The Antifungal Ability of Amino Acid Substitution of Antimicrobial Peptide Epinecidin-1 to <i>Botrytis cinerea</i> in Peach Fruit

Epinecidin-1 (EPI), an antifungal peptide, has specific anti-<i>Botrytis cinerea</i> activity <i>in vitro</i>, but <i>in vivo</i>, it requires a very high concentration to efficiently manage postharvest gray mold. Our work aimed to improve the antifungal ability of EPI <i>in vivo</i>. A set of EPI analogues, generated by arginine substitution on the polar surface of EPI, was acquired. The biological properties of EPI and its derivatives were evaluated. Among the tested EPI analogues, the modified peptide EPI-4 demonstrated the most favorable characteristics, with higher anti-<i>B. cinerea</i> activity (the MIC value was 6 μmol/L compared to 12.5 μmol/L for EPI) and the lowest cytotoxicity. <i>In vitro</i>, EPI-4 disrupted the ultrastructure and cell membrane integrity of <i>B. cinerea</i>, induced more ROS accumulation, and enhanced the DNA binding ability of <i>B. cinerea</i> by twofold. More importantly, <i>in vivo</i> experiments confirmed that EPI-4 could effectively control postharvest gray mold of peach fruit at 50 μmol/L, and its control effect was significantly better than that of the template peptide EPI. Our findings suggest that EPI-4 demonstrates considerable promise as a valid compound for regulating gray mold disease in postharvest peaches.