Isolation, Identification And Polyphasic Taxonomy Analysis For Rhizobium Kunmingense SP.Nov.

The bacterial strain LXD30T was isolated from rhizosphere soil of a plant of the species Camptotheca acuminata Decne which is native to warm, humid stream banks south of China. Strains were isolated using the dilution plating technique on LB agar. The systematic taxonomy of the isolates have been done using polyphasic technology including morphological and cultural characteristic observation, physiological and chemical tests, analysis of chemtaxonomy and G+C content, DNA-DNA homology,16S rDNA sequences and recA and atpD gene sequences etc. The taxonomy status of these isolates were determined from three levels includling morphology, genetics and systematic development. The strain LXD30T was isolated from a field soil sample from Kunming in Yunnan Province, which belong to the genus of Rhizobium, was named Rhizobium kunmingense sp. nov.Gram-negative, aerobic, non-spore-forming, rod-shaped cells (0.6×1.6-2.9μm) with a single monopolar flagellum. After3days incubated on YMA agar, colonies are2.0-3.0mm in diameter, convex, pearl white, circle, opaque and smooth. Grows at15-34℃(optimum,28℃), at pH6.0-10.0(optimum, pH7.0) and with0-2%NaCl (optimum,0-1%). Positive for catalase and phenylalanine deaminase, but negative for oxidase. In API20E tests, positive for urease. Negative for β-galactosidase, lysine decarboxylase, arginine dihydrolase, arginine decarboxylase, ornithine decarboxylase, indole and H2S production, Voges-Proskauer reaction and nitrate reduction. Hydrolyses aesculin, and Tweens20,60and80, but not starch, DNA, casein or gelatin. The following substrates are utilized for growth:D-glucose, D-fructose, maltose, D-mannose, L-arabinose, D-ribose, cellobiose, D-xylose, D-galactose, L-rhamnose, melibiose, D-mannitol, gluconate, glycerol and N-acetyl-D-glucosamine. The following substrates are not utilized for growth:trehalose, salicin, D-lactose, sucrose, turanose, melezitose, raffinose, L-sorbose, adonitol, L-arabitol, i-erythritol, xylitol, D-sorbitol, inositol, dextrin, acetate, inulin, amygdalin, dulcitol, pyruvate and methy1-α-glucoside. Resistant to lincomycin (2μg), but sensitive to streptomycin (10μg), penicillin G (10IU), ampicillin (10μg), chloramphenicol (30μg), erythromycin (15μg), tetracycline (30μg), ofloxacin (5μg), amoxicillin (10μg), cefamezin (30μg), rifampicin (5μg), gentamicin (10μg), ceftazidime (30μg), vancomycin (30μg), kanamycin (30μg), neomycin(30μg), carbenicillin (100μg) and polymyxin B (30μg). The major fatty acids are C16:0(17.5%) and summed feature7(C18:1ω7c/ω9t/ω12t,58.3%). The G+C content of the DNA is59.3mol%.