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Previously, 159 bacterial strains were isolated from the root nodules of wild perennial Glycyrrhiza legume species grown on 40 sites in central and north-western China, in which 57 strains were classified as "true symbionts" belonging to the genus Mesorhizobium based on amplified fragment length polymorphism (AFLP) genomic fingerprinting and partial sequences of the 16S rRNA gene [20]. In the present work, the phylogeny of Glycyrrhiza nodulating mesorhizobia was further examined by multilocus sequence analysis (MLSA). The concatenated gene tree of three housekeeping genes (16S rRNA, recA, and rpoB) of 59 strains including the 29 mesorhizobial test strains and 30 type mesorhizobial species, was constructed applying the maximum likelihood method and Bayesian inference. In the concatenated gene tree, the 29 test strains were distributed in seven separate clades. Seventeen test strains clustered with Mesorhizobium tianshanense, Mesorhizobium temperatum, Mesorhizobium muleiense, and Mesorhizobium alhagi with high bootstrap support (BS>85%). Eight test strains did not cluster with any of the described Mesorhizobium species. Based on the results, we proposed these eight test strains might belong to a putative new species of the genus Mesorhizobium. The sequences of three accessory genes (nodA, nodC, and nifH) of the test strains were also analyzed and were compared with those of representatives of the 30 described mesorhizobial species. The results showed that mesorhizobia involved in symbiosis with Glycyrrhiza plants probably have acquired some genetic material from other rhizobia in co-evolution with Glycyrrhiza and other legume species.

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. 15N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen.

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.

Eighty-seven rhizobial strains isolated from root nodules of field standing native and exotic woody legumes in southern Ethiopia were characterized using the Biolog method and AFLP fingerprinting technique. Cluster analysis of the metabolic and genomic fingerprints revealed 18 and 25 groups, respectively, demonstrating considerable diversity in rhizobial population indigenous to Ethiopian soils. While 25 strains (29%) were linked to members of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium or Sinorhizobium, the bulk of the strains formed several distinct groups in both methods and did not relate to reference species included in the study. In contrast to exotic species which formed symbiosis with strains of only one specific genomic group, indigenous host species nodulated by metabolically and genomically diverse groups. The results in this study support the view, that long-term association between the symbionts allows gradual differentiation and diversity in compatible rhizobial population resident in native soils. Lack of significant metabolic and genomic relatedness to the reference strains in our results suggested that test strains in our collection probably included 'unique' types, which belong to several yet undefined rhizobial species.

Twenty-six Rhizobium galegae strains, representing the center of origin of the host plants Galega orientalis and G. officinalis as well as other geographic regions, were used in a polyphasic analysis of the relationships of R. galegae strains. Phage typing, lipopolysaccharide (LPS) profiling, pulsed field gel electrophoresis (PFGE) profiling and rep-PCR (use of repetitive sequences as PCR primers for genomic fingerprinting) with REP and ERIC primers investigated nonsymbiotic properties, whereas plasmid profiling and hybridisation with a nif gene probe, and with nodB, nodD, nod box and an IS sequence from the symbiotic region as probes, were used to reveal the relationships of symbiotic genes. The results were used in pairwise calculations of distances between the strains, and the distances were visualised as a dendrogram. Indexes of association were compared for all tests pooled, and for chromosomal tests and symbiotic markers separately, to display the input of the different categories of tests on the grouping of the strains. Our study shows that symbiosis related genetic traits in R. galegae divide strains belonging to the species into two groups, which correspond to strains forming an effective symbioses with G. orientalis and G. officinalis respectively. We therefore propose that Rhizobium galegae strains forming an effective symbiosis with Galega orientalis are called R. galegae bv. orientalis and strains forming an effective symbiosis with Galega officinalis are called R. galegae bv. officinalis.

Twenty-two rhizobial strains isolated from the root nodules of two Chinese peanut cultivars (Arachis hypogaea L. Tianfu no. 3 and a local cultivar) growing at four different sites in the Sichuan province, Southwest China, were characterized by growth rate, rep-PCR, PCR-RFLP of 16S rDNA, partial sequencing of ribosomal genes, and fatty acid-methyl ester analysis (FAME), and compared with strains representing Bradyrhizobium japanicum, B. elkanii and other unclassified Bradyrhizobium sp. All peanut isolates from Sichuan were bradyrhizobia. Dendrograms constructed using the rep-PCR fingerprints grouped the strains mainly according to their geographic and cultivar origin. Based on PCR-RFLP and partial sequence analysis of 16S rDNA it appears that peanut bradyrhizobial strains from Sichuan are similar to peanut strains from Africa and Israel, and closely related to B. japonicum. In contrast, analysis of FAME data using two-dimensional principal component analysis indicated that Bradyrhizobium sp. (Arachis) were similar to, but slightly different from other bradyrhizobia. The presence and level of fatty acid 16:1 w5c was the distinguishing feature. The results of PCR-RFLP of the 16S rRNA gene, the partial sequence analysis of 16S rDNA, and FAME were in good agreement.

Summary The genetic diversity among 204 rhizobial isolates from field-grown Astragalus sinicus collected from 52 ecological different sites of seven southern provinces of China was investigated by using rep-PCR fingerprinting with REP primers and 16S rDNA PCR-RFLP and sequence analyses. Most isolates (78.4%) belonged to different 16S rDNA genotypes from that of Mesorhizobium huakuii type strain CCBAU2609 T . Rep-PCR fingerprinting analysis of 136 randomly chosen isolates categorized 27 REP groups at a similarity level of 60%. Genetically diverse rhizobial strains could be detected at single site, and strains with the same genetic type could be found in different geographic locations. Twelve representative strains were selected for identification using 16S and 23S rDNA analyses, which resulted in four 16S and five 23S rDNA PCR-RFLP genotypes. When both 16S and 23S rDNA PCR-RFLP data were combined together, seven different genotypes were detected among the 12 representative strains, and no strain shared a genotype with the M. huakuii type strain. Both 16S and 23S rRNA genes analyses clustered the 12 representative stains within the Mesorhizobium branch. The 16S rRNA results obtained by PCR-RFLP were confirmed by sequencing the first 600 bp of the 16S rRNA genes.

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The minimum growth temperature was 8 degrees C for C. milleri and also for most of sour dough yeasts.

Summary To assess the phylogenetic diversity of fast-growing rhizobium strains isolated from root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Kenya, a 230-nucleotide segment of the 16S ribosomal RNA gene was sequenced from each of thirty strains. Twelve different sequences were found: four were identical to those of previously described species, eight were novel. Sequence comparisons indicated that one strain belonged to the Rhizobium huakuii phylogenetic branch, while the rest were Sinorhizobium , as they were close to S. (Rhizobium) meliloti, S. f redii, S. teranga and S. saheli . Isolates from Acacia and from Prosopis often had the same sequence, but the sequence type was not very well correlated with the phenotypic characteristics determined previously for these strains. One isolate had two 16S rRNA sequences, differing at six positions, in approximately equal copy number. The type strain of S. saheli was also shown to have two different sequences.

Summary Repetitive sequences (repetitive extragenic palindromic [REP] and enterobacterial repetitive intergenic consensus [ERIC]) together with the polymerase chain reaction (PCR) were used to fingerprint pure DNA extracted from seven different Rhizobium galegae strains. The resulting PCR patterns were found to be highly specific for each strain and the REP PCR grouped the strains according to the host plant, Galega orientalis and Galega officinalis. The dendrogram generated from the REP PCR patterns correlated with the dendrogram derived from previous restriction fragment length polymorphism (RFLP) analysis. DNA was also extracted by sonication from R. galegae liquid cultures and root nodules and used in REP and ERIC PCR. The results obtained indicate that this technique coupled with the sonication of nodules can be a useful tool for genotypic characterization and identification of rhizobia as well as for diversity studies.