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

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.

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.

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.