Mitochondrial AOX Activation Drives Pyribencarb Resistance in <i>Sclerotinia sclerotiorum</i>: Cellular Trade-Offs and Nontarget Site Mechanisms

In this study, pyribencarb-resistant mutants were generated through fungicide mutagenesis, and the parental isolate SBH1 and three representative mutants (RH1-1, RH1-2, and RH1-3) were selected for comparative analysis. All mutants displayed reduced mycelial growth, impaired sclerotia formation, decreased respiration, and mitochondrial dysfunction, with elevated cell membrane permeability. No differences were detected in sclerotia weight, oxalate levels, or pathogenicity relative to the parental isolate. RH1-3 exhibited distinct morphological abnormalities, including distorted mycelia, damaged organelles, and enlarged mitochondria. Pyribencarb sensitivity showed a strong positive correlation with azoxystrobin but a negative correlation with dimetachlone. Sequence analysis revealed no mutations in the QoI-targeted cytochrome <i>b</i> (<i>Sscytb</i>) and alternative oxidase (<i>Ssaox</i>) genes. Quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed significant upregulation of <i>Ssaox</i> expression in mutants relative to SBH1, with unchanged <i>Sscytb</i> levels, as confirmed by protein expression analyses. Molecular docking identified a strong binding affinity of pyribencarb to SsAox domains. These results indicate that <i>Ssaox</i> overexpression confers pyribencarb resistance in <i>Sclerotinia sclerotiorum</i>.