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. Twelv e greenhouse isolates of B. cinerea were subcultured on potato dextrose aga r (PDA) for 20 generations and on geranium seedlings for 15 generations. Th ree isolates of each of the following four phenotypes were used: sensitive to the fungicides thiophanate-methyl (a benzimidazole) and vinclozolin (a d icarboximide) ((SSV)-S-T), resistant to both fungicides ((RRV)-R-T), resist ant to thiophanate-methyl and sensitive to vinclozolin ((RSV)-S-T), and sen sitive to thiophanate-methyl and resistant to vinclozolin ((SRV)-R-T). In t hree trials on PDA, 36 populations were subcultured; 8 populations changed phenotypes by the end of 20 generations, as determined by conidium germinat ion 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. Popula tions from eight other isolates exhibited temporary changes in phenotype du ring intermediate generations on PDA but reverted to initial phenotypes by the twentieth generation; five of these populations changed to phenotype (R RV)-R-T. In two geranium seedling trials, each of the 12 greenhouse isolate s was inoculated onto a set of three seedlings for each generation, and dis eased tissue that developed was used to initiate the next generation. There fore, a total of 72 populations of B. cinerea were subcultured in the two t rials; 5 of these populations changed phenotype at the end of 15 generation s. 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 popul ation resistant to thiophanate-methyl changed to a thiophanate-methyl-sensi tive phenotype, and no population changed to phenotype (SRV)-R-T. Random am plified polymorphic DNA (RAPD) fingerprints were generated with the 12 init ial isolates and 49 isolates subcultured on PDA or geranium seedlings. Clus ter analyses of RAPD markers showed that subcultured isolates exhibiting th e same phenotype clustered together and that subcultured isolates derived f rom a common greenhouse isolate but with different phenotypes were in diffe rent clusters. Some populations that did not change phenotype exhibited con siderable differences in RAPD marker patterns. The results of this study in dicate that, in the absence of fungicides, sensitive populations of B. cine rea can develop resistance to thiophanate-methyl and vinclozolin, and this resistance can be maintained in populations through multiple generations. P opulations resistant only to vinclozolin ((SRV)-R-T) exhibited a high frequ ency of phenotype change, and populations resistant to both fungicides ((RR V)-R-T) were stable.