Studies On The Fungal Diversity From The Soil Microbial Flora Of Ophiocordyceps Sinensis And Polyketide Synthase (PKS) And Non-ribosomal Peptide Synthetase (NRPS) Genes Diversity

Ophiocordyceps sinensis is results of parasitism of O.sinensis on underground ghost moth larvae on the Tibetan Plateau in China,which is well renowned in traditional Chinese medicine.It is mainly distributed in Qinghai-Tibetan Plateau,includeing Tibet,Qinghai,Gansu,Sichuan,Yunnan Province,and marginal region of Himalaya,such as Nepal,the Kingdom Bhutan and India.The anamorph of O.sinensis has been widely recognized as Hirsutella sinensis.However,there are still,numerous fungi in natural O.sinensis,which are important fungal resources.Several of these fungi have been found to have similar chemical components and pharmacological activities to O.sinensis,and fermented mycelial products have been developed and applied in clinical practice.These fungi are hypothesized to involve in the process of natural O.sinensis formation.Therefore,it is necessary to conduct a comprehensive investigation on the fungal diversity in natural O.sinensis.In this paper,the diversity of fungi from the soil in natural O.sinensis is studied,which aimed to fully understand the fungal groups that exist in them.O.sinensis mainly grows in alpine shrubs and meadows at an elevation of about 3500⁵000 m,especially in snow-covered and well-drained alpine meadows.Alpine meadow is a microorganism resource treasury with germplasm novelty and diversity.Due be discovered from to the distinct environment,various novel bioactive substances may be the polar microorganisms.Study on functional genes with secondary metabolic provide evidence for molecular biology of microbial secondary metabolites and that also could provide genetic resources for future the relevant functional genes of in-depth studies of the polar from the biosynthetic pathway,and that also could provide genetic resources for future development.The main results were as follows:1.Based on high-through sequencing method,the population compositions of fungi associated with the soil microbial flora O.sinensis indicated that there are significant differences in fungal community between soil and external mycelial cortices?EMC?,and 5 phylum,15 class,41 order,79 family and 112 genus.A total of 66 well-known genera fungal taxa were detected in the soil area.Among them,Inocybe,Archaeorhizomyces,Tomentella and Thelephora were four dominant groups in soil and accounting for 53.32%,8.69%,8.12%,7.21%,respectively.A total of 43 well-known genera fungal taxa were detected,of which Ophiocordyceps and sebacinia were dominant genera,and the proportion of them were 95.86% and 1.14%,respectively.Furthermore,A total of 34 genera are common in the soil and EMC,there are 32 genera in the soil and 9 genera in the EMC were unique.2.Seventy-seven fungal isolates were obtained from the soil microbial flora O.sinensis.on the basis of morphological characteristics and analysis of ITS gene sequence,the isolates from the soil microbial flora O.sinensis were assigned to 2 phylum,4 class,9 order,21 family and 33 genus.They were Ophiocordyceps,Trichoderma,Cytospora,Truncatella,Dactylonectria,Isaria,Cephalosporium,Fusarium,Cosmospora,Paecilomyces,Tolypocladium,Cercophora,Beauveria,Neonectria,Microdochium,Coniochaeta,Lecanicillium,Ilyonectria,Stagonosporopsis,Cladosporiu,Pleotrichocladium,Leptosphaeri,Chaetosphaeronema,Paraphaeosphaeria,Preussia,Neosetophoma,Leptodontidium,Chalara,Pseudogymnoascus,Geomyces,Tetracladium,Mucor,Mortierella.Among them,Mortierella and Trichoderma were two dominant groups,accounting for 21.33% and 10.67% of the total strains,respectively.3.PKS and NRPS genes were amplified from the 77 strains fungi by using PKS?KAF1/KAR2?and NRPS?AUG003/AUG006?primers which were designed according to the different biosynthetic pathway of bioactive substances.The results showed that PKS genetic fragments were amplified from 15 strains and the positive rate was 19.48%.NRPS genetic fragments were amplified from 31 strains and the positive rate was 40.26%.Two different PKS genetic fragments were respectively amplified from strain Fusarium verticillioides?JM22?,Hypocrea pachybasioides?JM112?and Helotiales sp.?JM125?.PKS and NRPS genetic fragments were amplified simultaneously from strain Trichoderma paraviridescens?JM31?,Trichoderma polysporum?JM100?,Hypocrea pachybasioides?JM112?,Leptosphaeria sclerotioides?TY110?,Trichoderma polysporum?TY128?,Pseudogymnoascus sp.?TY165?,Paecilomyces hepiali?TY187?and Ophiocordyceps sinensis?ZG₁?which deserved further study.