Elucidating the antifungal mechanism of Citrus reticulata L. peel extract against P. italicum: Membrane disruption and metabolic perturbation revealed by untargeted metabolomics.

Developing natural antifungal agents is critical for reducing reliance on synthetic fungicides in postharvest citrus preservation. This study aimed to evaluate the antifungal activity and potential mechanisms of citrus peel extract (CPE) against Penicillium italicum (P. italicum) by combining in vivo and in vitro assays with metabolomics. Our results showed that CPE at 25, 50, and 100 mg/L effectively inhibited fungal growth in citrus fruits, suppressed spore germination, and damaged mycelial ultrastructure. Compared with the control group (diameter = 66 mm), the colony diameter in the 100 mg/L treatment group was significantly reduced by approximately 70% (diameter = 20 mm). Furthermore, CPE disrupted cellular membrane integrity in a concentration-dependent manner, as evidenced by elevated propidium iodide uptake, extracellular conductivity, and malondialdehyde levels. CPE also severely impaired cellular metabolism, with high-concentration (100 mg/L) treatment significantly reducing total protein and total lipid contents in P. italicum cells by 47.22% and 42.72%, respectively. Metabolomic analysis identified 56 differential metabolites, revealing that metabolic alterations intensified as treatment concentration increased. Specifically, high-concentration CPE (100 mg/L) exposure resulted in the significant down-regulation of 24 metabolites, including key compounds involved in glycolysis (d-glucose and d-fructose), fatty acid metabolism (hexadecanoic acid and octadecanoic acid), and amino acid metabolism (L-leucine and L-alanine). In contrast, medium-concentration treatment (50 mg/L) enhanced the synthesis of the TCA cycle intermediate malic acid and the amino acid metabolic regulator glutamic acid. This study provides new insights into the potential mechanisms of action of plant-derived antifungal compounds against P. italicum.