Mycophagy‐driven virulence attenuation in serially subcultured pine wood nematode Bursaphelenchus xylophilus

BACKGROUND: The pine wood nematode Bursaphelenchus xylophilus, an invasive species, drives pine wilt disease epidemics in coniferous ecosystems. During long-term in vitro subculture, the virulence of this nematode strongly attenuated. In this study, we investigated how sustained mycophagous subculture drives adaptive shifts that reduce B. xylophilus virulence. RESULTS: In pathogenicity assays, the mortality of Pinus thunbergii seedlings reduced by 70% with 180-day subcultured nematodes compared to non-subcultured in vivo populations. Adult body size (25-49% decrease in length; female: from 1207 to 907 μm; male: from 1348 to 688 μm) and thrashing frequency (by 62.8%) decreased, but female-biased sex ratio increased (from 0.94:1 to 2.08:1). Integrated omics analyses identified 702 differentially expressed genes, with subcultured nematodes exhibiting up-regulation of spliceosome/ribosome biogenesis pathways and down-regulation of phytophagy-associated systems (e.g., G protein-coupled receptor activity and xenobiotic metabolism). Notably, 50 differentially expressed microRNAs target developmental and signal transduction genes, suggesting post-transcriptional regulation of adaptive traits. Metabolomics revealed consumption of defense-linked tyrosine derivatives in vivo and accumulation of growth-promoting metabolites in vitro. CONCLUSION: Mycophagy-driven adaptation attenuates phytophagous traits essential for host invasion, such as chemoreception and detoxification machinery, thereby offering promising targets for RNA interference to manage pine wilt disease. © 2026 Society of Chemical Industry.