Nodules on legumes and also the nonlegume Parasponia exhibit different morphologies. Nodule cells can be considered to be bathed in a medium containing C sources provided by plant photosynthesis. Sucrose is considered to be the main energy source transported into the nodules for bacterial nitrogen fixation. Legumes entering into N2-fixing symbioses can be broken down into two groups; those whose nodules export amides or ureides. Bacteroids are defined as the specialized stage of the life cycle of symbiotic bacteria, occurring when they inhabit host cells. The peribacteroid space (PBS) is defined as the space between the symbiotic partners. The peribacterioid membrane (PBM) forms a continuous envelope around the prokaryote, and various numbers of bacteroids are contained within each membrane, between 1 and 20, depending on species and the age of the nodule. Mitochondria in infected cells are cristae-rich through intensive folding of the inner mitochondrial membrane and move to the cell periphery.

ABSTRACT A highly supported maximum-likelihood species phylogeny for the genus Bradyrhizobium was inferred from a supermatrix obtained from the concatenation of partial atpD , recA , glnII , and rpoB sequences corresponding to 33 reference strains and 76 bradyrhizobia isolated from the nodules of Glycine max (soybean) trap plants inoculated with soil samples from Myanmar, India, Nepal, and Vietnam. The power of the multigene approach using multiple strains per species was evaluated in terms of overall tree resolution and phylogenetic congruence, representing a practical and portable option for bacterial molecular systematics. Potential pitfalls of the approach are highlighted. Seventy-five of the isolates could be classified as B. japonicum type Ia (USDA110/USDA122-like), B. liaoningense , B. yuanmingense , or B. elkanii , whereas one represented a novel Bradyrhizobium lineage. Most Nepalese B. japonicum Ia isolates belong to a highly epidemic clone closely related to strain USDA110. Significant phylogenetic evidence against the monophyly of the of B. japonicum I and Ia lineages was found. Analysis of their DNA polymorphisms revealed high population distances, significant genetic differentiation, and contrasting population genetic structures, suggesting that the strains in the Ia lineage are misclassified as B. japonicum . The DNA polymorphism patterns of all species conformed to the expectations of the neutral mutation and population equilibrium models and, excluding the B. japonicum Ia lineage, were consistent with intermediate recombination levels. All species displayed epidemic clones and had broad geographic and environmental distribution ranges, as revealed by mapping climate types and geographic origins of the isolates on the species tree.

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.

Soluble proteins from the salt-tolerant Rhizobium etli strain EBRI 26 were separated by two-dimensional (2D) gel electrophoresis and visualised by Commassie staining. Six proteins are highly expressed after induction by 4% NaCl compared to the non-salt-stressed cells. These proteins have pI between 5 and 5.5 and masses of approximately 22, 25, 40, 65, 70, and 95 kDa. These proteins were analysed by Matrix-assisted laser adsorption ionization time of flight (MALDI-TOF) after digestion with trypsin. Despite having very good peptide mass fingerprint data, these proteins could not be identified, because the genome sequence of R. etli is not yet published. In a second approach, soluble proteins from salt-induced or non-salt-induced cultures from R. etli strain EBRI 26 were separately labelled with different fluorescent cyano-dyes prior to 2D difference in gel electrophoresis. Results revealed that 49 proteins are differentially expressed after the addition of sodium chloride. Fourteen proteins are overexpressed and 35 were downregulated. The genome of Sinorhizobium meliloti, a closely related species to R. etli, has been published. Similar experiments using Sinorhizobium meliloti strain 2011 identified four overexpressed and six downregulated proteins. Among the overexpressed protein is a carboxynospermidin decarboxylase, which plays an important role in the biosynthesis of spermidin (polyamine). The enzyme catalase is among the downregulated proteins. These proteins may play a role in salt tolerance.

Rhizobium tropici CIAT899 is highly tolerant to several environmental stresses and is a good competitor for nodule occupancy of common bean plants in acid soils. Random transposon mutagenesis was performed to identify novel genes of this strain involved in symbiosis and stress tolerance. Here, we present a genetic analysis of the locus disrupted by the Tn5 insertion in mutant 899-PV9, which lead to the discovery of sycA, a homolog of the ClC family of chloride channels and Cl-/H+ exchange transporters. A nonpolar deletion in this gene caused serious deficiencies in nodule development, nodulation competitiveness, and N2 fixation on Phaseolus vulgaris plants, probably due to its reduced ability to invade plant cells and to form stable symbiosomes, as judged by electron transmission microscopy. A second gene (olsC), found downstream of sycA, is homologous to aspartyl/asparaginyl β-hydroxylases and modifies two species of ornithine-containing lipids in vivo, presumably by hydroxylation at a still-unknown position. A mutant carrying a nonpolar deletion in olsC is symbiotically defective, whereas overexpressed OlsC in the complemented strain provokes an acid-sensitive phenotype. This is the first report of a ClC homolog being essential for the establishment of a fully developed N2-fixing root nodule symbiosis and of a putative β-hydroxylase that modifies ornithine-containing membrane lipids of R. tropici CIAT899, which, in turn, are contributing to symbiotic performance and acid tolerance.

Sustainable management for existing Amazonian forests requires an extensive knowledge about the limits of ecosystem nutrient cycles. Therefore, symbiotic nitrogen (N 2 ) fixation of legumes was investigated in a periodically flooded forest of the central Amazon floodplain (Várzea) over two hydrological cycles (20 months) using the 15 N natural abundance method. No seasonal variation in 15 N abundance ( δ 15 N values) in trees which would suggest differences in N 2 fixation rates between the terrestrial and the aquatic phase was found. Estimations of the percentage of N derived from atmosphere (%Ndfa) for the nodulated legumes with Neptunia oleracea on the one side and Teramnus volubilis on the other resulted in mean %Ndfa values between 9 and 66%, respectively. More than half of the nodulated legume species had %Ndfa values above 45%. These relatively high N gains are important for the nodulated legumes during the whole hydrological cycle. With a %Ndfa of 4–5% for the entire Várzea forest, N 2 fixation is important for the ecosystem and therefore, has to be taken into consideration for new sustainable land-use strategies in this area.

Saline and alkaline soils are major problems contributing to the low productivity of common bean (Phaseolus vulgaris) in arid and semi-arid regions such as Egypt. Therefore our study was directed toward selecting strains more tolerant to these environmental stresses. Among seven Rhizobium etli strains isolated from Egyptian soils, we found a high degree of diversity. Strains EBRI 21 and EBRI 26 are highly tolerant to a salt concentration up to 4% NaCl. A positive correlation was found between the salt tolerance and the adaptation to alkaline pH (9). Strains EBRI 2 and EBRI 26 were adapted to elevated temperatures (42°C). The minimum level of low pH for the majority of Rhizobium etli strains from Egypt was pH 4.7 while the Colombian strain Rhizobium tropici CIAT 899 survived well at pH 4. At 0.4% NaCl, the symbiotic efficiency of the salt-tolerant strain EBRI 26 was superior in cultivar Giza 6 compared with the salt-sensitive strain EBRI 2 (18.2 compared with 13.9 nM C2H4 h−1 mg−1 nodule fresh weight). In the bean cultivar Saxa, nitrogen fixation was much more affected by high salt concentration (0.4% NaCl) than in the cultivar Giza 6 with both strains (3.9 and 3.8 nM C2H4 h−1 mg−1 nodule fresh weight, respectively). In general, stress of alkalinity had a less detrimental effect on nodulation and N2 fixation than stress of salinity.

A combination of population genetics and phylogenetic inference methods was used to delineate Bradyrhizobium species and to uncover the evolutionary forces acting at the population-species interface of this bacterial genus. Maximum-likelihood gene trees for atpD, glnII, recA, and nifH loci were estimated for diverse strains from all but one of the named Bradyrhizobium species, and three unnamed "genospecies," including photosynthetic isolates. Topological congruence and split decomposition analyses of the three housekeeping loci are consistent with a model of frequent homologous recombination within but not across lineages, whereas strong evidence was found for the consistent lateral gene transfer across lineages of the symbiotic (auxiliary) nifH locus, which grouped strains according to their hosts and not by their species assignation. A well resolved Bayesian species phylogeny was estimated from partially congruent glnII+recA sequences, which is highly consistent with the actual taxonomic scheme of the genus. Population-level analyses of isolates from endemic Canarian genistoid legumes based on REP-PCR genomic fingerprints, allozyme and DNA polymorphism analyses revealed a non-clonal and slightly epidemic population structure for B. canariense isolates of Canarian and Moroccan origin, uncovered recombination and migration as significant evolutionary forces providing the species with internal cohesiveness, and demonstrated its significant genetic differentiation from B. japonicum, its sister species, despite their sympatry and partially overlapped ecological niches. This finding provides strong evidence for the existence of well delineated species in the bacterial world. The results and approaches used herein are discussed in the context of bacterial species concepts and the evolutionary ecology of (brady)rhizobia.

With four different cultivars of Glycine max (soybeans) a 3-10 fold increase in specific activity of alpha-mannosidase activity was observed in roots, inoculated with Glomus mosseae compared to the control plants without mycorrhiza development. The increase in a-mannosidase activity after infection with Glomus intraradices was slightly less but still significant. No such effect was found for the beta-D-glucosidase activity. With the model legume Medicago truncatula cultivar Jemalong A17 a more than 3 fold increase in the mRNA levels of two glycosyltransferases compared to the control plants was detected with a macroarray technique. RNA levels of 16 other enzymes involved in membrane and lipid metabolism, such as choline kinase, glycerol-3-phosphate dehydrogenase, phospholipase D and galactosyltransferase were not significantly increased.

Highly diverse Bradyrhizobium strains nodulate genistoid legumes (brooms) in the Canary Islands, Morocco, Spain and the Americas. Phylogenetic analyses of ITS, atpD, glnII and recA sequences revealed that these isolates represent at least four distinct evolutionary lineages within the genus, namely Bradyrhizobium japonicum and three unnamed genospecies. DNA-DNA hybridization experiments confirmed that one of the latter represents a new taxonomic species for which the name Bradyrhizobium canariense is proposed. B. canariense populations experience homologous recombination at housekeeping loci, but are sexually isolated from sympatric B. japonicum bv. genistearum strains in soils of the Canary Islands. B. canariense strains are highly acid-tolerant, nodulate diverse legumes in the tribes Genisteae and Loteae, but not Glycine species, whereas acid-sensitive B. japonicum soybean isolates such as USDA 6(T) and USDA 110 do not nodulate genistoid legumes. Based on host-range experiments and phylogenetic analyses of symbiotic nifH and nodC sequences, the biovarieties genistearum and glycinearum for the genistoid legume and soybean isolates, respectively, were proposed. B. canariense bv. genistearum strains display an overlapped host range with B. japonicum bv. genistearum isolates, both sharing monophyletic nifH and nodC alleles, possibly due to the lateral transfer of a conjugative chromosomal symbiotic island across species. B. canariense is the sister species of B. japonicum, as inferred from a maximum-likelihood Bradyrhizobium species phylogeny estimated from congruent glnII+recA sequence partitions, which resolves eight species clades. In addition to the currently described species, this phylogeny uncovered the novel Bradyrhizobium genospecies alpha and beta and the photosynthetic strains as independent evolutionary lineages. The type strain for B. canariense is BTA-1(T) (=ATCC BAA-1002(T)=LMG 22265(T)=CFNE 1008(T)).

Twelve rhizobial isolates were recovered from nodules of common bean (Phaseolus vulgaris) grown in two different locations of Egyptian soils. The most effective strains for nodule formation and nitrogen fixation were selected. Strain specificity with the bean cultivars Saxa, Canoca and Giza 6 from Germany, Colombia and Egypt were studied. The strains were characterized by amplified rDNA restriction analysis of 16S and 23S rDNA (ARDRA), plasmid DNA content and 16S rDNA sequencing. A high degree of genetic diversity was observed among the strains used. The strains were separated into three genotype groups. Genotype A was displayed by seven isolates classified as Rhizobium etli, while genotype B was displayed by a single isolate, classified as R. gallicum. Genotype C included four isolates which were unable to re-nodulate Phaseolus vulgaris, which were drelated to Agrobacterium tumefaciens. Single strains were further characterized by specific physiological tests and measurements such as acetylene reduction activity, nodule/root biomass ratio, shoot and root fresh weight.

Two Rhizobium etli strains, EBRI 2 and EBRI 26, isolated from Egypt were tested for nodulation competitiveness on beans using Rhizobium tropici CIAT 899G as the competing strain. The insertion of the gus-reporter transposon mTn5ssgusA30 did not alter the nodulation or nitrogen fixation capacity of mutant strain CIAT 899G compared to the wild type. At neutral pH, R. etli strains EBRI 2 and EBRI 26 were more competitive than CIAT 899G with the bean cultivar Saxa. These two strains gave nodule occupancies of 52.1 and 61.1% competing with equal cell numbers of CIAT 899G. Nodule occupancies from these two native strains increased with the bean cultivar Giza 6 from Egypt to 66 and 67.5%. Based on these results, cultivar Giza 6 was used to select the most competitive strains under stress of salinity or alkalinity as a major problem for a large part of Egyptian soils. Under stress of salinity (0.2% NaCl or 34.2 mM NaCl), the salt-sensitive strain EBRI 2 was more competitive than the salt-resistant strain EBRI 26. Strain EBRI 2 gave 87.4% but strain EBRI 26 gave 63.7% nodule occupancy against CIAT 899G. The same trend of results was observed under stress of alkalinity (pH 8). Strain EBRI 2 occupied 83% while Strain EBRI 26 occupied 53.2%.

The production and application of biofertilizers for leguminous crops, oilseeds, rice, millets, and other important crops (besides forest nursery plants) are very common in India. Currently, more than 95 firms belonging to public and private sectors are involved in the production of biofertilizers, with the annual capacity of 18,000 metric tons (out of which substantial amount is accounted as Rhizobium) against the total potential demand of 3.4×105confirmed metric tons. Rhizobium is one of the important nitrogen-fixing bacteria, helping the legumes to maintain soil fertility by means of their symbiotic association and nitrogen fixation. Besides the efforts taken by the government and research institutions and agricultural universities, rhizobial technology has still a long way to go to obtain maximum benefit. This may be due to the varied reasons which limit the usage of the inoculant. However, the most important difficulty lies in the performance of the inoculated strain of rhizobia. This problem may be solved by using appropriate strains of rhizobia, i.e., crop/variety-specific, location-specific, and soil-specific, to be properly isolated and screened for effectiveness in nitrogen fixation. Still, variations among the local strains is possible and is evidenced through field experiments conducted at different locations in India. Some strains performed better than the best local strains, though they originated under different agroecological conditions. However, in most cases, the best performing strain in one location did not differ significantly at other locations tested. confirmed.

Rhizobium tropici CIAT899 is highly acid tolerant and a good competitor for Phaseolus vulgaris nodule occupancy at low pH values. Using Tn5 mutagenesis, we identified an operon required for acid tolerance and nodulation competitiveness. The insertion was mapped to the 5' end of atvA, encoding a product with high sequence identity to the agro-bacterial AcvB virulence protein. Complementation analyses indicated that atvA is an ortholog of acvB, both genes being required for acid tolerance. A Ser/Ala substitution in the LIPASE_SER motif of AtvA resulted in an acid sensitive Fix(+) but very poorly competing strain, demonstrating that Ser-313 is essential for AtvA function. atvA is the second gene in an operon that is transcriptionally upregulated by acid shock. The acid-responsive promoter was mapped to a 469-bp intergenic region located upstream of lpiA, the first gene in the operon. lpiA-like genes are found in several alpha, beta, and gamma Proteobacteria that interact with eukaryotic host cells, and they are predicted to encode membrane proteins related to the FmtC/MprF family from low G+C Firmicutes. The latter proteins are involved in resistance to cationic antimicrobial peptides. A nonpolar deletion in lpiA caused a sevenfold decrease in relative nodulation competitiveness.

To investigate whether leucine biosynthesis plays a role in survival and growth under acidic conditions, leucine auxotrophs were produced from acid-tolerant Rhizobium tropici CIAT899. An internal fragment of the R. tropici leuA gene was PCR amplified using degenerate primers and cloned into an integrative plasmid for mutagenesis of the locus. Sequence and homology analysis confirmed the identity of the 550-bp gene fragment, with highest similarity (86% identity) to leuA from Sinorhizobium meliloti . The resultant mutants were strict leucine auxotrophs when tested on solid and liquid media. The mutants could be divided into two groups dependent on their ability to grow in the presence of leucine at both pH 6.8 and pH 5.5, or only at pH 5.5. One mutant, HS20, which could only grow properly in the presence of leucine at pH 5.5, was tested for its acid tolerance. When inoculated into medium at pH 3.5, the mutant was able to survive and to alter the pH from 3.5 to 3.8 whereas the wildtype could not. The wildtype was able to grow at pH 4.0, 4.5 and 5.0 without raising the extracellular pH, indicating that alteration of extracellular pH is not one of the strategies used by the wildtype for growth under acidic conditions. The ability of the mutant to raise the extracellular pH from 3.5 to 3.8 may enable it to survive at the extreme of its pH range of growth.