Isolation And Identification Of The Endophytic Fungi Producing Resveratrol And Polydatin In Polygonurn Cuspidatum From Qin-Ba Mountain Area

The plant endophytic fungi is a kind of fungus that total or part of their life histories exist in healthy plant tissue without infecting their host plants. Since 1993,Strobel. et al. firstly isolated a species of endophytic fungus from the bark of Taxus brevifolia, which can produce taxol. It's a potential way to dissolve the problem that we are lack of plant medicine by microbiology fermentment of plant endophytic fungi to produce plant medicine and other active substances.Polygonurn cuspidatum distributed in the Mountains QIN-BA (Qinling and Bashan).Its active composition, resveratrol and polydatin, are of medicinal active function in anticancer,bacteriostasis, antiphlogistics, keeping liver and gallbladder in good order, antioxidation as well as reducing blood fat. Our study was prompted by isolating and identificating of Endophytic Fungi of Polygonurn cuspidatum and analysing its metabolites, obtaining Endophytic Fungi producing resveratrol and polydatin. The studies are summerized as follows:1.Isolation, Purification And the Basic Classification of Endophytic Fungi116 strains of endophytic fungus were obtained and isolated by tissue culture of fresh Polygonurn cuspidatum roots after surface sterilization and 3-7d incubation on the PDA plate with small piece of 0.5mm×0.5mm maintained at 28℃. The result shows that they are belonged to 9 genera(Aspergillus, Penicillium, Mycelia class, Fusarium, Mucor, Pestalotiopsis spp, Cladosporium, Alternaria and Trichoderma),5 orders (Moniliales, Discellaceae,Melanconiales,Mucorales,Agonomycetales).2. Endophytic Fungi screening of Polygonurn CuspidatumThe 116 strains were cultured in the promoted potato liquid medium under 28℃, shake culture with 160 r/min for 7d. Mixture of 1:2 ratio, (v/v or w/v), hypha and fermenting liquid was extracted for 15 min by ethyl acetate seperatedly. Extraction mixtures were dried by vacuum rotary evaporation in 40℃and dissolved in methanol to 25 mL.Then, contents of resveratrol were measured by HPLC.Two strains, M-56 and L-18 producing resveratrol and polydatin were found. M-56 only produced polydatin, and its yield was 1.029 mg/L, while L-18 could produce both polydatin and resveratrol, and their contents are 4.672 mg/L and 4.812 mg/L.3.The Identification of Endophytic Fungi produced Resveratrol and PolydatinBasing on our observation of the morphological characteristics of M-56 and L-18, we preliminarily identified them as fungi of Alternaria sp.and Penicillium sp. respectively. The 564 bp DNA were obtained by amplifying ITS fragments.The result shows that ITS sequence homology of M-56 and L-18 strains can reach 99%. We also compared the ITS sequence of M-56 and L-18 with that of the other species of Alternaria and Penicillium recorded in NCBI.,we identified strains M-56 and L-18 as Alternaria and Penicillium.