Antifungal potential of the novel antimicrobial peptide N1-6L7A against Penicillium digitatum in Citrus sinensis (L.) Osbeck and its mechanism of action

Green mold, caused by Penicillium digitatum , is one of the most destructive postharvest diseases of citrus fruits, leading to significant rot and economic losses. In this study, the antifungal activity and underlying mechanism of the novel antimicrobial peptide (AMP) N1–6L7A against P. digitatum were investigated. Additionally, the potential application of N1–6L7A for controlling postharvest green mold in Citrus sinensis (L.) Osbeck was examined. N1–6L7A exhibited potential antifungal efficacy by inhibiting both mycelial growth and spore germination of P. digitatum . Furthermore, it demonstrated excellent thermal stability, pH tolerance, and biosafety. The results of fluorescence microscopy assay, cell component leakage, and microscopic observations revealed that N1–6L7A severely damaged the fungal membrane of P. digitatum , leading to the release of intracellular nucleic acids and proteins. N1–6L7A treatment also induced excessive intracellular reactive oxygen species (ROS) production, collapse of mitochondrial membrane potential, and decreased activities of malate dehydrogenase (MDH) and ATPase. Gel retardation assay indicated that P. digitatum -DNA was a target of N1–6L7A. Furthermore, transcriptomic analysis suggested that N1–6L7A might inhibit the growth of P. digitatum by blocking the expression of genes related to the ribosome, amino acid metabolism, and pyrimidine metabolism. Meanwhile, the peptide potentially promoted the expression of genes involved in steroid biosynthesis. Notably, in vivo assays confirmed that N1–6L7A effectively controlled P. digitatum infection in citrus fruits and enhanced host resistance by activating defense-related enzymes. Collectively, our results suggested that N1–6L7A could be used as an alternative fungicide to prevent green mold in citrus fruits.