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A 260-bp segment of the DNA that encodes 16S rRNA, corresponding to positions 44 to 337 in the Escherichia coli sequence, was amplified by the polymerase chain reaction and sequenced frofn each of 13 bacteria (rhizobia and purple phototrophs) in the alpha subdivision of the class Proteobacteria. The phylogenetic tree calculated from differences in the sequenced segment conforms well to our expectations based on other previously published data. The sequence from BTAil (a recently described phototrophic symbiont of the legume Aeschynomene) and that from the free-living phototroph Rhodopseudomonas palustris both fall within the range of variation found among strains of the soybean symbiont Bradyrhizobium japonicum. This suggests that it would be appropriate to include all of these organisms in a single genus.

The current classification of the rhizobia (root-nodule symbionts) assigns them to six genera. It is strongly influenced by the small subunit (16S, SSU) rRNA molecular phylogeny, but such single-gene phylogenies may not reflect the evolution of the genome as a whole. To test this, parts of the atpD and recA genes have been sequenced for 25 type strains within the α-Proteobacteria, representing species in Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium, Agrobacterium, Phyllobacterium, Mycoplana and Brevundimonas. The current genera Sinorhizobium and Mesorhizobium are well supported by these genes, each forming a distinct phylogenetic clade with unequivocal bootstrap support. There is good support for a Rhizobium clade that includes Agrobacterium tumefaciens, and the very close relationship between Agrobacterium rhizogenes and Rhizobium tropici is confirmed. There is evidence for recombination within the genera Mesorhizobium and Sinorhizobium, but the congruence of the phylogenies at higher levels indicates that the genera are genetically isolated. rRNA provides a reliable distinction between genera, but genetic relationships within a genus may be disturbed by recombination. Rhizobium, Sinorhizobium, Mesorhizobium, recA, atpD Keywords : INTRODUCTIONThe rhizobia are root-nodulating bacteria responsible for a significant part of the global fixation of nitrogen. The ability of rhizobia to nodulate plants and reduce N # is conferred by genes that are plasmid-borne in many species (Pueppke, 1996), and the lateral transfer The atpD and recA sequences and details of the SimPlot analyses are available as supplementary material in IJSEM Online (http :// ijs.sgmjournals.org/ ).Abbreviations : HKY85, Hasegawa-Kishino-Yano model ; K2P, Kimura's two-parameter model ; SSU, small subunit rRNA.The EMBL accession numbers for the sequences reported in this study are AJ294386-AJ294409 (atpD) and AJ294363-AJ294385 (recA). of these genes is the most likely explanation for their occurrence within several distinct clades of subgroup 2 of the α-Proteobacteria (Dobert et al., 1994 ; Kaijalainen & Lindstro$ m, 1989 ;Lindstro$ m et al., 1995 ;Young, 1998 ;Young & Johnston, 1989). The phylogeny of the nodulation genes is quite different from that of the small subunit rRNA (SSU or 16S rRNA) genes in these bacteria. As in other bacterial groups, the SSU phylogeny has had a major influence on our current perception of evolutionary relationships among rhizobia ( Willems & Collins, 1993 ;Young, 1996 ;Young et al., 1991). More than 20 species have been described, and they are classified into the genera Rhizobium, Sinorhizobium, Allorhizobium, Mesorhizobium, Bradyrhizobium and Azorhizobium as well as ' Methylobacterium nodulans ' (de Lajudie et al., 1994' (de Lajudie et al., , 1998b Dreyfus et al., 1988 ; Jarvis et al., 1997 ; Jordan, 1982 ;Sy et al., 2001 M. W. Gaunt and others ing much slower growth on laboratory media (Dreyfus et al., 1988 ; Fred et al., 1932 ; Jordan, 1982). They are also fairly distant in the SSU p...

We used differences in small subunit ribosomal RNA genes to identify groups of arbuscular mycorrhizal fungi that are active in the colonisation of plant roots growing in arable fields around North Yorkshire, UK. Root samples were collected from four arable fields and four crop species, fungal sequences were amplified from individual plants by the polymerase chain reaction using primers NS31 and AM1. The products were cloned and 303 clones were classified by their restriction pattern with HinfI or RsaI; 72 were subsequently sequenced. Colonisation was dominated by Glomus species with a preponderance of only two sequence types, which are closely related. There is evidence for seasonal variation in colonisation in terms of both level of colonisation and sequence types present. Fungal diversity was much lower than that previously reported for a nearby woodland.

A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16s ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nod4 and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16s ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16s rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal.

The sequence of a 292 bp segment of the DNA encoding 16s rRNA (corresponding to positions 44-337 of the Escherichia coli 16s rRNA sequence) was determined for each of 40 Pseudomonas solanacearum, four banana Blood Disease Bacterium, three P. syzygii and two P. pickettii strains. Phylogenetic relationships derived from comparison of these sequences to each other, and to equivalent 16s rRNA gene sequences from other bacteria present in the EMBL databank, conform well with those obtained previously by DNA-DNA/rRNA hybridization experiments. The 16s rRNA sequence of the Blood Disease Bacterium was identical over the 292 bp to one of the four sequence groups of P. solanacearum, suggesting that these pseudomonads are more closely related to each other than to P. syzygii or P. pickettii. Sequence data comparisons allowed construction of an oligonucleotide specific for P. solanacearum, P. syzygii and the Blood Disease Bacterium. Use of the specific oligonucleotide with a non-specific oligonucleotide in the polymerase chain reaction enabled 1-10 cells of bacteria in this group to be detected after 50 rounds of amplification by visualizing a 287-288 bp product on agarose gels.

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. YThe taxonomy of the root-and stem-nodulating bacteria of legumes is in a state of transition. The classification of these organisms based on plant infection (3, 21) has been abandoned after extensive criticism (23, 78) and the demonstration that the genes coding for nodulation, host specificity, and nitrogen fixation in fast-growing rhizobia are located on transmissible symbiotic plasmids (6, 48, 53). Introduction of Adansonian and holistic approaches (10, 12, 23) led initially to a consolidation of species but more recently has resulted in the description of additional genera and species. In some cases, this has led to confusion as to the proper terminology for these organisms and how they should be distinguished.One of the functions of the subcommittees of the International Committee of Systematic Bacteriology is to recommend minimal standards for the valid publication of new taxa (38) and so to avoid the situation in which the literature includes many inadequately described bacteria for which no type strain is available. In this paper, the members of the International Subcommittee for the Taxonomy of Rhizobium and Agrobacterium briefly review recent developments in the taxonomy of root-and stem-nodulating bacteria and then propose minimal standards for their description.Current taxonomy of the root-nodule bacteria of legumes. After reviewing data on the numerical taxonomy, DNA mole percent G + C values, nucleic acid hybridizations, cistron similarities, serological relationships, extracellular polysaccharide composition, carbohydrate utilization patterns, and metabolic capacities, antibiotic sensitivities, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein-banding patterns, and speed of growth of rhizobia on laboratory media, Jordan (31, 32) separated the root-nodule bacteria into two genera, Rhizobium and Bradyrhizobium. Those organisms previously designated R . leguminosarum, R . trifolii, and R . phaseoli were combined into a single species, R. leguminosarum, with three biovar designations. Two other species, R. meliloti and R . loti, were listed in this genus, but slowly growing nodule bacteria were transferred to the genus Bradyrhizobium, with the designation of a single * Corresponding author. species, B . japonicum. This classification was clearly an interim one in that (i) it was based on rhizobia collected from only 15% of the 19,700 species of Leguminosae (1) and so was unlikely to be representative, (ii) it failed to consider the recently identified fast-growing rhizobia from soybean (34), and (iii) it grouped many slowly ...

The genetic diversity of symbiosis (Sym) plasmids was investigated in samples from two field populations of Rhizobiurn legurninosarurn biovar viceae that had previously been characterized for chromosomally-encoded enzyme electrophoretic polymorphism. Five overlapping cloned DNA fragments from the Sym plasmid pRLlJI were used as hybridization probes to identify restriction fragment variation in the homologous genes of the isolates. In addition, a clone of the P-galactosidase gene region was used as a probe, extending the data on chromosomal relatedness. The plasmid-encoded Sym region was very polymorphic (1 1.4% average DNA sequence divergence). Some isolates had the same Sym fragment pattern as pRLl JI, but most were very different. One was closely similar to pRL6J1, another widely-studied viceae plasmid. The distribution of plasmids across chromosomal backgrounds was far from random, as though the species were subdivided into compartments with largely separate plasmid pools. Nevertheless, indistinguishable plasmids were found in quite different genetic backgrounds, implying that plasmid transfer must occur in the field. This has implications for the population genetics and evolution of bacteria and for the release of genetically engineered strains.