Diversity Of Endophytic Bacteria In Bletilla Striata And Analysis Of Secondary Metabolites Of An Endophytic Bacterium BSR2010

Bletilla striata,an Orchidaceae plant,has medicinal value and developmental prospect.In recent years,due to the extensive application in pharmaceutical,medical,cosmetic,and even other fields,the demands for white blood have increased rapidly,resulting in the overexploitation of wild B striata,which led to a shortage of wild resources.Endophytes are highly diverse microbial resources,which parasitized in healthy plant tissues,organs as well as intercellular spaces without causing plant disease.Endophytes promote the growth and development of host and help host resisting the stress of pathogens and external conditions.In the long-term evolution process,endophytes produce the same,similar,or new active secondary metabolites as the host plants due to gene exchange between them.So far,a large number of metabolites produced by endophytes have been isolated and widely used in medicine and agriculture,which makes them with great development potential.In this paper,we sampled mature seedlings of B striata from Lijiang planting area of Yunnan Province.The object of this study is to analyze the diversity,composition,and functional characteristics of total endophytic bacteria in fibrous root,tuber,stem,and leaf of B striata by high-throughput sequencing technology with a traditional microbial isolation culture method.An endophytic bacterial(No.BSR2010)was isolated,which exhibited distinct antibacterial activity against four pathogenic bacteria strains,including Staphylococcus aureus,Escherichia coli,Micrococcus luteus,and Pectobacterium carolovorum.Therefore,the optimization of culture conditions,activity of metabolites,and isolation and purification were studied.(1)The diversity of total endophytic bacteria in fibrous root,tuber,stem,and leaf of B.striata was analyzed by Illumina Hi-seq high-throughput sequencing.The 16S rDNA of the all samples was investigated.The v5-v7 region in the samples showed that the bacterial flora in fibrous root was the most,followed by tuber,and leaf was the least.A total of 1168652 effective sequences were obtained and assigned to 30 phyla,79 classes,194 orders,369 families and 893genera.Bacteroides and Staphylococcus were the main dominant genera.The diversity of bacterial colonies was as follows:fibrous root>tuber>stem>leaf,and the composition,abundance and function of total endophytic bacteria were different among the four tissues.(2)Using a traditional method for microbial isolation and purification,204 strains of endophytic bacteria were isolated from tuber of B striata.The metabolites of the isolated endophytic bacteria were examined for bacteriostatic test and its related genes.Finally,an endophytic bacterium(BSR2010)with inhibitory effect on all four pathogenic bacteria was used for further analysis.In addition,metabolites of BSR2010 contained DAPG and NRPS related genes were deduced that the inhibitory effect of BSR2010 on 4 pathogens was due to the production of secondary metabolites with antibiosis,which provided ideas for the separation and analysis of later metabolites.(3)BSR2010 was identified and the culture conditions were optimized.The endophytic bacterium was initially identified as Pseudomonas.With the high antibacterial activity of its fermentation product,the optimal medium components contained 5g/L sucrose,10g/L soybean powder,and NaH₂PO₄.The optimal fermentation condition was as follows,5%inoculum size with initial pH 6.5,and 150 r/min rotation speed at 28?for 60 h.This condition for BSR2010will achieve the maximum yield of metabolites.(4)The types of secondary metabolites and extractant of strain BSR2010 were examined.BSR2010 likely produces lipopeptide secondary metabolites.The best extractant of secondary metabolites in our hands was ethyl acetate.BSR2010 was expanded and then the monomer compounds produced by strain BSR2010 were obtained by preparative HPLC.The compounds were identified as cyclo(tyr-pro),cyclo(pro-phe),(1H-indol-3-yl)-oxoacetamide,and 2',4',6'-trihydroxyacetophenone.All the four compounds isolated from endophytes of B striata were first reported,which provided medicinal value in the pharmaceutical industry.The results of this study enriched the research content of endophytic bacteria in B striata, and provided a good foundation for the development of endophytic bacteria resources and screening of new antibacterial compounds.It also provides a new way to obtain active components of B striata by endophytic fermentation.