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 resistance status after long-term use in the field is unclear. The baseline sensitivity to difenoconazole of 142 B. cinerea isolates from China with no history of DMI usage was characterized, with a mean effective concentration for 50% mycelial growth inhibition (EC₅₀) of 0.97 ± 0.50 μg/ml. EC₅₀ values for difenoconazole sensitivity 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 nonnormal distribution curve. Detached fruit studies revealed that isolates with EC₅₀ values of approximately 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 isolate and one triple resistant isolate 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.

Flumorph is a carboxylic acid amide (CAA) fungicide with high activity against oomycetes. However, evolution to CAAs from low resistance to high resistance has never been reported. This study investigated the basis of resistance evolution of flumorph in Phytophthora sojae. Total of 120 P. sojae isolates were collected and their sensitivity to flumorph was evaluated. Although no spontaneous resistance was found among the field isolates, adaptation on flumorph‐amended media resulted in the selection of five stable mutant types exhibiting varying degrees of resistance to CAAs. Type I, which exhibited the lowest resistance level, was obtained when the wild‐type isolate was exposed to a low concentration of flumorph, but no resistant mutants were obtained by direct exposure to higher concentrations. However, the more resistant types (Type II, III, IV and V) were obtained when Type I were exposed to higher concentrations of flumorph. Similar results were obtained when the entire screening process was repeated, which implied that evolution of resistance to flumorph in P. sojae could be a two‐step process, where high resistance phenotypes could develop gradually from low resistance ones. Further investigation into molecular mechanism strongly confirmed that evolution of isolates highly resistant to flumorph occurs in a stepwise process with Type I as intermediary, through accumulation of mutations in their target protein of CAAs (CesA3). Together, our findings indicate that application of low rates of flumorph in field could result in selection of low resistance Type I isolates, but that raising dosage to maintain comparable levels of control could elicit rapid evolution of more resistant Type II, III, IV and V isolates with stepwise accumulation of mutations in CesA3, which would render flumorph ineffective as a control method. Precautionary resistance management strategy should be implemented. The phenomenon described in the study could have broader biological significance.