Occurrence of Fungicide Resistance in Botrytis cinerea from Greenhouse Tomato in Hubei Province, China

During the early summer of 2012 and 2013, samples of gray mold were collected from greenhouse tomato at eight locations in Hubei Province, and 221 isolates of Botrytis cinerea were obtained and evaluated for the sensitivity to fungicides carbendazim, diethofencarb, boscalid, fludioxonil, and cyprodinil. Results showed that isolates with resistance to carbendazim and cyprodinil were widespread, whereas isolates with resistance to carbendazim and diethofencarb were found at only two locations. No isolates with resistance to boscalid or fludioxonil were detected. Altogether, four resistant phenotypes were determined (i.e., Car(R)Die(S)Cyp(S), Car(R)Die(R)Cyp(S), Car(R)Die(S)Cyp(S), and Car(R)Die(R)Cyp(R)). Among them, Car(R)Die(S)Cyp(S) and Car(R)Die(S)Cyp(R) were widely distributed, and there was a dominant resistant phenotype at each location. Interestingly, isolates resistant only to cyprodinil were not obtained because the resistance to cyprodinil was always associated with the resistance to carbendazim, demonstrating that a phenotype of multiple fungicide resistance of B. cinerea was more likely to have evolved from previously resistant subpopulations. Stability of resistance to carbendazim, diethofencarb, and cyprodinil was assessed, and the resistance was stable. Fitness tests showed that, as a group, the carbendazim-resistant isolates were not significantly different from sensitive isolates. However, the mycelial growth and virulence of the carbendazim, diethofencarb, and cyprodinil triple-resistant group were significantly lower than the sensitive group, indicating that the triple resistant isolates suffered from the disability of colonizing the hosts. It should be noted that there was no significant difference for other fitness components (e.g., sporulation or osmotic sensitivity to NaCl), suggesting that the triple-resistant isolates were still competitive in these traits. To investigate the mechanisms of resistance to carbendazim and diethofencarb, partial beta-tubulin genes of 10 carbendazim-resistant isolates and 5 isolates resistant to carbendazim and diethofencarb were sequenced, and all 10 carbendazim-resistant isolates harbored the mutation E198V or E198A. For the 5 isolates resistant to carbendazim and diethofencarb, all of them possessed the mutation E198K, and no other mutations were detected. The location-specific resistance profiles found in this study are crucial in designing proper gray mold management strategies in these areas.