G462S substitution of AaCYP51 confers moderate resistance to tebuconazole in Alternaria alternata

Tobacco brown spot (TBS) caused by Alternaria alternata is one of the most common diseases of tobacco in China, resulting in large loss in yield and quality. Demethylation inhibitors (DMIs) such as tebuconazole are commonly used pesticides to control TBS. However, their control effect has shown a downward trend in recent years. In this study, the occurrence and molecular mechanism of resistance to tebuconazole in Alternaria alternata were analyzed. The resistance of 63 strains of Alternaria alternata to tebuconazole was investigated with the concentration of 5 and 20 μg/mL as the identification standard, and the resistance frequency was as high as 93.65%. It was found that the target mutation from G to S at the 462nd amino acid position of CYP51 was the cause of moderate resistance to tebuconazole in A. alternata. Molecular docking analysis further confirmed that the G462S mutation of AaCYP51 decreased the binding affinity of tebuconazole to CYP51. The artificial AaCYP51-G462S transformants based on wild-sensitive GZA-24 showed resistance to tebuconazole and cross-resistance to metconazole and prothioconazole. In the present investigation, the virulence of the CYP51-G462S mutant was reduced, while mycelial growth, sporulation, and conidial germination did not change in comparison with the progenitor strain GZA-24. In addition, the mutants containing the G462S mutation in AaCYP51 exhibited decreased sensitivity to high osmotic stress stimulated by 1 M NaCl, and the capacity to respond to cell wall- and cytomembrane-damaging agents did not change in the mutants. The G462S substitution of CYP51 is the main factor for the moderate resistance to tebuconazole in A. alternata and mechanisms other than CYP51-target mutation might be involved in tebuconazole lowly resistant isolates. © 2025 Society of Chemical Industry.