Integrative cytology and proteome alterations unravel the multitarget 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 remains to be elucidated.This study aimed to investigate the antifungal mechanism of antofine against P italicum using scanning electron microscopy,transmission electron microscopy(TEM),propidium iodide staining,and tandem mass tag-labeled quantitative proteomic analysis.Antofine was found to exhibit its preeminent antifungal activity against P italicum with a minimum inhibitory concentration of 1.56 mg/L and a minimum fungicidal concentration of 6.25 mg/L.The challenge test revealed that antofine inhibited the development of citrus blue mold during a 6-d P italicum-infected period.Antofine acted on its potential multitargets to inhibit P italicum growth by synergistically activating oxidative stress through accumulating excess reactive oxygen species,impairing membrane integrity.inducing membrane lipid peroxidation,and disrupting mitochondrial function,thereby disrupting the membrane system and reducing cell via-bility.Moreover,antofine treatment downregulated most differentially expressed proteins involved in carbon metabolism,pyruvate metabolism,and the tricarboxylic acid cycle(TCA)in P italicum mycelia,which may explain the mitochondrial decomposition observed by TEM and the de-clines in ATP levels as well as the activities of TCA-related enzymes.These results indicate that antofine treatment inhibited P italicum growth by targeting the cell membrane and mitochondria.