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Aiming to investigate the diversity and distribution of rhizobia associated with Ammopiptanthus, an endangered evergreen legume widely distributed in deserts, we characterized a total of 219 nodule isolates from nine sampling sites in Northwest China with different soil characteristics based upon restriction fragment length polymorphism (RFLP) analysis of 16S ribosomal RNA (rRNA) and symbiotic genes (nodC and nifH). Ten isolates representing different 16S rRNA-RFLP types were selected for further sequence analyses of 16S rRNA and four housekeeping genes. As results, nine genospecies belonging to the genera Ensifer, Neorhizobium, Agrobacterium, Pararhizobium, and Rhizobium could be defined among the isolates. The nodC and nifH phylogenies of 14 isolates representing different symbiotic-RFLP types revealed five lineages linked to Ensifer fredii, Ensifer meliloti, Rhizobium leguminosarum, Mesorhizobium amorphae, and Rhizobium gallicum, which demonstrated the various origins and lateral transfers of symbiotic genes between different genera and species. The rhizobial diversities of Ammopiptanthus mongolicus varied among regions, and the community compositions of rhizobia associated with A. mongolicus were significantly different in wild and cultured fields. Constrained correspondence analysis showed that the distribution of A. mongolicus rhizobia could be explained by available potassium content and that the assembly of symbiotic types was mainly affected by available phosphorus content and carbon-nitrogen ratio.

The whole-genome sequences of three strains of Rhizobium gallicum reported here support the concept that the distinct nodulation host ranges displayed by the symbiovars gallicum and phaseoli can be largely explained by different symbiotic plasmids.

The legume species of Astragalus as traditional Chinese medicine source and environmental protection plants showed an extensive distribution in the arid region of northwestern China. However, few rhizobia associating with Astragalus have been investigated in this region so far. In this study, 78 endophytic bacteria were isolated from root nodules of 12 Astragalus species and characterized by the PCR-RFLP of 16S rRNA gene and symbiotic genes together with the phylogenetic analysis. Results showed that the majority (53%) of isolates are non-nodulating Agrobacterium sp. and the rest are Mesorhizobium genomic species (41%), Ensifer spp. and Rhizobium gallicum (6%), respectively. Mesorhizobium genomic species are broadly distributed in the Astragalus symbioses and most of them share similar symbiotic genes. It seems that horizontal gene transfer occurred frequently among different genomic species independent of their original hosts and sites. Astragalus adsurgens is nodulated by a widely range of rhizobial species in the nodulation test, revealing that it could play an important role in diversification of Astragalus symbionts and that might be a reason for its wide adaptation to diverse environments.

Aim To determine and compare the taxonomic identity and diversity of root nodule and rhizospheric microbial symbionts associated with Acacia pycnantha Benth. in its native (Australian) and invasive (South African) ranges, and to establish whether these associations are general or host specific. Location The native range of A. pycnantha in Australia and invasive ranges in South Africa and Western Australia. Methods Bacteria were isolated from root nodules collected from 18 populations of A. pycnantha. Repetitive element polymerase chain reaction (REP-PCR) fingerprinting was used to assess overall bacterial diversity and clustering. Molecular phylogenies for a subset of isolates representing major REP-PCR clades were reconstructed using maximum parsimony and Bayesian phylogenetic analyses of the nuclear 16S–23S rRNA intergenic spacer (IGS), 16S rRNA, and the symbiotic nodA genes. Results Twelve clusters were identified from the REP-PCR analysis; 11 included isolates from both the native range in Australia and invasive range in South Africa, while one cluster comprised only Australian isolates. Six rhizobial species were found in association with A. pycnantha: Bradyrhizobium japonicum, Rhizobium gallicum, R. lusitanum, R. miluonense, R. multihospitium and R. tropici. We also identified three plant-growth promoting bacteria isolated from root nodules of A. pycnantha: Burkholderia caledonica, B. graminis and B. phytofirmans. Phylogenetic analysis of the IGS gene retrieved clades containing symbionts from both Australia and South Africa while others comprised only South African taxa, suggesting the introduction of bacterial lineages from Australia to South Africa. Our phylogeographic analysis of the nodA gene confirmed that A. pycnantha was co-introduced with its symbionts to South Africa. Main conclusions Acacia pycnantha is a promiscuous legume, associated with at least six different rhizobial symbionts, and forms associations with plant-growth promoting rhizosphere bacteria from the genus Burkholderia. In the invasive range of A. pycnantha in South Africa, nodules contained some symbionts of South African origin while other symbionts appear to have been co-introduced from Australia. Acacia pycnantha is associated with a wider suite of symbionts in its invasive than native range.

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156T. S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602spT but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.

A total of 159 endophytic bacteria were isolated from surface-sterilized root nodules of wild perennial Glycyrrhiza legumes growing on 40 sites in central and northwestern China. Amplified fragment length polymorphism (AFLP) genomic fingerprinting and sequencing of partial 16S rRNA genes revealed that the collection mainly consisted of Mesorhizobium, Rhizobium, Sinorhizobium, Agrobacterium and Paenibacillus species. Based on symbiotic properties with the legume hosts Glycyrrhiza uralensis and Glycyrrhiza glabra, we divided the nodulating species into true and sporadic symbionts. Five distinct Mesorhizobium groups represented true symbionts of the host plants, the majority of strains inducing N2-fixing nodules. Sporadic symbionts consisted of either species with infrequent occurrence (Rhizobium galegae, Rhizobium leguminosarum) or species with weak (Sinorhizobium meliloti, Rhizobium gallicum) or no N2 fixation ability (Rhizobium giardinii, Rhizobium cellulosilyticum, Phyllobacterium sp.). Multivariate analyses revealed that the host plant species and geographic location explained only a small part (14.4%) of the total variation in bacterial AFLP patterns, with the host plant explaining slightly more (9.9%) than geography (6.9%). However, strains isolated from G. glabra were clearly separated from those from G. uralensis, and strains obtained from central China were well separated from those originating from Xinjiang in the northwest, indicating both host preference and regional endemism.

Twenty seven rhizobial strains associated with Acacia saligna grown in northern and southern Algeria were characterized, including generation time, host-range, the 16S rRNA gene and 16S–23S rRNA intergenic spacer restriction patterns, 16S rRNA gene sequence analysis and tolerance to salinity and drought. Cross inoculation tests indicated that 11 slow-growing isolates from northern nurseries were able to nodulate introduced Australian acacias exclusively, whereas 16 fast-growing isolates, mainly from southern nurseries, were capable of also nodulating native acacias. Restriction patterns and sequence analysis of the 16S rRNA gene showed that strains of the first group belonged to Bradyrhizobium while strains of the second group were related to Sinorhizobium meliloti and Rhizobium gallicum. Interestingly, five strains of the first group formed a distinct cluster phylogenetically close to Bradyrhizobium betae, a non-nodulating species causing tumour-like deformations in sugar beet roots. Bradyrhizobium strains were in general more sensitive to NaCl and PEG than the S. meliloti and R. gallicum representatives. Among the latter, strains S. meliloti BEC1 and R. gallicum DJA2 were able to tolerate up to 1 M NaCl and 20% PEG. This, together with their wide host-range among Acacia species, make them good candidates for developing inoculants for A. saligna and other acacia trees growing in arid areas.

Bacteria from nodules of the legume Acaciella angustissima native to the south of Mexico were characterized genetically and their nodulation and competitiveness were evaluated. Phylogenetic studies derived from rpoB gene sequences indicated that A. angustissima is nodulated by Sinorhizobium mexicanum, Rhizobium tropici, Mesorhizobium plurifarium and Agrobacterium tumefaciens and by bacteria related to Sinorhizobium americanum, Sinorhizobium terangae, Rhizobium etli and Rhizobium gallicum. A new lineage related to S. terangae is recognized based on the sequences of gyrA, nolR, recA, rpoB and rrs genes, DNA-DNA hybridization and phenotypic characteristics. The name for this new species is Sinorhizobium chiapanecum and its type strain is ITTG S70T. The symbiotic genes nodA and nifH were similar to those from S. mexicanum strains, which are Acaciella symbionts as well, with nodA gene sequences grouped within a cluster of nod genes from strains that nodulate plants from the Mimosoideae subfamily of the Leguminosae. Sinorhizobium isolates were the most frequently obtained from A. angustissima nodules and were among the best strains to promote plant growth in A. angustissima and to compete in interstrain nodule competition assays. Lateral transfer of symbiotic genes is not evident among the genera that nodulate A. angustissima (Rhizobium, Sinorhizobium and Mesorhizobium) but may occur among the sympatric and closely related sinorhizobia that nodulate Acaciella.

In the present study, a total of 154 bacterial strains isolated from nodules of eighteen Vicia species mainly grown in the temperate Chinese provinces were characterized by ARDRA, ITS PCR-RFLP, BOX-PCR, sequencing of 16S rDNA, nodC, nifH, atpD and glnII, and nodulation tests. The results demonstrated that most of the R. leguminosarum strains were effective microsymbionts of the wild Vicia species, while genomic species related to Rhizobium gallicum, Mesorhizobium huakuii, Ensifer meliloti and Bradyrhizobium spp. were symbiotic bacteria occasionally nodulating with Vicia species. In addition, fourteen strains related to Agrobacterium, Phyllobacterium, Ensifer, Shinella and R. tropici, as well as 22 strains of R. leguminosarum might be nodule endophytes without symbiotic genes. Diverse symbiotic gene lineages were found among the test strains and a strong association was found among the symbiotic gene types and genomic species, indicating the absence of lateral gene transfer. These results greatly enlarged the rhizobial spectrum of Vicia species.

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N2 fixation.

A novel Mesorhizobium group associated with Albizia kalkora [Wang et al. (2006), Syst Appl Microbiol 29, 502-517] was further characterized. The seven strains in this group showed similar protein patterns and were different from defined Mesorhizobium species in SDS-PAGE of whole-cell proteins. The representative strain CCBAU 61158(T) formed a novel Mesorhizobium lineage in phylogenetic analyses of 16S rRNA, atpD, glnII and nifH genes. However, its nodC gene sequence was more similar to that of Rhizobium gallicum R602sp(T) than to those of Mesorhizobium species. DNA-DNA relatedness between CCBAU 61158(T) and reference strains of defined Mesorhizobium species was lower than 34.1 %. These results indicated that this Mesorhizobium group was a unique genomic species. The subtropical distribution, host origin, PCR-RFLP patterns of 16S rRNA genes, fatty acid profile and a series of phenotypic characteristics could be used as distinctive features of this group. This group is therefore proposed as a novel species, Mesorhizobium albiziae sp. nov., with CCBAU 61158(T) (=LMG 23507(T)=USDA 4964(T)) as the type strain. Strain CCBAU 61158(T) could form effective nodules on Albizia julibrissin, Glycine max, Leucaena leucocephala and Phaseolus vulgaris.

In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions when using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum.

Comparative analysis of 70 16S rDNA sequences representing 20 Rhizobium species (including pathogenic Agrobacterium spp.) was conducted using Maximum Likelihood to establish relationships of species using multiple sequences. There is no significant internal division of the Rhizobium clade to suggest that it represents more than one genus. Plant pathogenic (Agrobacterium) species are distributed within the genus. The analysis supported the synonymy of some species (Rhizobium gallicum and Rhizobium mongolense) and the need for comparative investigations of the tumorigenic and nodulating properties of Rhizobium tropici and Rhizobium rhizogenes. Misidentification of some sequences may conceal one or more putative novel species. Some sequences appear to be misidentified because of faulty sequencing or incomplete or inadequate analysis.

The diversity and taxonomic relationships of 83 bean-nodulating rhizobia indigenous to Ethiopian soils were characterized by PCR-RFLP of the internally transcribed spacer (ITS) region between the 16S and 23S rRNA genes, 16S rRNA gene sequence analysis, multilocus enzyme electrophoresis (MLEE), and amplified fragment-length polymorphism. The isolates fell into 13 distinct genotypes according to PCR-RFLP analysis of the ITS region. Based on MLEE, the majority of these genotypes (70%) was genetically related to the type strain of Rhizobium leguminosarum. However, from analysis of their 16S rRNA genes, the majority was placed with Rhizobium etli. Transfer and recombination of the 16S rRNA gene from presumptively introduced R. etli to local R. leguminosarum is a possible theory to explain these contrasting results. However, it seems unlikely that bean rhizobia originating from the Americas (or Europe) extensively colonized soils of Ethiopia because Rhizobium tropici, Rhizobium gallicum, and Rhizobium giardinii were not detected and only a single ineffective isolate of R. etli that originated from a remote location was identified. Therefore, Ethiopian R. leguminosarum may have acquired the determinants for nodulation of bean from a low number of introduced bean-nodulating rhizobia that either are poor competitors for nodulation of bean or that failed to survive in the Ethiopian environment. Furthermore, it may be concluded from the genetic data presented here that the evidence for separating R. leguminosarum and R. etli into two separate species is inconclusive.

Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs , glnII , nifH , and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed.

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD+-dependent (DME: EC 1.1.1.39) and NADP+-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was perfor...

Three microbial strains isolated from common beans, 23C2T (Tunisia), Gr42 (Spain) and IE4868 (Mexico), which have been identified previously as representing a genomic group closely related to Rhizobium gallicum , are further studied here. Their 16S rRNA genes showed 98.5–99 % similarity with Rhizobium loessense CCBAU 7190BT, R. gallicum R602spT, Rhizobium mongolense USDA 1844T and Rhizobium yanglingense CCBAU 71623T. Phylogenetic analysis based on recA, atpD, dnaK and thrC sequences showed that the novel strains were closely related and could be distinguished from the four type strains of the closely related species. Strains 23C2T, Gr42 and IE4868 could be also differentiated from their closest phylogenetic neighbours by their phenotypic and physiological properties and their fatty acid contents. All three strains harboured symbiotic genes specific to biovar gallicum. Levels of DNA–DNA relatedness between strain 23C2T and the type strains of R. loessense , R. mongolense , R. gallicum and R. yanglingense ranged from 58.1 to 61.5 %. The DNA G+C content of the genomic DNA of strain 23C2T was 59.52 %. On the basis of these data, strains 23C2T, Gr42 and IE4868 were considered to represent a novel species of the genus Rhizobium for which the name Rhizobium azibense is proposed. Strain 23C2T ( = CCBAU 101087T = HAMBI3541T) was designated as the type strain.

The nodC and nifH genes were characterized in a collection of 83 rhizobial strains which represented 23 recognized species distributed in the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium, as well as unclassified rhizobia from various host legumes. Conserved primers were designed from available nucleotide sequences and were able to amplify nodC and nifH fragments of about 930 bp and 780 bp, respectively, from most of the strains investigated. RFLP analysis of the PCR products resulted in a classification of these rhizobia which was in general well-correlated with their known host range and independent of their taxonomic status. The nodC and nifH fragments were sequenced for representative strains belonging to different genera and species, most of which originated from Phaseolus vulgaris nodules. Phylogenetic trees were constructed and revealed close relationships among symbiotic genes of the Phaseolus symbionts, irrespective of their 16S-rDNA-based classification. The nodC and nifH phylogenies were generally similar, but cases of incongruence were detected, suggesting that genetic rearrangements have occurred in the course of evolution. The results support the view that lateral genetic transfer across rhizobial species and, in some instances, across Rhizobium and Sinorhizobium genera plays a role in diversification and in structuring the natural populations of rhizobia.

One hundred and sixty isolates of rhizobia were sampled from the root nodules of common bean (Phaseolus vulgaris L.) cultivated in Tunisian soil samples originating from three geographically distinct fields. Plasmid profiling was used as a primary method to rapidly screen the isolates, and then 38 plasmid types were recorded among the 160 isolates. A sample representing the majority of plasmid types was chosen for further characterization by restriction fragment length polymorphism (RFLP) analysis of genomic DNA using chromosomal and symbiotic gene probes, and by their ability to nodulate a potential alternative host, Leucaena leucocephala. One third of the isolates showed a high similarity to Rhizobium gallicum isolated from common bean in France, another third showed the same characteristics as the R. etli-R. leguminosarum group, while the remaining isolates could not be related to any of the five species nodulating bean. When reexamined for nodulation, some of these isolates, showing similarities to R. tropici and Agrobacterium with respect to colony morphology and growth in different media, failed to nodulate their original host. The R. gallicum-like isolates, R. etli-like isolates, and R. leguminosarum-like isolates were recovered from regions where bean is frequently grown, while in fields which had not been cultivated with beans for at least the 10 previous years, solely unrecognized taxa of ineffective isolates were recovered. We detected variations in the symbiotic regions, but certain pSym RFLP patterns for nifH were conserved between Tunisian, French, and Austrian populations of bean rhizobia. Evaluation of symbiotic effectiveness showed that R. gallicum-like isolates and R. etli-like isolates were effective, whereas some R. leguminosarum-like isolates were ineffective. Furthermore, effective isolates were also found among the unrecognized taxa.

A systematic analysis of the symbiotic phenotype on Phaseolus vulgaris of tropical rhizobia isolated from a variety of leguminous plant species and of classified type strains from the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium and Azorhizobium was performed. Depending on the bean cultivar, between 80 and 90% of the tested rhizobia were able to nodulate. A minority of these nodules were also nitrogen-fixing. A detailed microscopic analysis of the induced nodules or nodular structures revealed that the interaction between these strains and P. vulgaris was arrested at different stages of the interaction. Our results show that P. vulgaris is able to perceive signals for nodulation from many rhizobia although most of the interactions are not effective.

Medicago ruthenica [(L.) Ledebour] is native to inner Mongolia where rhizosphere samples were collected for the isolation of 106 rhizobial cultures. Besides nodulating the original trap host, the isolates formed nitrogen-fixing symbioses with Phaseolus vulgaris. Only half of the isolates nodulated alfalfa (Medicago sativa), but these did not form nitrogen-fixing symbioses. Rhizobium tropici also formed nitrogen-fixing symbioses with Medicago ruthenica. A total of 56 distinctive multilocus electrophoretic types (ETs) were identified among 94 of the 106 isolates which were analysed for variation in electrophoretic mobility of 12 enzyme loci. One isolate (USDA 1920) possessed a unique ET, while the ETs of the other isolates formed two weakly divergent subgroups approximately equal in size. It was concluded from small subunit rRNA gene sequences of eight isolates of Medicago ruthenica that they belonged to the genus Rhizobium and not to the genus Sinorhizobium which is more commonly associated with Medicago. Genomic similarity, determined from DNA hybridization analysis, between USDA 1920 and the strain representing the remaining isolates (USDA 1844) was lower than 20%. Based upon these observations it was concluded that at least three genomic species of rhizobia form nitrogen-fixing symbioses with Medicago ruthenica. One of these genomic species is R. tropici, another is represented by the single isolate USDA 1920 and the name Rhizobium mongolense is proposed for the third genomic species represented by USDA 1844.

The phylogenetic relationships among Rhizobium species that nodulate Phaseolus vulgaris (common bean) were determined by directly sequencing the amplified 16S ribosomal DNA genes of these organisms. The bean strains formed four separate clusters. One cluster was composed of Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, and R. leguminosarum bv. phaseoli. Two other clusters comprised Rhizobium etli and Rhizobium tropici, and the fourth cluster contained a single bean-nodulating strain. Data for species identification were obtained from DNA-DNA reassociation experiments. The levels of DNA relatedness among strains belonging to the three biovars of R. leguminosarum ranged from 58 to 67%. The levels of DNA relatedness between R. leguminosarum bv. phaseoli and R. etli and R. tropici ranged from 43 to 45% and 13 to 16%, respectively. The levels of DNA relatedness between the strain belonging to the fourth cluster and strains of the other three Rhizobium species that nodulate beans were less than 10%.

The strain designated as AB21(T) was isolated from chloroethylenes contaminated soil. Cells are gram-negative, aerobic, non-spore-forming, and motile rods. Phylogenetic analysis based on 16S rRNA gene sequence showed that it belonged to the genus Rhizobium, and was closely related to Rhizobium sullae IS 123(T) (97.4 %), Rhizobium yanglingense SH 22623(T) (97.2 %), Rhizobium gallicum R 602sp(T) (97.1 %), Rhizobium alamii GBV 016(T) (97.0 %), and Rhizobium monogolense USDA 1844(T) (97.0 %). It showed less than 97 % identity with the remaining Rhizobium species. This novel isolate grew optimally at 25-37 °C (optimum, 30 °C) and pH 6-9 (optimum, pH 8.0). It grew in the presence of 0-4 % (w/v) NaCl, tolerating a 4 % (w/v) NaCl. DNA-DNA hybridization experiment shows less than 53 % binding with closely related Rhizobium. Predominant quinone is ubiquinone (Q-10). The major fatty acids were summed feature 8 (composed of C(18:1) ω7c/C(18:1) ω6c), C(19:0) cyclo ω8c, and C(16:0). The G+C molar content is 62.5 mol%. Based on the polyphasic analysis, strain AB21(T) is referred to be a novel species of the genus Rhizobium for which the name Rhizobium halotolerans sp. nov. is proposed. The type strain is AB21(T) (=KEMC 224-056(T) = JCM 17536(T)).

A study of symbiotic bacteria from traditional agricultural legumes from Central Spain was performed to create a collection of rhizobia from soils differing in physicochemical, analytical and/or agroecological properties which could be well-adapted to the environmental conditions of this region, and be used for sustainable agricultural practices. Thirty-six isolates were obtained from root-nodules of fifteen legume species (including Cicer arietinum, Lathyrus sativus, Lens culinaris, Lupinus spp., Medicago sativa, Phaseolus vulgaris, Pisum sativum, and Vicia spp.) from three agriculture areas with soils of different pHs and from a forest area with undisturbed soils. Phenotypical characterization revealed uniformity across the thirty-six isolates, with important exceptions in terms of environmental tolerance (three isolates survived at high temperatures, three at high salinity and three at acid pH). The molecular analysis of 16S rRNA gene showed a close relationship of twenty-nine isolates to Rhizobium leguminosarum, one to Rhizobium gallicum, one to Mesorhizobium ciceri, two to Sinorhizobium (Ensifer) meliloti and three to Bradyrhizobium canariense. The sequence analysis of a symbiosis-specific gene, nod C, showed a correlation with the plant host and grouped twenty-six isolates with Rhizobium leguminosarum bv. viciae, establishing the diversity in relation to legume-host. The 16S-23S rRNA intergenic spacer (IGS) region allowed for intraspecific differentiation, so that strains with equal 16S rRNA were grouped by means of their soil origin. These results indicated that phenotypical and genetically related strains may be widely distributed in this region and that soil abiotic characteristics could have a substantial bearing on the selection of the strains living in each environment., Junta de Comunidades de Castilla‐La Mancha, Spain. Grant Numbers: POII09‐0182‐3834, POII10‐0211‐5015.--I3P program of CSIC

Two hundred and eighty seven isolates of Rhizobium nodulating Phaseolus vulgaris L. were sampled in France from four geographically distant field populations. They were characterized by their colony morphology and by plasmid profiles. A representative sample was further characterized: a) by the ability of each isolate to nodulate a potential alternative host Leucaena leucocephala and to grow on specific media, and b) by RFLP analysis of PCR amplified 16S rRNA genes. On the basis of their phenotypic and genetic characteristics the isolates could be assigned either to Rhizobium leguminosarum bv phaseoli, or to R. tropici. The two species co-occurred at three sites. R. leguminosarum bv phaseoli represented 2%, 4%, 72% and 100% of the population at the four different sites. Eighteen and 22 different plasmid profiles were identified within R. tropici and R. leguminosarum bv phaseoli, respectively. Some of them were conserved between distant geographical regions. The fact that R. tropici was found in France shows that this species is not limited to tropical regions and gives additional evidence of the multi-specific nature of the Phaseolus microsymbiont, even over a geographically limited area.

Presence of microcystin (MC), a predominant freshwater algal toxin and a suspected liver carcinogen, in Florida’s freshwaters poses serious health threat to humans and aquatic species. Being recalcitrant to conventional physical and chemical water treatment methods, biological methods of MC removal is widely researched. Water samples collected from five sites of Lake Okeechobee (LO) frequently exposed to toxic Microcystis blooms were used as inoculum for enrichment with microcystin LR (MC-LR) supplied as sole C and N source. After 20 days incubation, MC levels were analyzed using high performance liquid chromatography (HPLC). A bacterial consortium consisting of two isolates DC7 and DC8 from the Indian Prairie Canal sample showed over 74% toxin degradation at the end of day 20. Optimal temperature requirement for biodegradation was identified and phosphorus levels did not affect the MC biodegradation. Based on 16S rRNA sequence similarity the isolate DC8 was found to have a match with Microbacterium sp. and the DC7 isolate with Rhizobium gallicum (AY972457).

Low-molecular-weight (LMW) RNA molecules were analyzed to characterize rhizobial isolates that nodulate the common bean growing in Spain. Since LMW RNA profiles, determined by staircase electrophoresis, varied across the rhizobial species nodulating beans, we demonstrated that bean isolates recovered from Spanish soils presumptively could be characterized as Rhizobium etli , Rhizobium gallicum , Rhizobium giardinii , Rhizobium leguminosarum bv. viciae and bv. trifolii, and Sinorhizobium fredii .

Eighteen strains of common bean rhizobia from Konso (GI), Arbaminch (GII), Sodo (GIII) and Ziway-Awassa-Dilla (GIV) areas in southern Ethiopia were isolated. The strains were characterized by sixty-four phenotypic traits. These traits were tested for cluster analysis using unweighted pair group method with average (UPGMA) with NTSYS version 2.1. pH tolerance/sensitivity, antibiotic tolerance/sensitivity together with the numerical analysis results clearly showed diversity among the strains. The cluster analysis grouped the strains into two clusters that included 78% of the strains at 85% similarity level, except four strains which were not clustered. These strains were less related to the clustered strains and to each other. The clustered stains were with the presumed similar characters of Rhizobium leguminosarun/Rhizoium etli group except AUPR10. Two isolates, AUPR9 (unclustered) and AUPR10 (cluster I), exhibited rough colony appearance on Peptone Yeast Extract Agar medium (PY) that resemble the characteristics of Rhizobium gallicum . Strain AUPR8 (from unclustered strains) displayed creamy colony appearance on PY, tolerance to extreme pH, salt, and temperature and inability to utilize dulcitol and utilization of glycine that resembled with characters of Rhizobium tropici . This result, therefore, indicates the possibility of obtaining inoculant strains with tolerance to environmental conditions of common bean-producing areas of Ethiopia.

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29-20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.

Since the first volume of Bergey's Manual of Systematic Bacteriology was published, in 1984, two additional genera and several new species of stem- and root-nodulating bacteria have been proposed; further changes to the taxonomy of this group of organisms appear likely. This paper briefly reviews the current status of “Rhizobium” taxonomy and proposes minimal standards for the description of future genera and species belonging to this group of organisms.

A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.

A total of 103 root nodule isolates were used to estimate the diversity of bacteria nodulating Lotus tenuis in typical soils of the Salado River Basin. A high level of genetic diversity was revealed by repetitive extragenic palindromic PCR, and 77 isolates with unique genomic fingerprints were further differentiated into two clusters, clusters A and B, after 16S rRNA restriction fragment length polymorphism analysis. Cluster A strains appeared to be related to the genus Mesorhizobium , whereas cluster B was related to the genus Rhizobium . 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either Rhizobium or Mesorhizobium : the only exception was isolate BA135, whose 16S rRNA gene was closely related to the 16S rRNA gene of the genus Aminobacter. Most Mesorhizobium -like isolates were closely related to Mesorhizobium amorphae , Mesorhizobium mediterraneum , Mesorhizobium tianshanense , or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with Mesorhizobium loti type strain NZP2213. Rhizobium -like strains were related to Rhizobium gallicum , Rhizobium etli , or Rhizobium tropici , for which Phaseolus vulgaris is a common host. However, no nodC or nifH genes could be amplified from the L. tenuis isolates, suggesting that they have rather divergent symbiosis genes. In contrast, nodC genes from the Mesorhizobium and Aminobacter strains were closely related to nodC genes from narrow-host-range M. loti strains. Likewise, nifH gene sequences were very highly conserved among the Argentinian isolates and reference Lotus rhizobia. The high levels of conservation of the nodC and nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range Lotus symbionts, supporting the hypothesis that both intrageneric horizontal gene transfer and intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the Salado River Basin.

Thirty-three isolates of indigenous Rhizobium meliloti, either possessing cryptic plasmids that hybridize to probes for symbiotic genes or lacking a 1500-kb megaplasmid band in Eckhardt gels, were tested for infectivity on 10 legume species grown under axenic conditions. A previous study had shown that all but two isolates were symbiotically effective with Medicago sativa. All indigenous isolates and two reference strains of R. meliloti induced nodules which were symbiotically ineffective on Trigonella foenum-graecum (100% plants nodulated) and Phaseolus vulgaris (40 to 100% plants nodulated). Eighteen indigenous isolates of R. meliloti elicited ineffective nodules on Macroptilium atropurpureum (2 to 25% plants nodulated) and Leucaena leucocephala (11 to 75% plants nodulated). The identity of single colony nodule isolates from each R. meliloti inoculant and host combination was verified by phage typing and analysis of plasmid profiles; tests with subsamples of these isolates showed that all were capable of nodulating M. sativa. There was no apparent relationship between the host range of indigenous R. meliloti and either the presence of cryptic plasmids that hybridize to probes for symbiotic genes or the absence of a megaplasmid band in Eckhardt gels. The data suggest that nodulation promiscuity may be a relatively common characteristic of R. meliloti. Key words: host range, Rhizobium meliloti, Leucaena, Macroptilium, Phaseolus.

To screen high strength phenol degrading bacteria, we selected 108 rhizobial strains isolated from nodules of eight wild legumes species in the mining tailing region of Shaanxi province, northwest of China, and cultivated them in a basal salt (BS) medium supplemented with different phenol concentrations as a sole carbon source. The results showed that some of the strains could use phenol as sole carbon source. In order to study the characteristics of phenol degradation, the strain CCNWTB701 isolated from Astragalus chrysopteru was used as well, due to the fact that it was very efficient in phenol degradation. The phenol degradation was around 99.5 and 78.3%, with an initial concentration of 900 and 1000 mg/l phenol in 62 and 66 h, respectively. Kinetic studies indicated that the strain had a high KS (743.1 microM) and an extremely high KSI (10,469 microM) in Haldane's model. The phylogenetic analysis based on 16S rRNA gene sequences showed that CCNWTB701 belonged to the Rhizobium genus, and it was closely related to Rhizobium mongolense and Rhizobium gallicum.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Nodulation of common bean was explored in six oases in the south of Tunisia. Nineteen isolates were characterized by PCR-RFLP of 16S rDNA. Three species of rhizobia were identified, Rhizobium etli, Rhizobium gallicum and Sinorhizobium meliloti. The diversity of the symbiotic genes was then assessed by PCR-RFLP of nodC and nifH genes. The majority of the symbiotic genotypes were conserved between oases and other soils of the north of the country. Sinorhizobia isolated from bean were then compared with isolates from Medicago truncatula plants grown in the oases soils. All the nodC types except for nodC type p that was specific to common bean isolates were shared by both hosts. The four isolates with nodC type p induced N-2-fixing effective nodules on common bean but did not nodulate M. truncatula and Medicago sativa. The phylogenetic analysis of nifH and nodC genes showed that these isolates carry symbiotic genes different from those previously characterized among Medicago and bean symbionts, but closely related to those of S. fredii Spanish and Tunisian isolates effective in symbiosis with common bean but unable to nodulate soybean. The creation of a novel biovar shared by S. meliloti and S. fredii, bv. mediterranense, was proposed

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins.

Habitat fragmentation affects the biodiversity and function of aboveground organisms in natural ecosystems but has not been studied for effects on belowground species. In this paper, we consider the diversity of the rhizobia associated with the indigenous legume Dalea purpurea in 3 residual prairie areas in Minnesota and Iowa. Using Dalea purpurea as a trap host, 218 rhizobia were recovered from these soils then characterized using BOXA1R PCR. Three major and 13 minor groups were distinguished based on a similarity of greater than 75% in fingerprint patterns. Each major group consisted almost exclusively of rhizobia from a single prairie, with the diversity of Dalea rhizobia recovered from the Hayden Prairie less than that obtained with rhizobia from the other prairies. Based on 16S rRNA gene sequence analysis, isolates from the Hayden, Ordway, and Kellogg–Weaver Dunes prairies were most similar to Rhizobium etli and Rhizobium leguminosarum, Rhizobium gallicum, and Mesorhizobium amorphae and Mesorhizobium huakuii, respectively. This variation in the dominant microsymbiont species across the 3 prairies studied was unexpected but could have been influenced by the limited number of samples that we were allowed to take, by unanticipated cross-nodulation between native legumes, and by variation in the range of legume species present in each residual prairie area. While some of the rhizobia from Dalea nodulated Phaseolus vulgaris, Macroptilium atropurpureum, Leucaena leucocephala, and Onobrychis viciifolia in addition to the Dalea species tested, others nodulated Astragalus canadensis or Amorpha canescens.Key words: Rhizobium, Dalea, prairie, diversity, fragmentation, rep PCR.

Fifteen isolates from several nodulated tropical legumes from Puerto Rico (USA) were characterised by their phenotypic, molecular and symbiotic features. The identification of isolates was based on a polyphasic approach, including phenotypic characteristics, 16S rRNA sequencing, Low molecular weight (LMW) RNA profiles, Two Primers-RAPD patterns, and restriction patterns from 16S rDNA molecules. Despite of the variety of hosts included in this study the 15 isolates were separated into only two groups that corresponded to Rhizobium gallicum and Rhizobium tropici. This work shows that R. gallicum and R. tropici nodulate legume plants, such as Sesbania, Caliandra, Poitea, Piptadenia, Neptunia and Mimosa species, that were not previously considered as hosts for these rhizobia. Moreover, some of these host plants can be nodulated by both species. The results confirm the great promiscuity of R. tropici and also support the hypothesis that the species R. gallicum may be native from America or cosmopolitan and worldwide spread.

Previous studies have demonstrated that cellular fatty acid analysis is a useful tool for identifying unknown strains of rhizobia and establishing taxonomic relationships between the species. In this study, the fatty acid profiles of over 600 strains belonging to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were evaluated using the gaschromatography-based Sherlock Microbial Identification System (MIS). Data collected with the MIS showed that the three phylogenetically defined biovars of the genus Agrobacterium formed discrete clusters, whilst species belonging to the genus Mesorhizobium formed three subclusters which were easily distinguished. These three subclusters contained Mesorhizobium ciceri and Mesorhizobium mediterraneum, Mesorhizobium tianshanense fatty acid group I and Mesorhizobium plurifarium, and Mesorhizobium huakuii and Mesorhizobium loti. The genus Sinorhizobium was composed of an individual position for Sinorhizobium meliloti and a large cluster comprising Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium terangae, Sinorhizobium kostiense and Sinorhizobium arboris. S. meliloti contained significantly higher levels of the fatty acid 19:0 cyclo omega 8 cis and clustered with Rhizobium sp. (Hedysarum coronarium). However, discrimination between the species of genera Sinorhizobium and Rhizobium was a function of the concentration of 16:0 3-OH. The genus Rhizobium contained a single cluster containing Rhizobium sp. (Hedysarum coronarium), Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium etli, along with individual positions for Rhizobium giardinii, Rhizobium tropici, Rhizobium galegae and Rhizobium hainanense. R. tropici and R. hainanense exhibited similarity to Agrobacterium biovar 2, whilst R. galegae was similar to Agrobacterium biovar 1. R. giardinii appeared unique, with comparatively little similarity to the other species. Analysis of the genus Bradyrhizobium revealed large differences from the other genera studied. Two subgroups of Bradyrhizobium elkanii were detected and easily distinguished from Bradyrhizobium japonicum. Bradyrhizobium liaoningense and Bradyrhizobium sp. (Arachis hypogaea), a group isolated from Chinese peanut plants, showed similarities to B. japonicum, whilst a subgroup of M. tianshanense appeared identical to Bradyrhizobium sp. (Arachis hypogaea).

The genetic diversity of 44 rhizobial isolates from Astragalus, Oxytropis, and Onobrychis spp. originating from different geographic locations was evaluated by mapped restriction site polymorphism (MRSP) analysis of 16S rRNA genes and by PCR DNA fingerprinting with repetitive sequences (REP-PCR). A comparison of tree topologies of reference strains constructed with data obtained by MRSP and by 16S rRNA gene sequence analyses showed that the topologies were in good agreement, indicating that the MSRP approach results in reasonable estimates of rhizobial phylogeny. The isolates were distributed into 14 distinct 16S rRNA gene types clustering into three major groups which corresponded with three of the genera within the legume symbionts. Most of the isolates were within the genus Mesorhizobium. Five were identified with different genomic species nodulating Lotus spp. and Cicer arietinum. Three Astragalus isolates were classified as Bradyrhizobium, one being similar to Bradyrhizobium elkanii and another being similar to Bradyrhizobium japonicum. Six of the isolates were related to species within the genus Rhizobium. Two were similar to Rhizobium leguminosarum, and the remainder were identified as Rhizobium gallicum. DNA fingerprinting by REP-PCR revealed a high level of diversity within single 16S ribosomal DNA types. The 44 isolates were distributed into 34 REP groups. Rhizobial classification at the genus and probably also the species levels was independent of geographic origin and host plant affinity.

The phylogenetic positions of four rhizobial strains obtained from nodules of common bean plants (Phaseolus vulgaris L.) grown in an Austrian soil and of the Mexican bean isolate FL27 are described. Analysis of the 16S rRNA genes revealed sequences almost identical to that of the Rhizobium gallicum type strain, R602sp, with a maximum of two nucleotide substitutions. Comparison of the 16S rRNA gene sequences with those from other bacteria indicated highest similarity to Rhizobium sp. strain OK-50, Rhizobium leguminosarum IAM 12609, and Rhizobium etli. DNA homology determined by DNA-DNA hybridization was high among the Austrian isolates and R602spT (45 to 90%) and ranged from 21 to 65% with FL27, but hybridization analysis revealed very low homology to the recognized common bean-nodulating species, R. leguminosarum bv. phaseoli, R. etli, and Rhizobium tropici. Ribosomal gene organization was studied by Southern hybridization with the 16S rRNA gene and temperature gradient gel electrophoresis, indicating identical organizations and the presence of three identical 16S rRNA copies in the genome of this species. The six strains investigated showed different plasmid profiles based on their geographical origins. We propose that the Austrian isolates and the Mexican strain FL27 are members of the species R. gallicum.

Seventy-two rhizobial strains were isolated from the root nodules of french beans (Phaseolus vulgaris). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nifH from Rhizobium etli bv. phaseoli) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nifH and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co-occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.

Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.

Plant specificities and deoxyribonucleic acid homologies were studied among 122 strains of Rhizobium. Some strains were assigned to species on the basis of their source of isolation and present nodulation capabilities, but many did not fit into one of the six currently recognized species of the genus Rhizobium. Among those strains assigned to species were many which also nodulated plants outside their species-specific, cross-inoculation group. Conversely, isolates from a wide variety of plants could be designated Rhizobium phaseoli since they were capable of nodulating Phaseolus vulgaris. Acid production and growth rate on yeastmannitol agar were tested for all strains. Some strains grew rapidly but did not produce an acid reaction; these were grouped with the fast growing acid producers. Deoxyribonucleic acid homology was used to identify four genetic groups of fastgrowing, acid-producing rhizobia. Group 1 included strains of Rhizobium trifolii (except strains obtained from Trifolium lupinaster, Rhizobium leguminosarum, Rhizobium phaseoli (obtained from Phaseolus vulgaris), and two strains obtained from Neptunia gracilis. Group 2 comprised six American strains obtained from crown vetch (Coronilla varia), sainfoin (Onobrychis vicifolia), and Sophora spp. Species status for this group should remain tentative until further strains have been studied. Group 3 corresponded with Rhizobium meliloti as presently defined. Group 4 included fast-growing Lotus rhizobia, two strains obtained from T. lupinaster, and a wide variety of previously unclassified strains. Nine fastgrowing strains could not be included in any of these groups. The nine slowgrowing, non-acid producing strains included in this study showed < 10% homology with DNAs from seven fast-growing reference strains. The relationships between subgroups in group 1 are discussed, and the genetic diversity of strains obtained from Phaseolus vulgaris is examined. It is proposed that fast-growing rhizobia comprise at least four species corresponding with the four genetic groups described.

Summary: We have previously reported that Rhizobium phaseoli has multiple copies of nitrogen-fixation gene sequences. In this work we extend our analysis to cover a broader range of R. phaseoli strains and other rhizobia isolated from different legumes. Our results indicate that most R. phaseoli strains have reiterated nifH sequences. Reiterations are also found in rhizobia from nodules of other Phaseolus species and from the close relative of Phaseolus, Pachyrhizus. However, nifH gene reiteration is not always found in the rhizobia able to nodulate and fix nitrogen in Phaseolus, since some strains isolated from nodules of Phaseolus vulgaris as well as from other legumes are able to establish an effective symbiosis with Phaseolus vulgaris and do not show reiterations. We propose that there are different evolutionary lines of R. phaseoli. The reiteration of nif genes may be considered a marker for the most abundant and specialized of these evolutionary lines.