Field assay of LDH-formulated dsRNA specific to vATPase against the pine wood nematode, Bursaphelenchus xylophilus, via trunk injection.

The pine wood nematode, Bursaphelenchus xylophilus, causes severe damage to pine trees. Current chemical control methods have raised environmental concerns and the resistance issue of the nematode. Due to its high specificity, RNA interference (RNAi) has emerged as a promising approach for controlling pests, including plant-parasitic nematodes, through the application of double-stranded RNA (dsRNA). This study has developed an effective dsRNA-based nematicide for controlling B. xylophilus under field conditions via trunk injection. First, the core RNAi machinery of B. xylophilus was identified and confirmed to be inducibly expressed in response to dsRNA treatment. Using a fluorescently labeled dsRNA probe, the translocation of dsRNA from the mouth to internal tissues was observed following a diet-soaking treatment. Second, a 12-well plate assay using Botrytis cinerea cultured on potato dextrose agar was employed to screen candidate genes, leading to the selection of vATPase subunit B (Bx-vATPaseB) as the optimal RNAi target. Third, the presence of dsRNase activity reduced the nematicidal efficacy of the dsRNA. To overcome dsRNA degradation and enhance delivery within pine trees, dsRNA was formulated with layered double hydroxide (LDH). A semi-field assay using pine branches inoculated with B. xylophilus demonstrated that LDH-dsRNA significantly enhanced nematicidal activity compared to naked dsRNA. Finally, field trials via trunk injection of LDH-dsRNA into infested pine trees showed significantly higher control efficacy (> 84%) against B. xylophilus than that (64% at maximum) of abamectin treatment at the same dose (1.5 mg per tree), suggesting dsRNA as a practical control technique in field conditions.