Studies On The Isolation And Secondary Metabolites Of Endophytes In Dioscorea Opposite

Plant entophyte is a kind of microbial living in plant tissues in a period of its life history and does not cause obvious symptoms of disease on its host. Modern research has shown that plant endophyte is one of the ways in forming geoherbs. Dioscorea opposite Thunb, one of Henan known geoherbs, belonging to the family of Dioscorea, is one of "Four Huaiqing Chinese Medicines". However, there are few reports about entophytes from Dioscorea opposite Thunb currently. To better understand and make full use of the resources of entophytes from Dioscorea opposite Thunb, this thesis studied on the isolation and secondary metabolites of endophytes in Dioscorea opposite.Using Dioscorea opposite Thunb as the experimental materials, 41 endophytes were isolated from roots of the samples treated by the method of tissue and grinding separation. The results showed that 18 bacteria strains which belong to Bacillus, Staphylococcus, Aneurinibacillus, Agrobacterium, Leclercia, Chryseobacterium, Leifsonia, Streptomyces represented 8 genera of endophytic bacteria; Based on the molecular identification, 23 fungi strains were identified as Fusarium, Penicillium, Aspergillus, Geotrichum, Curvularia, Alternaria representing 6 genera of endophytic fungi. The results showed the advantage populations were Bacillus and Aspergillus, respectively.According to the investigation morphology and molecular biological methods, the Doef-1 strain was identified as a strain of Fusarium chlamydosporum. The Fusarium chlamydosporum were fermented and then extracted with methanol and ethyl acetate at room temperature respectively. The concentrated extracts were separated by silica gel, then further separated by other kinds of column chromatographies. The structures of the compounds from Fusarium chlamydosporum were elucidated by spectroscopic analysis and physicochemical characters. As a result, 8 compounds were isolated and identified as ergosterol-5α, 8α-peroside(1), ergosta-4, 22-dien-3-one(2), di-n-butyl phthalate(3), di-isobutyl phthalate(4), 3-pyridinecarboxylic acid(5), amber acid(6), pentanedioic acid(7) and Nb-Acetyltrytamine(8). All these compounds were isolated from Fusarium chlamydosporum for the first time.