Polyphasic Taxonomy Study Of Endophytic Bacteria Isolated From Robinia Pseudoacacia Nodules With Lead-zinc Soil

Four potential novel bacteria species, which needed further analysis and identification, were found using 16 S r DNA phylogenetic analysis when researching on the endophytes diversity of Robinia pseudoacacia nodules collected from contaminated soil in lead-zinc mining area in Hanzhong by our group. Therefore, this study is to determine their taxonomic status by the phenotypic characteristics, chemical characteristics and molecular analysis of polyphasic taxonomical method.The results of phenotypic characteristics: Strain HZ7, colonies of strain HZ7 grown for 2~3days were 2~5mm in diameter, semitransparent, circular and protuberant, with a dulate margin. Cells were long rod with a size range of 0.3×1.5~2 μm and motile by means of a single polar flagellum; Gram-negative; MR-negative; catalase-positive; The range of growth p H is 5~10; The range of growth temperature is 10~37℃, but was very weak at 4 or 45℃; The strain was able to grow on YMA with 0%~1% Na Cl; The strain was sensitive to levofloxacin and cefotaxime. Strain HZ10, colonies of strain HZ10 grown for 2~3days were 3~5mm in width, circular and protuberant, with a dulate margin. Cells were long rod with a size range of 0.3×1.0~1.5 μm and motile by means of two polar flagella; Gram-negative; MR-negative(non-enterobacterial rods); catalase-positive; The range of growth p H is 5~9; The range of growth temperature is 10~37℃, but was very weak at 4 or 45℃; The strain was able to grow on YMA with 0% ~1% Na Cl; The strain was sensitive to Cefepime, kanamycin and ceftazidime; H2 S is not produced. Strain HZ28 and HZ34, colonies of strains grown for 2~3days were 2~3mm in width, circular and protuberant, with a dulate margin. Cells were rod with a size range of 0.3×1.0 μm and motile by means of a single polar flagellum; Gram-negative; MR-negative(non-enterobacterial rods); catalase-positive; The range of growth p H is 5~9; The range of growth temperature is 20~37℃, but was very weak at 10℃; The strain HZ28 was able to grow on YMA with 0%~1% Na Cl and the strain HZ34 coule grow on YMA with 0%~3% Na Cl; The strains were sensitive to Kanamycin and midecamycin.The reaults of fatty acid composition: Strain HZ7, the major cellular hydroxy fatty acids are C16:0(29.9%) and Sum In Feature 3(C16:1 w7 c and/or C16:1 w6 c,34.44%). Strain HZ10, the major cellular hydroxy fatty acids are C16:0(20.87%), Sum In Feature 3(36.55%)and Sum in feature 8(maybe C18:1 w7 c,23.08%). Strain HZ28, the major cellular hydroxy fatty acids are C16:0(36.99%), C17:0 cyclo(9.86%) and Sum In Feature 3(30.03%), Strain HZ34, the major cellular hydroxy fatty acids are C16:0(32.9%), C17:0 cyclo(13.18%) and Sum In Feature 3(25.17%).The results of molecular phylogeny:The 16 S r DNA gene sequences of strain HZ7 exhibited most similar with Mitsuaria chitosanitabida(99.05%), HZ7 is a number of Roseateles, combination with the results of G+C% and housekeeping gens rec A, photosynthetic genes, IGS. The 16 S r DNA gene sequences of strain HZ10 exhibited most similar with Herbaspirillum chlorophenolicum(98.51%), strain HZ10 is a number of Herbaspirillum, combination with the results of G+C% and housekeeping gens rec A, nif H, IGS. The 16 S r DNA gene sequence of strains HZ28 and HZ34 exhibited most similar with Achromobacter insolitus(98.22%), HZ28 and HZ34 are number of Achromobacter, combination with the results of G+C% and housekeeping gens rec A, IGS.The results of indicators analysis: Strain HZ7 might be a new number of Roseateles. Strain HZ10 might be a new number of Herbaspirillum. Strain HZ28 and HZ34 might be new number of Achromobacter. The DNA homology analysis is necessary to determine its taxonomic position.