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To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.

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.

A total of 115 endophytic bacteria were isolated from root nodules of the wild legume Sphaerophysa salsula grown in two ecological regions of Loess Plateau in China. The genetic diversity and phylogeny of the strains were revealed by restriction fragment length polymorphism and sequencing of 16S rRNA gene and enterobacterial repetitive intergenic consensus-PCR. Their symbiotic capacity was checked by nodulation tests and analysis of nifH gene sequence. This is the first systematic study on endophytic bacteria associated with S. salsula root nodules. Fifty of the strains found were symbiotic bacteria belonging to eight putative species in the genera Mesorhizobium, Rhizobium and Sinorhizobium, harboring similar nifH genes; Mesorhizobium gobiense was the main group and 65 strains were nonsymbiotic bacteria related to 17 species in the genera Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus, which were universally coexistent with symbiotic bacteria in the nodules. Differing from other similar studies, the present study is the first time that symbiotic and nonsymbiotic bacteria have been simultaneously isolated from the same root nodules, offering the possibility to accurately reveal the correlation between these two kinds of bacteria. These results provide valuable information about the interactions among the symbiotic bacteria, nonsymbiotic bacteria and their habitats.

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.

We recently showed that Rhizobium galegae strains had two different lipopolysaccharide types, short and long O-chain. In the present study we observed that the lipopolysaccharide type was a stable feature of the strain. Both types persisted in cells at all phases of the growth cycle, during differentiation of bacteria into bacteroids and when the cells were grown under environmental stress. When R. galegae strains were grown at low pH or in medium containing aluminum or salt, simulating conditions in acid soils of temperate regions and osmotic stress, respectively, the tolerance of low pH was associated with the long O-chain lipopolysaccharide and abundant acidic polysaccharide production.

Rhizobial nodD genes produce transcriptional regulators that, together with appropriate inducer compounds, activate the other symbiotic nodulation (nod) genes and initiate the nodule formation process. Two nodD homologues, nodD1 and nodD2, are present in the Rhizobium galegae strain HAMBI 1174. In this work we analysed their ability to induce the nodA promoter with synthetic inducers known to activate nod genes in other rhizobia. According to phylogenetic analysis, the inducer-specific carboxy-terminal part of the R. galegae nodD protein sequence groups together with those of Rhizobium leguminosarum and Sinorhizobium meliloti. However, the respective inducer compounds for their NodD proteins are not highly effective with R. galegae nodD products. The best inducer discovered with R. galegae nodD1 was the root exudate of the host plant of R. galegae, Galega orientalis. HPLC analyses revealed the presence of many divergent flavonoid compounds in the G. orientalis root exudate. The most effective HPLC fractions induced R. galegae nodD1 up to the level obtained by intact G. orientalis root exudate while apigenin and luteolin, which were also present in the root exudate, were only moderate inducers. A UV-Vis diode array spectrum of the most active peak indicated that the main inducer present in the G. orientalis root exudate is an unidentified chalcone-type compound. In the Galega-R. galegae interaction the first recognition between the NodD protein and the flavonoid inducer secreted from the roots of Galega is specific for these organisms, and thus partly responsible of the strict host specificity of this symbiosis.

Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.

The effect of heat stress on the growth, physiological state, cell activity and cell morphology of the tropical Sinorhizobium arboris strain HAMBI 2190 was studied. The cells were chromosomally tagged with the firefly luciferase gene, luc. Since the bioluminescence phenotype is dependent on cellular energy reserves it was used as an indicator of the metabolic status of the cell population under various heat conditions. Variations in the numbers and lengths of growth phases between individual cultures indicated that the growth pattern at 40°C was disturbed compared to growth at 37 or 28°C. In addition, the cell morphology was changed radically. The number of culturable cells and the luciferase activity declined when the cultures were incubated at 40°C. By contrast, under all conditions studied, the cells could be stained with 5-(and 6-)sulfofluorescein diacetate, indicating esterase activity. This demonstrated that although the culturability and cellular energy reserves decreased considerably during heat stress, a majority of the of S. arboris cell population maintained basal enzyme activity.

Sinorhizobium sp. strain HAMBI 2180, marked with the β-glucuronidase (GUS) encoding gusA gene, was used for studying the effect of heat stress on the symbiotic interaction between Acacia senegal seedlings and Sinorhizobium sp. in growth cabinet conditions. The infection process, observed during 18 days after inoculation, was delayed at a root temperature of 40°C compared to a root temperature of ≤38°C, but nodulation was retarded already at 38°C. At 42°C no nodules were formed. At this root temperature the development of lateral roots and root hairs was one week delayed. Although the roots had normally deformed root hairs in addition to abnormally deformed ones, only disintegrated infection threads were present. However, the ability to form symbiosis was reversible. Nitrogen-fixing nodules developed on those parts of the roots which were formed after completion of the thermal stress at 42°C. A root temperature of 42°C did not affect the numbers of culturable rhizobia in the A. senegal rhizosphere.

Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, XbaI. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.

Lipopolysaccharide (LPS) patterns were obtained from fast-growing Rhizobium strains after silver staining of proteinase K treated cells lysates, run in SDS-PAGE. The rhizobia came from root nodules of Acacia senegal and Prosopis chilensis, collected in differents part of the Sudan. The LPS profiles of all strains were typical of rhizobia. Two different LPS region with lower and higher electrophoretic mobility (region I and region II, respectively) coulld be dinguished in the gels, and based on the profiles the strains were divided into three groups. Strains isolated from A. senegal showed a wider range of different profiles than strains isolated from P. chilensis, even though the plants belong to the same cross-infection group. Otherwise there was no clear correlation between the taxonomic relatedness or site of isolation of the strains and their LPS profiles.

Seven bacteriophage (Φ1R, Φ10W, Φ3R, Φ30W, Φ1261 M, Φ1261 V and Φgor3V), specific for the Rhizobium galegae species and representing three morphotypes, were isolated from different locations in Finland and in New Zealand. DNA was isolated from these phage and from phage Φ1R-3 and Φ1R', which were derived from Φ1R in the laboratory, and analyzed by restriction endonuclease digestion and dot blot DNA hybridization. The sizes of the phage DNAs were estimated to range from 45.1–114.6 kb. The restriction patterns revealed four different phage genotypes, which correlated with the isolation hosts. DNA hybridization showed that the four genotypes were distantly related. The genotypes were distinguished when purified phage protein was analyzed in SDS-PAGE gels.

Strains of Rhizobium sp. (Galega) (R. galegae), R. meliloti, R. leguminosarum, and R. loti were compared for their lipopolysaccharide (LPS) and whole cell protein patterns. Antigenic properties of these LPS and proteins were tested by immunoblotting with rabbit antiserum raised against R. galegae strain HAMBI 540. The LPS and protein patterns of R. galegae strains differed from those of the other rhizobia tested. By immunoblotting, a species-specific R. galegae LPS antigen and two proteins specific for R. galegae were identified. Our results support the suggestion that R. galegae strains form a distinct taxonomic group within the genus Rhizobium.

Rhizobium strains nodulating Galega species were characterized by metabolic tests, maximum growth temperature determinations in a temperature gradient incubator and phage typing, and compared with other fast-growing rhizobia. By numerical taxonomy it was shown that the Galega Rhizobium strains are closely related to each other and unrelated to the recognized species of Rhizobium. The maximum growth temperature of rhizobia nodulating G. orientalis was 33.0–34.0°C and of rhizobia nodulating G. officinalis 35.0–37.0°C. The Galega rhizobia were only lysed by their own phages, and not by typing phages for other Rhizobium species. The G + C% of Rhizobium sp. (Galega) strainswas 63%.44 previously unclassified fast-growing rhizobia from tropical plants, which were included in the experiments, were shown to form a heterogenous group with diverse properties. The results confirm and extend previous findings, and suggest that Rhizobum sp. (Galega) should be considered a new species of Rhizobium.