Molecular Mechanisms and Biological Characteristics of Botrytis cinerea Field Isolate Resistance to Pyrisoxazole in Liaoning Province

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. One hundred fifty-seven isolates of B. cinerea were collected from tomato greenhouses in fourteen cities of Liaoning Province from 2016 to 2021 and examined for sensitivity to pyrisoxazole, with a mean EC50 of 0.151 µg/mL. Three highly resistant isolates, XD-5, DG-4 and GQ-3, were screened, and the EC50 values were 0.734, 0.606 and 0.639 µg/mL with corresponding resistance factors of 12.88, 10.63 and 11.21, respectively. Compared to field-sensitive strains, the highly resistant isolate XD-5 exhibited fitness defects in traits including mycelial growth, conidial production, and pathogenicity, but the DG-4 and GQ-3 did not experience fitness costs. Positive cross-resistance was observed only between pyrisoxazole and the DMIs tebuconazole and prochloraz but not between pyrisoxazole and the none-DMIs iprodione, procymidone, pyrimethanil, fludioxonil, fluazinam and fluopyram. Sequence alignment of the CYP51 gene indicated that three point mutations were observed in the highly resistant mutant, namely, V24I in XD-5, G461S in GQ-3 and R464K in DG-4. When exposed to pyrisoxazole, the induced expression levels of the ABC transporter AtrD and MFS transporter Mfs1 increased in the resistant isolates compared to the sensitive isolates, while the expression level of the CYP51 gene did not change significantly. Molecular docking suggested that the G461S and R464K mutations both led to a decrease in the binding energy between CYP51 and pyrisoxazole, while no change was found with the V24I mutation. Thus, two point mutations in the CYP51 protein combined with induced expression of the Mfs1 and AtrD genes appeared to mediate the pyrisoxazole resistance of the highly resistant mutants DG-4 and GQ-3, while the overexpression of the Mfs1 and AtrD genes was responsible for the highly resistant mutant XD-5.