Isolation And Identification Of Endophytic Fungi From Ginkgo Biloba And Study On Antimicrobial And Antioxidant Activity

Endophytic fungi,microorganisms that reside in the tissues of living plants,are relatively unstudied.Those plants growing in unique environmental settings,having ethnobotanical uses,having extreme age or interesting endemic locations generally contain novel endophytic microorganisms.Novel taxonomy of an endophyte or the acquisition of one that is only rarely seen,generally offers a prospect for finding novel bioactive natural products.Ginkgo biloba is a precious and rare plant endemic to China,named as "a living fossil". It is scarcely infected with plant pathogens and insect pests,and has a long life,so it has potential to isolate novel endophytic fungi.In our study,branches and leaves were collected from old Ginkgo biloba in Taixing and Pizhou,Jiangsu province.Endophytic fungi were isolated,and those with antimicrobial activity were screened and subsequently identified by morphological features or ITS sequence similarity analysis.Mycelia sterilia YX-28 with significant and stable antimicrobial activity was identified on the basis of morphological features and molecular analysis.Growth conditions and medium compositions were optimized,and compound(s) with antimicrobial activity was/were isolated and elucidated. In addition,conditions of solid fermentation for the growth of the stromata of the strain and its antioxidant activity were studied.The main results were given as follows:1.55 endophytic fungi were obtained from the branches and leaves of Ginkgo biloba. Among them,29 strains,mostly spore-producing fungi,were isolated on plates containing tetrachlorotetraiodo fluorescein di sodium(TFDS),including 1 Alternaria sp.,6 Aspergillus spp.,10 Penicillium spp.,1 Mucor sp.,9 mycelia sterilia and 1 yeast.26 strains were obtained on water agar,including 1 Penicillium sp.,3 Alternaria spp.,3 Chromosporium spp.and 19 Mycelia Sterilia.The results indicated that water agar was more suitable for the isolation of endophytic fungi.2.Endophytic fungi with antimicrobial activity were screened using agar diffusion method,resulting in 23 bioactive strains,mostly mycelia sterilia,accounting for 41.82%of the total.The amount of strains with antimicrobial activity by water agar method was highly more than that by TFDS method.3.11 bioactive strains isolated on water agar plate were identified preliminarily.Among them,4 spore-producing strains were identified by the morphological features,resulting in YX-2 as Chromosporium sp.,YX-9 as Acremoniella sp.,and YX-30 and YX-45 as Alternaria spp.The other 7 mycelia sterilia were conducted sequence similarity analysis of ITS1-5.8S rDNA-ITS2,indicating that YX-13 has a high similarity with Fusarium lateritium,YX-16 and YX-44 with Alternaria tenuissima,YX-27,YX-28 and YX-41 with Xylaria,and YX-47 with a foliar endophyte from Picea glauca,respectively.The results of sequence analysis were in agreement with those of morphological features.4.Nonspore-producing fungus YX-28 with great and stable antimicrobial activity was further studied.YX-28 produced black and carbon-like colony on agar plates,and finger-like stromata on different media.However,neither sexual nor asexual spores produced through different spore promoting methods.According to molecular analysis of 18S rDNA and ITS1-5.8S rDNA-ITS2 sequences,YX-28 could be determined to be a similarity species of Xylaria hypoxylon.5.Custom Design was used to determine the conditions for the growth of stromata of Xylaria sp.YX-28.The results showed that the stromata grow well when 15g cotton seedcoat-wheat bran(3:1) as solid media with broth at a ratio of 30:70(W/V),sealed the bottle with plastics,growing mycelia in the dark,and then in lightness.The production of the stomata was 441.96g/m².6.One-factor-at-a-time method was used to determine the suitable conditions for the growth and antimicrobial activity of Xylaria sp.YX-28.The optimal temperature is 25℃, while YX-28 can grow at a range from 5℃to 37℃,or stop growing beyond the range. YX-28 can grow at pH 3-12,with the optimal original pH 6.Medium volume of 150 mL in a 500 mL flask and inoculation of 2.5 g wet mycelia were suitable for Xylaria sp.YX-28.7.Fractional Factorial Design and Response Surface Methodology were used for the medium optimization of Xylaria sp.YX-28.Peptone and ZnSO₄ were key factors for the antimicrobial activity of Xylaria sp.YX-28.The optimized medium compositions were 1.500%glucose,0.200%NH₄Cl,0.500%soybean powder,0.158%peptone and 0.168% ZnSO₄,leading to an enhancement of the antimicrobial activity by 1.44-fold against S.aureus and 2.02-fold against E.coli,respectively.8.Membrane filtration was used to deal with large scale of fermentation broth of Xylaria sp.YX-28.The filtration part with the greatest antimicrobial activity was extracted by ethyl acetate.The extract was isolated through thin layer chromatography,column chromatography and preparative high performance liquid chromatography.A compound obtained was elucidated as 7-amino-4-mythylcourium.9.The antimicrobial test indicated that 7-amino-4-mythylcourium showed good inhibitory against the tested 10 bacteria,1 yeast and 2 moulds.Compared to the positive controls,7-amino-4-mythylcourium showed comparative activity,and had broader inhibitory spectrum.10.Antioxidant activity of the extracts of Xylaria sp.YX-28 was evaluated.A positive relation existed between the total phenolics and flavonoids content and the antioxidant activity,with the coefficients R² 0.7336 and 0.9392,respectively.The methanol extract had the highest total phenolics and flavonoids content and the DPPH scavenging activity.In theβ-carotene/linoleic assay,the methanol extract of Xylaria sp.YX-28 had a 72.9%bleaching activity at a concentration of 400μg·mL^-1,significantly higher than those of the positive controls BHT and Asc A,while exhibited moderate activity in DPPH scavenging assay.11.GC/MS were used to analyze the compositions of the methanol extract of Xylaria sp. YX-28.Several phenolics with antioxidant activity were identified,including 2-hydrazino-8-hydroxy-4-phenylquinoline,3,4-dimethoxy-phenol,2,4-bis(1,1-dimethyl ethyl)-Phenol,3,4-dihydro-8-hydroxy-3-methyl-isocoumarin,and ferruginol.