Diversity And Polyphasic Taxonomy Of Actinomycetes From Hunan Yanling Alpine Wetland

Actinomycetes have great application prospects in many fields such as medicine,food and agriculture due to their ability to produce beneficial metabolites.Recent studies have indicated that certain strains,especially actinobacteria strains,from natural wetland ecosystem may show promising potential for the production of bioactive secondary metabolites.The Yanling alpine wetland in Hunan Province is an undeveloped natural environment and there are few reports on soil actinomycetes diversity and functional metabolism in this area.Therefore,a study on the resources of actinomycetes from Yanling alpine wetland soil makes theoretical and practical sense.In this study,high-throughput sequencing technology was used for identifying the diversity of actinomycetes in soil samples from the Yanling alpine wetland.Then,the culturable actinomycetes were obtained by different isolation methods from the soil sample with the highest abundance of actinomycetes,and the potential novel species were identified by polyphasic taxonomy.Finally,the antibacterial activity of isolated strains was evaluated for gram-positive bacteria and gram-negative bacteria.The main results obtained are as follows:(1)High-throughput sequencing results showed that the effective sequence of actinomycete primer amplification was 157,236.For actinobacteria,14 populations were detected at the level of order,including Frankiale(41.9%),Acidimicrobiales(28.0%),and Solirubrobacteralse.(8.6%),Corynebacteriales(4.8%),Micrococcales(4.0%),Gaiellales(3.3%),Micromonosporles(2.9%),Propionibacteriales(1.7%),Streptomycetales(0.9%),Kineosporiales(0.8%),Catenulisporales(0.6%),others(0.4%),norank-Actinobacteria(0.4%)and Pseudonocardiales(0.2%).The wetland soil environment is rich in Frankiale,while Streptomycetales accounts for only 0.9% of the soil in these samples.(2)46 strains of actinomycetes were isolated from the alpine wetland of Yanling Province.The diversity of 46 strains was researched by classical taxonomy.46 strains of actinomycetes were initially classified into 10 different color groups based on the color of aerial mycelia or the soluble pigment produced;Through the culture characteristics,morphological characteristics and 16 s r RNA sequence analysis of these 46 strains,the results showed that 39 strains belonged to Streptomyces and 3 strains belonged to Kitasatospora,4 strains belonged to Nocardiospsis,Pseudonocardia,Amycolatopsis,and Micromonospora,respectively.The evolutionary status of 39 strains of Streptomyces and 7 strains of non-Streptomyces were confirmed through 16 S r RNA sequence analysis of system development.The results showed that Streptomyces strains GSSD-08,GSSD-12,GSSD-14,GSSD-23,GSSD-25,GSSD-28,GSSD-30 and GSSD-45,non-Streptomyces strains GSSD-48 and GSSD-50 may be a potential new species.(3)Strain GSSD-12 T was identified by polyphasic taxonomy.Strain GSSD-12 T,aerobic,gram-stain positive actinobacterium which forms an extensively branched substrate mycelium and aerial mycelium that differentiates into loose spiral spore chains.The cell wall contains alanine,glycine,glutamic acid and LL-diaminopimelic acid.The whole cell sugar pattern contains glucose.The predominant menaquinones are MK-9(H4),MK-9(H6)and MK-9(H8).The major cellular fatty acids were identified as iso-C16:0,C16:0 and iso-C14:0.The diagnostic phospholipid is diphosphatidyl glycerol,phosphatidyl ethanolamine,phsphotidylinositol mannosides and phosphotidyl inositol.The G+C content of the genomic DNA of the strain GSSD-12 T is 71.2 mol%.BLAST search of the full-length 16 S r RNA gene sequence of strain GSSD-12 T indicated it was within the genus Streptomyces,and displayed highest similarity with Streptomyces scopuliridis NRRL B-24574T(99.2%)and less than 98.6% of the similarity with other recognized species of the genus Streptomyces.Phylogenetic analysis based on 16 S r RNA gene sequences showed that strain GSSD-12 T was most closely related to S.scopuliridis NRRL B-24574 T,S.odonnellii NRRL B-24891 T and S.lushanensis NRRL B-24994 T.However,the digital DNA–DNA hybridization value,the average nucleotide identity value and the MLSA evolutionary distance between this strain and the closest relatives showed that it belonged to a distinct species.Furthermore,strain GSSD-12 T was also distinctly differentiated from them by morphological,physiological and biochemical characteristics.Therefore,strain GSSD-12T(=CICC 11051 T =JCM 33018T)represents a novel species of the genus Streptomyces,for which the name Streptomyces palus sp.nov.is proposed.(4)The antibacterial activity of strains isolates in this study showed that 7 strains of Streptomyces in preliminary test have antagonistic effects on gram-positive bacteria(Staphylococcus aureus)or negative bacteria(Escherichia coli),except GSSD-02.Strain GSSD-02 only inhibits Staphylococcus aureus.However the other 6 strains have inhibitory effect on both Staphylococcus aureus and Escherichia coli.The same strains have different resistance effects in different media.In yeast-malt extract agar(ISP2),oatmeal agar(ISP3)and peptone-yeast extract iron agar(ISP6)have better antagonistic activity,followed by Gause's agar medium.In the repeated experiments,the antagonistic resistance of the strains showed different degrees of degradation.The antagonistic strains capable of stabilizing were GSSD-07,GSSD-21,GSSD-43 and GSSD-49.These results indicate that some strains grown in the alpine wetland soil of Hunan have antibacterial activity,which may be the valuable resources for potential antibiotics.