Isolation And Identification Of The Antagonistic Substance Of The Endophytic Fungi From Sophora Flavescens And Its Function Mechanism

This research screened 5 strains endophytic fungi from Sophora flavescens that have antagonistic activity, and studied the classification and the optimum condition of the active substance BS001 by using 16Sr-DNA method, response surface methodology, eight kinds of solvent system,1H-NMR,13C-NMR and LC-MS. The antagonistic activity of the BS001 was purified and identified in this paper, also the mechanism. After identifying, the strain of BS001 was confirmed as Aspergillu terreus. This study confirmed the conditions of liquid fermentation of BS001 strain and isolated a new antibacterial active component from its fermentation liquor. And the antagonistic mechanism and the effect on soil microorganism of the active component were dealt with. These results provided a solid foundation for the application of the endophytic fungi from Sophora flavescens and its antagonistic active components.The antifungal activity of 5 strain endophytic fungi to 25 agricultural pathogenic fungi and 14 bacterial were studied. The strain of BS001 could inhibited the growth of Sclerotinia sclerotiorum, Alternaria mali Roberts, Colletotrichum coccodes Hughes., Fusarium oxysporum f.sp. cucumerinum, Rhizoctonia cerealis, Phytophthora capsici, Pseudomonas aeruginosa, Bacillus thuringiensis, Herba Pogostemonis, Bacillus cereus. Frankland, Enterobacter aerogenes, Proteusbacillus vulgaris, Ralstoia solanacearum Smith, Bacillus subtilis (Ehrenberg) Cohn and Erwinia carotovora subsp, in which the inhibition to Fusarium oxysporum f.sp. cucumerinum and Enterobacter aerogenes were the biggest with the antibacterial diameters of 25 mm and 14 mm, respectively.The optimum condition for BS001 fermentation was obtained by using the Placket Burman analysis, and then selected the main effect factor, analyzed the data through Duncan Analysis, RSM (Response surface methodology) and Design expert 8.0 Soft. The best condition for fermenting BS001 was 100 mL medium volume by adding 107 cfu/mL inoculums concentration,1 mmol/mL KH2PO4,5 pH value,335 g/L potato,35 g/L glucose, 1.80 g/L NH4NO3, with the revolution of 150 rpm and the culture temperature of 25℃.The determination of chemical contents of BS001 through HPLC with the DiamonsilTM-C18 chromatographic column, the mobile phase was methanol:2% phosphate =2:8, the flow rate was 0.5mL/min, the detection wavelength was 220 nm and the linear range was 10μL. The oxymatrine and matrine, main components of Sophora flavescens, were presented in secondary metabolite of BS001 with the retention time of 9.282 min and 13.298 min, respectively.The structure of antifungal substance, had inhibition to F. oxysporum f.sp. cucumerinum, in BS001 fermentation was identified by using macrospore absorbed resin and 10-20% ethanol gradient elution. The component was a new compound, (2E,2E')- ((Z)-2,3-dihydroxy-5,8-dioxocyclooct-6-ene-1,4-diyl)dibut2-enoate, named Qianshanmycin, primarily.The mechanism of active substance to fusarium wilt was defined. In this study, the active substance inhibited the mycelia growth and the spore germination, destroyed the cell membrane structure. But this substance could not produce chitosanase to hydrolyzing the chitin or preventing the synthesis of the chitin. Qianshanmycin had no significant effect on the protein synthesis, while inhibited the synthesis of the carboxymethylcellulose enzyme.The efficacy of biological control of Qianshanmycin on cucumber seedlings was investigated. The results showed that at the concentration of 160μg/mL and 320μg/m, the control effect on fusarium wilt of cucumber were 80.1% and 81.82% after treating 15 d, which has the same result with 30% hymexazo at 40μg/mL. At F=0.01 level, the concentration of 160μg/mL of Qianshanmycin had better effect, and that was considered the best test concentration.The active substance increased the number of bacteria and actinomycetes, while decreased fungi number. From this result we concluded that Qianshanmycin could increase the number of beneficial flora, while decrease the deleterious number to prevent the fusarium wilt.