Engineered Pine Endophytic Fungus Expressing Double-Stranded RNA Targeting Lethal Genes to Control the Plant-Parasitic Nematode Bursaphelenchus xylophilus

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most serious invasive forest pests, responsible for pine wilt disease. Currently, there are no effective, environmentally friendly control methods available. RNA interference technology has been extensively utilized to screen functional genes in eukaryotes and to explore sustainable pest management approaches through genetic engineering. In this study, we identified 353 predicted lethal genes in PWN by comparing its genome with those of lethal genes from Caenorhabditis elegans. We selected five predicted lethal genes ( Bxy1177, Bxy1239, Bxy1104, Bxy667, and BxyAK1) with identification values exceeding 60% to evaluate their nematicidal effects on PWN. We tested the double-stranded RNA (dsRNA) of these genes using two methods: first, soaking in a synthesized dsRNA solution in vitro, and second, feeding on a dsRNA-engineered endophytic fungus, Fusarium babinda. Following dsRNA ingestion, either through soaking or fungal feeding, the expression of genes Bxy1177, Bxy667, Bxy1104, and BxyAK1 was significantly suppressed. Notably, nematode populations that consumed fungi expressing dsL1177 and dsAK1 showed substantial declines over time. These findings provide novel insights and a practical foundation for employing endophytic fungi-expressed dsRNA in sustainable pest management strategies.