Nanovaccine for Plants from Organic Waste: D‑Limonene-Loaded Chitosan Nanocarriers Protect Plants against Botrytis cinerea

ABSTRACT: Chitosan and D-limonene are bioactive molecules derived from organic waste. We repurposed them in the form of a drug delivery system for agricultural applications in crop protection to combatBotrytis cinerea, a broad host-range necrotrophic (BHN) pathogen that causes economic losses worldwide. Synthetic fungicide application remains the most common method to control this pathogen, but this comes with a significant environmental cost. Drug delivery systems from naturally occurring biomaterials can offer efficient treatment options to combat this pathogen. We hypothesized that engineered chitosan nanoparticles (CSNPs) loaded with D-limonene can trigger an ondemand systemic defense response in plants that enhances its quantitative disease resistance against BHN. Chitosan nanocarriers encapsulated with D-limonene as cargo were fabricated through a rational formulation via ionic gelation and the spontaneous emulsification method, respectively, to produce chitosan D-limonene nanoparticles (CdlNPs). The therapeutic effect of the Dlimonene nanoemulsion, CSNPs, and CdlNPs on the plants’ defense response against necrotrophic fungal pathogens was evaluated by monitoring the dynamic morpho-physiological changes via multispectral image-based phenotyping in an Arabidopsis thaliana (Col-0)−B. cinerea model system. Functional analysis of the differentially expressed genes revealed that at the concentration of significant disease resistance (0.5%), CSNPs downregulated the biological process involved in plant growth and development, but upregulated the main process controlling response to stress..