Targeting V‐ATPase subunits via RNA interference disrupts ultrastructural and normal life activities in Bursaphelenchus xylophilus: a novel strategy for sustainable pine wilt disease management

Pine wood nematode (Bursaphelenchus xylophilus, PWN), a devastating pine parasite, induces widespread mortality in host trees. Chemical pesticides have been conventionally used for PWN control; however, their prolonged use drives the evolution of pesticide resistance in PWN and poses environmental risks. RNA interference (RNAi)-based biopesticides represent a promising alternative, offering species-specific targeting, high efficacy and a reduced ecological footprint. To engineer RNAi biopesticides against PWN, we systematically investigated eight V1-domain subunits (A-H) of the V-type proton ATPase (V-ATPase) family using integrated transcriptomic profiling and bioinformatic analyses. Eight corresponding double stranded (ds) RNAs were designed, synthesized, and functionally evaluated. RNAi targeting these subunits significantly impaired PWN viability, motility, feeding efficiency, and fecundity. Notably, dsV-ATPase-H exhibited the most pronounced interference efficacy: 72 h exposure to 700 ng μL-1 dsRNA induced 80.12% corrected mortality in J4-stage nematodes. This treatment reduced egg production from 7 to 1 egg per nematode and population density from 8136 to 705 individuals. Transmission electron microscopy further revealed that RNAi disrupted nematode ultrastructure, manifesting as altered cuticle thickness and reduced intestinal microvilli density, thereby compromising structural and functional integrity. Our study demonstrates the potential use of V-ATPase subunits (A-H) as molecular targets for RNAi-based PWN control. This study is the first to reveal, at the ultrastructural level, the regulatory effects of RNAi targeting V-ATPase (A-H) subunits in PWN on their critical life activities. It proposes a sustainable RNAi-based strategy for controlling pine wilt disease. © 2025 Society of Chemical Industry.