| To understand the ecology diversity and distribution characteristics of endophytic fungal from Juglans regia L.woody tree in different areas,three sampling sites(Yijun,Lantian and Shanyang of Shaanxi Province)were chosen,and the fungal endophytes from 6 tissues(root,annual stem,biennial stem,bark,leave and green husk)of walnut tree in the four seasons were isolated,which were classified by morphology and similarity of internal transcribed spacer(ITS)sequence.To screen the antimicrobial endophytic fungi of J.regia L.,the antagonistic activity of endophytic fungus and the antimicrobial activity of its metabolites were determined by confrontation culture method,growth rate method and Kirby-Bauer method.The fermentation metabolites of highly active strains were polarized separated,bioactive constituents in the metabolite were traced by growth rate method and vitro experiment,then the bioactive substances in the target strains were detected by metabolomics.The main conclusions are as follows:(1)A total of 2485 endophytic fungal strains in 65 genera were isolated,in which Alternaria were the dominant genera.In different tissues of walnut,endophytic fungal from biennial stems and annual stems had the most quantity,the isolation frequency was 27.73% and 26.04%,respectively;endophytic fungal from barks showed the highest diversity,and there are 445 strains in 36 genera.Among different seasons,endophytic fungi obtained from autumn samples displayed the highest diversity that contained 780 strains and distributed in 35 genera,which was nearly 2-fold of that in spring.The walnut endophytic fungi showed a dynamic change law of ‘exuberant autumn and declining spring’ during the year.Among different habitats,endophytic fungal from Lantian showed the highest diversity,from which 676 strains in 45 genera were isolated;endophytic fungal from Shanyang displayed the most abundant,in which 1011 strains were gained that they were comprised the 40.68% of all the strains.(2)11 strains(LTL-G3 etc.)with outstanding antagonistic activity from the 176 endophytic fungus strains were screened by confrontation culture method.It’s also found that Phytophthora capsici and Alternaria sonali were more suitable as antimicrobial agents than other ones.Antimicrobial test of fermentation broth showed that Fusarium camptoceras LTL-G3,Trichoderma YJL-3 and Drechslera YJL-14 synthesized unique antimicrobial active substances in their secondary metabolites,which deserve further studying.The antimicrobial effect of the metabolites of endophytic fungus were much more distinctly on Alternaria sonali and Clostridium perfringens than others.(3)LTL-G3(Fusarium camptoceras Wollenw.& reink.)could fermentate maturely in 12 days cultivation under the condition of 28 ℃ and 180 r/min in potato-dextrose broth(PDB)medium.The fermentation products were extracted by petroleum ether(PET),ethyl acetate(EAC)and n-butanol(NBA).The quality of each phase extracts was determined as follows: NBA extracts > EAC extracts > PET extracts.The antimicrobial test of metabolites extracts with different polarities suggested that the antifungal activity of the 10 mg/mL of EAC extracts and NBA extracts were strongest,which could reach 100% inhibition of Valsa ceratosperma and Fusarium graminearum,while the relative control effects of NBA extracts and EAC extracts in vitro experiment to Valsa ceratosperma were only 40.74% and 33.33%,respectively.(4)307 compounds in 17 class were detected from LTL-G3 fermentation product by metabolomics analysis,among which the most abundant substances were flavonoids,amino acids and its derivatives,lipids.The highest relative content class of components were amino acids and its derivatives,phytohormones,nucleotides and its derivatives.The salicylic acid,chinese bittersweet alkaloid and N-feruloyl cadaverine are presumed to be the major active compounds in the metabolite of LTL-G3. |
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