| Huperzia serrata,also known as the Melaleuca serrata,is a perennial herbaceous plant of Huperziaceae.Huperzine A is an acetylcholinesterase(AchE)inhibitor,which has a significant effect on the prevention and treatment of Alzheimer's disease.At present,wild Huperzia serpentina resources have been overharvested,and the development of artificial cultivation is relatively lagging behind,which can not meet the market demand.According to the diversity analysis of endophytic fungi in Huperzia serrata,it was found that Huperzia serrata has abundant endophytic fungi.A large number of endophytic fungi were isolated by tissue isolation method.The endophytic fungi were identified by morphology and molecular biology.And the strain producing huperzine A was screened by HPLC and ESI-MS.At the same time,endophytic fungi have biological control effect.Through screening test of dual cultures,producing volatile antifungal organic compounds and PDB cultural filtrates,the strains for controlling Botrytis cinerea and Sclerotinia sclerotiorum were screened out.These results lay a foundation for obtaining endophytic fungi producing huperzine A with high efficiency and industrialized production of huperzine A,and provided germ resources for biological control agents of Botrytis cinerea and Sclerotinia sclerotiorum.The specific research results are as follows:1.Using Illumina MiSeq high throughput sequencing technology,the amplified ITS sequences were detected.Through OTU and species community analysis,Alpha Diversity analysis,Beta Diversity analysis and other analysis methods,the diversity of endophytic fungi in Huperzia serrata was analyzed.There were 1,800 strains endophytic fungi in the Huperzia serrata,and 824 strains,1045 strains and 1167 strains in the roots,stems and leaves,respectively.Among them,295 strains were distributed in roots,stems and leaves,but only endophytic fungi contained specific tissue roots,stems and leaves of 311 strains,240 strains and 308 strains,respectively.The abundance of endophytes fungi in the roots stems and leaves of Huperzia serrata: leaf>stem>root.And the endophytic fungi community structure of the organs was significantly different.The diversity of endophyticfungi in Huperzia serrata provided abundant information and resources for screening endophytic fungi producing huperzine A efficiently.2.Through PERMANOVA analysis,eleven genus(Derxomyces,Lophiostoma,Cyphellophora,Devriesia,Serendipita,Kurtzmanomyces,Mycosphaerella,Conoideocrella,Brevicellicium,Piskurozyma,Trichomerium)were found to be positive relationship with the content of Huperzine A.Derzomyces had the highest correlation index with Huperzine A(Maximum correlation index CI=0.92).It is speculated that endophytic fungi of these 11 genera has the potential to replace Huperzia serrata in producing Hup A.At the same time,the results of inter-species correlation analysis showed there are positive correlations between various endophytic fungi and endophytic fungi of these 11 genera.The beneficial effects of these endophytic fungi on Huperzine A-producing fungi provided information for excavating endophytic fungi that produce Huperzine A efficiently.3.180 endophytic fungi were isolated and cultured from Huperzia serrata.50 endophytic fungi were identified by morphological observation and ITS sequence analysis.Colletotrichum boninense HS7-1,a huperzine-producing strain,was screened by HPLC and ESI-MS,which laid a foundation for the industrialization of huperzine-producing endophytic fungi.4.There were 7 strains of endophytic fungi of Huperzia serrata which inhibited the growth of Botrytis cinerea through dual cultures.Producing volatile antifungal organic compounds and PDB cultural filtrates showed that Alternaria alternate HR38-7 had a stable inhibition of Botrytis cinerea.and the resultes provided strain resources for obtaining biocontrol agents against Botrytis cinerea.5.There were 9 strains of endophytic fungi of Huperzia serrata which inhibited the growth of Sclerotinia sclerotiorum through dual cultures.Producing volatile antifungal organic compounds and PDB cultural filtrates showed that Colletotrichum boninense HL33-6-1,Colletotrichum gloeosporioides HL33-12-3,Alternaria alternate HR38-7 had a stable inhibition of Sclerotinia sclerotiorum.The inhibition of Colletotrichum boninense HL33-6-1 wsa the best.Which provides strain resources for obtaining biocontrol agents against Sclerotinia sclerotiorum. |
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