Intraspecific chemical variation of Tanacetum vulgare affects plant growth and reproductive traits in field plant communities.

The study investigated the impact of intraspecific plant chemodiversity on plant growth and reproductive traits at both the plant and plot levels. It also aimed to understand how chemodiversity at stand level affects ecosystem functioning and plant-plant interactions. We describe a biodiversity experiment in which we manipulated intraspecific plant chemodiversity at the plot level using six different chemotypes of common tansy (Tanacetum vulgare L., Asteraceae). We tested the effects of chemotype identity and plot-level chemotype richness on plant growth and reproductive traits and plot-level headspace emissions. The study found that plant chemotypes differed in growth and reproductive traits and that traits were affected by the chemotype richness of the plots. Although morphological differences among chemotypes became less pronounced over time, reproductive phenology patterns persisted. Plot-level trait means were also affected by the presence or absence of certain chemotypes in a plot, and the direction of the effect depended on the specific chemotype. However, chemotype richness did not lead to overyielding effects. Lastly, chemotype blends released from plant communities were neither richer nor more diverse with increasing plot-level chemotype richness, but became more dissimilar as they became more dissimilar in their leaf terpenoid profiles. We found that intraspecific plant chemodiversity is crucial in plant-plant interactions. We also found that the effects of chemodiversity on plant growth and reproductive traits were complex and varied depending on the chemotype richness of the plots. This long-term field experiment will allow further investigation into plant-insect interactions and insect community assembly in response to intraspecific chemodiversity.

Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation.

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency approximately 68 %) but was recovered infrequently ( approximately 1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species 'Sinorhizobium morelense'. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

The genetic diversity of rhizobia associated with Dalea purpurea Vent. in fragmented grasslands of west-central Minnesota.

The increase in human population and the spread of agriculture over the past 150 years have transformed the landscape in west-central Minnesota into a mosaic of agricultural fields and urban land, leaving only remnants of the once dominant prairie ecosystem. Limited natural habitat in this fragmented landscape threatens the diversity and abundance of native legumes and could impact the size and function of associated belowground microbial populations. In this study, BOXA1R PCR and 16S rRNA gene sequence analyses were used to assess the genetic diversity of rhizobia associated with Dalea purpurea (Vent.) in nine prairie remnants ranging in size from 0.04 to 3.5 ha. The variation in soil properties was also determined. While 53 different genotypes of rhizobia were identified, four of these accounted for 84% of the 1029 rhizobia characterized using BOXA1R PCR. Representatives from three of the four dominant genotypes had a 16S rRNA gene sequence similar to that of Rhizobium gallicum, with two of these genotypes recovered at all sites. The fourth genotype was similar to that of Rhizobium etli and occurred with frequency at only two sites. Rhizobium genotype richness and site area were positively correlated. The implications of these results are discussed.

Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil.

The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR-RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.

Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale.

Forty-six bacterial cultures, including one culture collection strain, thirty from the rhizosphere of Alyssum murale and fifteen from Ni-rich soil, were tested for their ability to tolerate arsenate, cadmium, chromium, zinc, mercury, lead, cobalt, copper, and nickel in their growth medium. The resistance patterns, expressed as minimum inhibitory concentrations, for all cultures to the nine different metal ions were surveyed by using the agar dilution method. A large number of the cultures were resistant to Ni (100%), Pb (100%), Zn (100%), Cu (98%), and Co (93%). However, 82, 71, 58 and 47% were sensitive to As, Hg, Cd and Cr(VI), respectively. All cultures had multiple metal-resistant, with heptametal resistance as the major pattern (28.8%). Five of the cultures (about of 11.2% of the total), specifically Arthrobacter rhombi AY509239, Clavibacter xyli AY509235, Microbacterium arabinogalactanolyticum AY509226, Rhizobium mongolense AY509209 and Variovorax paradoxus AY512828 were tolerant to nine different metals. The polymerase chain reaction in combination with DNA sequence analysis was used to investigate the genetic mechanism responsible for the metal resistance in some of these gram-positive and gram-negative bacteria that were, highly resistant to Hg, Zn, Cr and Ni. The czc, chr, ncc and mer genes that are responsible for resistance to Zn, Cr, Ni and Hg, respectively, were shown to be present in these bacteria by using PCR. In the case of, M. arabinogalactanolyticum AY509226 these genes were shown to have high homology to the czcD, chrB, nccA, and mer genes of Ralstonia metallidurans CH34. Therefore, Hg, Zn, Cr and Ni resistance genes are widely distributed in both gram-positive and gram-negative isolates obtained from A. murale rhizosphere and Ni-rich soils.

Chromate-tolerant bacteria for enhanced metal uptake by Eichhornia crassipes (Mart.).

A total of 85 chromate-resistant bacteria were isolated from the rhizosphere of water hyacinth grown in Mariout Lake, Egypt, as well as the sediment and water of this habitat. Only 4 (11%), 2 (8%), and 2 (8%) of isolates from each of the environments, respectively, were able to tolerate 200 mg Cr (VI) L(-1). When these eight isolates were tested for their ability to tolerate other metals or to reduce chromate, they were shown to also be resistant to Zn, Mn, and Pb, and to display different degrees of chromate reduction (28% to 95%) under aerobic conditions. The isolates with the higher chromate reduction rates from 42% to 95%, (RA1, RA2, RA3, RA5, RA7, and RA8) were genetically diverse according to RAPD analysis using four differentprimers. Bacterial isolates RA1, RA2, RA3, RAS, and RA8 had 16 S rRNA gene sequences that were most similar to Pseudomonas diminuta, Brevundimonas diminuta, Nitrobacteria irancium, Ochrobactrum anthropi, and Bacillus cereus, respectively. Water hyacinth inoculated with RA5 and RA8 increased Mn accumulation in roots by 2.4- and 1.2-fold, respectively, compared to uninoculated controls. The highest concentrations of Cr (0.4 g kg(-1)) and Zn (0.18 g kg(-1)) were accumulated in aerial portions of water hyacinth inoculated with RA3. Plants inoculated with RA1, RA2, RA3, RA5, RA7, and RA8 had 7-, 11-, 24-, 29-, 35-, and 21-fold, respectively, higher Cr concentrations in roots compared to the control. These bacterial isolates are potential candidates in phytoremediation for chromium removal.

Characteristics of the rhizobia associated with Dalea spp. in the Ordway, Kellogg-Weaver Dunes, and Hayden prairies.

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.

Rhizobial 16S rRNA and dnaK genes: mosaicism and the uncertain phylogenetic placement of Rhizobium galegae.

The phylogenetic relatedness among 12 agriculturally important species in the order Rhizobiales was estimated by comparative 16S rRNA and dnaK sequence analyses. Two groups of related species were identified by neighbor-joining and maximum-parsimony analysis. One group consisted of Mesorhizobium loti and Mesorhizobium ciceri, and the other group consisted of Agrobacterium rhizogenes, Rhizobium tropici, Rhizobium etli, and Rhizobium leguminosarum. Although bootstrap support for the placement of the remaining six species varied, A. tumefaciens, Agrobacterium rubi, and Agrobacterium vitis were consistently associated in the same subcluster. The three other species included Rhizobium galegae, Sinorhizobium meliloti, and Brucella ovis. Among these, the placement of R. galegae was the least consistent, in that it was placed flanking the A. rhizogenes-Rhizobium cluster in the dnaK nucleotide sequence trees, while it was placed with the other three Agrobacterium species in the 16S rRNA and the DnaK amino acid trees. In an effort to explain the inconsistent placement of R. galegae, we examined polymorphic site distribution patterns among the various species. Localized runs of nucleotide sequence similarity were evident between R. galegae and certain other species, suggesting that the R. galegae genes are chimeric. These results provide a tenable explanation for the weak statistical support often associated with the phylogenetic placement of R. galegae, and they also illustrate a potential pitfall in the use of partial sequences for species identification.

Characteristics of rhizobia nodulating beans in the central region of Minnesota.

Until recently, beans (Phaseolus vulgaris L.) grown in Minnesota were rarely inoculated. Because of this, we hypothesized that bean rhizobia collected in Minnesota would either share characteristics identifiable with Rhizobium etli of Mesoamerican or Andean origin, introduced into the region as seed-borne contaminants, or be indigenous rhizobia from prairie species, such as Dalea spp. The latter organisms have been shown to nodulate and fix N2 with Phaseolus vulgaris. Rhizobia recovered from the Staples, Verndale, and Park Rapids areas of Minnesota were grouped according to the results of BOXA1R-PCR fingerprint analysis into 5 groups, with only one of these having banding patterns similar to 2 of 4 R. etli reference strains. When representative isolates were subject to fatty acid - methyl ester analysis and 16S rRNA gene sequence analysis, the results obtained differed. 16S rRNA gene sequences of half the organisms tested were most similar to Rhizobium leguminosarum. Rhizobia from Dalea spp., an important legume in the prairie ecosystem, did not play a significant role as the microsymbiont of beans in this area. This appears to be due to the longer time needed for them to initiate infection in Phaseolus vulgaris. Strains of Rhizobium tropici IIB, including UMR1899, proved tolerant to streptomycin and captan, which are commonly applied as seed treatments for beans. Local rhizobia appeared to have very limited tolerance to these compounds.

A unique Mycobacterium species isolated from an epizootic of striped bass (Morone saxatilis).

We isolated a Mycobacterium sp. resembling Mycobacterium marinum and M. ulcerans from diseased striped bass (Morone saxatilis) during an epizootic of mycobacteriosis in the Chesapeake Bay. This isolate may represent an undescribed Mycobacterium species, based on phenotypic characteristics and comparative 16S rRNA gene sequence.

Characterization of soybean bradyrhizobia for which serogroup affinities have not been identified.

The USDA, ARS National Rhizobium Germplasm Collection contains 143 accessions of slow-growing soybean strains among which there are 17 distinct serological groups. However, 11 strains appear to have no serological affinity with the 17 serogroups. Therefore, we determined whether these strains were diverse and examined their phylogenetic placement. Nine strains formed nitrogen-fixing symbioses with soybean indicating that these accessions were not contaminants. We concluded from results of amplified fragment length polymorphism (AFLP) analysis, using 3 selective primers with 8 strains, that they were genetically dissimilar. Nine strains were examined for their fatty acid composition using fatty acid methyl ester (FAME) derivatives. The FAME results with 5 strains and serotype strains of Bradyrhizobium elkanii were similar, while results with each of the remaining 2 pairs were either similar to the type strain of Bradyrhizobium japonicum (USDA 6) or to USDA 110. Evolutionary history of 9 strains was reconstructed from sequence divergence of a combination of the complete 16S rRNA gene, the internally transcribed spacer region, and about 400 bases of the 5' end of the 23S rRNA gene. Placement of 5 strains was nested within B. elkanii, 2 with USDA 110, and the other 2 with USDA 6. We concluded that soybean isolates that cannot be placed within one of the 17 established serogroups are phenotypically and genetically as diverse as the serotype strains.

Variability in plant-microbe interaction between Lupinus lines and Bradyrhizobium strains.

Even though lupin (Lupinus albus L.) is known to potentially fix 150-200 kg/ha nitrogen for the use of a succeeding crop, precise information about lupinxBradyrhizobium strain interaction under the climatic conditions prevalent in the mid-Atlantic region of the United States is unknown. We conducted two greenhouse experiments with the objective of characterizing this symbiotic relationship and to evaluate potential interaction between Bradyrhizobium strains and lupin lines. In the first experiment, performance of 60 bradyrhizobial strains was evaluated by inoculating three lupin cultivars and using combined score, which consisted of an arithmetic total of plant vigor, nodulation scores from crown root, nodulation scores from fibrous roots, shoot dry weight, and root dry weight. In the second experiment, performance of 80 lupin lines was evaluated by inoculating with three selected Bradyrhizobial strains and using the combined score, which consisted of an arithmetic total of plant vigor, acetylene reduction activity, nodule number per plant, nodule weight per plant, and dry shoot weight. Significant variation existed for all traits in both experiments except for nodule number in the second experiment. Significant Bradyrhizobial strain by lupin line interaction existed for nodulation score, shoot and root dry weights, and the combined scores. Comparison of relative ranks indicated that nodulation effectiveness was dependent on specific strain and lupin line combinations. It was concluded that specific Bradyrhizobial strain and lupin line combinations would need to be identified for successful utilization of lupin's capability to fix atmospheric nitrogen for use in low-input and sustainable agriculture.

Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence.

From sequence divergence of 16S rRNA genes and the internally transcribed spacer (ITS) region it is reported that variation in phylogenetic placement exists among the 17 different serotype strains of Bradyrhizobium that have been isolated from nodules of soybean. Evolutionary relationships among the bradyrhizobia were more resolved using reconstructions derived from ITS than from 16S rRNA gene sequence divergence. Strain USDA 129 was placed together with USDA 62, 110, 122 and 126, but did not cluster with USDA 123 and 127, with which it shares antigenic determinants. The results from the phylogenetic analysis were supported with data from determinations of genetic diversity among additional strains within each of these serogroups using amplified fragment length polymorphism analysis. From these results it was concluded that strains of serogroup 129 were more similar to strains of serogroups 62, 110 and 122 than they were to strains of serogroups 123 and 127. The serotype strain of Bradyrhizobiumjaponicum USDA 135 and the type strain for Bradyrhizobium liaoningense possessed identical 16S rRNA gene and ITS region sequences. Also, the type strain for B. liaoningense cross-reacted with antisera prepared against somatic antigens of USDA 135. Therefore, it was not possible to distinguish B. liaoningense from serogroup 135 in our analysis of B. japonicum and Bradyrhizobium elkanii.

Discrimination between two Perkinsus spp. isolated from the softshell clam, Mya arenaria, by sequence analysis of two internal transcribed spacer regions and the 5.8S ribosomal RNA gene.

The internal transcribed spacer (ITS-1 and ITS-2) regions and the 5.8S ribosomal RNA gene of 2 Perkinsus spp. (G117 and H49) originating from the softshell clam, Mya arenaria, of the Chesapeake Bay were cloned and sequenced to obtain evidence for their genetic divergence. A high level of heterogeneity in both regions, probably resulting from deletions, insertions, and base substitutions, was evident from alignments of the sequences of the 2 isolates with published sequences of other Perkinsus spp. The isolate G117 and other Perkinsus spp. were highly divergent (13-26% and 19-20% sequence divergence in ITS-1 and ITS-2, respectively). These regions in the isolate H49 and Perkinsus marinus were similar (99.07% and 99% for ITS-1 and ITS-2, respectively). Evidence obtained from a phylogenetic analysis using the aligned sequences suggests that G117 and H49 belong to 2 distinct species of Perkinsus. The isolate G117 possibly belongs to an as yet undescribed species of Perkinsus, and H49 belongs to the species P. marinus. The conclusions drawn from the genetic analysis of H49 and G117 are supported by previously reported morphological characteristics (McLaughlin & Faisal, 1998b). Isolates H49 and G117 originated from the same molluscan species demonstrating that at least 2 different species of Perkinsus can co-exist in 1 host.

Characterization of two Perkinsus spp. from the softshell clam, Mya arenaria using the small subunit ribosomal RNA gene.

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.

Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and description of Mesorhizobium amorphae sp. nov.

Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-fixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplified 16S rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 16S rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16S rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 16S rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 16S rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov.

Rhizobium huautlense sp. nov., a symbiont of Sesbania herbacea that has a close phylogenetic relationship with Rhizobium galegae.

The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve at the Sierra de Huautla, Mexico, were isolated and characterized. All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 16S rRNA genes as revealed by PCR-RFLP analysis. Similarity in the sequences of the 16S rRNA genes placed the isolates on a phylogenetic branch shared with R. galegae. Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R. galegae. A new species Rhizobium huautlense, represented by the Sesbania isolate SO2T, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 16S rRNA genes, and their distinct ETs compared with R. galegae. The description of R. huautlense is significant because in the reconstruction of the phylogeny at R. huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R. galegae. The revised phylogenetic tree would tend to indicate common ancestry between R. galegae and Rhizobium leguminosarum.

Identification and sequencing of a cytochrome P450 gene cluster from Bradyrhizobium japonicum.

Sequencing of a region from Bradyrhizobium japonicum previously shown to encode for cytochromes P450 revealed a cluster of three complete P450 genes (CYP112, CYP114, and CYP117) plus a partial P450 gene fragment (CYP115P). Present also are five additional open reading frames. The close positioning of the genes suggests that they comprise an operon. Although the biochemical function of the gene products is uncertain, the similarities to other genes suggests an operon involved in terpenoid synthesis. ORF3 has similarity to a [3Fe-4S] ferredoxin from Streptomyces griseolus. ORF4 has strong similarity to members of the short chain alcohol dehydrogenase family, including sterol dehydrogenases from enteric bacteria and to some plant 3-oxoacyl-(acyl carrier protein) reductases. ORF6 has strong similarity to prenyl transferases, including dimethylallyltranstransferase from Escherichia coli. ORF7 bears some similarity to plant genes for ent-kaurene synthase (a precursor of gibberellins), and to bacterial squalene-hopene cyclases. ORF8 has some similarity to a Streptomyces gene for synthesis of the cyclic sesquiterpene pentalenene. The 5' end of the mRNA transcript is 38-39 nucleotides downstream from the center of a motif that bears sequence homology to bacterial fnr promoters. A gus operon fusion to the promoter was expressed anaerobically and symbiotically 6-10-fold greater than aerobically.

Rhizobium mongolense sp. nov. is one of three rhizobial genotypes identified which nodulate and form nitrogen-fixing symbioses with Medicago ruthenica [(L.) Ledebour].

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.