Reasons are advanced for removal of Rhizobium ciceri, Rhizobium huakuii, Rhizobium loti, Rhizobium mediterraneum, and Rhizobium tianshanense from the genus Rhizobium and for establishment of Mesorhizobium gen. nov. for these species. A description of the genus Mesorhizobium and amended descriptions of Mesorhizobium ciceri, Mesorhizobium huakuii, Mesorhizobium loti, Mesorhizobium mediterraneum, and Mezorhizobium tianshanense are provided.In a review of root nodule symbioses by Vincent (22), the fast-growing rhizobia associated with Lotus comiculatus and Lupinus densiflorus were recognized as a separate group which merited a specific designation, and the name Rhizobium loti was tentatively proposed for it. Approval for the designation of R. loti as a new species was also voiced at a roundtable discussion on Rhizobium taxonomy associated with the 4th International Congress on Nitrogen Fixation at Canberra, Australia, in 1980. The subsequent publication of the new species in 1982 (9) enabled it to be included in a revised taxonomy of the Rhizobiaceae presented by Jordan in Bergey's Manual of Systematic Bacteriology (12).At that time R. loti was distinguished from other fast-growing rhizobia on the basis of flagellation (l), esterase patterns (14), response to isoflavonoids (18), plant nodulation (11, 12), internal antigens (23), electrophoresis of soluble cellular proteins (19,20), and DNA relatedness (4, 8). More recently, cellular fatty acid analysis was used to reveal differences, useful for identification purposes, between strains of R. loti and strains of the genus Agrobacterium and other Rhizobium or Sinorhizobium species (10). All of these methods differentiate species but give little indication of the relationships between species and genera.A n early indication of the relationship among R. loti, other Rhizobium species, and Sinorhizobium and Agrobacterium species was obtained by studying the intergeneric similarities of rRNA cistrons (7). The results of this analysis indicated that Rhizobium leguminosarum, Rhizobium galegae, Sinorhizobium meliloti, Sinorhizobium fredii, Agrobacterium tumefaciens (biovar l), and Agrobacterium rhizogenes (biovar 2) were all more closely related to one another than they were to R. loti. Subsequent analyses of 16s rRNA gene sequences of species in these genera have confirmed, refined, and extended this observation (21,24,25); the levels of 16s ribosomal DNA sequence similarity between R. loti and other Rhizobium and Agrobacterium species are around 93.5%. Consequently, there has been considerable support for the establishment of a separate genus for R. loti and related root nodule bacteria (5, 13, 27).The original description of R. loti (9) clearly indicated that fast-growing Lotus rhizobia formed part of an extensive plant cross-inoculation group involving plant species in the genera Lupinus, Ornithopus, Lotus, Anthyllis, Caragena, Astragalus, Ononis, Genista, and Mimosa. Jarvis et al. also indicated that the fast-growing Lotus rhizobia were related to rhizobia obtained from s...

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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 Bradyrhizobium japonicum 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.Keywords : Bradyrhizobium, phylogeny, soybean, Glycine max, serology INTRODUCTIONTraditional classification schemes of living organisms rely upon variation in morphological characters and a fossil record. Since there are only few useful morphological characters for rhizobia, classification schemes based on variation in genetic characters are often more informative than schemes based on phenotypic variation. An advantage of this approach is that DNA sequences and gene products can be compared The GenBank accession numbers for the ITS region sequences of strains USDA 122, 123, 124, 125, 126, 127, 129, 130, 135, 31, 3622 T (Bradyrhizobium liaoningense), 38, 4, 46, 62 and 94 are AF208503-AF208518, respectively. The GenBank accession numbers U35000 and U69638 were modified to reflect the sequencing results obtained with strains USDA 76 T and 6 T , respectively.in an evolutionary context (molecular systematics). Usually evolutionary history (or phylogeny) is then represented as a bifurcating hierarchical tree. It has become acceptable to reconstruct such evolutionary relationships among bacteria from data of sequence divergence among their 16S rRNA genes (Maidak et al., 1994).Published investigations of evolutionary relationships and genetic diversity in rhizobia has been focused more on the genera Rhizobium, Sinorhizobium and Mesorhizobium rather than Bradyrhizobium. Most reported data of diversity in Bradyrhizobium are centred on the symbionts of soybean. Initially, Bradyrhizobium (Rhizobium) japonicum was the only recognized species (Fred et al., 1932 ; Jordan, 1982). Later, Hollis et al. (1981) separated B. japonicum into three DNA homolog...

Summary• Alyssum murale has a remarkable ability to hyperaccumulate Ni from soils containing mostly nonlabile Ni. Here, rhizobacteria are shown to play an important role in increasing the availability of Ni in soil, thus enhancing Ni accumulation by A. murale .• Three bacteria, originally isolated from the rhizosphere of A. murale , were examined for their ability to solubilize Ni in soil and for their effect on Ni uptake into Alyssum . Sphingomonas macrogoltabidus , Microbacterium liquefaciens , and Microbacterium arabinogalactanolyticum were added to both sterile and nonsterile Ni-rich Serpentine soil from OR, USA.• Sphingomonas macrogoltabidus significantly reduced Ni extraction by 10 mM Sr(NO 3 ) 2 from soil, M. arabinogalactanolyticum significantly increased Ni extraction, whereas M. liquefaciens had no effect. Extractability of few other metals was affected by inoculation. When these bacteria were added to surface-sterilized seeds of A. murale grown in nonsterile soil, they increased Ni uptake into the shoot by 17% ( S. macrogoltabidus ), 24% ( M. liquefaciens ), and 32.4% ( M. arabinogalactanolyticum ), compared with uninoculated controls.• These results show that bacteria are important for Ni uptake, which from a commercial perspective, could significantly increase revenue generated during phytomining of Ni from soil.

It is evident from complete genome sequencing results that lateral gene transfer and recombination are essential components in the evolutionary process of bacterial genomes. Since this has important implications for bacterial systematics, the primary objective of this study was to compare estimated evolutionary relationships among a representative set of ␣-Proteobacteria by sequencing analysis of three loci within their rrn operons. Tree topologies generated with 16S rRNA gene sequences were significantly different from corresponding trees assembled with 23S rRNA gene and internally transcribed space region sequences. Besides the incongruence in tree topologies, evidence that distinct segments along the 16S rRNA gene sequences of bacteria currently classified within the genera Bradyrhizobium, Mesorhizobium and Sinorhizobium have a reticulate evolutionary history was also obtained. Our data have important implications for bacterial taxonomy, because currently most taxonomic decisions are based on comparative 16S rRNA gene sequence analysis. Since phylogenetic placement based on 16S rRNA gene sequence divergence perhaps is questionable, we suggest that the proposals of bacterial nomenclature or changes in their taxonomy that have been made may not necessarily be warranted. Accordingly, a more conservative approach should be taken in the future, in which taxonomic decisions are based on the analysis of a wider variety of loci and comparative analytical methods are used to estimate phylogenetic relationships among the genomes under consideration.Rhizobia are nitrogen-fixing bacterial symbionts of legumes that are of economic importance in low-input sustainable agriculture, agroforestry, and land reclamation. Descriptions of phenotypic and genetic variation among rhizobia have been extensive. Currently there are six genera of rhizobia, Allorhizobium, Azorhizobium, Bradyrhizobium, Rhizobium, Mesorhizobium, and Sinorhizobium, that have been proposed (65), and there is a report of a single species of Methylobacterium (of the family Methylobacteriaceae), Methylobacterium nodulans, which forms a symbiosis with specific species of Crotolaria (59). The primary criterion by which these genera are defined is analysis of 16S rRNA gene sequence (65).Sequencing the 16S rRNA gene has profoundly affected how relationships among the bacteria are portrayed (45). The 16S rRNA gene sequence is useful for this purpose because it is slowly evolving and the gene product is both universally essential and functionally conserved. However, basing bacterial phylogeny on 16S rRNA gene sequence variation not only presupposes that evolution throughout the genome progresses at a constant rate by mutation and Darwinian selection but also assumes that the evolution of the genome and of the 16S rRNA gene is strictly hierarchical. From a practical point of view this approach also requires each genome to harbor a single copy of the 16S rRNA gene or that multiple alleles within single genomes have identical sequences.Although seemingly convenient for c...

The phylogenetic relationships among 44 isolates representing 16 species of Stemphylium were inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase (gpd) sequence data. The results generally agree with morphological species concepts. There was strong support for monophyly of the genus Stemphylium. Analysis of the gpd fragment in particular was useful for establishing well-supported relationships among the species and isolates of Stemphylium. Species of Stemphylium that appear to have lost the ability to produce a sexual state are scattered among the species with the ability to reproduce sexually (Pleospora spp.). Species that are pathogenic to alfalfa are resolved into two groups. Stemphylium botryosum and two isolates with morphological characters similar to S. globuliferum had identical sequences at both loci. These two loci in S. vesicarium, S. alfalfae and S. herbarum are nearly identical but differ from S. botryosum. The separation of S. vesicarium, S. herbarum and S. alfalfae into separate species by morphometric evidence was not supported by the molecular data. Morphological and developmental characters such as size and shape of conidia, conidiophores, and ascospores, and size and time of maturation of pseudothecia are useful for diagnosing species. However, other morphological characters such as septum development and small variations in conidial wall ornamentation are not as useful.

Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213 T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.

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-f ixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplif ied 165 rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 165 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 165 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 165 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.