Genetic Diversity And Phylogeny Of The Rhizobia Isolated From Leguminous Tree In Arid-hot River Valley Area In Panxi, Sichuan, China

The rhizobial strains isolated from the nodules of Leucaena leucocephala, other leguminous trees including Robinia pseudoacacia, Amorpha fruticosa, Dalbergia, Acacia confusa and Desmodium, growing in arid-hot river valley area in Panxi, Sichuan, China, were characterized by the genetic diversity including analysis of 16S,IGS,23S rDNA PCR-RFLP and phylogeny of 16S rDNA,atpD,recA genes sequences. Besides, the symbiotic efficiency and nifH genes sequences of L. leucocephala rhizobia were also characterized.The results of the inoculation test on their original host plants showed that the majority (85%) of 42 Leucaena rhizobia were ineffective, even about 15% of them were hurtfully parasitic rhizobia. High-effective rhizobia in nitrogen fixing efficency nodulated moderate nodules per plant, whereas rhizobia which formed a good many of nodules were usually ineffective strains.The majority strains of 42 Leucaena rhizobia or 56 other leguminous tree rhizobia were in good agreement with genus level among four methods 16S, IGS,23S RFLP and 16S-IGS-23S PCR RFLP. A total of 11,11,30 (or 17,17,32) genotypes of 42 Leucaena rhizobia (or 56 other leguminous tree rhizobia) were acquired from the RFLP analysis of the 16S, IGS,23S rRNA genes with same restriction endonucleases, respectively. The RFLP analyses and phylogenetic analyses based on the 16S rRNA gene sequences of representative strains showed that 32 (76.2%),5 (11.9%),2 (4.8%),2 (4.8%) of L. leucocephala rhizobia apart from SCAU193 belonged to Sinorhizobium, Bradyrhizobium, Rhizobium and Mesorhizobium, and that 19 (34.5%),18 (32.7%),10 (18.2%),8 (14.6%) of 56 rhizobia apart from SCAU257 isolated from other leguminous tree belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Agrobacterium, respectively. Inoculation test and phylogenetic analysis of 16S rRNA genes sequence indicated that SCAU193 were able to nodulate Leucaena leucocephala plants and the closest related genus is Herbaspirillum within theβ-proteobacteria. This is the first record that Herbaspirillum ofβ-proteobacteria can be the specific symbionts of leguminous plant. Phylogenetic analysis of 16S rRNA genes sequence indicated that SCAU257 were the closest related genus is Herbaspirillum within theβ-proteobacteria as well, but nodulation ability of SCAU257 were not tested by plant inoculation assay on their original host plants.Phylogenetic analysis of the 16S rRNA, atpD, recA or nifH genes of representative rhizobia apart from SCAU202,SCAU209 and SCAU211 were in good agreement at the genus level. SCAU202 which shared 99.9% with defined Rhizobium elti in 16S rDNA phylogenetic tree, were members of the genus Mesorhizobium in three phylogenetic trees of atpD, recA and nifH genes. SCAU209 were members of the genus Rhizobium in three phylogenetic trees of 16S rDNA atpD, and recA genes, but was member of the genus Sinorhizobium in the nifH phylogenetic tree. SCAU211 were members of the genus Bradyrhizobium, Rhizobium in three phylogenetic trees of 16S rDNA, recA and nifH genes, of atpD gene, respectively. The incongruent phylogenies of the genes studied suggest that horizontal gene transfer and recombination shape Leucaena rhizobia populations in the gene center of the host plants.NifH Sequences of 7 representative strains isolated from Leucaena leucocephala of SCAU191, SCAU192, SCAU196, SCAU197, SCAU213, SCAU226, SCAU228 were wholly the same. But SCAU191, SCAU196 were significantly effective, the other 5 strains were ineffective on the symbiotic efficiency. Moreover, The similarity of SCAU191 and SCAU192 sequences of 16S rDNA, atpD, recA genes respectively, were all 100.0%. The above results implied that the symbiotic efficiency of rhizobia had nothing to do with 16S rDNA, atpD, recA and nifH sequences, and were possibly connected with fix gene or other genes of regulating and controling nifH.