Search

Rhizobia represent a diverse group of gram-negative bacteria capable of fixing atmospheric nitrogen in symbiosis with leguminous plants. Mechanisms of symbiotic efficiency are important to study not only to reveal the “fine tuning” of the host–symbiont supra-organismal genetic system emergence, but also to develop agriculture with minimal environmental risks. In this paper we demonstrate that among seven genes whose inactivation by Tn5 insertions results in an increased efficiency of rhizobia (Sinorhizobium meliloti) symbiosis with alfalfa (Eff++ phenotype), six genes are involved in the metabolism of small molecules. One of them (SMc04399) encodes for acetate-CoA transferase catalyzing the formation of acetyl-CoA from acyl-CoA. Since acetyl-CoA is required for operation of the Krebs cycle, providing ATP for symbiotic N2 fixation, we suggest that a significant portion of this coenzyme utilized by bacteroids is provided by the plant cell supporting the energy-consuming nitrogenase reaction. Proteomic data analysis allow us to reveal the lability of enzymatic pathways which are involved in bacteroids in the production and catabolism of acetyl-CoA and which should be modified to obtain the Eff++ phenotype. This phenotype was developed also after inactivation of NoeB protein which is involved in the host-specific nodulation and is characterized by an elevated production in wild type S. meliloti bacteroids, suggesting a multifunctional role of noeB in the symbiosis operation.

R. leguminosarum bv. trifolii strains are characterized by narrow host specificity. We have identified a strain that forms nodules on several types of clover and studied the genetic organization of its symbiotic region.

—The importance of root nodule bacteria in biotechnology is determined by their distinctive feature: symbiotic nitrogen fixation resulting in the production of organic nitrogen-containing compounds. While interacting with host legume plants, the cells of these bacteria undergo global changes at all levels of expression of genetic information leading to the formation in root nodules of so-called bacteroids functioning as nitrogen fixation factories. The molecular mechanisms underlying plant-microbial symbiosis are actively investigated, and one of the most interesting and poorly studied aspects of this problem is the species-specificity of interaction between root nodule bacteria and host plants. In this work we have performed the proteomic analysis of the Sinorhizobium meliloti bacteroids isolated from two legume species: alfalfa (Medicago sativa L.) and yellow sweet clover (Melilotus officinalis L.). It has been shown that the S. meliloti bacteroids produce a lot of proteins (many of them associated with symbiosis) in a host-specific manner, i.e., only in certain host plant species. It has been demonstrated for the first time that the levels of expression in bacteroids of the genes encoding the ExoZ and MscL proteins responsible for the synthesis of surface lipopolysaccharides and formation of a large conductance mechanosensitive channel, respectively, depend on a host plant species that confirms the results of proteomic analysis. Overall, our data show that the regulation of bacteroid development by the host plant has species-specific features.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

We propose the Evolutionary Moulding hypothesis that population diversities of partners in nitrogen-fixing rhizobium-legume symbiosis are matched, and tested it in nucleotide polymorphism of symbiotic genes encoding two components of the plant-bacteria signalling system. The first component is the rhizobialnodA acyltransferase involved in the fatty acid tail decoration of Nod factor (rhizobia signalling molecule). The second component is the plantNFR5receptor, putatively required for Nod-factor binding.We collected three wild growing legume species together with soil samples adjacent to the roots (soil pool) from one large 25-year fallow:Vicia sativa, Lathyrus pratensisandTrifolium hybridumnodulated by one of the twoRhizobium leguminosarumbiovars (viciaeandtrifolii). For each plant species we prepared three pools for DNA extraction: the plant pool (30 plant indiv.), the nodule pool (90 nodules) and the soil pool (30 samples).NFR5gene libraries from the plant pool andnodA gene libraries from nodule and soil pools were sequenced by Sanger technology and High-throughput pyrosequencing, respectively. Analysis of the data demonstrated concordance in population diversities of one symbiotic partner (rhizobia) the second partner (legume host), in line with the Evolutionary Moulding hypothesis. This effect was evinced by the following observations for each plant species: (1) significantly increased diversity in the nodulenodA popset (set of gene sequences derived from the nodule population) compared to the soil popset; (2) a monotonic relationship between the diversity in the plantNFR5gene popset and the nodule rhizobialnodA gene popset; and (3) higher topological similarity of theNFR5gene tree with thenodA gene tree of the nodule popset, than with thenodA gene tree of the soil popset. Both nonsynonymous diversity and Tajima’s D were increased in the nodule popsets compared to the soil popsets, consistent with relaxation of negative selection and/or admixture of balancing selection underlying the Evolutionary Moulding effect. We propose that the observed genetic concordance arises from the selection of particular characteristics of the nodulenodA genes by the host plant.

Using the example of N2-fixing legume-rhizobial symbiosis, we demonstrated that the origin and evolution of bacteria symbiotic for plants involve: (i) the formation of novel sym gene systems based on reorganizations of the bacterial genomes and on the gene transfer from the distant organisms; (ii) the loss of genes encoding for functions that are required for autonomous performance but interfere with symbiotic functions (negative regulators of symbiosis). Therefore, the construction of effective rhizobia strains should involve improvement of sym genes activities (for instance, nif, fix, and dct genes encoding for nitrogenase synthesis or for the energy supply of N2 fixation), as well as the inactivation of negative regulators of symbiosis identified in our lab (eff genes encoding for the transport of sugars and the production of polysaccharides and storage compounds, as well as for oxidative-reductive processes).

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (&gt, 90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (&lt, 60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive &ldquo, Afghan&rdquo, peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the &ldquo, gain-and-loss of sym genes&rdquo, and the &ldquo, compaction of sym cluster&rdquo, strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae.

In the N2-fixing symbionts of legumes (rhizobia), the evolution of (altruistic) characters that are useful for a host occurs in the populations colonizing the subscellular compartments of nodules (infection threads, symbiosomes). These compartments appear as a result of the coevolution of partners, which is associated with the complication of the trophic and regulatory interactions determining the ecological efficiency of symbiosis. Their analysis allows us to study the correlation of the mechanisms of the adaptive and progressive evolution of symbiosis, which is still unclear for free-living organisms.

The mathematical model is constructed which describes the impacts of chaotically changing environment on the frequencies and productivity of partners in the legume-rhizobia symbiosis. The most sensitive for external impacts are the adaptively prospective bacteria strains which are specific with respect to hosts and are capable for the intensive evolution towards an improved symbiotic efficiency. An increased stability of these strains in symbiotic system may be an important factor of its evolution for the improved efficiency of partners’ interaction.

The review summarizes the results of studies on the comigration of tubercular bacteria and bean plants to new habitats, which is often accompanied by a decrease in the symbiosis efficiency due to a loss of the diversity of genes responsible for the interaction. This migration may lead to a rise in new symbionts as a result of gene transfers from initial symbionts to local bacteria. It was demonstrated that typically new symbionts lack an ability for N2 fixation but are highly competitive, blocking the inoculation of bean cultures by industrial strains. The design of coadapted systems of recognition and signal interaction of partners is a perspective approach to ensure competitive advantages of efficient rhizobia strains introduced into agrocenoses, together with host plants, over inactive local strains.

Factor analysis has been conducted for the data on the interaction between the genes of the root nodule bacteria (rhizobia), which influence the efficiency of symbiosis with leguminous plants, including dctA (encoding succinate permease), dctBD (activating the dctA gene due to binding its enhancer in the presence of succinate), rpoN (activating the promoters of dctA and nitrogenase genes nifHDK), and nifA (activating the nitrogenase genes due to binding their enhancers). The analysis of the alfalfa rhizobia (Sinorhizobium meliloti) recombinants that contain additional copies of these genes suggested the antagonistic (epistatic) interaction between nifA and rpoN. It may be associated either with the competition for C compounds imported into the nodules between the energy production and nitrogen assimilation processes or with the competition for redox potentials between the oxidative phosphorylation and nitrogen fixation processes. Since the phenotypic effects of the studied genes depend on the activity of nitrogen export into the aerial parts of plants, we suppose that its accumulation in bacteroids impairs the activation of the nifHDK genes by the NifA protein due to its interaction with the GlnB protein (the nitrogen metabolism regulator) or with the FixLJ and ActSR proteins (the redox potential regulators).

In N2-fixing symbionts of leguminous plants (rhizobia) evolution of the host-beneficial (“altruistic”) traits occurs in populations colonizing the subcellular compartments in nodules (infection threads, symbiosomes). These compartments are developed as a result of partners’ coevolution related to complications of trophic and regulatory interactions elevating the ecological efficiency of symbiosis. Their analysis enables us to study correlations between genetic mechanisms of adaptive and progressive symbiosis evolution which remain obscure in free-living organisms

Using the example of nodular legume-rhizobia symbiosis (LRS), we discuss the evolution in plant micro-symbionts of mutualistic traits that are apparently host-beneficial and therefore the products of inter-species evolution. These traits include: in planta activation of N2 fixation machinery; exporting the products of nitrogenase reaction into the plant cells/tissues; and the terminal differentiation of bacteria into non-reproductive N2-fixing bacteroids. It seems probable that such adaptive traits evolved by natural selection within the populations of endosymbiotic bacteria that colonize the extra- and intra-cellular compartments provided by the hosts (i.e., infection threads and symbiosomes). This evolution would occur under the impacts of group (inter-deme, kin) selection pressures induced by the partners’ metabolic and regulatory feedbacks that ensure the high activity of symbiotic N2 fixation. These important feedbacks include: progressive allocation of C compounds into N2-fixing nodules; maintenance of micro-aerobic intracellular environments that are indispensable for intensive N2 fixation; and stringent control by the host over bacterial reproduction in planta. A computational simulation of the associated co-evolutionary processes reveals the trade-off between inter-species and individual species components of progressive and adaptive LRS evolution. This is expressed as a correlated increase of ecological efficiency, functional integrity and genotypic specificity of mutualistic symbiosis. Thus, the evolution of rhizobia in symbiosis may be represented by a progressive multi-level scenario based on increasing the dependency of bacteria on the host-provided nutrients accompanied by increasing complexity of the bacterial genomes and of the symbiosis-encoding gene networks.

Polymorphism analysis was performed in Rhizobium leguminosarum bv. viceae populations isolated from geographically distant regions of Ukraine and Middle Asia. Examination of cultural, biochemical, and symbiotic traits revealed interpopulation differences, which were attributed to the difference in conditions between natural ecosystems and agrocenoses. Vetch has high species diversity and is not cultivated in Middle Asia, and the corresponding rhizobial population displayed higher genetic diversity and higher polymorphism of adaptive traits ensuring saprophytic development in soil and the rhizosphere, including melanin synthesis (35%) and active exopolysaccharide production (90%). Strains of the Ukrainian population had a lower exopolysaccharide production (10%), did not produce melanin, had higher herbicide resistance, and utilized glucose and succinate (main components of plant root exudation) as carbon sources. Strains capable of efficient symbiosis with Vicia villosa Roth. had a higher frequency in the Middle Asian than in the Ukrainian population, especially among strains isolated from soil (80 and 35%, respectively). In addition, strains of the Middle Asian population better competed for nodulation. It was assumed that the formation of rhizobial populations in vetch cultivation regions (Ukraine) is aimed at adaptation to ectosymbiotic (rhizospheric) interactions with plants and anthropogenic stress factors, while strains of the vetch original center (Middle Asia) are mostly adapted to the endosymbiotic interaction and to natural edaphic stress factors.

Legumes develop different mutually beneficial microbial-root symbioses such as arbuscular mysorrhiza (AM), rhizobium-legume symbiosis (RLS) and epiphytic or endophytic associations with plant growth-promoting bacteria (PGPB) which are distinguished in level of integration of the partners. Evidences of the role of AM as ancestral form of symbiosis which might be a source of the legume pre-adaptation to form some RLS are demonstrated. The RLS is supposed to evolve for a few times in ancient legumes in parallel ways based on the universal organization and regulatory mechanisms of the plant genetic material. Associations of plant roots with PGPB probably are the vestige of the early stages of evolution in morphologically differentiated RLS. Also, it is quite possible that 'first' rhizobia have originated from bacterial endosymbionts of AM fungi; then AM fungi might operate as effective vectors for introducing bacteria into the plants. Thus, the legume root symbioses may be considered as a single 'evolutionary plant-microbial continuum'. The acquired knowledge about evolution of plantmicrobe symbioses would contribute to the creation of new commercial varieties of plants with the use of both bio-engineered methods and traditional plant breeding. An original conception of legume breeding to improve their symbiotic effectiveness is proposed.

Simulation of evolution in the legume-rhizobial symbiosis allowed us to suggest the mathematical criterion (index) for the functional integrity of symbiosis (FIS). Calculation of FIS index is based on analysis of the covariance matrix of partners’ frequencies influenced by fluctuations of the systemic parameters. The positive correlations of FIS with ecological efficiency of symbiosis (its impact on the seed productivity of plants) and a negative correlation with the partners’ diversity indices were demonstrated. The revealed dependences allow us to explain the increasing of structural-functional integrity in mutualistic symbiosis byoperation of the natural selection in favor of an improved ecological efficiency of partners’ interactions.

The model is suggested for evolution of Nsub2/sub-fixing legume-rhizobia symbiosis implemented under the conditions of multi-strain bacteria competition for inoculation of symbiotic habitats (rhizosphere, nodules). Competitiveness of each strain is characterized by the power coefficients which reflect the operation of frequency-dependent selection in the rhizobia population. When polymorphic bacteria populations are interacting with the dimorphic plant population, the selective pressures in favor of hostspecific symbionts (forming Nsub2/sub-fixing nodules only with one of the available plant genotypes) are higher than the pressures in favor of non-host-specific symbionts (forming these nodules with both plant genotypes). The highest mutualism efficiency is reached under an intermediate level of plant population diversity.

The molecular mechanism of the agronomically important nutritional and defensive plantmicrobe symbioses are reviewed. These symbioses are based on the signaling interactions which result in the development of novel tissue/cellular structures and of extended metabolic capacities in the partners which improve greatly the adaptive potential of plants due to an increased tolerance to biotic or abiotic stresses. The molecular, genetic and ecological knowledge on plant-microbe interactions provide a strategy for a sustainable crop production based on substituting the agrochemicals (mineral fertilizers, pesticides) by the microbial inoculants. An improvement of plantmicrobe symbioses should involve the coordinated partners' modifications resulted in complementary combinations of their genotypes. The research presented in this paper is supported by grants from Russian Foundation of Basic Research (04-04-48457, 06-04-48800, 06-04-89000NWO); NWO Centre of Excellence: 047. 018. 001.

The mathematical model for evolution of legume-rhizobia mutualism based on the partners' positive feedbacks resulted from their metabolic (C-N) exchange is presented. Negative FDS in rhizobia population, combined with the partners' positive feedbacks ensure anchoring or even domination of the mutants which either acquired the mutualistic traits or changed the specificity in their expression with different host genotypes. The created model allows us to consider the mutualistic symbiosis as of a finely balanced population system in which the equilibrium may be shifted in favor of beneficial microbial genotypes due to natural selection for an improved symbiotic efficiency implemented in plant population. Research is supported by RFBR grant 06-04-48800a.

Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains SXM1, DM7-R, and P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation experiment (120 days) was studied. The isolated clones of strains SXM1 and DM7-R manifested stable inheritance of plasmids, whereas 12.7–14.0% of clones with changed plasmid profile were detected in a population of clones from strain P108. These segregants were designated as P108c. Segregants P108c exhibited significantly decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, compared to the original strain P108. It was established that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230 kb) of segregants P108c. It was concluded that genetic rearrangements are possible in rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the natural vegetation period of alfalfa.

Nodulation, rhizospheral, and saprophytic types of competitiveness (NC, RC, and SC, respectively) were studied in the highly active strains CXM1-105 and CXM1-188 of the alfalfa rhizobium Sinorhizobium meliloti.The competitiveness was estimated with the use of markers of antibiotic resistance. It was found that the mutant strain T37, which was characterized by a drastically decreased NC, had higher SC and RC than the parental strain. The mutant T107 (with a moderately decreased NC) did not differ from the parental strain with respect to RC but had a higher SC. The mutant T27 (with the lowest NC) did not differ from the parental strain with respect to SC or RC. In the mutant Tb1, the NC and RC were decreased and the SC was the same as in the parental strain. In Tb7, the SC was decreased and RC was increased. In the mutant T795, all of the three types of competitiveness were decreased. The difference between the mutants studied and the parental strain with respect to NC and RC was confirmed using an indirect method (the ability to form effective symbiosis after mixed inoculation together with the an ineffective tester strain CXM1-48) and the X-Gluc staining method (using the S. melilotiRmM4gustester strain carrying the gene of β-glucuronidase). However, the decreased SC that the mutants exhibited when they were cultivated together with parental strains in a plant-growth substrate (vermiculite) was not observed in the case of their cocultivation in liquid media. The independent variation of different types of competitiveness indicate that rhizobia have several separate gene systems determining their survival in in plantaandex plantaecological niches.

The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarum bv. viceae 1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mob into the plasmidless Agrobacterium tumefaciens Gm1-9023 strain. Plasmid pSym1-32 was transferred into R. leguminosarum bv. viceae strains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen). Transconjugants formed Fix+ nodules on roots of V. villosa and had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains. Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected. Moreover, the donor strain R. leguminosarum bv. viceae 1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain. Both these properties were transmitted upon transfer of pSym1-32 into the recipient. Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R. leguminosarum bv. viceae.

Mutualistic symbioses represent the favorable models for analyzing the tradeoff between progressive and adaptive evolution which remains unclear for the unitary (“free-living”) organisms. Using the model of N2-fixing legume-rhizobia symbiosis we demonstrate that acquisition by plants of the novel extracellular and intracellular compartments (infection threads, symbiosomes) for hosting the bacteria induce in their populations novel selective pressures in favor of host-beneficial (“altruistic”) traits (intensive in planta N2 fixation, export of fixed nitrogen into the plant tissues, differentiation into the non-reproducible bacteroids). Due to colonization of extracellular infection threads, the degree of clonality is increased in the endosymbiotic bacterial populations which opens prospects for the interdeme selection for an increased activity of symbiotic N2 fixation. When bacteria colonize the intracellular symbiosomes, this activity is further elevated due to the kin selection in favor of irreversible bacteroid differentiation. The revealed feedbacks between the macroevolutionary events occurring in hosts and the selective pressures induced in microsymbionts may be responsible for the rapid filiations from regulatory-type anodular rhizospheric/endophytic associations (based on stimulation of the growth and assimilatory capacities of roots by the bacteria-produced auxins) to the nutritional-type nodular symbioses (based on the novel organogenesis ensuring conditions optimal for the symbiotic N2 fixation).

Symbiosis, or “coexistence of diverse organisms”, is the term which was proposed firstly by Anton de Bary (1879). Roughly, there are three types of symbiosis: commensalism (an interaction which is beneficial for one symbiotic partner but neutral for the other), antagonism (or parasitism, where one symbiont develops to the detriment of another – see elsewhere in this Book) and mutualism (where both symbionts benefit from mutual coexistence). Mutualism is a special ecological and evolutionary strategy for two (or more) dissimilar organisms to share (allocate) all of their physiological functions between them rather than them being separated. This is distinguished principally from individual adaptations which are dependent on morpho-physiological and behavioural reactions of one free-living organism. Here there is an evolutionary opportunity to develop certain functions and possibly lose others leading to genetic and metabolic integration according to a dialectical law “negation of negation” which may be applicable to evolution. As a result some intimate genetic and metabolic cooperation between the symbiotic partners of complicated super-organismic systems develop resulting in new adaptations (functions) with their environments. In mutualistic (mutually beneficial) symbioses between plants and microbes, novel types of cells and tissues (physiologically and/or structurally) and even organs have often evolved (Douglas 1994; Tikhonovich and Provorov 2007)

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.

High-throughput sequencing of the amplicon gene library revealed variations in the population structure of clover rhizobia (Rhizobium leguminosarum bv. trifolii) upon transition from soil into the root nodules of the host plant (Trifolium hybridum). Analysis of rhizobial diversity using the nodA gene revealed 3258 and 1449 nucleotide sequences (allelic variants) for the soil and root nodule population, respectively. They were combined into 29 operational taxonomic units (OTU) according to the 97% identity level; 24 OTU were found in the soil population, 12 were present in the root nodule population, and 7 were common. The predominant OTE13 (77.4 and 91.5% of the soil and root nodule populations, respectively) contained 155 and 200 variants of the soil and root nodule populations, respectively, with the nucleotide diversity increasing significantly upon the “soil → root” transition. The “moving window” approach was used to reveal the sites of the nodA gene in which polymorphism, including that associated with increased frequency of non-synonymous substitution frequency, increased sharply upon transition from soil into root nodules. PCR analysis of the IGS genotypes of individual strains revealed insignificant changes in rhizobial diversity upon transition from soil into root nodules. These results indicate that acceleration of rhizobial evolution in the course of symbiosis may be associated with development of highly polymorphic virulent subpopulations subjected to directional selection in the “plant-soil” system.