Targeting the SCP2 domain of Bx‐ DAF ‐22 to identify nematicidal compounds against Bursaphelenchus xylophilus

BACKGROUND The pine wood nematode (PWN), Bursaphelenchus xylophilus , causes pine wilt disease (PWD), a devastating forest disease lacking effective and environmentally sustainable control measures. Commercial nematicides face resistance risks and application limitations, demanding novel molecular targets with distinct mechanisms. The thiolase‐encoding gene daf‐22 participates in ascaroside pheromone biosynthesis, which constitutes a signaling pathway that modulates nematode development in B. xylophilus . RESULTS Here, we report that B. xylophilus Bx‐DAF‐22 uniquely contains a C‐terminal sterol carrier protein type‐2 (SCP2) domain. In silico molecular docking predicted that the four candidate compounds displayed binding poses and theoretical affinities comparable to those of cholesterol for the SCP2 domain. These small molecules are hypothesized to possess potential competitive binding activity. RNAi‐mediated gene knockdown of Bx‐daf‐22 significantly inhibited nematode growth, reduced neutral lipid accumulation, and increased total cholesterol content in mixed‐stage PWN, suggesting a role for this protein in both fatty acid metabolism and sterol homeostasis. Screening an insect SCP2 inhibitor library identified four compounds exhibiting nematicidal activity; at 50 μM for 96 h, all four reduced nematode survival to below 15%. Two compounds (C2 and C3) interacted with recombinant Bx‐DAF‐22 in 1,8‐ANS competitive displacement assays and significantly reduced cholesterol content in vivo . CONCLUSION These findings suggest that Bx‐DAF‐22 is a potential molecular target for nematicide discovery, although further rigorous validation is required, and identify two preliminary lead scaffolds from cross‐species testing of an insect‐derived compound library. Importantly, pharmacological evidence confirming on‐target engagement remains absent; substantial structural optimization and multi‐level biological validation are needed before any practical consideration against PWD.