Abstract
In this research, a new ecofriendly and sustainable fungicide agent, with the ability to control Verticillium wilt, was developed. To this purpose, a green extract of olive leaf (OLE) was prepared by ultrasound-assisted extraction (UAE) and characterized in terms of polyphenol content and antioxidant activity. Then, OLE was loaded in chitosan nanoparticles (CTNPs) to combine the antifungal activity of CTNPs and phenolic compounds to obtain an important synergic effect. Nanoparticles were synthetized using the ionic gelation technique and characterized in terms of sizes, polydispersity index, Z-potential, encapsulation efficiency, and release profile. Qualitative and quantitative analyses of OLE were performed by the HPLC method. OLE-loaded CTNPs exhibited good physicochemical properties, such as a small size and positive surface charge that significantly contributed to a high antifungal efficacy against Verticillum dahliae. Therefore, their antifungal activity was evaluated in vitro, using the minimal inhibition concentration (MIC) assay in a concentration range between 0.071 and 1.41 mg/mL. Free OLE, blank CTNPs, and OLE-loaded CTNPs possessed MIC values of 0.35, 0.71, and 0.14 mg/mL, respectively. These results suggest an important synergic effect when OLE was loaded in CTNPs. Thereafter, we tested the two higher concentrations on tomato plants inoculated with V. dahliae, where no fungal growth was observed in the in vitro experiment, 0.71 and 1.41 mg/mL. Interestingly, OLE-loaded CTNPs at the higher concentration used, diminished the symptoms of Verticillium wilt in tomato plants inoculated with V. dahliae and significantly enhanced plant growth. This research offers promising results and opens the possibility to use OLE-loaded CTNPs as safe fungicides in the control strategies of Verticillium wilt at open field.