Integrative cytology and proteome alterations unravel the multi-target effect of a novel alkaloid antofine against Penicillium italicum

Penicillium italicum is the causal agent of citrus blue mold, which is a major threat to the global citrus fruit industry. Antofine, a natural phenanthroindolizidine alkaloid, is water-soluble and exhibits a broad range of biological activities. However, whether it can inhibit P. italicum growth and the potential inhibitory mechanism remain to be elucidated. This study aimed to investigate the antifungal mechanism of antofine against P. italicum using SEM, TEM, PI staining, and TMT-labeled quantitative proteomic analysis. Antofine was found to exhibit its preeminent antifungal activity against P. italicum with a MIC of 1.56 mg/L and a MFC of 6.25 mg/L. The challenge test revealed that antofine inhibited the development of citrus blue mold during a 6 day P. italicum-infected period. Antofine acted on its potential multi-targets to inhibit P. italicum growth by synergistically activating oxidative stress through accumulating excess ROS, impairing membrane integrity, inducing membrane lipid peroxidation, and disrupting mitochondrial function, thereby disrupting the membrane system and reducing cell viability. Moreover, antofine treatment down-regulated most DEPs involved in carbon metabolism, pyruvate metabolism, and the TCA cycle in P. italicum mycelia, which may explain the mitochondrial decomposition observed by TEM and the declines in ATP level as well as the TCA cycle-related enzymes activities. These results indicate that antofine treatment inhibited P. italicum growth by targeting the cell membrane and mitochondria.