Case of localized recombination in 23S rRNA genes from divergent bradyrhizobium lineages associated with neotropical legumes

Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5' portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5' 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii.     

Case of Localized Recombination in 23S rRNA Genes from Divergent Bradyrhizobium Lineages Associated with Neotropical Legumes

Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes (Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis, and Desmodium axillare) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes (Platypodium and Machaerium) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii.     

Diversity and relationships of bradyrhizobia from legumes native to eastern North America

DNA sequencing and polymerase chain reaction (PCR) assays with lineage-specific primers were used to analyze the diversity of 276 isolates of Bradyrhizobium sp. nodule bacteria associated with 13 native legumes species in the northeastern United States, representing eight genera in six legume tribes. A PCR screen with two primer pairs in the rRNA region indicated that seven of the legume species were exclusively associated with strains having markers resembling Bradyrhizobium elkanii, while the remaining six host species harbored strains related to both B. elkanii and Bradyrhizobium japonicum. Sequence analysis of 22 isolates for portions of 16S rRNA and 23S rRNA yielded congruent phylogenetic trees and showed that isolates from different legume genera often shared similar or identical sequences. However, trees inferred from portions of two other genes (alpha-ketoglutarate dioxygenase gene (tfdA), the alpha-subunit of nitrogenase (nifD)) differed significantly from the rRNA phylogeny. Thus, for Bradyrhizobium populations in this region, lateral gene transfer events appear to have altered genealogical relationships of different portions of the genome. These results extend the number of likely cases of gene transfer between divergent taxa of Bradyrhizobium (from members of the B. elkanii lineage to the B. japonicum group) and suggest that transfers have also occurred among separate subgroups of the B. elkanii lineage.     

RRNA and dnaK relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legume trees in Costa Rica

Enzyme electrophoresis and sequencing of rRNA and dnaK genes revealed high genetic diversity among root nodule bacteria from the Costa Rican trees Andira inermis, Dalbergia retusa, Platymiscium pinnatum (Papilionoideae tribe Dalbergieae) and Lonchocarpus atropurpureus (Papilionoideae tribe Millettieae). A total of 21 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 36 isolates analyzed, and no ETs were shared in common by isolates from different legume hosts. However, three of the ETs from D. retusa were identical to Bradyrhizobium sp. isolates detected in prior studies of several other legume genera in both Costa Rica and Panama. Nearly full-length 16S rRNA sequences and partial 23S rRNA sequences confirmed that two isolates from D. retusa were highly similar or identical to Bradyrhizobium strains isolated from the legumes Erythrina and Clitoria (Papilionoideae tribe Phaseoleae) in Panama. rRNA sequences for five isolates from L. atropurpureus, P. pinnatum and A. inermis were not closely related to any currently known strains from Central America or elsewhere, but had affinities to the reference strains Bradyrhizobium japonicum USDA 110 (three isolates) or to B. elkanii USDA 76 (two isolates). A phylogenetic tree for 21 Bradyrhizobium strains based on 603 bp of the dnaK gene showed several significant conflicts with the rRNA tree, suggesting that genealogical relationships may have been altered by lateral gene transfer events.     

Diverse rhizobia associated with woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the temperate zone of China

Fifty-nine bacterial isolates from root nodules of the woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the central and eastern regions of China were characterized with phenotypic analysis, PCR-based 16S and 23S rRNA gene RFLP, Box PCR and 16S rRNA gene sequencing. Seven main phena were defined in numerical taxonomy, which corresponded to distinct groups within the genera Agrobacterium, Bradyrhizobium, Mesorhizobium and Rhizobium in 16S and 23S rRNA gene PCR-RFLP. The phylogenetic relationships of the 16S rRNA genes supported the grouping results of PCR-RFLP. Most of the isolates from Amorpha fruticosa were classified into two groups closely related to Mesorhizobium amorphae. Seventeen of the 21 isolates from Wisteria sinensis were identified as two groups related to Rhizobium and Agrobacterium. Six out of 10 isolates from Cercis racemosa were identified as a group related to Bradyrhizobium. Our results indicated that each of the investigated legumes nodulated mainly with one or two rhizobial groups, although isolates from different plants intermingled in some small bacterial groups. In addition, correlation between geographic origin and grouping results was found in the isolates from Amorpha fruticosa. These results revealed that the symbiotic bacteria might have been selected by both the legume hosts and the geographic factors.     

Prevalence of Burkholderia sp. nodule symbionts on four mimosoid legumes from Barro Colorado Island, Panama

Sequences of 16S rRNA and partial 23S rRNA genes and PCR assays with genotype-specific primers indicated that bacteria in the genus Burkholderia were the predominant root nodule symbionts for four mimosoid legumes (Mimosa pigra, M. casta, M. pudica, and Abarema macradenia) on Barro Colorado Island, Panama. Among 51 isolates from these and a fifth mimosoid host (Pithecellobium hymenaeafolium), 44 were Burkholderia strains while the rest were placed in Rhizobium, Mesorhizobium, or Bradyrhizobium. The Burkholderia strains displayed four distinct rRNA sequence types, ranging from 89% to 97% similarity for 23S rRNA and 96.5-98.4% for 16S rRNA. The most common genotype comprised 53% of all isolates sampled and was associated with three legume host species. All Burkholderia genotypes formed nodules on Macroptilium atropurpureum or Mimosa pigra, and sequencing of rRNA genes in strains re-isolated from nodules verified identity with inoculant strains. Sequence analysis of the nitrogenase alpha-subunit gene (nifD) in two of the Burkholderia genotypes indicated that they were most similar to a partial sequence from the nodule-forming strain Burkholderia tuberum STM 678 from South Africa. In addition, a PCR screen with primers specific to Burkholderia nodB genes yielded the expected amplification product in most strains. Comparison of 16S rRNA and partial 23S rRNA phylogenies indicated that tree topologies were significantly incongruent. This implies that relationships across the rRNA region may have been altered by lateral gene transfer events in this Burkholderia population.     

Genetic markers for analysing symbiotic relationships and lateral gene transfer in Neotropical bradyrhizobia

Assays with seven sets of lineage-specific polymerase chain reaction (PCR) primers in the ribosomal RNA region were performed on 96 isolates of the Bradyrhizobium sp. nodule bacteria from Barro Colorado Island, Panama. The isolates were derived from 10 legume host species in six genera (Centrosema, Desmodium, Dioclea, Inga, Machaerium and Vigna). The PCR assays differentiated 13 composite genotypes, and sequencing of a 5' 23S rRNA region indicated that all but one had a unique sequence. The most common genotype (seen in 44% of the isolates) was associated with all six legume host genera, and had a marker profile and 5' 23S rRNA sequence identical to a Bradyrhizobium lineage associated with several other legume genera in Panama and Costa Rica. Another 46% of the isolates had genotypes found to be associated with two to three legume genera. Bradyrhizobium strains with low host specificity thus appear to be prevalent in this tropical forest. Based on 16S rRNA and 5' 23S rRNA markers, most of the isolates had clear affinities to either B. japonicum or B. elkanii. However, one strain (Cp5-3) with a B. elkanii-type 16S rRNA marker had a 5' 23S rRNA region resembling B. japonicum. A partition homogeneity test indicated that relationships of strain Cp5-3 were significantly discordant for 16S rRNA vs. 23S rRNA sequences, and a runs test detected significant mosaic structure across the rRNA region. Lateral gene transfer events have therefore played a role in the evolution of symbiotic bacteria in this environment.     

Relationships of bradyrhizobia from the legumes Apios americana and Desmodium glutinosum.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.     

Symbiotic Relationships of Legumes and Nodule Bacteria on Barro Colorado Island,Panama: A Review

New data on 129 bacterial isolates were analyzed together with prior samples to characterize community-level patterns of legume–rhizobial symbiosis on Barro Colorado Island (BCI), Panama. Nodules have been sampled from 24 BCI legume species in 18 genera, representing about one quarter of the legume species and one half of the genera on the island. Most BCI legumes associated exclusively with nodule symbionts in the genus Bradyrhizobium, which comprised 86.3% of all isolates (315 of 365). Most of the remaining isolates (44 of 365) belonged to the β-proteobacterial genus Burkholderia; these were restricted to two genera in the legume subfamily Mimosoideae. Multilocus sequence analysis indicated that BCI Bradyrhizobium strains were differentiated into at least eight lineages with deoxyribonucleic acid divergence of the same magnitude as found among currently recognized species in this bacterial genus. Two of these lineages were widely distributed across BCI legumes. One lineage was utilized by 15 host species of diverse life form (herbs, lianas, and trees) in 12 genera spanning two legume subfamilies. A second common lineage closely related to the taxon B. elkanii was associated with at least five legume genera in four separate tribes. Thus, BCI legume species from diverse clades within the family frequently share interaction with a few common lineages of nodule symbionts. However, certain host species were associated with unique symbiont lineages that have not been found on other coexisting BCI legumes. More comprehensive sampling of host taxa will be needed to characterize the overall diversity of nodule bacteria and the patterns of symbiont sharing among legumes in this community.     

Diversity and relationships of Bradyrhizobia from Amphicarpaea bracteata based on partial nod and ribosomal sequences.

Partial sequences of three nod genes (nodC, nodD1, and nodA 5' flanking region) and of 16S and 23S rDNA were obtained from isolates of Bradyrhizobium sp. associated with the native North American legume Amphicarpaea bracteata. Isolates from Amphicarpaea had identical sequences in the three nod gene regions, but differed from all other Bradyrhizobium taxa at > 10% of nucleotide sites. Parsimony analysis of all nod gene segments indicated a phylogenetic relationship of these bacteria to B. elkanii, with B. japonicum diverging prior to the diversification of these taxa. All Bradyrhizobium isolates from Amphicarpaea were also identical to B. elkanii in the size of the intervening sequence (IVS) in the 5' region of the 23S rRNA gene, while B. japonicum had an IVS length variant with 29 additional nucleotides. Parsimony analysis of both 16S and 23S partial rDNA sequences grouped Bradyrhizobium sp. isolates from Amphicarpaea into a clade together with B. elkanii, consistent with the relationships inferred from nod sequences.     

The large mimosoid genus Inga Mill. (tribe Ingeae,Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.     

Bradyrhizobia from wild Phaseolus, Desmodium, and Macroptilium species in northern Mexico

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (Amphicarpaea) that are closely related to Bradyrhizobium elkanii.     

Bradyrhizobia from Wild Phaseolus, Desmodium, and Macroptilium Species in Northern Mexico

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (Amphicarpaea) that are closely related to Bradyrhizobium elkanii.     

Distinct Bradyrhizobium [corrected] communities nodulate legumes native to temperate and tropical monsoon Australia

Geographic isolation and growing climate aridity played major roles in the evolution of Australian legumes. It is likely that these two factors also impacted on the evolution of their root-nodule bacteria. To investigate this issue, we applied a multilocus sequence analysis (MLSA) approach to examine Bradyrhizobium isolates originating from temperate areas of Western Australia (WA) and the tropical-monsoon area of the Northern Territory (NT). The isolates were mostly collected from the nodules of legumes belonging to tribes, genera and species endemic or native to Australia. Phylogenetic analyses of sequences for the housekeeping atpD, dnaK, glnII, gyrB, recA and 16S rRNA genes and nodulation nodA gene revealed that most isolates belonged to groups that are distinct from non-Australian Bradyrhizobium isolates, which is in line with earlier studies based on 16S rRNA gene sequence analyses. Phylogenetic analysis of the nodA data allowed identification of five major Clades among the WA and NT isolates. All WA isolates grouped in a subgroup I.1 of Clade I with strains originating from temperate eastern Australia. In contrast, the NT isolates formed part of Clades I (subgroup I.2), III (subgroup III.3), IV, V and X. Of these nodA clades, Clade I, Clade IV, Clade X presumably have an Australian origin. Overall, these data demonstrate that the impact of geographic isolation of the Australian landmass is manifested by the presence of numerous unique clusters in housekeeping and nodulation gene trees. In addition, the intrinsic climate characteristics of temperate WA and tropical-monsoon NT were responsible for the formation of distinct legume communities selecting for unrelated Bradyrhizobium groups.     

Relationships of Bradyrhizobia from the Legumes Apios americana and Desmodium glutinosum

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.     

Genetic diversity of bradyrhizobial populations from diverse geographic origins that nodulate Lupinus spp. and Ornithopus spp

The genetic diversity of 45 bradyrhizobial isolates that nodulate several Lupinus and Ornithopus species in different geographic locations was investigated by 16S rDNA PCR-RFLP and sequence analysis, 16S-23S rDNA intergenic spacer (IGS) PCR-RFLP analysis, and ERIC-PCR genomic fingerprinting. Reference strains of Bradyrhizobium japonicum, B. liaoningense and B. elkanii and some Canarian isolates from endemic woody legumes in the tribe Genisteae were also included. The 16S rDNA-RFLP analysis resolved 9 genotypes of lupin isolates, a group of fourteen isolates presented restriction-genotypes identical or very similar to B. japonicum, while another two main groups of isolates (69%) presented genotypes that clearly separated them from the reference species of soybean. 16S rDNA sequencing of representative strains largely agreed with restriction analysis, except for a group of six isolates, and showed that all the lupin isolates are relatives of B. japonicum, but different lineages were observed. The 16S-23S IGS-RFLP analysis showed a high resolution level, resolving 19 distinct genotypes among 30 strains analysed, and so demonstrating the heterogeneity of the 16S-RFLP groups. ERIC-PCR fingerprint analysis showed an enormous genetic diversity producing a different pattern for each but two of the isolates. Phylogeny of nodC gene was independent from the 16S rRNA phylogeny, and showed a tight relationship in the symbiotic region of the lupin isolates with isolates from Canarian genistoid woody legumes, and in concordance, cross-nodulation was found. We conclude that Lupinus is a promiscuous host legume that is nodulated by rhizobia with very different chromosomal genotypes, which could even belong to several species of Bradyrhizobium. No correlation among genomic background, original host plant and geographic location was found, so, different chromosomal genotypes could be detected at a single site and in a same plant species, on the contrary, an identical genotype was detected in very different geographical locations and plants.     

Genetic diversity of native bradyrhizobia isolated from soybeans (Glycine max L.) in different agricultural-ecological-climatic regions of India

Fifty isolates from root nodules of soybean plants sampled in five agricultural-ecological-climatic regions of India were analyzed by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene, the intergenic spacer region between the 16S and 23S rRNA genes (IGS), and the nifH and nodC genes. Eight haplotypes assigned to the Bradyrhizobium genus were identified, and the genetic diversity was conserved across regions. Sequence analyses of the IGS and the dnaK, glnII, recA, and nifH genes revealed three groups. One of them (26% of isolates) was assigned to Bradyrhizobium liaoningense. A second group (36% of isolates) was identified as B. yuanmingense but likely forms a new biovar able to nodulate soybean plants. The third lineage (38% of isolates) was different from all described Bradyrhizobium species but showed the same symbiotic genotype as B. liaoningense and B. japonicum bv. glycinearum.     

Contrasting nifD and ribosomal gene relationships among Mesorhizobium from Lotus oroboides in northern Mexico

PCR screens for length variation in a 5' portion of 23S ribosomal RNA and in the 3' end of the 16S rRNA-23S rRNA internal transcribed spacer (ITS) region indicated that nodule bacteria from a Mexican population of Lotus oroboides were diverse on a local scale. Three 23S rRNA length variants and five ITS length variants were detected among the 22 isolates. Sequencing of nearly full-length 16S rRNA genes in three isolates indicated that they fell into the genus Mesorhizobium, but comprised two distinct groups. Two isolates were closely related to M. loti LMG 6125T, while the other isolate clustered with an assemblage of Mesorhizobium taxa that included M. amorphae, M. plurifarium and M. huakuii. However, a phylogenetic tree based on 715 bp of the nitrogenase alpha-subunit (nifD) gene was significantly discordant with the relationships inferred from rRNA sequences. Two isolates that were nearly identical for 16S rRNA had nifD genes that varied at 2% of sites, and one of these nifD sequences was identical to that of another isolate with a strongly divergent 16S rRNA gene. A plasmid screen followed by Southern hybridization indicated that only one of these strains harbored a plasmid-borne nifD gene. These results imply that gene transfer events have altered the distribution of nifD sequences among lineages within this natural population of Mesorhizobium strains.     

A novel symbiotic nitrogen-fixing member of the Ochrobactrum clade isolated from root nodules of Acacia mangium

Ten strains of root nodule bacteria were isolated from the nodules of Acacia mangium grown in the Philippines and Thailand. Partial sequences (approx. 300 bp) of the 16S rRNA gene of each isolate were analyzed. The nucleotide sequences of strain DASA 35030 indicated high homology (>99%) with members of the genus Ochrobactrum in Brucellaceae, although the sequences of other isolates were homologous to those of two distinct genera Bradyrhizobium and Rhizobium. The strain DASA 35030 was strongly suggested to be a strain of Ochrobactrum by full length sequences of the 16S rRNA gene, fatty acids composition, G+C contents of the DNA, and other physiological characteristics. Strain DASA 35030 induced root nodules on A. mangium, A. albida and Paraserianthes falcataria. The nodules formed by strain DASA 35030 fixed nitrogen and the morphology of the nodules is the same as those of nodules formed by the other isolates. This is the first report that the strain of Ochrobactrum possesses complete symbiotic ability with Acacia.     

Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China

Rhizobia are soil bacteria with the capacity to induce nitrogen-fixing nodules on the roots or stems of legume plants. A total of 40 bacterial isolates from the root nodules of Caragana microphylla growing in desert soil in Ningxia, China, were analyzed for genetic diversity and phylogenetic position. These isolates were classified into 7 types of 16S ribosomal DNA (rDNA) using polymerase chain reaction-restriction fragment length polymorphism analysis. They were grouped into 4 clades, Rhizobium-Agrobacterium, Sinorhizobium, Phyllobacterium, and Bradyrhizobium, when the phylogenies of 16S rDNA, recA, and atpD genes were applied. Phylogenetic analysis showed that the tree generated from the 16S rDNA sequencing agreed with that produced from the recA and atpD genes. By analyzing phylogenetic relationship using the 3 loci, the isolates in the branches of Phyllobacterium and Sinorhizobium could be identified as P. brassicacearum and S. meliloti. The isolates in the branch of Rhizobium-Agrobacterium were the most abundant microsymbiont of C. microphylla and were designated R. leguminosarum, R. galegae, R. alamii, and A. tumefaciens. Two isolates with low sequence similarity to the known species of Bradyrhizobium might be novel species in this genus.