Preliminary Study Of Endogenous Fungal Biological Activity And Host Cells Co-cultured. Yam

Endophytic fungus can produce many kinds of bioactive substance and a large number of endophytic fungi exists in plant, so it is a new microorganism resource which has latent value for exploitation.But until now, the research for endophytic fungus is not thorough and systematic, there are only hundreds of plant which had been studied for it, so it needs more research. Dioscorea is an important plant genus of medicinal material which faces being exhausted because of too much plucking. One way to deal with this problem is to synthesize diosgenin by the calli and suspension cultured cells of Dioscorea, but it did not realize industrialization because of its little production. Resent research showed that endophytic fungi were co-evoluted with its host as the result of symbiosis, they may produce the same or similar compound as their host. So based on above-mentioned, it is impossible to improve the production of diosgenin by the co-culture of Dioscorea and its endophytic fungi in one culture solution, which simulates their Symbiosis. In this paper, endophytic fungi were isolated from the leaf, stem, seed and rootstalk of Dioscorea zingiberensis and identified based on morphological taxonomy. The antioxidant activity and the fungistasis of the metabolite of these endophytic fungi were preliminary studied and the endophytic fungi which could produce flavonoids and dioscin were searched. At the same time, the calli were induced from the stem of D. zingibernsis and D. zingibernsis cells line under suspension culture were formed. One strain of these endophytic fungi and D. zingibernsis suspension cultured cells were successfully cultivated together in the same culture solution. The diosgenin production and antioxidant activity and antioxidase activity of co-culture system and suspension cultured cells line of D. zingibernsis and the endophytic fungi of D. zingibernsis were compared.50 strains of endophytic fungi were isolated from D. zingibernsis. They were identified as Aspergillus Micheli ex Fr. , Pyrenochaeta de Not, Chaetomella Fuckel, Sporotrichum Lk. ex Fr , Isaria Pers. ex Fr. , Coniosporium Lk. ex Fr. and Chromosporium Corda except 31 strains which did not be identified for they could notproduce spores. These endophytic fungi were cultured under 27°C, 120r/min for 7d, then the mycelia and the fermented broth were gained for detection. The antioxidant activity of compound extracted by ethanol from the mycelia and the fermented broth of four strains were found by detecting POV. The extract from the fermented broth of EDT5j which was isolated from the rootstalk of D. zingibernsis, and EDS4, which was isolated from the seed of D. zingibernsis, showed very strong antioxidant activity, and within certain range, its antioxidant activity were enhanced when more extract were added. This suggested that EDT5 and EDS4 had latent value used for exploiting natural antioxidant component. The fermented broth of six strains had different inhibiting rate to the mycelia growth and the sprouting of six kinds of plant pathogenic fungi such as Verticillium dahliae, they were EDT3, EDT4 and EDT5. which were isolated from the rootstalk of D. zingibernsis, and EDSi, EDS3 and EDS4, which were isolated from the seed. E-y, which was mixture of the fermented broth of EDS3 and EDS4, had higher inhibiting rates to the mycelia growth and the sprouting of six plant pathogenic fungi. One strain (EDS4) which produces flavonoids and two strains (EDS4 and EDT5) which produce dioscin were found by detecting the extract of the mycelia and the fermented broth of those endophytic fungi by TLC and HPLC and color reaction analysis. The result of HPLC showed that the compound extracted by ethanol from the fermented broth of EDS4 had one peak which tallied with Rutin and the result of color reaction was coincide to flavonoids, so it was infered preliminarily that the fermented broth of EDS4 contained flavonoids or its similar compound. The result of TLC showed that Rf of the extract from the mycelia of EDT5 and EDS3 tallied with diosgenin and the result of color reaction was coincide to dioscin, so it was infered preliminarily that the extract of the mycelia of EDT5 and EDS3 contained dioscin or its similar compound. The calli induced from the stem of D. zingibernsis were fresh and tender and the content of diosgenin in 30-day-culture of it was 2.713u.g/gdw. The suspension cultured cells line of D. zingibernsis was good and the content of diosgenin in 30-day-culture of it was 1.732ug/gdw. EDT5 could be successfully cultivated with D. zingibernsis suspension cultured cells which had been cultured for 7d firstly. The content of diosgenin in 30-day-culture of co-culture was2.167(xg/gdw. The activity of antioxidase in the cells of co-culture system was higher than that in D. zingibernsis suspension cultured cells line and EDT5, but it was contraty for the fermented broth of co-culture system. POV (meq/kg) of lard coped with the extract of the fermented broth of co-culture system was lower than D. zingibernsis suspension cultured cells line. It showed that what decide the antioxidantactivity was not antioxidase but certain compound endophytic fungi secreted.