A Trichoderma milRNA cross-species targeting chitin synthases genes is delivered by carbon nanomaterials to suppress Botrytis cinerea.

Botrytis cinerea is a globally destructive plant pathogen that threatens major agricultural crops. Although biocontrol agents are widely used against this fungus, their inconsistent efficacy reflects a limited mechanistic understanding, hindering the development of effective disease management strategies. In this study, we investigated the in-depth biocontrol mechanism of Trichoderma breve against B. cinerea on tomato plants and various fruits. We identified a new microRNA Tri-milR29 from T. breve. Tri-milR29 is delivered into B. cinerea via extracellular vesicles, enabling cross-species RNA interference (RNAi). Once inside B. cinerea, Tri-milR29 targets the chitin synthase genes BcCHS7 and BcCHS6, thereby impairing fungal growth and reducing its pathogenicity. This cross-species miRNA phenomenon significantly enhanced the antifungal and disease-suppressing efficacy of T. breve against B. cinerea. Moreover, to optimize the antifungal and disease-control effects of Tri-milR29, we engineered polyethyleneimine-modified carbon dot nanocomposites (CPP) as nanocarriers using coconut water as a raw material. This green, biomass-based nanocarrier system improved the delivery and uptake of Tri-milR29 in B. cinerea. Pot experiments indicated that CPP-loaded Tri-milR29 improved disease control efficacy by 37.5%. A six-month field trial further confirmed that its field control rate reached 78.71%, which was close to the commercial fungicide procymidone (82.84%). Understanding the targets and mechanism of Tri-milR29, along with its enhanced performance via CPP facilitation, could support the broader application of dsRNA-CPP in other plant-pathogen systems, leading to reduced chemical inputs and encouraging safer, more environmentally-friendly alternatives.