AaCyt b Point Mutation and Overexpression of the Alternative Oxidase (AOX) Gene Conferred Moderate to High Level Resistance to Azoxystrobin in Alternaria alternata, the Causal Agent of Ginseng Leaf and Stem Blight Disease

Ginseng Alternaria leaf and stem blight (GALSB), mainly caused by Alternaria alternata (Aa), is a devastating aboveground disease of ginseng. Azoxystrobin (azo), a preferred fungicide for GALSB control, faces emerging resistance issues due to A. alternata adaptation. This study elucidated the resistance mechanisms of A. alternata to azoxystrobin through integrated approaches, including targeted cytochrome b (Cyt b) sequencing, molecular docking, transcriptomics, RT-qPCR, inhibitor (salicylhydroxamic acid, SHAM) addition, and validation of gene function assays. The results revealed that azo-resistant strains harbored a G143A mutation in the AaCyt b gene, reducing the binding energy between Cyt b protein and azoxystrobin from -8.31 to -7.08 kcal/mol and weakening their interaction. Transcriptomic analysis of resistant (TYC8-1 and TYC8-2) and wild-type (YC8) strains identified significant upregulation of the alternative oxidase (AOX) gene (involved in tyrosine metabolism) and key energy metabolism genes in the azo-resistant strains. RT-qPCR confirmed a 4.45–6.91-fold AOX overexpression in TYC8-2 versus YC8. AOX inhibition with SHAM increased sensitivity of A. alternata strains to azoxystrobin by 11.66-fold. AOX deletion enhanced azoxystrobin sensitivity by 2.7×104-fold, concurrently increasing sensitivity to NaCl, H2O2, and NaAc, and attenuating ginseng leaf pathogenicity, indicating AOX not only confers azo-resistance but also modulates stress tolerance and virulence. Collectively, the AaCyt b G143A point mutation and AOX upregulation are primary mechanisms driving A. alternata resistance to azoxystrobin. The findings provide critical insights for Aa-resistance monitoring, management, and precision control of GALSB.