Abstract: Plant gray mold disease, caused by the necrotrophic fungus Botrytis cinerea, threatens global food security by infecting over 1,400 species. The resistance of B. cinerea to fungicides poses a major challenge for its control. RNA interference (RNAi) shows promise for pathogen management, but high production costs, poor stability, and inefficient delivery of double-stranded RNA (dsRNA) restrict its practical use. In this study, we developed Yarrowia lipolytica strain MP181-2 as a novel RNAi platform, leveraging its ability to colonize the phyllosphere of crop plants (tomato and alfalfa) to enable simultaneous dsRNA production and pathogen targeting via microbe-induced gene silencing (MIGS). Through target screening, we identified three essential fungal genes, BcRpd3, BcNat1, and BcArd1, whose silencing significantly reduced the pathogenicity of B. cinerea. We further constructed a chimeric dsRNA molecule (BcANR-dsRNA) targeting these three genes, which demonstrated superior disease suppression compared to single-gene targeting. The engineered Y. lipolytica strains that produce dsRNA could effectively interfere with the expression of target genes in B. cinerea, and the strain expressing BcANR-dsRNA (designated Yl-dsANR) acquired the ability to suppress the growth of B. cinerea. In planta tests confirmed that Yl-dsANR successfully interfered with the expression of three target genes, leading to decreased fungal biomass and reduced lesion development on different hosts. Notably, foliar application of Yl-dsANR provided durable protection (over 70% efficacy for 5 d) in potted plants. Our integrated approach combines the advantages of microbial biocontrol with RNAi precision, establishing Y. lipolytica as a versatile chassis for sustainable crop protection. This study also provides an effective solution to current limitations in RNA pesticide development and offers a scalable, eco-friendly alternative to chemical fungicides for gray mold management.

Key words: Botrytis cinerea, chimeric dsRNA, microbe-induced gene silencing, plant disease control, RNA interfere, Yarrowia lipolytica