Biocontrol of Root‐Knot Nematodes via <scp>siRNA</scp> ‐Loaded Extracellular Vesicles From a Nematophagous Fungus <i>Arthrobotrys oligospora</i>

Root-knot nematodes (Meloidogyne spp.) represent a major threat to global crop production, and current chemical nematicides pose serious environmental and health risks. RNA interference (RNAi) offers a promising gene-specific strategy for nematode control. However, the efficient and sustainable delivery of RNA molecules into nematodes remains a significant challenge. In this study, we developed an innovative RNA delivery platform using extracellular vesicles (EVs) derived from the nematode-trapping fungus Arthrobotrys oligospora. EVs were either exogenously loaded with synthetic siRNAs targeting the Mi-flp-18 gene of M. incognita or harvested from engineered fungal strains expressing short hairpin RNAs (shRNAs) or double-stranded RNAs (dsRNAs) against multiple nematode neuropeptide genes (flp and nlp families). The engineered EVs efficiently delivered RNA cargos into nematodes, leading to significant downregulation of target gene expression. Functional assays and greenhouse experiments revealed the biocontrol potential of the engineered fungal strains, with reductions in nematode motility, root invasion and infectivity. This is the first demonstration in a nematophagous fungus that EVs can serve as effective RNA delivery vehicles for the control of root-knot nematodes. The use of engineered A. oligospora strains provides a scalable, eco-friendly alternative to synthetic delivery systems and transgenic crops. Our findings establish fungal EVs as a powerful tool in cross-kingdom RNAi applications and open new avenues for sustainable pest management in agriculture.