Species Separation And Phylogenetic Analysis Of Actinomycetes In Nihewan Ancient Site

In order to analyze the species diversity of cultivable actinomycetes isolated from Nihewan Ancient Site and establish the information repository of actinomycetes of this region,samples from eight areas of Nihewan Ancient Site were separated by the dilution coated plate method,and coated on the separation medium of R₂A⁺?G⁺and Bennett.Finally,1088 pure cultures of actinomycetes were isolated,numbered and preserved,then analyse the phylogeny and acquire the research results as follow:1.410 strains of these actinomycetes were randomly selected,and preliminarily identified by the specific primers of Streptomyces.The results show that the bands of specific PCR amplification of 275 strains actinomycetes were around 840 bp.Therefore,275 strains actinomycetes belong to Streptomyces.2.16S rDNA sequence determination was used for the phylogenetic analysis of the remaining 135 unidentified strains.Finally,the 135 strains belong to 8 suborders,10 families,16 genera and two unknown genera of Actinomycetales,Actinobacteridae,Actinobaceteria,Actinobaceteria.The genera of the strains are Saccharopolyspora?Pseudonocardia?Microbacterium?Amycolatopsis?Actinoplanes?Saccharothrix?Glycomyces?Kribbella?Promicromonospora?Streptomyces?Nocardiopsis?Micromonospora?Micrococcus?Lentzea?Nocardia and Nocardioides,respectively.3.In this study,two new genera were discovered.(1)Among of these strains,the 16S rDNA sequence similarity of the strain 18-64 and the most similar strain Haloglycomyces albus YIM 92370^T is 93.64%,which suggests that it may belong to a potential new genus.(2)The 16S rDNA sequence similarity of the strain 12-9 and the most similar strain Nocardioides albus KCTC 9186^T is 93.73%,which suggests that it may be a potential new genus.4.In this study,four new species were discovered.(1)The 16S rDNA sequence similarity of strain 1-22 and the most similar strain Streptomyces pratensis ch24^T is 96.78%,which suggests that it may belong to a potential new species of Streptomyces.(2)The 16S rDNA sequence similarity of strain 3-27 and the most similar strain Streptomyces iconiensis BNT558^T is 96.66%,which suggests that it may be a potential new species of Streptomyces.(3)The 16S rDNA sequence similarity of strain 3-29 and the most similar strain Streptomyces iconiensis BNT558^T is 96.34%,which suggests to be a potential new species of Streptomyces.(4)The 16S rDNA sequence similarity of strain 33 and the most similar strain Lentzea flaviverrucosa AS4.0578^T is 95.72%,which suggests that it may belong to a potential new species of Lentzea.5.Among the 16 genus of Actinomyces isolated from Nihewan Ancient Site,there were12 genus to be consistent with the result of Illumina MiSeq high-throughput sequencing,and the other 4 genus were not detected by high-throughput sequencing,while 25 of 37 genus detected by Illumina MiSeq high-throughput sequencing were not separated;9 of 16 genus Actinomyces isolated from Nihewan Ancient Site were detected by 16S rDNA clone library technology,and the other 7 genus were not detected by 16S rDNA clone library technology,while 33 of 42 genus detected by 16S rDNA clone library technology were not separated.There has been difference between the results of high-throughput sequencing analysis and clone library technology with species separation,so it's necessary to continuously improve the technology and method.