Genetic Diversity And Horizontal Transfer Of NifH Gene From Pongamia Pinnata In Guangdong, China

Pongamia pinnata is widely distributed in Southeast Asia to North Australia, and also in Guangdong, Hainan, Fujian, Taiwan provinces in China. The genetic phylogeny and horizontal gene transfer of12rhizobium strains isolated from Pongamia pinnata in Guanzhou and Zhuhai were analyzed by16S rDNA, housekeeping genes (atpD, glnII, recA) and nifH sequences. In addition, two Agrobacterium strains were analyzed by the DNA homology analysis, cell fatty acid composition analysis, physiological and biochemical characteristics analysis.16S rDNAPCR-RFLP analysis divided12Pongamia rhizobial strains into genus Sinorhizobium, Ochrobactrum, Rhizobium, and they were belonged to four species, i.e. Ensfer adhaerens, S.morelense, R.alamii, R.multihospitium and O.lupini; and the result is consistance with housekeeping genes (atpD, glnll, recA) analysis.The phylogenic tree constructed by nifH gene sequences divided12rhizobial strains into three groups. Group1contained PIN13, PIN14, PIN27, Azotobacter chroococcum and Pseudomonas, Group2including strain PIN4, PIN6, PIN12, PIN23and Klebsiella pneumoniae NG14T; Group3consists of strain PIN5, PIN8, PIN10, PIN19, PIN26, Bradyhizobium, and some uncultured bacterium. Obviously, the nifH gene horizontal transfer and recombination was occured during the evolution of Pongamia strains.Two Agrobacterium strains were characterized by16S rDNA, atpD, recA, glnll, biophysiologic aspects,(G+C) mol%content determination, and DNA-DNA hybridization.16S rDNA sequence analyses showed that, these two strains were closely related to the Ag. tumefaciene NCPPB2437T, the similarity was99.6%between the two strains and Ag.tumefaciense. The similarity of housekeeping genes (atpD, glnlI, recA) was94.4%,93.8%and91.4%with Ag. tumefaciense IAM13129T. DNA hybridization tests showed that between PIN15and PIN17and Ag. mmefaciense IAM13129T was48.9%and49.3%, respectively.Based on the above results, a new species was determined, and the description of the new species was as follow:rods, G-,1.6～2.5×0.8μm, round slime colony when grow on the yeast-mannitol agar plate, the optimal growth temperature is28℃, they can utilizate includes sucrose, Sodium acetate trihydrate, lactose, D-fructose, L(+) rhamnopyranose, D-Mannose, inositol, L-threonine, L-aspartic element, L-proline, L-Cysteine, L-Tyrosine. This species could grow on YMA medium at original pH5, and was tolerant with4%and5%NaCl in YMA, but was sensitive to5μg/ml of kanamycin. The fatty acid composition of PIN15, PIN17showed that they have no C15:0iso, C15:0anteiso, C18:0, C18:1w9c, C19:010-methyl, compared with Agrobucterium tumefaciens NCPPB2437T. The G+C mol%content of PIN15and PIN17were57.6%,57%, respectively. The new species was termed as Agrobacterium dafushanense sp. nov., and the type strain was PIN15.