method using arithmetic average and cladograms were constructed. Isolates were clustered primarily by fungicide-sensitivity phenotype. In one set of greenhouse isolates, 6 of 10 STSV isolates clustered together with a bootstrap confidence value of 91%. In the other fingerprint set of greenhouse isolates, 9 of 11 STSV isolates clustered together and had a bootstrap confidence value of 98%. Isolates resistant to thiophanate-methyl, vinclozolin, or both fungicides usually were not clustered with other isolates or were clustered with isolates of the same phenotype. To further elucidate these relationships, variant isolates resistant to one or both fungicides were produced on fungicide-amended agar medium from 14 STSV greenhouse isolates. These 14 STSV parent isolates, 57 resistant variant isolates, and 11 resistant greenhouse isolates were analyzed as three independent RAPD fingerprint sets. Variants selected from eight STSV parent isolates were resistant to both thiophanate-methyl and vinclozolin even though parent isolates were exposed to only one of the fungicides. Isolates resistant only to vinclozolin (STRV) had fingerprint patterns similar to and clustered with those of parent isolates, while fingerprint patterns of isolates resistant to thiophanate-methyl (i.e., RTRV or RTSV), regardless of sensitivity to vinclozolin, clustered differently from both those of STSV parent isolates and those of STRV isolates derived from the same parent. RTRV and RTSV variant isolates derived from the same fungicide-sensitive parents only clustered with other variants having the same phenotype.