Microfluidic-Directed Self-Assembly of dsRNA-Fungicide Nanocomplexes toward Synergistically Enhanced Disease Control

Abstract Botrytis cinerea (B. cinerea) causes severe agricultural losses. RNA interference (RNAi) offers precise control but is limited by dsRNA instability and inefficient delivery. Here, dsRNA targeting an ergosterol biosynthesis gene (dsERG) and berberine (BBR) self-assembled into carrier-free nanocomplexes (NCsdsERG-BBR) via microfluidics. The nanocomplexes showed efficient foliar and spore uptake and strongly inhibited spore germination and hyphal growth. Exogenous ergosterol fully rescued the dsERG-induced phenotype but only partially rescued NCsdsERG-BBR, confirming ergosterol biosynthesis inhibition by dsERG and an additional antifungal contribution from BBR. In a grape protective assay, the Bliss-predicted additive inhibition was calculated to be 80.0%, whereas NCsdsERG-BBR achieved 96.9% inhibition, demonstrating a synergistic effect under this condition. These findings indicate that the enhanced antifungal performance of NCsdsERG-BBR arises from improved delivery, BBR activity, and synergy, providing a scalable, solvent-free platform for sustainable crop protection.