| Macrofungi resource,a kind of important microorganism resource in nature,has the extremely high development and utilization value,many kinds of these has higher medicinal and food value.Gymnopus sp.0612-9,a macrofungi for marasmius rotula,was collected in Nanchang Meiling,its fermentation broth has the broad spectrum antimicrobial activity,It’s a potential preservative for citrus.This study aimed to investigate the bacteriostatic mechanism of the fermentation activity products to the two pathogens and the effects of fermentation activity products on the biocontrol effect to pathogens and its resistance induction,in addition,the active monomer compounds of fermentation broth were traced and identified.The main results are as follows:(1)Different concentrations of crude extracts were selected to investigate the effect on fruit quality and prevention.The result suggested that different concentration of GSFE treatment could significantly reduce the incidence and disease index of citrus and 6 d after treatment,the 5.5 mg/mL treatment group was 48.98%and 62%lower than that of the control group,respectively.while the GSFE with concentrations of 0.55 and 5.5 mg/mL can significantly inhibit the decline rate of nutrient components in the fruit,and maintain the better quality of the fruit.(2)the active substances were separated by silica gel column chromatography,ODS column chromatography,and Semi-prepared liquid chromatography method and the active monomer compounds was traced by TLC-bioautography and HPLC,the result showed that the active substance Ⅰ’ and the active substance Ⅱ’ were separated by silica gel column chromatography,and then the three kinds of active monomer compounds(A1,A2,A3)were obtained from the active substance Ⅰ’,while the compound II were obtained from active substance Ⅱ’,the retention time was 7.681 min,7.669 min,8.209 min,4.522 min,respectively,and the structure of monomer compounds A3 was analyzed by spectral analysis,which was named Methyl gymnabloate the other three compounds need further analysis.The inhibitory effect of 50μg dose of monomer compound on P.italicum was determined by TLC-BA.It was found that the inhibitory effect A2 monomer compound was the strongest,with an inhibitory area of 50.67±1.9 mm2,A1 was 21.81±.1 mm2,A3 was 39.96±1.7 mm2,Ⅱwas15.92 ±1.4 mm2。(3)Virulence equation of the active substance Ⅰ’ to blue and green mold was determined by the method of mycelium growth rate,and effects of different concentrations of active substances Ⅰ’ on growth and development of mycelium,cell membrane,membrane lipid peroxidation,antioxidant enzymes,tricarboxylic acid cycle,and energy metabolism were studied by microscopic observation,fluorescence staining,spectrophotometry and liquid chromatography.The result revealed that:the EC50 of the active substance Ⅰ’ to blue and green mold was 43.23,44.26 μg/mL,respectively,and the MIC of both were 125μg/mL.When the treatment concentration was 125 μg/mL,the mycelium of blue and green mold showed irregular twisting and winding,and the cell membrane was damaged under fluorescence microscope.Furthermore,the permeability of hyphae cell membrane treated with different concentration of active substance Ⅰ’ increased,the lipid content of cell membrane decreased,the synthesis and metabolism of protein and total sugar were inhibited,and the peroxidation of membrane lipid increased,meanwhile,the active substances Ⅰ’ had obvious inhibitory effect on CS,IDH,α-KGDH,SDH,MDH activity and ATP,ADP,AMP content,among which 125 μg/mL of the active substance Ⅰ’ had the best inhibitory effect.As a result,the active substance Ⅰ’ can achieve bacteriostatic effect by damage the cell membrane of chloromycetes and then destroy the defense system,tricarboxylic acid cycle and energy metabolism.(4)In order to research the effects of GSFE on Nanfeng mandarin disease-resistance,citrus fruit was treated with GSFE and inoculated with blue and green mold pathogens,and then determined the changes of citrus defense mechanism.The result suggested that the activities of phenylalanine ammonialyase(PAL),superoxide dismutase(SOD),peroxidase(POD)of Citrus fruits were promoted remarkably by GSFE treatment.The contents of total phenolic compounds and flavonoid were also increased significantly.Besides,the content of malondialdehyde(MDA)and lipoxygenase(LOX)activity was decreased by GSFE treatment.It can be concluded that the fermentation product of the Gymnopus sp.0612-9 can enhance the protective effect of fruit body by inducing the resistance of pericarp. |
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