Phylogenetic Diversity Of Endophytic Bacteria From The Populus Euphratica At The Tarim River And Taxonomic Identification Of A Novel Bacteirum

In this study, we investigated the community diversity of the culturableendophytic bacteria in Populus euphratica at the Tarim river by usingcultured-dependent method and16S rDNA sequencing. A total of167phenotypicallydifferent isolates were obtained from the six Populus euphratic’s storage liquidsample at the two sites by using five types of medium (LB, TSA, NB, KA, R2A). Thepopulation numbers of endophytic bacteria were8×10~7cfu/ml (TSA),1.6×10~7cfu/ml(NB),1×10~7cfu/ml (LB),2.2×10~7cfu/ml (KA) and0.64×10~7cfu/ml (R2A),respectively.16S rRNA gene sequence analysis and phylogenetic analysis showedthat167strains belonged to Gamma-proteobacteria, Alpha-proteobacteria, Firmicutesand Actinobacteria, respectively. Among them108(64.7%) strains belonged toGamma-proteobacteria, including Pseudomonas, Planococcus and other six genera;39(23.3%) strains belonged to Firmicutes, including Bacillus, Aerococcus and otherfour genera;11(6.6%) strains belonged to Actinobacteria, including genus ofNesterenkonia, Kocuria and Sanguibacter;9(5.3%) strains belonged toAlpha-proteobacteria, including genus of Rhizobium and Roseomonas. All of thestrains were classified into19genera and39different species. Pseudomonas waspredominant genus and account for38.8%of the total isolates. Strain RK7-2, W9,W13, RN7, W22, W17, RL5, RT17-2, RN3-2, RT4, RT41, RN23-4and RL21havelower than98.7%sequence similarities with their closest type strain, so they initiallyare considered as potential new species. The obtained data demonstrated that bacteriadiversity in the storage liquid of Populus euphratica at the Tarim river was abundant.Polyphasic analysis of strain ML-64showed that, strain ML-64cells areGram-positive, endospore-forming and rod-shaped. Colonies are pale-yellow, circular,entire margin and2-4mm in diameter after48h incubation at37°C on TSA agar.Temperature range for growth is10–45°C, with optimal growth occuring at37°C. ThepH range for growth is6.0–9.0(optimum pH7.0). The NaCl concentration range for growth is0–6%(optimum3%). Cells are positive for Alkalin phosphatase,,Acidphosphatase, Naphthol-AS-BI-phosphohydrolase,β-Galactosidase,α-Galactosidase,β-Glycosidases, Arginine dihydrolased, urease, Tryptophan deaminase, catalase,oxidase, Voges–Proskauer test. No sugars were fermented in the API50CH strips.L-Serine, Methyl Pyruvate, α-Keto-Butyric, Acetoacetic Acid are oxidized. Resistantto polymyxin b (30μg), novobiocin (30μg), peillin G (10U).16S rRNA genesequence demonstrated that the strain ML-64was closely related to Lysinibacillusmassiliensis CIP108446T(97.14%), Lysinibacillus odysseyi NBRC100172T(96.68%),Lysinibacillus xylanilyticus XDB9T(97.11%). DNA-DNA relatedness were22.2%and50.9%with Lysinibacillus xylanilyticus and Lysinibacillus massiliensis,respectively. The genomic DNA G+C content of the strain ML-64was36.8mol%.Major fatty acids were iso-C15:0(55.05%), anteiso-C15:0(20.70%). The predominantmenaquinone is MK-7. Based on the phylogenetic and genotypic analyses, the strainML-64is concluded to represent a novel species in the genus Lysinibacillus, forwhich the name Lysinibacillus Tograkense sp. nov. is proposed.