Effect Of Streptomyces N2 On Penicillium Citricum And Its Preliminary Study On Postharvest Fresh-keeping Of Citrus

Streptomyces sp.N2 is a new species that can produce new antifungal active substances isolated(3-methyl-3,5-amino-4-ene-pyran-2-one,molecular formula is C6H702N,antifungalmycin N2)and screened by the Engineering Laboratory for Development and Utilization of Agricultural Microbial Resources in Jiangxi Province.It has a good inhibitory effect on Penicillium italicum and Penicillium digitatum.This article provides an important theoretical basis for the development of a new type of fruit and vegetable preservatives.It mainly explores the mechanism of antagonism and induced resistance of antifungalmycin N2 against P.italicum and P.digitatum,and explores the preliminary application of biocontrol effects on citrus after harvest.The main results are as follows:(1)The antagonistic activity of antifungalmycin N2 against P.italicum and P.digitatum was determined by the tube-and-dish method,and its effect on P.italicum and P.digitatum spore germination,colony morphology and bacterial ultrastructure was explored.The results show that antifungalmycin N2 has a significant growth inhibition effect on P.italicum and P.digitatum,which can effectively reduce the spore germination of P.italicum and P.digitatum(inhibition rates are as high as 91.3%and 90.6%);the colony growth after antifungalmycin N2 treatment is significantly slower,And the color of the colony was significantly lighter than the control group,indicating that antifungalmycin N2 seriously interfered with the normal development of the colony;and the mycelium under antifungalmycin N2 treatment was seriously deformed,the cytoplasmic wall separation phenomenon was obvious,the organelles were broken,the cytoplasmic outflow,and the cavity phenomenon appeared antifungalmycin N2 can produce bacteriostatic effect by destroying the cell wall and cell membrane of the mycelium.(2)Afterwards,we further explored the effect of antifungalmycin N2 on the cell wall and cell membrane structure of P.italicum and P.digitatum and its effect on membrane lipid peroxidation.The results show that antifungalmycin N2 can induce the production of chitinase and β-1,3 glucanase by pathogenic fungi,promote the hydrolysis of chitin and polysaccharides and lead to the destruction of the cell wall.The content of ergosterol and total lipids in the cells treated with antifungalmycin N2 was significantly reduced,causing an imbalance in cell osmotic pressure.At the same time,the intracellular reactive oxygen species in the bacteria increased significantly,which exacerbated the degree of membrane lipid peroxidation of the mycelium,causing the polymerization and damage of intracellular macromolecules.Through fluorescence microscope observation,it was found that the fluorescence of the bacterial cells after a treatment was significantly enhanced,indicating that the cell membrane of the pathogenic bacteria was damaged,and the permeability and fluidity of the cell membrane were affected,thereby inhibiting the growth and metabolism of the bacterial cells.(3)Then,we explored the effect of antifungalmycin N2 on P.italicum and P.digitatum’s antioxidant system and physiological metabolism.The results show that antifungalmycin N2 can significantly enhance the cell’s peroxidation reaction,allowing it to generate a large amount of reactive oxygen species,destroying the antioxidant enzyme system of pathogens,and affecting the ability of the bacteria to remove ROS.At the same time,antifungalmycin N2 can cause the protein and nucleic acid to leak out of the cell by destroying the carbohydrate anabolic system,protein anabolic system and nucleic acid synthesis and decomposition of the cell,resulting in an imbalance in the energy metabolism of the bacterial material.(4)The effect of antifungalmy’cin N2 on the respiratory energy metabolic system of pathogenic bacteria was investigated by measuring the activity of enzymes related to respiration in the energy metabolism system,calcium ion concentration,and the opening of mitochondrial membrane conversion pores after treatment of P.italicum and P.digitatum by antifungalmycin N2.The results show that it can not only significantly inhibit the activities of malate dehydrogenase(MDH),succinate dehydrogenase(SDH)and ATPase in the bacteria,but also activate fructose 6-phosphate(PFK)and initiate glycolysis way.At the same time,the Ca2+ concentration inside the mitochondria of the bacteria increases,causing the Ca2+in and out of the bacteria to lose balance.At the same time,antifungalmycin N2 can increase the MPTP opening degree of the mitochondria,affect the respiration of the mitochondria,and destroy the respiratory energy metabolism of the bacteria.(5)In order to investigate whether Streptomyces sp.N2 as an antagonistic antibacterial agent can induce postharvest citrus to develop disease resistance.Using distilled water as a control,changes in antioxidant enzyme activities,phenol metabolism-related enzyme activities,and disease-related proteins in citrus were measured.The results showed that the incidence of citrus and the diameter of the lesions in the Streptomyces+Penicillium treatment group were significantly lower than those in the Penicillium treatment group,and Streptomyces sp.N2 was able to activate the antioxidant enzyme Superoxide dismutase(SOD)in citrus.,The activity of catalase(CAT),peroxidase(POD),remove the active oxygen and other substances in the body of the fruit,and reduce the toxic effect of the fruit.Streptomyces sp.N2 can also induce citrus to produce polyphenol oxidase(PPO)and phenylalanine ammonia-lyase(PAL),enhance its ability to oxidize phenols,while enhancing β-1,3-Glucanase(GUN)and Chitinase(CHT)activity,induce resistance in citrus fruit.(6)In order to explore the effectiveness of antifungalmycin N2 on the biocontrol of post-harvest citrus,experiments showed that the water loss rate and decay rate of citrus after antifungalmycin N2 coating treatment were significantly reduced,and the total sugar consumption was reduced.At the same time,antifungalmycin N2 can effectively slow down the decomposition rate of soluble solids,ascorbic acid and titratable acid in citrus.At the same time,the content of total phenols and total flavonoids in citrus fruits increased significantly,which shows that antifungalmycin N2 can significantly extend the shelf life of citrus after harvest.