Functional Characterization of Two Adjacent Oxygenase Genes, SsOxy1 and SsOxy2, Reveals Their Critical Roles in Sclerotial Development, Stress Tolerance, and Pathogenicity of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a notoriously destructive, broad-host-range necrotrophic fungus responsible for devastating crop losses worldwide. Although genes encoding oxygenases are ubiquitous in fungal genomes, and the encoded enzymes play instrumental roles in driving diverse biochemical cascades, their specific functional repertoire in S. sclerotiorum pathogenesis remains poorly defined. Here, we characterize a contiguous pair of early-infection-induced oxygenase genes: SsOxy1 (encoding a 2-oxoglutarate-dependent oxygenase) and SsOxy2 (encoding a non-heme iron-dependent oxygenase). Targeted gene disruption demonstrated distinct developmental roles: ΔSsOxy1 mutants exhibited compromised vegetative growth (colony diameter reduced by approximately 18% to 27% at 24 h) and significantly diminished sclerotial biomass (ranging from approximately 8% to 28% reduction), whereas the absence of SsOxy2 selectively uncoupled the spatial regulation of sclerotial biogenesis without restricting radial growth. Importantly, both mutants displayed a severe decline in virulence across multiple hosts, with lesion diameters reduced by approximately 30% to 67% in ΔSsOxy1 and 62% to 66% in ΔSsOxy2 (p < 0.01).This attenuation was fundamentally linked to their inability to elaborate mature infection cushions, compounded by hypersensitivity to exogenous oxidative (H2O2) and hyperosmotic (NaCl) stresses. Untargeted metabolomics uncovered a profound metabolic reprogramming shared by both mutants, characterized by severe depletion of purine metabolism intermediates, including hypoxanthine (4.6–4.9-fold), xanthine (6.1–8.1-fold), and guanine (2.5–3.7-fold), alongside a disrupted glutathione redox pool (elevated GSSG: 3.0-fold in ΔSsOxy1 and 5.7-fold in ΔSsOxy2). Genomic colocalization combined with these metabolomic shifts suggests that SsOxy1 and SsOxy2 may function in a coordinated manner within a putative secondary metabolite biosynthetic gene cluster, although direct evidence for co-transcription and a shared product is currently lacking. Our findings provide novel mechanistic insights into how these paired oxygenases govern redox homeostasis, energy allocation, and the critical morphological transitions required for successful host colonization in S. sclerotiorum.