Botrytis cinerea is the second most economically important fungal phytopathogen causing gray mold disease. Multiple fungicide-resistant B. cinerea strains have also been reported, especially in strawberries, raspberries, grapes, and tomatoes. RNA interference (RNAi) is a post-transcriptional gene silencing mechanism in all known eukaryotes. The exogenous application of dsRNAs to knock down the target organism's essential genes is called spray-induced gene silencing (SIGS). This non-transgenic SIGS-based approach has emerged as an appealing alternative biofungicide. Despite the great potential of sprayable RNAi-based pesticides, this innovative technology encountered challenges. The low stability and the limited uptake efficiency of dsRNA are significant challenges facing SIGS. Nanomaterials-based delivery systems and structured modification of dsRNA molecules could be innovative SIGS approaches for improving its stability, uptake efficiency, and biofungicidal efficacy. This study aims to develop sprayable RNAi (SIGS) solutions by modifying dsRNA structure and using chitosan-based nanoparticles to control fungicide-resistant B. cinerea. Chitosan nanoparticles (CNPs) were generated using ionic gelation, and different forms of double-stranded RNA (dsRNA), either linear or secondary-structured, were loaded into them. The positive charges from the amine groups present in chitosan facilitated the self-assembly of the CNPs-dsRNA complex through electrostatic attraction. The stability of CNPs-dsRNAs complexes was evaluated ex-vivo by incubating naked-dsRNAs and complex-dsRNAs with the RNase A. Gel retardation assay revealed that CNPs-dsRNA complex of either linear or secondary structured-dsRNAs exhibited substantial protection of dsRNA from RNase A degradation for up to 72 hours, suggesting its potential for improving stability and long-lasting efficacy. The CNPs-dsRNAs significantly reduced the mycelial growth of wild-type and fungicide-resistant B. cinerea isolates. The results from this study indicated that chitosan-based polymer could be an effective delivery technology for both linear and secondary-structured dsRNA and hold great promise for the management of gray mold diseases.
