Diversity Of Endophytic Fungi From Gingko Biloba L. And Their Activities

Endophytes are microorganisms such as fungi and bacteria (including actinomycete) that live inter- and/or intra-cellularly inside the healthy plant tissues causing asymptomatic infections for all or part of their life cycle. Endophytic fungi have been investigated to have anti-fungal, anti-tumor and anti-oxidation activities, and evolve with the plant. Research results in recent years show that endophytes can produce some metabolites same or similar to those from the host plant, and metabolites with the novel structure and specific bioactivity.In the study, materials of Ginkgo biloba L. were selected to isolate and identify their endophytic fungi, then screen the bioactive strains and specific metabolites of endophytic fungi in different areas. The main results are as follows:The optimum surface sterilization procedure for leaves of G. biloba was 75% ethanol 1 min, 3% sodium hypochlorite 1 min, 75% ethanol 0.5 min, for breaches of G. biloba was 75% ethanol 1 min, 5% sodium hypochlorite 3 min, 75% ethanol 0.5 min, for barks of G. biloba was 75% ethanol 1 min, 5% sodium hypochlorite 5 min, 75% ethanol 0.5 min.There was a rich diversity of endophytic fungi of Gingko biloba. A total of 822 isolates were recovered from tissue of G. biloba, of which 440 were from Tianmu mountain and 382 were from Jiande. Five genera (Fusarium, Phomopsis, Alternaria, Bjerkandera and Phoma) were common at Tianmu mountain, while five genera (Fusarium, Phomopsis, Diaporthe, Mucor and Pestalotiopsis) were common at Jiande. 805 strains were isolated from 40 branches and barks of male and female G. biloba L. (416 from male plants and 389 from female plants). The colonization rate and the number of the strains isolated from male plants were both higher than those from female plants.The isolated stains are divided into 43 morphotypes according to their cultural characteristics. Using phylogenetic analysis and similarity comparison of ITS sequences, 29 of 43 morphotypes were identified into species, nine were grouped into genus level. Five morphotypes were grouped into family level. To compare the isolated strains of endophytic fungi from male and female Gingko biloba L. We found that Trichoderma atroviride and Leptosphaeria microscopica were only separated from branches of male G. biloba L. Strains of Fusarium tricinctum isolated from male plants were more than that isolated from female ones. Phomopsis fukushii isolated from the female plants were more than strains isolated from male ones. The reason may be due to the different chemical composition of the host plant.The bioactivity of the ethyl acetate extracts of the fermentation broths of 56 strains from Gingko biloba were studied. Biological model Pyricularia oryzae was efficiently applied to the primary screening of the antifungal in vitro agents from the secondary metabolites of the microorganisms. Nine strains (8.9 %) of endophytes from G. biloba could induce morphological abnormality of P. oryzae. Antimicrobial activity against four fungi of Candida albicans, Cryptococcus neoformans, Trichophyton rubrum and Aspergillus fumigatus were done. The ethylacetate extracts of endophytes isolated from Ginkgo biloba were screened for their antioxidant, and T-3-2-2 (Chaetomium globosum), T-6-5-7 (Xylaria sp.) showed exellent antioxidant activity. These results indicate that endophytic fungi possess bioactivities, further study should be done with them.Methods of molecular biology were used to find the characteristic chemicals of Gingko biloba L. from its endophytic fungi. Four gene of key enzyme were cloned and found no specific target band. Although the target was not found in any strains, a fast, efficient and convenient way method of screening the target compounds was found.