Inhibition of induced citrus peel disease-resistant components against Penicillium digitatum and Geotrichum citri-aurantii

Abstract: Citrus are popular worldwide fruits due to the high nutritional value and special flavor, but they are easy to decay during storage and transportation, particularly on green mold, blue mold and sour rot. Alternatively, disease controlling methods are also necessary to protect from the bandage of synthetic chemical fungicides due to the health and environmental concerns. In a previous study, the fruits cannot decay when red pigment substances were accumulated in the fruit peels around wounding sites after inoculation with Actinomucor elegan, indicating the Citrus Peel Red-substances Extract (CPRE) has strong antifungal activity against Penicillium italicum. Here, this study aims to assess antifungal effects of the partially purified CPRE on Penicillium digitatum and Geotrichum citri-aurantii, the casual agents for green mold and sour rot, respectively. To further explore the possible inhibition mechanism of the substance against the pathogens, the damage on cell wall was evaluated by chitin content and distribution, and leakage of alkaline phosphatase, while the interferences on cell membrane were investigated to assess membrane integrity, release of proteins and nucleic acids, extracellular electronic conductivities and contents of lipids in the hyphae cells of both paghogens. The results showed that at the concentration of 50 to 200 μg/mL, CPRE exhibited strong inhibitory effects on spore germinations, elongations of germ tubes, and mycelia growth of P. digitatum and G. citri-aurantii, in a concentration dependent manner, with the Minimal Inhibitory Concentration (MIC) at 200 μg/mL for both pathogens. Calcofluor white staining, an agent which specifically binds to chitin exposed, revealed that the chitin distribution and cell wall integrities of the hyphae of both pathogens were markedly interfered when incubated with CDPC at 1/2 MIC for 24 hours. CPRE treatments also made chitin contents decrease by 30.29%, 52.64%, and accelerate the release of alkaline phosphatase in the mycelia of both pathogens, indicating that cell wall structures and functions were disturbed by the active components in CPRE. Furthermore, membranes of the hyphae cells of both pathogens can be deteriorated after CPRE treatments at the concentration of 100 μg/mL, as indicated by propidium iodide staining, which emits red fluorescence when entered into dead cells and bind with nucleic acids. The extracellular electronic conductive of the hyphae cells of both pathogens increased significantly after 6 hours of incubation with CPRE within the concentrations tested, indicating that the metal ion balance was disrupted in the treated cells. There were significant differences in leakages of soluble proteins and nucleic acids from hyphae cells with CPRE treatments, showing a consistence with that of extracellular conductivity assays. Moreover, CPRE treatments reduced the total contents of cell lipid in hyphae of both pathogens. These results demonstrated that CPRE can effectively inhibit both pathogens by damaging or interfering with the cell membranes and cell walls of the hyphae. The powerful antifuangal effects of CPRE against P. digitatum and G. citri-aurantii can offer the great potential application prospects in control of postharvest decay of citrus fruits, and futher provide a theoretical basis for the development of natural products with antifungal effects.