Integrated multi-omics analysis revealed the response mechanism of Osmanthus fragrans leaves against the infection by Botryosphaeria dothidea

Osmanthus fragrans Lour. (Oleaceae) , a perennial woody species, faces severe threats from Botryosphaeria dothidea , which causes severe diseases in economically important woody plants, leading to substantial agricultural losses. However, the stage-specific molecular and metabolic mechanisms underlying osmanthus resistance to B. dothidea remain unclear. Here, we performed integrated transcriptomic and metabolomic analyses combined with Weighted Gene Co-expression Network Analysis (WGCNA) to systematically clarify the dynamic defense responses of the previously screened resistant genotype 'No. 35' and susceptible genotype 'No. 37' at early (0 days post inoculation, dpi), middle (3 dpi), and late (7 dpi) stages of B. dothidea infection. A total of 30,125 DEGs and 356 DAMs were identified, respectively. WGCNA revealed that stage-specific defense modules with hub genes from ERF5, RVE and WRKY transcription factor families were significantly correlated with disease resistance at early, middle and late infection stages respectively, forming a sequential multi-layered defense regulatory network. Metabolome profiling revealed the DAMs enriched in flavonoid biosynthesis with significantly upregulated 4CL and F3H genes and enriched luteolin/apigenin and phenylpropanoid synthesis metabolism at 7dpi in enhancing the tolerance of B. dothidea infection. Our research spotlights the genes 4CL, F3H , and accumulation of luteolin/apigenin, which showed a strong positive correlation. Its defense mechanism primarily functions by upregulating key genes in the flavonoid biosynthesis, which drives the accumulation of defensive metabolites, and ultimately leading to the enrichment of flavonoid precursors in leaves. These findings provide a theoretical basis for understanding the multi-omics synergistic mechanism of biotic stress resistance, molecular breeding, and biological control, offering theoretical support for sustainable disease management in Osmanthus cultivation.