Structural and functional organization of the plasmid regulons of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> symbiotic genes

The structure of the plasmid locus containing the sym-genes (nod-, nif-, and fix-operons) was investigated in eight Rhizobium leguminosarum strains differing in their origin and host specificity, including five strains of the viciae biovar—symbionts of pea (3), forage beans (1), and Vavilovia (1)—as well as three strains of the biovar trifolii (clover symbionts). Strains of R. leguminosarum bv. viciae, which possess the nodX gene (controlling acetylation of the Nod factor, which is responsible for the ability of rhizobia to form symbioses with a broad spectrum of hosts, including the “Afghan” pea lines, homozygous by the allele sym^2A), are characterized by a less compact location of the sym-genes than the strains lacking the nodX gene. The size of the symbiotic cluster in the strains possessing nodX was 94.5 ± 3.5 kb, with the share of the sym-genes of 36.5 ± 1.5%, while for the strains lacking nodX these values were 61.7 ± 3.7 kb and 56.3 ± 1.4%, respectively (significant difference at P ₀ < 0.01). Syntenic structures were revealed in the symbiotic regions of strains Vaf12, UPM1131, and TOM, as well as syntenic structures of non-symbiotic regions in strains Vaf12, TOM, and WSM1689. The correlation coefficients between the matrices of genetic distances in the analyzed strains for the nodABC, nifHDK, and fixABC operons were on average 0.993 ± 0.002, while their values for the plasmid sites located between the sym-genes were considerably less (0.706 ± 0.010). In these regions, 21 to 27% of the genes were involved in amino acid transport and metabolism, which was substantially higher than the average for the genome of R. leguminosarum bv. viciae (11–12%). These data suggest that the evolution of R. leguminosarum bv. viciae, defined by narrowing of the host specificity (associated with a loss of the nodX gene), was accompanied by reduction of the regions of plasmids located between the sym-genes, as well as by specialization of these areas to perform the functions related to symbiotic nitrogen fixation. The observed increase of density in the cluster of sym-genes may be associated with intensification of their horizontal transfer in the populations of rhizobia, which determines the speed of evolution of the symbiotic system.     

Breeding for better symbiosis

The present review gives a critical assessment of the literature dealing with symbiosis between rhizobia and legumes and between AM fungi and most plants. Associative N₂ fixation (even though strictly speaking not a symbiotic relationship) does have some characteristics of symbiosis due to mutualistic dependence and usefulness of the relationship, and is therefore covered in this review. Nodulation in the rhizobia–legume symbiosis may be limited by an insufficient amount of the nod-gene inducers released from seed and/or roots. However, there is genotypic variation in the germplasm of legume species in all components of the signalling pathway, suggesting a prospect for improving nodulation by selecting and/or transforming legume genotypes for increased exudation of flavonoids and other signalling compounds. Deciphering chromosomal location as well as cloning nod, nif and other genes important in nodulation and N₂ fixation will allow manipulation of the presence and expression of these genes to enhance the symbiotic relationship. Increased efficacy of symbiotic N₂ fixation can be achieved by selecting not only the best host genotypes but by selecting the best combination of host genotype and nodule bacteria. As flavonoids exuded by legume seedlings may not only be nod-gene inducers, but also stimulants for hyphal growth of the AM fungi, selecting and/or transforming plants to increase exudation of these flavonoids may result in a double benefit for mycorrhizal legumes. Mutants unable to sustain mycorrhizal colonisation are instrumental in understanding the colonisation process, which may ultimately pay off in breeding for the more effective symbiosis. In conclusion, targeted efforts to breed genotypes for improved N₂ fixation and mycorrhizal symbiosis will bring benefits in increased yields of crops under a wide range of environmental conditions and will contribute toward sustainability of agricultural ecosystems in which soil-plant-microbe interactions will be better exploited.     

Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N₂-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N₂-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N₂ fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C₄ metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic β-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.     

sym 13-A Gene Conditioning Ineffective Nodulation in Pisum sativum

Treatment of Pisum sativum (L.) cv. `Sparkle' with ethyl methanesulfonic acid (EMS) produced a stable mutant, E135F, which forms small, white, ineffective nodules. These nodules exhibit histological zonation typical of an indeterminant nodule, e.g. meristematic, early symbiotic, late symbiotic, and senescent zones. Compared with the nitrogen fixing nodules of the parent, the zones are smaller and the nodules senesce prematurely. Bacteroids in E135F are less elongated and less differentiated than those in `Sparkle.' The E135F mutant forms ineffective nodules when inoculated with nine different effective strains of Rhizobium leguminosarum and also when grown in a soil containing effective strains. The ineffective phenotype of E135F is under monogenic recessive control; the gene is designated sym 13. sym 13 was located on chromosome 2 by linkage with genes for shikimic dehydrogenase and esterase-2. The original selection E135F carried another mutation in heterozygous form at a separate locus, yielding some homozygous recessive nonnodulating progeny, E135N, in later generations. This indicates that EMS treatments may cause mutations at more than one sym gene. The gene conditioning non-nodulation in E135N was designated sym 14. It mapped to a locus on a different part of chromosome 2 by linkage to the gene for fumarase. The data demonstrate that sym genes are not necessarily closely linked.     

Identification and phenotypical characterization of a cluster of fix genes, including a nif regulatory gene, from Rhizobium leguminosarum PRE

Summary A nif regulatory gene in R. leguminosarum PRE was identified by interspecies DNA hybridization and site-directed Tn5 mutagenesis. Significant homology was found with the K. pneumoniae nifA locus, a R. meliloti symbiotic regulatory gene and E. coli ntrC; Tn5 insertions within this nifA gene inhibit the expression of the nifHDK operon, encoding synthesis of the nitrogenase polypeptides.Specific DNA hybridization also was detected between a downstream adjacent part of the PRE sym plasmid and the R. leguminosarum 248 fixZ gene, a homologue of the K. pneumoniae nifB locus. To detect further fix genes we investigated a region of the sym plasmid which is localized within a short distance upstream from the nifA gene and is transcribed selectively at a high rate during symbiosis. This approach revealed the existence of a fix cluster in which Tn5-mutations cause a Fix^- phenotype although wild-type levels of nitrogenase synthesis were detectable. In a sym plasmid fragment, which is immediately upstream adjacent to the nifA locus and only moderately expressed in Rhizobium bacteroids, a second fix gene conferring the same symbiotic phenotype was detected.     

Rhizobium phaseoli: A molecular genetics view

We have used molecular genetics techniques to analyze the structural and functional organization of genetic information ofRhizobium phaseoli, the symbiont of the common bean plantPhaseolus vulgaris. As in otherRhizobium species, the genome consists of the chromosome and plasmids of high molecular weight. Symbiotic determinants, nitrogen fixation genes as well as nodulation genes, are localized on a single replicon, the symbiotic (sym) plasmid. Thesym plasmid of differentR. phaseoli strains was transferred to anAgrobacterium tumefaciens strain cured of its native plasmids. In all cases, Agrobacterium transconjugants able to nodulate bean plants were obtained. Some of the transconjugants had the capacity to elicit an effective symbiosis. The genome ofR. phaseoli is complex, containing a large amount of reiterated DNA sequences. In mostR. pahseoli strains one of such reiterated DNA families corresponds to the nitrogenase structural genes (nif genes). A functional analysis of these genes suggested that the presence of reiteratednif genesis is related to the capacity of fixing atmospheric nitrogen in the symbiotic state. The presence of several repeated sequences in the genome might provide sites for recombination, resulting in genomic rearrangements. By analyzing direct descendants of a single cell in the laboratory, evidence of frequent genomic rearrangements inR. phaseoli was found. We propose that genomic rearrangements constitute the molecular basis of the frequent variability and loss of symbiotic properties in different Rhizobium strains.     

Evidence of Horizontal Transfer of Symbiotic Genes from a Bradyrhizobium japonicum Inoculant Strain to Indigenous Diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah Soil

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N₂-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.     

Characterization of four defense-related genes up-regulated in root nodules of Casuarina glauca

Actinorhizal plants are capable of high rates of nitrogen fixation, due to their capacity to establish a root-nodule symbiosis with N₂-fixing actinomycetes of the genus Frankia. Nodulation is an ontogenic process which requires a sequence of highly coordinated events. One of these mechanisms is the induction of defense-related events, whose precise role during nodulation is largely unknown. In order to contribute to the clarification of the involvement of defense-related genes during actinorhizal root-nodule symbiosis, we have analysed the differential expression of several genes with putative defense-related functions in Casuarina glauca nodules versus non-inoculated roots. Four genes encoding a chitinase (CgChi1), a glutathione S-transferase (CgGst), a hairpin-inducible protein (CgHin1) and a peroxidase (CgPox4) were found to be up-regulated in mature nodules compared to roots. In order to find out to which extend were the encoded proteins involved in nodule protection, development or both, gene regulation studies in response to SA and wounding as well as phylogenetic analysis of the protein sequences were performed. These were further characterized through expression studies after SA-treatment and wounding, and by phylogenetic analysis. We suggest that CgChi1 and CgGst are involved in defense or microsymbiont control and CgPox4 is involved in nodule development. For CgHin1 the question “defense, development or both” remains open.     

ROS production during symbiotic infection suppresses pathogenesis-related gene expression

Leguminous plants have exclusive ability to form symbiotic relationship with soil bacteria of the genus Rhizobium. Symbiosis is a complex process that involves multiple molecular signaling activities, such as calcium fluxes, production of reactive oxygen species (ROS) and synthesis of nodulation genes. We analyzed the role of ROS in defense gene expression in Medicago truncatula during symbiosis and pathogenesis. Studies in Arabidopsis thaliana showed that the induction of pathogenesis-related (PR) genes during systemic acquired resistance (SAR) is regulated by NPR1 protein, which resides in the cytoplasm as an oligomer. After oxidative burst and return of reducing conditions, the NPR1 undergoes monomerization and becomes translocated to the nucleus, where it functions in PR genes induction. We show that ROS production is both stronger and longer during symbiotic interactions than during interactions with pathogenic, nonhost or common nonpathogenic soil bacteria. Moreover, root cells inoculated with Sinorhizobium meliloti accumulated ROS in the cytosol but not in vacuoles, as opposed to Pseudomonas putida inoculation or salt stress treatment. Furthermore, increased ROS accumulation by addition of H₂O₂ reduced the PR gene expression, while catalase had an opposite effect, establishing that the PR gene expression is opposite to the level of cytoplasmic ROS. In addition, we show that salicylic acid pretreatment significantly reduced ROS production in root cells during symbiotic interaction.     

Roles of the Structural Symbiosis Polysaccharide (syp) Genes in Host Colonization,Biofilm Formation,and Polysaccharide Biosynthesis in Vibrio fischeri

The symbiosis polysaccharide locus, syp, is required for Vibrio fischeri to form a symbiotic association with the squid Euprymna scolopes. It is also required for biofilm formation induced by the unlinked regulator RscS. The syp locus includes 18 genes that can be classified into four groups based on putative function: 4 genes encode putative regulators, 6 encode glycosyltransferases, 2 encode export proteins, and the remaining 6 encode proteins with other functions, including polysaccharide modification. To understand the roles of each of the 14 structural syp genes in colonization and biofilm formation, we generated nonpolar in-frame deletions of each gene. All of the deletion mutants exhibited defects in their ability to colonize juvenile squid, although the impact of the loss of SypB or SypI was modest. Consistent with their requirement for colonization, most of the structural genes were also required for RscS-induced biofilm formation. In particular, the production of wrinkled colonies, pellicles, and the matrix on the colony surface was eliminated or severely decreased in all mutants except for the sypB and sypI mutants; in contrast, only a subset of genes appeared to play a role in attachment to glass. Finally, immunoblotting data suggested that the structural Syp proteins are involved in polysaccharide production and/or export. These results provide important insights into the requirements for the syp genes under different environmental conditions and thus lay the groundwork for a more complete understanding of the matrix produced by V. fischeri to enhance cell-cell interactions and promote symbiotic colonization.     

Phylogeny and distribution of Bradyrhizobium symbionts nodulating cowpea (Vigna unguiculata L. Walp) and their association with the physicochemical properties of acidic African soils

In the N₂-fixing symbiosis, the choice of a symbiotic partner is largely influenced by the host plant, the rhizobial symbiont, as well as soil factors. Understanding the soil environment conducive for the survival and multiplication of root-nodule bacteria is critical for microbial ecology. In this study, we collected cowpea-nodules from acidic soils in Ghana and South Africa, and nodule DNA isolates were characterized using 16S–23S rRNA-RFLP, phylogenetic analysis of housekeeping and symbiotic genes, and bradyrhizobial community structure through canonical correspondence analysis (CCA). The CCA ordination plot results showed that arrow of soil pH was overlapping on CCA2 axis and was the most important to the ordination. The test nodule DNA isolates from Ghana were positively influenced by soil Zn, Na and K while nodule DNA isolates from South Africa were influenced by P. The amplified 16S–23S rRNA region yielded single polymorphic bands of varying lengths (573–1298 bp) that were grouped into 28 ITS types. The constructed ITS-dendrogram placed all the nodule DNA isolates in five major clusters at low cut-off of approx. 0.1 Jaccard’s similarity coefficient. The phylogenetic analysis of 16S rRNA and housekeeping genes (glnII, gyrB, and atpD) formed distinct Bradyrhizobium groups in the phylogenetic trees. It revealed the presence of highly diverse bradyrhizobia (i.e. Bradyrhizobium vignae, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense) together with novel/unidentified bradyrhizobia in the acidic soils from Ghana and South Africa. Discrepancies noted in the phylogenies of some nodule DNA isolates could be attributed to horizontal gene transfer or recombination.     

Following phylogenetic tracks of <Emphasis Type="Italic">Astragalus cicer</Emphasis> microsymbionts

A multilocus phylogenetic approach was applied to elucidate the phylogeny of Astragalus cicer rhizobia derived from Poland, Ukraine, and Canada. The strains selected for the studies represented three main geographically different phenons of these bacteria. Phylogenetic analyses were performed with three chromosomal housekeeping loci (16S rRNA, atpD, glnII) and three symbiotic genes located on a plasmid (nodA, nodC, nifH). The “core” and “auxiliary” gene trees revealed that A. cicer nodule isolates were intermingled with the strains of Mesorhizobium species, which implies that they are descendents of the same ancestor as mesorhizobia and fall into the Mesorhizobium genus. The noted congruence of the housekeeping and symbiotic gene phylogenies of A. cicer microsymbionts indicates that sym loci are transferred to these bacteria through vertical transmission without a significant participation of intergeneric horizontal gene spread. All the three sym gene sequences of the Polish and Ukrainian A. cicer nodule isolates were more closely related to one another than to the corresponding sequences of the Canadian isolates. The phylogeographic patterns of the sym genes of intercontinental strains point to their relatively long, separate, evolutionary history.     

Influence of salt stress on the genetically polymorphic system of Sinorhizobium meliloti-Medicago truncatula

The impacts of salt stress (75 mM NaCl) on the ecological efficiency of the genetically polymorphic Sinorhizobium meliloti-Medicago truncatula system were studied. Its impact on a symbiotic system results in an increase of the partners’ variability for symbiotic traits and of the symbiosis integrity as indicated by: (a) the specificity of the partners’ interactions-the nonadditive inputs of their genotypes into the variation of symbiotic parameters and (b) the correlative links between these parameters. The structure of the nodD1 locus and the plasmid content correlates to the efficiency of the symbiosis between S. meliloti and M. truncatula genotypes under stress conditions more sufficiently than in the absence of stress. Correlations between the symbiotic efficiency of rhizobia strains and their growth rate outside symbiosis are expressed under stress conditions, not in the absence of stress. Under salt stress symbiotic effectiveness was decreased for M. truncatula line F83005.5, which was salt sensitive for mineral nutrition. The inhibition of symbiotic activity for this line is linked with decreased nodule formation, whereas for Jemalong 6 and DZA315.16 lines it is associated with repressed N₂-fixation. It was demonstrated for the first time that salt stress impairs the M. truncatula habitus (the mass: height ratio increased 2- to 6-fold), which in the salt-resistant cultivar Jemalong 6 is normalized as the result of rhizobia inoculation.     

Phosphate Limitation Alters <Emphasis Type="Italic">Medicago–Sinorhizobium</Emphasis> Signaling: Flavonoid Synthesis and AHL Production

The legume plant Medicago truncatula Gaertn. can establish a symbiotic interaction with Sinorhizobium meliloti. One of the most limiting factors for symbiosis is phosphate (P) deficiency. Therefore, legumes and their symbiotic partners, rhizobia, have developed mechanisms to adapt to P restriction. In the non-symbiotic state, plants would up-regulate flavonoid biosynthesis via increasing the expression of chalcone synthase (chs), catalyzing the first step of flavonoid synthesis. Simultaneously, bacterial quorum sensing (QS) pathway can regulate the expression of certain genes involved in symbiotic functions of bacteria in response to P availability as well as bacterial population. Since both flavonoids and QS signaling molecules (N-acyl homoserine lactones, AHL) play important roles in the rhizobia-legume symbiosis, we evaluated these processes in the symbiotic state under different P concentrations and bacterial populations. In this study, by using real-time PCR and HPLC, we showed the expression of pt1 (phosphate transporter 1) and chs as well as luteolin production increased, in a time dependent manner, in plants following P limitation. Nod gene inducing flavonoids can up-regulate the bacterial QS pathway which results in an increase in AHL production, possibly to enhance symbiotic behaviors of rhizobia. It has been estimated that there is a feedback loop from bacterial AHL to flavonoid production pathway in legume plants.     

Cyanobacterial biofertilizers in rice agriculture

Floodwater and the surface of soil provide the sites for aerobic phototrophic nitrogen (N) fixation by free-living cyanobacteria and theAzolla-Anabaena symbiotic N₂-fixing complex. Free-living cyanobacteria, the majority of which are heterocystous and nitrogen fixing, contribute an average of 20–30 kg N ha^-1, whereas the value is up to 600 kg ha^-1 for theAzollaAnabaena system (the most beneficial cyanobacterial symbiosis from an agronomic point of view). Synthesis and excretion of organic/growth-promoting substances by the cyanobacteria are also on record. During the last two or three decades a large number of studies have been published on the various important fundamental and applied aspects of both kinds of cyanobacterial biofertilizers (the free-living cyanobacteria and the cyanobacteriumAnabaena azollae in symbiotic association with the water fernAzolla), which include strain identification, isolation, purification, and culture; laboratory analyses of their N₂-fixing activity and related physiology, biochemistry, and energetics; and identification of the structure and regulation of nitrogenfixing (nif) genes and nitrogenase enzyme. The symbiotic biology of theAzolla-Anabaena mutualistic N₂-fixing complex has been clarified. In free-living cyanobacterial strains, improvement through mutagenesis with respect to constitutive N₂ fixation and resistance to the noncongenial agronomic factors has been achieved. By preliminary meristem mutagenesis inAzolla, reduced phosphate dependence was achieved, as were temperature tolerance and significant sporulation/spore germination under controlled conditions. Mass-production biofertilizer technology of free-living and symbiotic (Azolla-Anabaena) cyanobacteria was studied, as were the interacting and agronomic effects of both kinds of cyanobacterial biofertilizer with rice, improving the economics of rice cultivation with the cyanobacterial biofertilizers. Recent results indicate a strong potential for cyanobacterial biofertilizer technology in rice-growing countries, which opens up a vast area of more concerted basic, applied, and extension work in the future to make these self-renewable natural nitrogen resources even more promising at the field level in order to help reduce the requirement for inorganic N to the bare minimum, if not to zero.     

Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.