Location-Specific Fungicide Resistance Profiles and Evidence for Stepwise Accumulation of Resistance in Botrytis cinerea.

The fungicide resistance profiles to seven chemical classes of fungicides were investigated in 198 Botrytis cinerea isolates from five blackberry fields and 214 B. cinerea isolates from 10 strawberry fields of North and South Carolina. Populations of B. cinerea tended to have a single dominant, location-specific resistance profile that consisted of resistance to multiple fungicides in fields sprayed weekly with site-specific fungicides. The most prevalent profile in blackberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, and boscalid. The most prevalent resistance profile found in conventional strawberry fields consisted of resistance to thiophanate-methyl, pyraclostrobin, boscalid, and cyprodinil. A statistical model revealed that multifungicide resistance patterns did not evolve randomly in populations from both crops. Instead, strains resistant to thiophanate-methyl were more likely to acquire resistance to pyraclostrobin, the resulting dual-resistant population was more likely to acquire resistance to boscalid, the resulting triple-resistant population was more likely to acquire resistance to cyprodinil, and the resulting quadruple-resistant population was more likely to acquire resistance to fenhexamid (strawberry population only) compared with random chance. Resistance to iprodione and fludioxonil evolved from a pool of strains with different fungicide resistance profiles. Resistance to thiophanate-methyl, pyraclostrobin, boscalid, and fenhexamid in blackberry isolates was, without exception, based on target gene mutations, including E198A and E198V in β-tubulin, G143A in cytochrome b, H272Y and H272R in SdhB, and F412I in Erg27, respectively. A new genotype associated with fenhexamid resistance was found in one strain (i.e., Y408H and deletion of P298). Fungicide-resistant strains were present but rare in an unsprayed blackberry field, where some unique phenotypes, including low and medium resistance to fludioxonil, had emerged in the absence of fungicide pressure. The isolates resistant to fludioxonil had effective dose that inhibited 50% of mycelial growth values of 0.16 μg/ml (low resistance) and 0.32 and 0.38 μg/ml (medium resistance) and were also resistant to the anilinopyrimidine fungicide cyprodinil, indicating that this and similar phenotypes will eventually be selected by continued applications of the fludioxonil + cyprodinil premixture Switch. This study shows that multifungicide-resistant phenotypes are common in conventionally maintained strawberry and blackberry fields and that resistance to multiple fungicides evolved from stepwise accumulation of single resistances.