Phylogenetic Diversity Of Endophytic Bacteria From Populus Euphratica At The Original Forest Of Chakhilikh County And Taxonomic Identification Of A Novel Bacterium

The aim of this study was investigated the community phylogenetic diversity ofthe endophytic bacteria from Populus euphratica at the original forest of ChakhilikhCounty by using cultured-dependent method and454high-throughput sequencingtechnologies. The storage liquid samples were collected from15Populus euphraticatrees. A total of233bacteria strains were isolated by using seven types of differentcultural medium (LB，TSA、TSB、MA、BPA、R2Aand GLB). The most of bacterialcolony color were white (27.5%); Simple staining results showed that most of thebacteria cells form for rods (48.1%); gram staining results showed that among them144strains were gram-positive (60.9%),89strains were gram-negative (38.2). The16S rRNA gene sequence analysis showed that those isolates belonged toGammaproteobacteria (129/233,55.36%), Firmicutes (83/233,35.62%),Alphaproteobacteria (11/233,4.72%), Actinobacteria (8/233,3.43%) andBacteroidete (2/233,0.86%), respectively, encompassing29genera and67species.The Bacillus (12.02%) and Pseudomonas (14.59%) were the dominant genera inPopulus euphratica stem liquid. There were65isolates with16S rDNA similarityvalues≤98.65%, belonging to25species within12genera that could be representedas the indigenous potential new species.In this study for the first time to diversity analysis of the endophytic bacteriafrom Populus euphratica at the original forest of Chakhilikh County was carried outby using454high throughput sequencing technological process.621OTUs have beendetected based on the standard of16s rDNA similarity--97%and the method ofmothur software. Phylogenetic analysis results show that621OTU were belongs to18phylum (Acidobacteria, Actinobacteria, Bacteroidetes, Chlorobi, Chloroflexi,Cyanobacteria, Deferribacteres, Fibrobacteres, Firmicutes, Lentisphaerae,Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericute,Verrucomicrobia, no_rank_Bacteria, unclassified_Bacteria). Among them,Bacteroidetes was the dominant phylum that account for the total OTU’s42.92%. Total OTU belongs to34class,125genera and153species, the Clostridia (26.46%)is dominant class; the Proteiniphilum (15.22%) is dominant genera and theProteiniphilum_uncultured_bacterium (11.78%) is dominant specie. The resultsdemonstrated that the primers used in454high throughput sequencing could revealhigh diversity.Strain MA-69Tisolated from Populus euphratica in Ugan ancient river wascharacterized using a polyphasic approach. Strain MA-69Tcells are Gram-negative,non-motile, non-spore-forming, strictly aerobic short rods bacterium. After2-3daysof incubation on MA agar, colonies are1.0-2.0mm in diameter, circular, yellow andsmooth with entire margins.0.5-0.7μm wide and1.5-2.0long. Growth occursbetween25and37℃; the optimum temperature for growth is30℃. The pH rangefor growth is6.0-9.0, with an optimum pH7.5. Growth occurs in the presence of0-3%(w/v) NaCl, Optimum NaCl concentration for growth is1%. Strains MA-69Tarepositive for catalase and oxidase activities, resistant to streptomycin (10μg),kanamycin (30μg), chloramphenicol (5μg), tetracycline (30μg), gentamicin (10μg).Based on16S rRNA gene sequences analysis showed that that Strains MA-69Tbelonged to the genus Pseudomonas, showing the highest sequence similarities toPseudomonas azotifgens JCM12708T(97.46%), Pseudomonas balearica DSM6083T(97.05) and Pseudomonas indica DSM14015T(95.93%), respectively. Strain MA-69Texhibited mean DNA-DNA relatedness to the type strains of Pseudomonas azotifgensJCM12708T(62.9%), Pseudomonas balearica DSM6083T(49.0%). The G+C contentof the genomic DNA was64.1mol%. The major fatty acids were C19:0cyclo w8c，C16:0，C12:0, summed feature3(comprising C16:1w6c and/or C16:1w7c), summedfeature8(comprising C18:1w7c and/or C18:1w6c). Polar lipids of strain MA-69Tincludephosphatidylethanolamine (PE), phosphatidyl choline (PC), phosphatidylglycerol (PG),diphosphatidylglycerol (DPG) and four unidentified phospholipids (PL1-4). Thepredominant isoprenoid quinone was Q-9. Based on phenotypic, chemotaxonomic andphylogenetic properties, strain MA-69Trepresents a novel species of the genusPseudomonas, for which the name Pseudomonas tarimensis is proposed. The typestrain is MA-69T(=CCTCC AB2013065T=JCM19137T).