Difenoconazole Resistance Shift in Botrytis cinerea from Tomato in China Associated with Inducible Expression of CYP51.

Gray mold caused by Botrytis cinerea is one of the most important diseases in tomato. It can be controlled effectively by demethylation inhibitor (DMI) fungicides, but their resistant status is unclear after long-term use in the field. Baseline sensitivity to difenoconazole of 142 B. cinerea isolates from China with no history of DMI usage was characterized, with a mean EC50 of 0.97 ± 0.50 μg/mL. EC50 values to difenoconazole of another 248 isolates collected in 2011 and 2016 ranged from 0.04 to 11.99 μg/mL, and the frequency of difenoconazole sensitivity formed a non-normal distribution curve. Detached fruit studies revealed that isolates with EC50 values of ~6.00 μg/ml were not controlled effectively. The mean EC50 of the resistant isolates changed from 6.74 to 8.65 μg/mL between 2011 and 2016. Positive cross-resistance was only observed between difenoconazole and two DMIs. One dual resistant and one triple resistant isolates were found among the difenoconazole-resistant isolates collected in 2016, associated with point mutations in corresponding target proteins of the fungicides azoxystrobin and fludioxonil. This indicated that B. cinerea not only showed higher difenoconazole resistance levels but gradually changed from single to multiple fungicide resistance over time. No amino acid variation was found in the CYP51 protein. In the absence of difenoconazole, the relative expression of CYP51 was not significantly different in sensitive and resistant isolates. Induced expression of CYP51 is an important determinant of DMI resistance in B. cinerea from tomato. However, nucleotide variants found in the upstream region had no association with the fungicide resistance phenotype. These results will be helpful for the management of B. cinerea in the field.