Phenotype instability in Botrytis cinerea in the absence of benzimidazole and dicarboximide fungicides

Stability of phenotypes of isolates of Botrytis cinerea that were sensitive or resistant to benzimidazole and dicarboximide fungicides was examined in the absence of fungicides in laboratory and growth room experiments. Twelve greenhouse isolates of B. cinerea were subcultured on potato dextrose agar (PDA) for 20 generations and on geranium seedlings for 15 generations. Three isolates of each of the following four phenotypes were used: sensitive to the fungicides thiophanate-methyl (a benzimidazole) and vinclozolin (a dicarboximide) ((SSV)-S-T), resistant to both fungicides ((RRV)-R-T), resistant to thiophanate-methyl and sensitive to vinclozolin ((RSV)-S-T), and sensitive to thiophanate-methyl and resistant to vinclozolin ((SRV)-R-T). In three trials on PDA, 36 populations were subcultured; 8 populations changed phenotypes by the end of 20 generations, as determined by conidium germination on fungicide-amended medium. Five of the eight initially were (SRV)-R-T; the resulting phenotypes were (SSV)-S-T, (RSV)-S-T, and (RRV)-R-T. Populations from eight other isolates exhibited temporary changes in phenotype during intermediate generations on PDA but reverted to initial phenotypes by the twentieth generation; five of these populations changed to phenotype (RRV)-R-T. In two geranium seedling trials, each of the 12 greenhouse isolates was inoculated onto a set of three seedlings for each generation, and diseased tissue that developed was used to initiate the next generation. Therefore, a total of 72 populations of B. cinerea were subcultured in the two trials; 5 of these populations changed phenotype at the end of 15 generations. Three of the five initially were (SRV)-R-T; these changed to phenotypes (SSV)-S-T or (RRV)-R-T. In each of the two trials on geranium seedlings, a population subcultured from one (SSV)-S-T isolate changed phenotype one to phenotype (RRV)-R-T and one to phenotype (RSV)-S-T. In all trials, no population resistant to thiophanate-methyl changed to a thiophanate-methyl-sensitive phenotype, and no population changed to phenotype (SRV)-R-T. Random amplified polymorphic DNA (RAPD) fingerprints were generated with the 12 initial isolates and 49 isolates subcultured on PDA or geranium seedlings. Cluster analyses of RAPD markers showed that subcultured isolates exhibiting the same phenotype clustered together and that subcultured isolates derived from a common greenhouse isolate but with different phenotypes were in different clusters. Some populations that did not change phenotype exhibited considerable differences in RAPD marker patterns. The results of this study indicate that, in the absence of fungicides, sensitive populations of B. cinerea can develop resistance to thiophanate-methyl and vinclozolin, and this resistance can be maintained in populations through multiple generations. Populations resistant only to vinclozolin ((SRV)-R-T) exhibited a high frequency of phenotype change, and populations resistant to both fungicides ((RRV)-R-T) were stable.