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), caused by Alternaria alternata, poses a severe threat to ginseng cultivation. Although azoxystrobin is a cornerstone fungicide for GALSB management, the emergence of widespread adaptive resistance has severely curtailed its field efficacy. This study integrated molecular, transcriptomic, and genetic approaches to unravel the underlying resistance mechanisms. Targeted gene sequencing and molecular docking revealed that resistant strains harbor a conserved G143A point mutation in the AaCyt b protein. This mutation weakens the azoxystrobin–AaCyt b protein binding affinity by elevating the binding energy from −8.31 to −7.08 kcal/mol. Additionally, comparative transcriptomics and RT-qPCR demonstrated pronounced upregulation of the alternative oxidase gene (AaAOX) and core energy metabolism pathways in resistant strain TYC8-2, with AaAOX expression increasing 4.45–6.91-fold. Fungicidal inhibition of AOX via salicylhydroxamic acid (SHAM) restored fungal sensitivity, increasing azoxystrobin sensitivity by 11.66-fold. Crucially, genetic knockout of AaAOX enhanced sensitivity by approximately 2.7 × 104-fold. Phenotypic assays further established AaAOX as a multifunctional regulator; the AaAOX mutant exhibited attenuated virulence on ginseng leaves and increased sensitivity to oxidative and osmotic stresses (NaCl, H2O2, NaAc). The G143A mutation in AaCyt b and the transcriptional overexpression of AaAOX contribute independently to drive azoxystrobin resistance in A. alternata. These findings provide comprehensive mechanistic insights to guide resistance surveillance, rational fungicide application, and precision prevention of GALSB in ginseng cultivation. We conclude that the G143A mutation in AaCyt b and the transcriptional overexpression of AaAOX act independently to drive azoxystrobin resistance in A. alternata. These findings provide comprehensive mechanistic insights to guide resistance monitoring, optimize fungicide applications, and develop precision strategies for GALSB management.