Three point mutations in AaCYP51 combined with induced overexpression of AaCYP51 conferred low-level resistance to mefentrifluconazole in Alternaria alternata.

Tomato early blight is a significant disease that causes substantial losses to tomato yield and quality. Mefentrifluconazole, an isopropanol-azole subgroup of triazole fungicides, has been registered in China for controlling various plant diseases, including tomato early blight, grape anthracnose, and apple brown spot. However, limited information is available on the mefentrifluconazole resistance risk and mechanism in plant pathogens. The sensitivity to mefentrifluconazole of 122 isolates of Alternaria alternata, one of the causal agents of tomato early blight, collected from different provinces in China, was evaluated. The results showed a unimodal curve for the sensitivity frequency, with an average EC of 0.306 μg/mL. Through fungicide adaption, six resistant mutants (N4, N5, T4, T5, NG1, and NG10) were obtained from three parental isolates, with a mutation frequency of 3.28 × 10 and resistance factors ranging between 19 and 147. The survival fitness of the resistant mutants, except for NG1, was significantly lower than that of their parental isolates. Positive cross-resistance was observed between mefentrifluconazole and difenoconazole or fenbuconazole, whereas no cross-resistance was found with three non-DMI fungicides. Furthermore, three distinct point mutations were detected in the AaCYP51 protein of the resistant mutants: I300S in T4 and T5; A303T in N4, NG1, and NG10; and A303V in N5. Compared to the parental isolates, the AaCYP51 gene was overexpressed in all six resistant mutants when treated with mefentrifluconazole. In summary, the resistance risk of A. alternata to mefentrifluconazole was low, and point mutations and overexpression of the AaCYP51 gene were identified as contributing factors to mefentrifluconazole resistance in A. alternata.