Axenic Bursaphelenchus xylophilus retains pathogenicity across a multi‐tiered host system: evidence from callus, axenic seedlings, and potted pine trees

BACKGROUND: Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is a devastating threat to coniferous forests worldwide. Whether B. xylophilus requires associated bacteria for pathogenicity remains debated, with significant implications for defining primary targets of disease management. RESULTS: Although autonomous pathogenicity of axenic B. xylophilus has been reported previously, the principal advance of this study lies in its systematic validation across a multi-tiered host system, encompassing Pinus massoniana calli, 2-year-old axenic seedlings, and potted Pinus thunbergii seedlings up to 8 years old. Axenic B. xylophilus consistently induced typical wilt symptoms and completed its life cycle within host tissues. On pine calli, axenic nematodes caused extensive necrosis and loss of cell viability. In 2-year-old sterile seedlings, inoculation led to 90-100% wilting and mortality, with nematode reproduction confirmed in the absence of bacterial associates. Similar results were observed in 2- to 8-year-old potted seedlings under greenhouse conditions, with no significant difference in disease severity between axenic and non-axenic inoculations. Axenic nematodes also exhibited enhanced fitness in vitro, with significantly extended longevity, suggesting that the native bacterial community may impose a metabolic burden under sterile conditions. CONCLUSION: These findings bring clarity to a long-standing debate by demonstrating that B. xylophilus is inherently capable of autonomous pathogenicity, and that associated bacteria are not a prerequisite for disease initiation. This establishes an evidence-based management framework: prioritizing phytosanitary measures, vector disruption, and nematode-targeted interventions as frontline defenses, while positioning microbiome-based strategies as secondary modulators of disease progression in natural settings. © 2026 Society of Chemical Industry.