Preliminary Study On The Effects Of Berberine Hydrochloride On Penicillium Digitatum And Geotrichum Citri-aurantii On Postharvest Citrus Fruit

Citrus is an important economic crop in China,and it is popular with its unique flavor and nutrition.Citrus fruit is usually infected by pathogenic fungi in the process of transportation and storage.So far,the utilization of chemical fungicides is the most effective way in disease control.However,the problems have arisen,such as human health,environmental problems and the increase in chemical resistance of pathogens.Therefore,it is urgent to develop new control strategies.Berberine hydrochloride(BH)is a quaternary ammonium salt from the group of isoquinoline alkaloid.The possibility of its application in the control of postharvest diseases of fruits and vegetables was preliminary revealed by some studies.In this paper,the antifungal ability of BH against Penicillium digitatum and Geotrichum citri-aurantii and the control effect on citrus green mold and sour rot were evaluated in vitro and in vivo.The effects of BH on the surface morphology of mycelia and spores were observed by SEM.The possible antifungal mechanism of BH on pathogenic fungi was further explored from cell wall,cell membrane,reactive oxygen species and energy metabolism.The main results are as follows:1.Antifungal activity and mechanism of BH against P.digitatumThe minimum inhibitory concentration(MIC)and the minimum fungicidal concentration(MFC)of BH against P.digitatum were 0.16 and 0.32 g/L,respectively.BH treatment can significantly inhibit the germination of spores.In addition,5×MFC,10×MFC BH can effectively reduce the disease incidence and delay the process of citrus green mold.Scanning electron microscope(SEM)results showed that the mycelia of P.digitatum became dry,wrinkled and distorted after BH treatment.Further study showed that BH could affect the cell wall integrity of spores,but the membrane permeability of spores was not damaged.The cell wall integrity of mycelia was destroyed at 30 min.The fluorescence intensity decreased after staining with calcofluor white(CFW)and the activity of extracellular alkaline phosphatase increased significantly.The membrane permeability was also damaged.The damaged membrane presented red fluorescence after staining with pyridinium iodide(PI),and the conductivity and soluble protein content increased sharply.Meanwhile,the energy metabolism of mycelia was also affected at 60 min.ATP content and energy charge decreased sharply,but the level of active oxygen and malondialdehyde content in MIC BH group increased significantly,thus the membrane lipid peroxidation occurred.Meanwhile,the mitochondrial membrane potential decreased,ATP content and energy charge decreased further than 30 min.In conclusion,BH damaged the cell wall integrity and membrane permeability of P.digitatum,which led to the leakage of intracellular substances and the influence of energy metabolism.Subsequently,ROS accumulation caused membrane lipid peroxidation and mitochondrial damage,which further caused energy metabolism imbalance.2.Antifungal activity and mechanism of BH against G.citri-aurantiiThe MIC and MFC of BH against G.citri-aurantii were 0.08 and 0.16 g/L,respectively.BH treatment can significantly inhibit the germination of spores,effectively reduce the disease incidence and delay the process of citrus sour rot.BH exhibited different antifungal effects on mycelia and spores of G.citri-aurantii.SEM results showed that BH could not affect the surface morphology of mycelia,but caused the collapse and deformity of spores.CFW and PI staining results showed that BH could not affect the cell wall integrity of mycelia and the membrane permeability of spores,but the cell wall integrity of spores was damaged at3 h.It is suggested that BH inhibited the growth of G.citri-aurantii mainly through destroying the cell wall integrity of spores.Further experiments showed that the chitin content in spore cell wall of G.citri-aurantii was significantly lower than that of control group after BH treatment for3 h,and decreased with the treatment time.At 9 h,the expression of chitin synthase gene CHS1,CHS2 and CHS3 were down regulated,while the relative expression of chitin deacetylase gene CDA1 was up regulated.In addition,the ?-1,3-glucan content was significantly lower than that in control group at 9 h,and the expression of FKS1,a ?-1,3-glucan synthesis related gene,and EXG1,a exo-beta-1,3-glucanase gene were down regulated,but there was no significant difference in the relative expression of endo-beta-1,3-glucanase gene BGL2.It is suggested that BH inhibited the spores germination of G.citri-aurantii through affecting the synthesis of chitin and?-1,3-glucan.