Dual roles of the G462S mutation of AaCYP51 in regulating self‐overexpression and DMI resistance in Alternaria alternata

BACKGROUND Alternaria alternata is an important fungal pathogen responsible for leaf black spot and fruit rot disease in sweet cherry ( Prunus avium ), leading to significant preharvest and postharvest losses. Demethylation inhibitor (DMI) fungicides are widely used for its control. However, reduced sensitivity has been reported in field populations. RESULTS In this study, we analyzed 109 A. alternata isolates collected from cherry orchards across four regions in China during 2022 and 2023. The mean half‐maximal effective concentration (EC 50 ) value for sensitive isolates was 2.978 ± 0.861 and 12.375 ± 1.412 μg/mL for resistant isolates, respectively. We investigated the molecular basis of DMI resistance in A. alternata by examining both the promoter and coding regions of the AaCYP51 gene. A consistent G462S mutation was identified in resistant isolates, along with many point mutations in the promoter region. Expression analysis showed significantly higher AaCYP51 expression levels in resistant isolates. Functional validation through reciprocal transformation revealed that the G462S mutation alone was sufficient to confer resistance and also promoted AaCYP51 gene overexpression, whereas promoter mutations only elevated overexpression without altering resistance phenotype. CONCLUSION These findings demonstrate dual roles of the coding mutation in regulating resistance and self‐overexpression. This mechanistic understanding of DMI resistance provides molecular targets for resistance monitoring and management, particularly in cherry orchards where A. alternata poses a major threat to fruit quality and yield.