Biodiversity And Bioactive Metabolites Of Endophytic Fungi From Medicinal Plants Taxus And Huperzia Serrata

Endophytic fungi are a group of microborganisms with great diversity and become a research hotspot of ecology, agriculture and medicine. Many of them have capacities to produce plant secondary metabolites or even new compound. Hence, endophytic fungi, as microbial cell factories, have great potential for producing plant secondary metabolites and an important source of new medicine.Firstly,609and155strains of endophytic fungi were isolated from Taxus and Huperzia serrata, respectively, by conventional isolation method. According to their morphological characteristics and molecular identification,67strains with typical phenotype from Taxus were classified into15genera, and19strains from Huperzia serrata were identified as8genera and1unclassified Sordariomycetes. Among them, Glomerella and Peyronellaea were firstly isolated from Taxus, and Neurospora and Mucor were first report from Huperzia serrata.Direct PCR method using metagenome, as a proof-of-concept, were firstly employed to study the diversity of endophytic fungi from Taxus. The total DNA (metagenome) were extracted from Taxus, and ITS squences of fungi were amplified using the specific primers ITS1and ITS4. Considering≥99%of sequence identity as a standard,41genetype were obtained from107positive clones including5genera isolated by conventional method such as Fusarium and Cladosporium. Other genera were only detected by direct PCR method such as Mycosphaerella、Mrakiella and unclassified fungi. Therefore, combination of these two methods could more accurately estimate the diverstiy of endophytic fungi.Taxol-producing fungi were screened by the method of gene detecting. The conserved sequences of three key genes (bapt, dbat and ts) were selected as molecular probes. Forty strains were detected as PCR positive hits. After fermentation and HPLC-MS detection, Guignardia mangiferae HAA11, Fusarium proliferatum HBA29and Colletotrichum gloeosporioides TA67were proved to have capability of producing taxol. Strain HAA11was the first Guignardia from Taxus that enables to produce taxol.Finally, alkaloid-producing fungi were screened by chemical method based on the reaction between Dragendorff’s reagent and alkaloid. Four strains (B14, SSA18, SSA5and DW42) gave positive hits with Dragendorff’s reagent, and may produce alkaloid. The Dragendorff-positive compound was isolated from the culture of strain B14, and is a new compound with M/Z549and formula C23H43N5O10. The bioactivity of compound B14is under further study.