Isolation,Identification And Antibacterial Metabolites Of Mangrove Symbiotic Actinomycetes On The West Coast Of Hainan

Marine actinomycetes are an important part of marine microorganisms and one of the important sources for the production of new biologically active compounds.Because of its unique habitat,mangrove plants containing abundant marine actinomycetes have become an important research object for the isolation of marine actinomyceteet resources.Related research shows that actinomycetes from mangrove habitats can produce a variety of active secondary metabolites including antibacterial activity.Therefore,the actinomycetes from mangrove plants have become an important source for exploring new antibacterial drugs.In this paper,63 strains of actinomycetes were isolated and purified from 10 mangrove plant samples collected from the western coast of Hainan.16 representative strains of actinomycetes were selected for 16S rRNA sequence analysis.16 strains of actinomycetes were identified and belonged to genus Streptomyces and Micromonospora.Among them,14strains of actinomycetes belonged to the genus Streptomyces and 2 strains of actinomycetes belonged to the genus Micromonospora.The antibacterial activity of fermentation products from the isolated actinomycetes was analyzed by the filter paper diffusion method.The results showed that the crude fermentation extracts of 17 actinomycetes had antibacterial activity,of which 11 exhibited antibacterial activity against at least one of the four tested bacteria and 10 displayed antifungal activity against at least one of the four tested plant pathogenic fungus.The polyphasic approach was used to identify two novel actinomycete species HNM0645 and HNM0663.The strain HNM0645 was isolated from the mangrove plant Kandelia obovata in Chengmai,Hainan.Through 16S rRNA sequence analysis,strain HNM0645 belonged to the genus Streptomyces and shared the highest 16S rRNA gene sequence similarity of 99.51%with known Streptomyces species.the average nucleotide identity values(ANI)between strain HNM0645 and its close phylogenetic neighbours found to be 90.77%for S.wuyuanensis CGMCC 4.7042^T,89.46%for S.tirandamycinicus HNM0039^T,and 89.36%for S.spongiicola HNM0071^T,which were clearly below the95%-96%threshold used for prokaryotic species delineation.The cell wall of HNM0645contains LL-diaminopimelic acid.Whole cell sugar are galactose,glucose,and mannose.The main phospholipids include diphosphatidylglycerol,phosphatidylethanolamine,phosphatidylinositol,and phosphatidylcholine.The main cellular fatty acids are iso-C_16:0(27.70%),anteiso-C_15:0(17.34%)and anteiso-C_17:0(11.74%).The main menaquinone are MK-9(H₆)(74.90%),MK-9(H₄)(19.20%)and MK-9(H₈)(5.90%).The GC content of the genome was 71.65 mol%.Therefore,a combination of genotypic,chemotaxonomic and phenotypic data obtained above clearly shows that the strain HNM0645 should be considered as a new species within the genus Streptomyces and its name is proposed as Streptomyces kandeliae sp.nov.The strain HNM0663 was isolated from the mangrove plant Avicennia marina in Chengmai,Hainan.Through 16S rRNA sequence analysis,strain HNM0663 belonged to the genus Streptomyces and shared highest 16S rRNA gene sequence similarities to the type strains S.lichenis LCR6-01^T(98.67%),S.nanningensis YIM 33098^T(98.12%)and S.palmae CMU-AB204^T(97.93%),which were lower than the 98.70%threshold used for prokaryotic species delineation.The cell wall of HNM0663 contains LL-diaminopimelic acid.Whole cell sugar are galactose and mannose.The main phospholipids include diphosphatidylglycerol,phosphatidylethanolamine,phosphatidylinositol,and phosphatidylcholine.The main cellular fatty acids are iso-C_16:0(36.14%),iso-C_15:0(10.85%)and anteiso-C_15:0(9.67%).The main menaquinone are MK-9(H₆)(37.24%),MK-9(H₄)(36.91%)and MK-8(H₄)(25.84%).The genome GC content was 70.98mol%.The datas obtained above comprehensively shows that the strain HNM0663 was identified as a new species within the genus Streptomyces and its name is proposed as Streptomyces avicenniae sp.nov.A total of 4 compounds were isolated and identified from the crude fermentation extract of the novel strain HNM0645 using natural product isolation and identification technology,of which one was an indolocarbazole compound and three were anthracycline compounds.Compound CX-01 had antifungal activity against yam anthracnose pathogens and watermelon blight pathogens,with EC₅₀values of 286.61 and 439.43?g/m L,respectively.Compound CX-02 had antibacterial activity against the four test bacteria.