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The present study demonstrated the differences in the seed metabolome and mycobiome of two Aegilops tauschii Coss accessions with different resistance to brown rust and powdery mildew. We hypothesized that the seeds of resistant accession k-1958 Ae. tauschii ssp. strangulata can contain a larger number of metabolites with antifungal activity compared with the seeds of susceptible Ae. tauschii ssp meyeri k-340, which will determine differences in the seed fungal community. Our study emphasizes the differences in the seed metabolome of the studied Ae. tauschii accessions. The resistant accession k-1958 had a higher content of glucose and organic acids, including pyruvic, salicylic and azelaic acid, as well as pipecolic acids, galactinol, glycerol and sitosterol. The seeds of Ae. tauschii -resistant accession k-1958 were found to contain more active substances with antifungal activity. The genera Cladosporium and Alternaria were dominant in the seed mycobiome of the resistant accession. The genera Alternaria , Blumeria and Cladosporium dominated in seed mycobiome of susceptible accession k-340. In the seed mycobiome of the resistant k-1958, a higher occurrence of saprotrophic micromycetes was found, and many of the micromycetes were biocontrol agents. It was concluded that differences in the seed metabolome of Ae. tauschii contributed to the determination of the differences in mycobiomes.

The search for active cellulolytic consortia among soil microorganisms is of significant applied interest, but the dynamics of the formation of such communities remain insufficiently studied. To gain insight into the formation of an active cellulolytic community, the experiment was designed to examine the colonization of a sterile substrate (cellulose) by microorganisms from two soil types: sod-podzolic and chernozem. To achieve this, the substrate was placed in the soil and incubated for six months. To assess microbiome dynamics, the experiment employed sequencing of 16S rRNA gene fragment and ITS2 amplicon libraries at four time points. It was demonstrated that, from the second month of the experiment, the prokaryotic component of the communities reached a state of stability, with a community composition specific to each soil type. The results demonstrated no relationship between changes in community diversity and soil respiration. There also was no significant shift in the community diversity throughout the chronosequence. Furthermore, the taxonomic composition of the community shifted towards a decrease in the proportion of Pseudomonadota and an increase in representatives of the Bacteroidota, Bacillota, and Verrucomicrobiota phyla. The network analysis of the community demonstrated that, in contrast to sod-podzolic soil, chernozem is distinguished by a higher modularity, with the formation of taxon-specific groups of microorganisms at each stage of the chronoseries. These differences are attributed to the alterations in the eukaryotic component of the community, particularly in the prevalence of nematodes and predatory fungi, which in turn influenced the cellulolytic community.

Cryoconites are the deposits on the surface of glaciers that create specific ecological niches for the development of microorganism communities. The sediment material can vary in origin, structure, and nutrient content, creating local variations in the growth conditions. An additional factor of variability is the location of the glaciers, as they are found in different climatic zones in the high mountain regions and closer to the poles. Here, using the analysis of amplicon sequencing of the 16S rRNA gene, we studied the taxonomic composition of the prokaryotic communities from glaciers from remote regions, including the Arctic (Mushketova on the Severnaya Zemlya, IGAN in Polar Ural), Antarctic (Pimpirev on the Livingstone Island) and Central Caucasus (Skhelda and Garabashi) and connected it with the variation of the physicochemical characteristics of the substrate: pH, carbon, nitrogen, macro- and microelements. The cryoconite microbiomes were comprised of specific for this environment phyla (mostly Pseudomonadota, Cyanobacteria, Bacteroidota, Acidobacteriota, and Actinobacteriota), but each glacier had a unique taxonomic imprint. The core microbiome between regions was composed of only a few ASVs, among which the most likely globally distributed ones attributed to Polaromonas sp., Rhodoferax sp., Cryobacterium sp., and Hymenobacter frigidus. The WGSNA defined clusters of co-occurring ASVs between microbiomes, that significantly change their abundance corresponding with the variation of chemical parameters of cryoconites, but do not fully coincide with their regional separation. Thus, our work demonstrates that the chemical characteristics of the sediment material can explain the variation in the cryoconite prokaryotic community which is not always linked to geographic isolation., (© 2024. The Author(s).)

Nodule bacteria (rhizobia) represent a suitable model to address a range of fundamental genetic problems, including the impacts of natural selection on the evolution of symbiotic microorganisms. Rhizobia possess multipartite genomes in which symbiotically specialized ( sym ) genes differ from core genes in their natural histories. Diversification of sym genes is responsible for rhizobia microevolution, which depends on host-induced natural selection. By contrast, diversification of core genes is responsible for rhizobia speciation, which occurs under the impacts of still unknown selective factors. In this paper, we demonstrate that in goat's rue rhizobia ( Neorhizobium galegae ) populations collected at North Caucasus, representing two host-specific biovars orientalis and officianalis (N 2 -fixing symbionts of Galega orientalis and G. officinalis ), the evolutionary mechanisms are different for core and sym genes. In both N. galegae biovars, core genes are more polymorphic than sym genes. In bv. orientalis , the evolution of core genes occurs under the impacts of driving selection (dN/dS > 1), while the evolution of sym genes is close to neutral (dN/dS ≈ 1). In bv. officinalis , the evolution of core genes is neutral, while for sym genes, it is dependent on purifying selection (dN/dS < 1). A marked phylogenetic congruence of core and sym genes revealed using ANI analysis may be due to a low intensity of gene transfer within and between N. galegae biovars. Polymorphism in both gene groups and the impacts of driving selection on core gene evolution are more pronounced in bv. orientalis than in bv. officianalis , reflecting the diversities of their respective host plant species. In bv. orientalis , a highly significant (P 0 < 0.001) positive correlation is revealed between the p-distance and dN/dS values for core genes, while in bv. officinalis , this correlation is of low significance (0.05 < P 0 < 0.10). For sym genes, the correlation between p-distance and dN/dS values is negative in bv. officinalis but is not revealed in bv. orientalis . These data, along with the functional annotation of core genes implemented using Gene Ontology tools, suggest that the evolution of bv. officinalis is based mostly on adaptation for in planta niches while in bv. orientalis , evolution presumably depends on adaptation for soil niches. New insights into the tradeoff between natural selection and genetic diversity are presented, suggesting that gene nucleotide polymorphism may be extended by driving selection only in ecologically versatile organisms capable of supporting a broad spectrum of gene alleles in their gene pools.

Burial mounds represent a challenge for microbiologists. Could ancient buried soils preserve microbiomes as they do archaeological artifacts? To investigate this question, we studied the soil microbiome under a burial mound dating from 2500 years ago in Western Kazakhstan. Two soil profile cuts were established: one under the burial mound and another adjacent to the mound surface steppe soil. Both soils represented the same dark chestnut soil type and had the same horizontal stratification (A, B, C horizons) with slight alterations. DNA samples isolated from all horizons were studied with molecular techniques including qPCR and high throughput sequencing of amplicon libraries of the 16S rRNA gene fragment. The taxonomic structure of the microbiome of the buried horizons demonstrated a deep divergence from ones of the surface, comparable to the variation between different soil types (representatives of the soil types were included in the survey). The cause of this divergence could be attributed to diagenetic processes characterized by the reduction of organic matter content and changes in its structure. Corresponding trends in the microbiome structure are obvious from the beta-diversity pattern: the A and B horizons of the buried soils form one cluster with the C horizons of both buried and surface soil. This trend could generally be designated as 'mineralization'. Statistically significant changes between the buried and surface soils microbiomes were detected in the number of phylogenetic clusters, the biology of which is in the line of diagenesis. The trend of 'mineralization' was also supported by PICRUSt2 functional prediction, demonstrating a higher occurrence of the processes of degradation in the buried microbiome. Our results show a profound shift in the buried microbiome relatively the "surface" microbiome, indicating the deep difference between the original and buried microbiomes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

The process of straw decomposition is dynamic and is accompanied by the succession of the microbial decomposing community, which is driven by poorly understood interactions between microorganisms. Soil is a complex ecological niche, and the soil microbiome can serve as a source of potentially active cellulolytic microorganisms. Here, we performed an experiment on the de novo colonization of oat straw by the soil microbial community by placing nylon bags with sterilized oat straw in the pots filled with chernozem soil and incubating them for 6 months. The aim was to investigate the changes in decomposer microbiota during this process using conventional sequencing techniques. The bacterial succession during straw decomposition occurred in three phases: the early phase (first month) was characterized by high microbial activity and low diversity, the middle phase (second to third month) was characterized by low activity and low diversity, and the late phase (fourth to sixth months) was characterized by low activity and high diversity. Analysis of amplicon sequencing data revealed three groups of co-changing phylotypes corresponding to these phases. The early active phase was abundant in the cellulolytic members from Pseudomonadota, Bacteroidota, Bacillota, and Actinobacteriota for bacteria and Ascomycota for fungi, and most of the primary phylotypes were gone by the end of the phase. The second intermediate phase was marked by the set of phylotypes from the same phyla persisting in the community. In the mature community of the late phase, apart from the core phylotypes, non-cellulolytic members from Bdellovibrionota, Myxococcota, Chloroflexota, and Thermoproteota appeared. Full metagenome sequencing of the microbial community from the end of the middle phase confirmed that major bacterial and fungal members of this consortium had genes of glycoside hydrolases (GH) connected to cellulose and chitin degradation. The real-time analysis of the selection of these genes showed that their representation varied between phases, and this occurred under the influence of the host, and not the GH family factor. Our findings demonstrate that soil microbial community may act as an efficient source of cellulolytic microorganisms and that colonization of the cellulolytic substrate occurs in several phases, each characterized by its own taxonomic and functional profile.

Restoration of anthropogenically disturbed soils is an urgent problem in modern ecology and soil biology. Restoration processes in northern environments are especially important, due to the small amounts of fertile land and low levels of natural succession. We analyzed the soil microbiota, which is one of the indicators of the succession process is the soil. Samples were obtained from three disturbed soils (self-overgrown and reclaimed quarries), and two undisturbed soils (primary and secondary forests). Primary Forest soil had a well-developed soil profile, and a low pH and TOC (total organic carbon) amount. The microbial community of this soil had low richness, formed a clear remote cluster in the beta-diversity analysis, and showed an overrepresentation of Geobacter (Desulfobacteriota). Soil formation in clay and limestone abandoned quarries was at the initial stage, and was caused by both a low rate of mineral profile formation and severe climatic conditions in the region. Microbial communities of these soils did not have specific abundant taxa, and included a high amount of sparse taxa. Differences in taxa composition were correlated with abiotic factors (ammonium concentration), which, in turn, can be explained by the parent rock properties. Limestone quarry reclaimed by topsoil coverage resulted in an adaptation of the top soil microbiota to a novel parent rock. According to the CCA analysis, the microbial composition of samples was connected with pH, TOC and ammonium nitrogen concentration. Changes in pH and TOC were connected with ASVs from Chloroflexota, Gemmatimonadota and Patescibacteria. ASVs from Gemmatimonadota also were correlated with a high ammonium concentration.

Crop rotation is one of the oldest and most effective methods of restoring soil fertility, which declines when the same plant is grown repeatedly. One of the reasons for a reduction in fertility is the accumulation of pathogenic and unfavorable microbiota. The modern crop rotation schemes (a set of plant species and their order in the crop rotation) are highly effective but are designed without considering soil microbiota dynamics. The main goal of this study was to perform a short-term experiment with multiple plant combinations to access the microbiological effects of crop rotation. It could be useful for the design of long-term crop rotation schemes that take the microbiological effects of the crop rotation into account. For the analysis, five plants (legumes: vetch, clover, and cereals: oats, wheat, and barley) were used. These five plants were separately grown in pots with soil. After the first phase of vegetation, the plants were removed from the soil and a new crop was planted. Soil samples from all 25 possible combinations of primary and secondary crops were investigated using v4-16S rDNA gene sequencing. It was shown that the short-term experiments (up to 40 days of growing) are effective enough to find microbial shifts in bulk soil from different plants. Both primary and secondary cultures are significant factors for the microbial composition of microbial soil communities. Changes are the most significant in the microbial communities of vetch soils, especially in the case of vetch monoculture. Growing clover also leads to changes in microbiota, especially according to beta-diversity. Data obtained can be used to develop new crop rotation schemes that take into account the microbiological effects of various crops.

A single universal open protocol RIAM (named after Research Institute for Agricultural Microbiology) for the isolation of high purity DNA from different types of soils and other substrates (high and low in humic, clay content, organic fertilizer, etc.) is proposed. The main features of the RIAM protocol are the absence of the sorption-desorption stage on silica columns, the use of high concentrations of phosphate in buffers, which prevents DNA sorption on minerals, and DNA precipitation using CTAB. The performance of RIAM was compared with a reference commercial kit and showed very good results in relation to the purity and quantity of DNA, as well as the absence of inhibitory activity on PCR. In all cases, the RIAM ensured the isolation of DNA in quantities much greater than the commercial kit without the effect of PCR inhibition up to 50 ng DNA per reaction in a volume of 15 µL. The latter circumstance along with the ability of the protocol to extract low molecular weight DNA fractions makes the method especially suitable for those cases where quantitative assessments, detection of minor components of soil microbiota, and completeness of isolation of all DNA fractions are required.

Drought and heavy metals seriously affect plant growth and the biodiversity of the associated rhizosphere microbiomes, which, in turn, could be involved in the adaptation of plants to these environmental stresses. Rhizosphere soil was collected from a three-factor pot experiment, where pea line SGE and its Cd-tolerant mutant SGECd t were cultivated under both optimal and limited water conditions and treated with a toxic Cd concentration. The taxonomic structure of the prokaryotic rhizosphere microbiome was analyzed with the high-throughput sequencing of 16S rRNA amplicon libraries. A permutation test demonstrated statistically significant effects of Cd and water stress but not of pea genotype on the rhizosphere microbiome structure. Phylogenetic isometric log-ratio data transformation identified the taxonomic balances that were affected by abiotic factors and pea genotypes. A small number of significant (log ratio [-3.0:+3.0]) and phylogenetically deep balances characterized water stress, while a larger number of weak (log ratio [-0.8:+0.8]) phylogenetically lower balances described the influence of the plant genotype. Stress caused by cadmium took on an intermediate position. The main conclusion of the study is that the most powerful factor affecting the rhizosphere microbiome was water stress, and the weakest factor was plant genotype since it demonstrated a very weak transformation of the taxonomic structure of rhizosphere microbiomes in terms of alpha diversity indices, beta diversity, and the log ratio values of taxonomic balances.

Nodule bacteria (rhizobia), N 2 -fixing symbionts of leguminous plants, represent an excellent model to study the fundamental issues of evolutionary biology, including the tradeoff between microevolution, speciation, and macroevolution, which remains poorly understood for free-living organisms. Taxonomically, rhizobia are extremely diverse: they are represented by nearly a dozen families of α-proteobacteria (Rhizobiales) and by some β-proteobacteria. Their genomes are composed of core parts, including house-keeping genes ( hkg ), and of accessory parts, including symbiotically specialized ( sym ) genes. In multipartite genomes of evolutionary advanced fast-growing species (Rhizobiaceae), sym genes are clustered on extra-chromosomal replicons (megaplasmids, chromids), facilitating gene transfer in plant-associated microbial communities. In this review, we demonstrate that in rhizobia, microevolution and speciation involve different genomic and ecological mechanisms: the first one is based on the diversification of sym genes occurring under the impacts of host-induced natural selection (including its disruptive, frequency-dependent and group forms); the second one-on the diversification of hkg s under the impacts of unknown factors. By contrast, macroevolution represents the polyphyletic origin of super-species taxa, which are dependent on the transfer of sym genes from rhizobia to various soil-borne bacteria. Since the expression of newly acquired sym genes on foreign genomic backgrounds is usually restricted, conversion of resulted recombinants into the novel rhizobia species involves post-transfer genetic changes. They are presumably supported by host-induced selective processes resulting in the sequential derepression of nod genes responsible for nodulation and of nif / fix genes responsible for symbiotic N 2 fixation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Provorov, Andronov, Kimeklis, Onishchuk, Igolkina and Karasev.)

Recycling plant matter is one of the challenges facing humanity today and depends on efficient lignocellulose degradation. Although many bacterial strains from natural substrates demonstrate cellulolytic activities, the CAZymes (Carbohydrate-Active enZYmes) responsible for these activities are very diverse and usually distributed among different bacteria in one habitat. Thus, using microbial consortia can be a solution to rapid and effective decomposition of plant biomass. Four cellulolytic consortia were isolated from enrichment cultures from composting natural lignocellulosic substrates-oat straw, pine sawdust, and birch leaf litter. Enrichment cultures facilitated growth of similar, but not identical cellulose-decomposing bacteria from different substrates. Major components in all consortia were from Proteobacteria, Actinobacteriota and Bacteroidota, but some were specific for different substrates-Verrucomicrobiota and Myxococcota from straw, Planctomycetota from sawdust and Firmicutes from leaf litter. While most members of the consortia were involved in the lignocellulose degradation, some demonstrated additional metabolic activities. Consortia did not differ in the composition of CAZymes genes, but rather in axillary functions, such as ABC-transporters and two-component systems, usually taxon-specific and associated with CAZymes. Our findings show that enrichment cultures can provide reproducible cellulolytic consortia from various lignocellulosic substrates, the stability of which is ensured by tight microbial relations between its components.

High-throughput sequencing of amplicon libraries is the most widespread and one of the most effective ways to study the taxonomic structure of microbial communities, even despite growing accessibility of whole metagenome sequencing. Due to the targeted amplification, the method provides unparalleled resolution of communities, but at the same time perturbs initial community structure thereby reducing data robustness and compromising downstream analyses. Experimental research of the perturbations is largely limited to comparative studies on different PCR protocols without considering other sources of experimental variation related to characteristics of the initial microbial composition itself. Here we analyse these sources and demonstrate how dramatically they effect the relative abundances of taxa during the PCR cycles. We developed the mathematical model of the PCR amplification assuming the heterogeneity of amplification efficiencies and considering the compositional nature of data. We designed the experiment-five consecutive amplicon cycles (22-26) with 12 replicates for one real human stool microbial sample-and estimated the dynamics of the microbial community in line with the model. We found the high heterogeneity in amplicon efficiencies of taxa that leads to the non-linear and substantial (up to fivefold) changes in relative abundances during PCR. The analysis of possible sources of heterogeneity revealed the significant association between amplicon efficiencies and the energy of secondary structures of the DNA templates. The result of our work highlights non-trivial changes in the dynamics of real-life microbial communities due to their compositional nature. Obtained effects are specific not only for amplicon libraries, but also for any studies of metagenome dynamics., Competing Interests: The authors declare there are no competing interests., (©2022 Korvigo et al.)

The rhizosphere community represents an "ecological interface" between plant and soil, providing the plant with a number of advantages. Despite close connection and mutual influence in this system, the knowledge about the connection of plant and rhizosphere diversity is still controversial. One of the most valuable factors of this uncertainty is a rough estimation of plant diversity. NGS sequencing can make the estimations of the plant community more precise than classical geobotanical methods. We investigate fallow and crop sites, which are similar in terms of environmental conditions and soil legacy, yet at the same time are significantly different in terms of plant diversity. We explored amplicons of both the plant root mass (ITS1 DNA) and the microbial communities (16S rDNA); determined alpha- and beta-diversity indices and their correlation, and performed differential abundance analysis. In the analysis, there is no correlation between the alpha-diversity indices of plants and the rhizosphere microbial communities. The beta-diversity between rhizosphere microbial communities and plant communities is highly correlated (R = 0.866, p = 0.01). ITS1 sequencing is effective for the description of plant root communities. There is a connection between rhizosphere communities and the composition of plants, but on the alpha-diversity level we found no correlation. In the future, the connection of alpha-diversities should be explored using ITS1 sequencing, even in more similar plant communities-for example, in different synusia.

The difference in symbiotic specificity between peas of Afghanistan and European phenotypes was investigated using molecular modeling. Considering segregating amino acid polymorphism, we examined interactions of pea LykX-Sym10 receptor heterodimers with four forms of Nodulation factor (NF) that varied in natural decorations (acetylation and length of the glucosamine chain). First, we showed the stability of the LykX-Sym10 dimer during molecular dynamics (MD) in solvent and in the presence of a membrane. Then, four NFs were separately docked to one European and two Afghanistan dimers, and the results of these interactions were in line with corresponding pea symbiotic phenotypes. The European variant of the LykX-Sym10 dimer effectively interacts with both acetylated and non-acetylated forms of NF, while the Afghanistan variants successfully interact with the acetylated form only. We additionally demonstrated that the length of the NF glucosamine chain contributes to controlling the effectiveness of the symbiotic interaction. The obtained results support a recent hypothesis that the LykX gene is a suitable candidate for the unidentified Sym2 allele, the determinant of pea specificity toward Rhizobium leguminosarum bv. viciae strains producing NFs with or without an acetylation decoration. The developed modeling methodology demonstrated its power in multiple searches for genetic determinants, when experimental detection of such determinants has proven extremely difficult., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Solovev, Igolkina, Kuliaev, Sulima, Zhukov, Porozov, Pidko and Andronov.)

Aim: This study aimed to determine the bacterial composition at various stages of the temperature regime in a recirculating aquaculture system (RAS) to assess the pathological risk of a group of opportunistic pathogenic microflora., Materials and Methods: Water temperature, incidences of illnesses, and fish mortality were monitored, during the research period to identify the causes of pathogens in sturgeons. Analysis of the nucleotide sequences was performed using the quantitative insights into microbial ecology module. Sequence alignment in the analysis of the distribution of gene libraries was performed using the Unclust method. The RDP database was used for the taxonomic identification of operational taxonomic units., Results: The pattern of the contraction of infection among sturgeons bred in the RAS was established. A detailed analysis of the microbiome structure's taxonomic features showed dominant taxa during the "artificial wintering" period and at a temperature optimum in industrial aquaculture. It was found that the main outbreaks of pseudomonosis occurred during this period in the RAS. With a decrease in temperature of the aquatic environment, the incidence of illness increased by 75% compared with the optimum temperature period. Pseudomonas, Cetobacterium , and Lactococcus were specific taxa characteristic for the "artificial wintering" period. Xanthomonadaceae and Flavobacterium were specific taxa characteristic for the optimum temperature., Conclusion: Consequently, the microbial structure was determined at different temperature regimes in a RAS, and the dominant communities were identified. The pattern of the contraction of infection caused by an opportunistic microflora (pseudomonosis) among sturgeons was established, allowing for the development and correction of treatment and preventive measures., (Copyright: © Sergaliev, et al.)

Rendzic Leptosols are intrazonal soils formed on limestone bedrock. The specialty of these soils is that parent rock material is more influential in shaping soil characteristics than zonal factors such as climate, especially during soil formation. Unlike fast evolving Podzols due to their leaching regime, Leptosols do not undergo rapid development due to the nature of the limestone. Little is known how microbiome reflects this process, so we assessed microbiome composition of Rendzic Leptosols of different ages, arising from disruption and subsequent reclamation. The mountains and foothills that cover much of the Crimean Peninsula are ideal for this type of study, as the soils were formed on limestone and have been subjected to anthropogenic impacts through much of human history. Microbiomes of four soil sites forming a chronosequence, including different soil horizons, were studied using sequencing of 16S rRNA gene libraries and quantitative PCR. Dominant phyla for all soil sites were Actinobacteria, Proteobacteria, Acidobacteria, Bacteroidetes, Thaumarchaeota, Planctomycetes, Verrucomicrobia and Firmicutes. Alpha diversity was similar across sites and tended to be higher in topsoil. Beta diversity showed that microbiomes diverged according to the soil site and the soil horizon. The oldest and the youngest soils had the most similar microbiomes, which could have been caused by their geographic proximity. Oligotrophic bacteria from Chitinophagaceae, Blastocatellaceae and Rubrobacteriaceae dominated the microbiome of these soils. The microbiome of 700-year old soil was the most diverse. This soil was from the only study location with topsoil formed by plant litter, which provided additional nutrients and could have been the driving force of this differentiation. Consistent with this assumption, this soil was abundant in copiotrophic bacteria from Proteobacteria and Actinobacteria phyla. The microbiome of 50-year old Leptosol was more similar to the microbiome of benchmark soil than the microbiome of 700-year old soil, especially by weighted metrics. CCA analysis, in combination with PERMANOVA, linked differences in microbiomes to the joint change of all soil chemical parameters between soil horizons. Local factors, such as parent material and plant litter, more strongly influenced the microbiome composition in Rendzic Leptosols than soil age., Competing Interests: The authors declare there are no competing interests., (©2021 Kimeklis et al.)

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense . We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a 'natural' unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis , "R. indicum" and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Aluminium being one of the most abundant elements is very toxic for plants causing inhibition of nutrient uptake and productivity. The aim of this study was to evaluate the potential of microbial consortium consisting of arbuscular mycorrhizal fungus (AMF), rhizobia and PGPR for counteracting negative effects of Al toxicity on four pea genotypes differing in Al tolerance. Pea plants were grown in acid soil supplemented with AlCl 3 (pH KCl = 4.5) or neutralized with CaCO 3 (pH KCl = 6.2). Inoculation increased shoot and/or seed biomass of plants grown in Al-supplemented soil. Nodule number and biomass were about twice on roots of Al-treated genotypes after inoculation. Inoculation decreased concentrations of water-soluble Al in the rhizosphere of all genotypes grown in Al-supplemented soil by about 30%, improved N 2 fixation and uptake of fertilizer 15 N and nutrients from soil, and increased concentrations of water-soluble nutrients in the rhizosphere. The structure of rhizospheric microbial communities varied to a greater extent depending on the plant genotype, as compared to soil conditions and inoculation. Thus, this study highlights the important role of symbiotic microorganisms and the plant genotype in complex interactions between the components of the soil-microorganism-plant continuum subjected to Al toxicity.

High-throughput 16S rRNA sequencing was performed to compare the microbiomes inhabiting two contrasting soil types-sod-podzolic soil and chernozem-and the corresponding culturome communities of potentially cellulolytic bacteria cultured on standard Hutchinson media. For each soil type, soil-specific microorganisms have been identified: for sod-podzolic soil-Acidothermus, Devosia, Phenylobacterium and Tumebacillus, and for chernozem soil-Sphingomonas, Bacillus and Blastococcus. The dynamics of differences between soil types for bulk soil samples and culturomes varied depending on the taxonomic level of the corresponding phylotypes. At high taxonomic levels, the number of common taxa between soil types increased more slowly for bulk soil than for culturome. Differences between soil-specific phylotypes were detected in bulk soil at a low taxonomic level (genus, species). A total of 13 phylotypes were represented both in soil and in culturome. No relationship was shown between the abundance of these phylotypes in soil and culturome., Competing Interests: The authors have declared that no competing interests exist.

A collection of rhizobial strains isolated from root nodules of the narrowly endemic legume species Oxytropis erecta , O. anadyrensis , O. kamtschatica , and O. pumilio originating from the Kamchatka Peninsula (Russian Federation) was obtained. Analysis of the 16S ribosomal RNA gene sequence showed a significant diversity of isolates belonging to families Rhizobiaceae (genus Rhizobium ), Phyllobacteriaceae (genera Mesorhizobium , Phyllobacterium ), and Bradyrhizobiaceae (genera Bosea , Tardiphaga ). A plant nodulation assay showed that only strains belonging to genus Mesorhizobium could form nitrogen-fixing nodules on Oxytropis plants. The strains M. loti 582 and M. huakuii 583, in addition to symbiotic clusters, possessed genes of the type III and type VI secretion systems (T3SS and T6SS, respectively), which can influence the host specificity of strains. These strains formed nodules of two types (elongated and rounded) on O. kamtschatica roots. We suggest this phenomenon may result from Nod factor-dependent and -independent nodulation strategies. The obtained strains are of interest for further study of the T3SS and T6SS gene function and their role in the development of rhizobium-legume symbiosis. The prospects of using rhizobia having both gene systems related to symbiotic and nonsymbiotic nodulation strategies to enhance the efficiency of plant-microbe interactions by expanding the host specificity and increasing nodulation efficiency are discussed.

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, gln II, glt A, and dna K) and symbiotic ( sym : nod A, nod C, nod D, and nif H) 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 nod X 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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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 (>90 kb cluster on p Sym ), with the location of nod T gene outside of the other nod operons, the presence of nod X and fix W, and the absence of chromosomal fix NOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (<60 kb) on a single p Sym , lacking nod X and fix W, with nod T between nod N and nod O, and possessing chromosomal fix NOPQ copies. The TOM strain, obtained from nodules of the primitive "Afghan" peas, occupied an intermediate position because it has the chromosomal fix NOPQ copy, while the other features, the most important of which is presence of nod X and fix W, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the "gain-and-loss of sym genes" and the "compaction of sym cluster" 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 ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Two Gram-stain-negative strains, RCAM04680 T and RCAM04685, were isolated from root nodules of the relict legume Caragana jubata (Pall.) Poir. originating from the south-western shore of Lake Khuvsgul (Mongolia). The 16S rRNA gene ( rrs ) sequencing data showed that these novel isolates belong to the genus Bosea and are phylogenetically closest to the type strains Bosea lathyri LMG 26379 T , Bosea vaviloviae LMG 28367 T , Bosea massiliensis LMG 26221 T and Bosea lupini LMG 26383 T (the rrs -similarity levels were 98.7-98.8 %). The recA gene of strain RCAM04680 T showed the highest sequence similarity to the type strain B. lupini LMG 26383 T (95.4 %), while its atpD gene was closest to that of B. lathyri LMG 26379 T (94.4 %). The ITS, dnaK and gyrB sequences of this isolate were most similar to the B. vaviloviae LMG 28367 T (86.8 % for ITS, 90.4 % for the other genes). The most abundant fatty acid was C 18 : 1 ω7 c (40.8 %). The whole genomes of strains RCAM04680 T and RCAM04685 were identical (100 % average nucleotide identity). The highest average nucleotide identity value (82.8 %) was found between the genome of strain RCAM04680 T and B. vaviloviae LMG 28367 T . The common nod ABC genes required for legume nodulation were absent in both strains; however, some other symbiotic nol , nod , nif and fix genes were detected. Based on the genetic study, as well as analyses of the whole-cell fatty acid compositions and phenotypic properties, a new species, Boseacaraganae sp. nov. (type strain RCAM04680 T (=LMG 31125 T ), is proposed.

We hypothesized that population diversities of partners in nitrogen-fixing rhizobium-legume symbiosis can be matched for "interplaying" genes. We tested this hypothesis using data on nucleotide polymorphism of symbiotic genes encoding two components of the plant-bacteria signaling system: (a) the rhizobial nod A acyltransferase involved in the fatty acid tail decoration of the Nod factor (signaling molecule); (b) the plant NFR5 receptor required for Nod factor binding. We collected three wild-growing legume species together with soil samples adjacent to the roots from one large 25-year fallow: Vicia sativa , Lathyrus pratensis , and Trifolium hybridum nodulated by one of the two Rhizobium leguminosarum biovars ( viciae and trifolii ). For each plant species, we prepared three pools for DNA extraction and further sequencing: the plant pool (30 plant indiv.), the nodule pool (90 nodules), and the soil pool (30 samples). We observed the following statistically significant conclusions: (a) a monotonic relationship between the diversity in the plant NFR5 gene pools and the nodule rhizobial nod A gene pools; (b) higher topological similarity of the NFR5 gene tree with the nod A gene tree of the nodule pool, than with the nod A gene tree of the soil pool. Both nonsynonymous diversity and Tajima's D were increased in the nodule pools compared with the soil pools, consistent with relaxation of negative selection and/or admixture of balancing selection. We propose that the observed genetic concordance between NFR5 gene pools and nodule nod A gene pools arises from the selection of particular genotypes of the nod A gene by the host plant., Competing Interests: None declared., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

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 lipopolysaccha-rides 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.

Reliance on pure cultures was introduced at the beginning of microbiology as a discipline and has remained significant although their adaptive properties are essentially dissimilar from those of mixed cultures and environmental populations. They are needed for (i) taxonomic identification; (ii) diagnostics of pathogens; (iii) virulence and pathogenicity studies; (iv) elucidation of metabolic properties; (v) testing sensitivity to antibiotics; (vi) full-length genome assembly; (vii) strain deposition in microbial collections; and (viii) description of new species with name validation. Depending on the specific task there are alternative claims for culture purity, i.e., when conventional criteria are satisfied or when looking deeper is necessary. Conventional proof (microscopic and plating controls) has a low resolution and depends on the observer's personal judgement. Phenotypic criteria alone cannot prove culture purity and should be complemented with genomic criteria. We consider the possible use of DNA high-throughput culture sequencing data to define criteria for only one genospecies, axenic state detection panel and only one genome. The second and third of these are preferable, although their resolving capacity (depth) is limited. Because minor contaminants may go undetected, even with deep sequencing, the reliably pure culture would be a clonal culture launched from a single cell or trichome (multicellular bacterium). Although this type of culture is associated with technical difficulties and cannot be employed on a large scale (the corresponding inoculums may have low chances of growth when transferred to solid media), it is hoped that the high-throughput culturing methods introduced by 'culturomics' will overcome this obstacle.

The main goal of modern microbial ecology is to determine the key factors influencing the global diversity of microorganisms. Because of their complexity, soil communities are largely underexplored in this context. We studied soil genesis (combination of various soil-forming processes, specific to a particular soil type) that is driven by microbial activity. To investigate the interrelation between soil type and microbial diversity, we analyzed six soil types that are common in Russia, the Crimea, and Kazakhstan using 16S rDNA pyrosequencing. Soils of different types varied in the taxonomic composition of microbial communities. Their core microbiomes comprised 47 taxa within the orders Solirubrobacteriales and Hyphomicrobiaceae and the Gaiellaceae family. Two species from Bradyrhizobiaceae and Solirubrobactriaceae were present in all samples, whereas most other taxa were soil-type specific. Multiple resampling analysis revealed that two random soil samples from the same soil type shared more taxa than two samples from different types. The differences in community composition were mostly affected by the variation in pH values and exchangeable potassium content. The results show that data on the soil microbiome could be used for soil identification and clarification of their taxonomic position., (Copyright © 2018. Published by Elsevier B.V.)

Ten rhizobial strains were isolated from root nodules of a relict legume Oxytropis popoviana Peschkova. For identification of the isolates, sequencing of rrs, the internal transcribed spacer region, and housekeeping genes recA, glnII, and rpoB was used. Nine fast-growing isolates were Mesorhizobium-related; eight strains were identified as M. japonicum and one isolate belonged to M. kowhaii. The only slow-growing isolate was identified as a Bradyrhizobium sp. Two strains, M. japonicum Opo-242 and Bradyrhizobium sp. strain Opo-243, were isolated from the same nodule. Symbiotic genes of these isolates were searched throughout the whole-genome sequences. The common nodABC genes and other symbiotic genes required for plant nodulation and nitrogen fixation were present in the isolate Opo-242. Strain Opo-243 did not contain the principal nod, nif, and fix genes; however, five genes (nodP, nodQ, nifL, nolK, and noeL) affecting the specificity of plant-rhizobia interactions but absent in isolate Opo-242 were detected. Strain Opo-243 could not induce nodules but significantly accelerated the root nodule formation after coinoculation with isolate Opo-242. Thus, we demonstrated that taxonomically different strains of the archaic symbiotic system can be co-microsymbionts infecting the same nodule and promoting the nodulation process due to complementary sets of symbiotic genes.

Gram-negative strains Tri-36, Tri-38, Tri-48 T and Tri-53 were isolated from root nodules of the relict legume Oxytropis triphylla (Pall.) Pers. originating from Zunduk Cape (Baikal Lake region, Russia). 16S rRNA gene sequencing showed that the novel isolates were phylogenetically closest to the type strains Phyllobacterium sophorae LMG 27899 T , Phyllobacterium brassicacearum LMG 22836 T , Phyllobacterium endophyticum LMG 26470 T and Phyllobacterium bourgognense LMG 22837 T while similarity levels between the isolates and the most closely related strain P. endophyticum LMG 26470 T were 98.8-99.5 %. The recA and glnII genes of the isolates showed highest sequence similarities with P. sophorae LMG 27899 T (95.4 and 89.5 %, respectively) and P. brassicacearum LMG 22836 T (91.4 and 85.1 %, respectively). Comparative analysis of phenotypic properties between the novel isolates and the closest reference strains P. sophorae LMG 27899 T , P. brassicacearum LMG 22836 T and P. endophyticum LMG 26470 T was performed using a microassay system. Average nucleotide identities between the whole genome sequences of the isolates Tri-38 and Tri-48 T and P. sophorae LMG 27899 T , P. brassicacearum LMG 22836 T and P. endophyticum LMG 26470 T ranged from 79.23 % for P. endophyticum LMG 26470 T to 85.74 % for P. sophorae LMG 27899 T . The common nodABC genes required for legume nodulation were absent from strains Tri-38 and Tri-48 T , although some other symbiotic nod and fix genes were detected. On the basis of genotypic and phenotypic analysis, a novel species, Phyllobacterium zundukense sp. nov. (type strain Tri-48 T =LMG 30371 T =RCAM 03910 T ), is proposed.

Sandwich-like docking configurations of the heterodimeric complex of NFR5 and K1 Vicia sativa receptor-like kinases together with the putative ligand, Nod factor (NF) of Rhizobium leguminosarum bv. viciae , were modeled and two of the most probable configurations were assessed through the analysis of the mutual polymorphisms and conservatism. We carried out this analysis based on the hypothesis that in a contact zone of two docked components (proteins or ligands) the population polymorphism or conservatism is mutual, i.e., the variation in one component has a reflected variation in the other component. The population material of 30 wild-growing V. sativa (leaf pieces) was collected from a large field (uncultivated for the past 25-years) and pooled; form this pool, 100 randomly selected cloned fragments of NFR5 gene and 100 of K1 gene were sequenced by the Sanger method. Congruence between population trees of NFR5 and K1 haplotypes allowed us to select two respective haplotypes, build their 3D structures, and perform protein-protein docking. In a separate simulation, the protein-ligand docking between NFR5 and NF was carried out. We merged the results of the two docking experiments and extracted NFR5-NF-K1 complexes, in which NF was located within the cavity between two receptors. Molecular dynamics simulations indicated two out of six complexes as stable. Regions of mutual polymorphism in the contact zone of one complex overlapped with known NF structural variations produced by R. leguminosarum bv. viciae . A total of 74% of the contact zone of another complex contained mutually polymorphic and conservative areas. Common traits of the obtained two stable structures allowed us to hypothesize the functional role of three-domain structure of plant LysM-RLKs in their heteromers.

This study is a comparative analysis of samples of archived (stored for over 70-90 years) and modern soils of two different genetic types-chernozem and sod-podzolic soils. We revealed a reduction in biodiversity of archived soils relative to their modern state. Particularly, long-term storage in the museum exerted a greater impact on the microbiomes of sod-podzolic soils, while chernozem samples better preserved the native community. Thus, the persistence of microbial DNA in soil is largely determined by the physico-chemical characteristics that differ across soil types. Chernozems create better conditions for the long-term DNA preservation than sod-podzolic soils. This results in supposedly higher levels of biodiversity conservation in the microbiomes of chernozem with preservation of major microbial taxa dominant in the modern (control) soil samples, which makes archived chernozems a promising object for paleosoil studies.

Gram-stain-negative strains V5/3MT, V5/5K, V5/5M and V5/13 were isolated from root nodules of Vicia alpestris plants growing in the North Ossetia region (Caucasus). Sequencing of the partial 16S rRNA gene (rrs) and four housekeeping genes (dnaK, gyrB, recA and rpoB) showed that the isolates from V. alpestris were most closely related to the species Microvirga zambiensis (order Rhizobiales, family Methylobacteriaceae) which was described for the single isolate from root nodule of Listia angolensis growing in Zambia. Sequence similarities between the Microvirga-related isolates and M. zambiensis WSM3693T ranged from 98.5 to 98.7 % for rrs and from 79.7 to 95.8 % for housekeeping genes. Cellular fatty acids of the isolates V5/3MT, V5/5K, V5/5M and V5/13 included important amounts of C18 : 1ω7c (54.0-67.2 %), C16 : 0 (6.0-7.8 %), C19 : 0 cyclo ω8c (3.1-10.2 %), summed feature 2 (comprising one or more of iso-C16 : 1 I, C14 : 0 3-OH and unknown ECL 10.938, 5.8-22.5 %) and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 02-OH, 2.9-4.0 %). DNA-DNA hybridization between the isolate V5/3MT and M. zambiensis WSM3693T revealed DNA-DNA relatedness of 35.3 %. Analysis of morphological and physiological features of the novel isolates demonstrated their unique phenotypic profile in comparison with reference strains from closely related species of the genus Microvirga. On the basis of genotypic and phenotypic analysis, a novel species named Microvirga ossetica sp. nov. is proposed. The type strain is V5/3MT (=LMG 29787T=RCAM 02728T). Three additional strains of the species are V5/5K, V5/5M and V5/13.

Genomes of 184 Sinorhizobium meliloti native isolates were studied to test the occurence of islands Sme21T, Sme19T, and Sme80S previously described in the model strain Rm1021. This analysis was conducted using PCR methodology involving specific primers. It was demonstrated that, in the examined geographically distinct populations of S. meliloti from the Northern Caucasus (NCG) and the Aral Sea region (PAG), the strains containing genomic islands were observed with similar frequency (0.55 and 0.57, respectively). Island Sme80S, denoted as an island of “environmental adaptivity,” was identified predominantly (frequency of 0.38) in genomes of strains which exhibited a lower level of salt tolerance and was isolated in PAG, a modern center of introgressive hybridization of alfalfa subjected to salinity. Island Sme21T designated as “ancestral” was observed in genomes of strains isolated in NCG, the primary center of host-plant biodiversity, 10-fold more often than in strains from PAG. An island Sme19T, which predominantly carries genes encoding transposases, was observed in genomes of strains in both populations with average frequency of 0.10. The analysis of linkage disequilibrium (LD) based on the assessment of probability for detection of different islands combinations in genomes revealed an independent inheritance of islands in salt-sensitive strains of various geographic origin. In contrast, the absence of this trend was noted in the majority of the examined combinations of salt-tolerant strains. It was concluded that the structure of chromosome in PAG strains which predominantly possessed a salt-sensitive phenotype was subjected to active recombinant processes, which could predetermine the intensity of microevolutionary processes in bacterial populations and facilitate an adaptation of bacteria in adverse environmental effect.

Effects of long-term application ofvarious fertilizers and crops on soil microbiomes an a long-term field experiment were investigated using the library of the 16S rRNA gene sequences obtained by high-throughput sequencing of the total DNA. The communities exhibited high diversity, with 655 microbial genera belonging to 34 phyla detected (31 bacterial and 3 archaeal ones). For analysis of the effect of the studied factors on community structure, a linear model was developed in order to simplify interpretation of the data of high-throughput sequencing and to obtain biologically important information. Liming was shown to modulate the effect of mineral fertilizers on the structure of microbial populations. The differences in the structure and alpha-diversity of microbial communities were shown to depend more on the crops and liming, rather than on the fertilizers applied. Interaction between the crop factor and liming expressed as an ambiguous effect of liming on the microbiome in the presence of different plants was reliably demonstrated. Thus, in the case of barley and clover, liming resulted in increased taxonomic diversity of the community, while in the case of potato and flax it had an opposite effect.

The processes of speciation and macroevolution of root nodule bacteria (rhizobia), based on deep rearrangements of their genomes and occurring in the N₂-fixing symbiotic system, are reconstructed. At the first stage of rhizobial evolution, transformation of free-living diazotrophs (related to Rhodopseudomonas) to symbiotic N₂-fixers (Bradyrhizobium) occurred due to the acquisition of the fix gene system, which is responsible for providing nitrogenase with electrons and reducing equivalents, as well as for oxygen-dependent regulation of nitrogenase synthesis in planta, and then of the nod genes responsible for the synthesis of the lipo- chito-oligosaccharide Nod factors, which induce root nodule development. The subsequent rearrangements of bacterial genomes included: (1) increased volume of hereditary information supported by species, genera (pan-genome), and individual strains; (2) transition from the unitary genome to a multicomponent one; and (3) enhanced levels of bacterial genetic plasticity and horizontal gene transfer, resulting in formation of new genera, of which Mesorhizobium, Rhizobium, and Sinorhizobium are the largest, and of over 100 species. Rhizobial evolution caused by development and diversification of the Nod factor synthesizing systems may result in both increased host specificity range (transition of Bradyrhizobium from autotrophic to symbiotrophic carbon metabolism in interaction with a broad spectrum of legumes) and to its contraction (transition of Rhizobium and Sinorhizobium to "altruistic" interaction with legumes of the galegoid clade). Reconstruction of the evolutionary pathway from symbiotic N₂-fixers to their free-living ancestors makes it possible to initiate the studies based on up-to-date genome screening technologies and aimed at the issues of genetic integration of organisms into supracpecies complexes, ratios of the macro- and microevolutionary mechanisms, and developmetn of cooperative adaptations based on altruistic relationship between the symbiotic partners.

Using high throughput sequencing of the nodA gene, we studied the population dynamics of Rhizobium leguminosarum (bv. viciae, bv. trifolii) in rhizospheric and nodular subpopulations associated with the leguminous plants representing different cross-inoculation groups (Vicia sativa, Lathyrus pratensis of the vetch/vetchling/pea group and Trifolium hybridum of the clover group). The "rhizosphere-nodules" transitions result in either an increase or decrease in the frequencies of 10 of the 23 operational taxonomic units (OTUs) (which were identified with 95% similarity) depending on the symbiotic specificity and phylogenetic positions of OTUs. Statistical and bioinformatical analysis of the population structures suggest that the type of natural selection responsible for these changes may be diversifying at the whole-population level and frequency-dependent at the OTU-specific level, ensuring the divergent evolution of rhizobia interacting with different host species.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6% minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

In the present work, the potential for the enhancement of the adaptive capacity of microbe-plant systems (MPSs) through the integration of the symbiosis partners' genomes is considered on the example of different types of symbiotic relationships. The accumulated data on the genetic control of interactions for both the plant and microbe, which are discussed in the paper with respect to signaling genes, suggest that it is the complementarity of genetic determinants that underlies the successful formation of MPSs. A eukaryotic genome with limited information content, which is stable throughout a generation, is complemented by a virtually unlimited prokaryotic metagenome. The microsymbiont's ability to adapt to different living conditions is based on the restructuring of the accessory genome by different mechanisms, which are likely to be activated under the influence of plants, although the details of such a regulation remain unknown. Features of the genetic control of the interaction, particularly its universal character for different symbionts, allow us to formulate a principle of genome-complementarity with respect to interacting organisms and consider it an important factor, an adaptation that enhances the abilities of M PSs for their sustainable development in natural ecosystems and for high plant productivity in agrocenoses.

The Gram-negative, rod-shaped slow-growing strains Vaf-17, Vaf-18(T) and Vaf-43 were isolated from the nodules of Vavilovia formosa plants growing in the hard-to-reach mountainous region of the North Ossetian State Natural Reserve (north Caucasus, Russian Federation). The sequencing of 16S rDNA (rrs), ITS region and five housekeeping genes (atpD, dnaK, recA, gyrB and rpoB) showed that the isolated strains were most closely related to the species Bosea lathyri (class Alphaproteobacteria, family Bradyrhizobiaceae) which was described for isolates from root nodules of Lathyrus latifolius. However the sequence similarity between the isolated strains and the type strain B. lathyri LMG 26379(T) for the ITS region was 90 % and for the housekeeping genes it was ranged from 92 to 95 %. All phylogenetic trees, except for the rrs-dendrogram showed that the isolates from V. formosa formed well-separated clusters within the Bosea group. Differences in phenotypic properties of the B. lathyri type strain and the isolates from V. formosa were studied using the microassay system GENIII MicroPlate BioLog. Whole-cell fatty acid analysis showed that the strains Vaf-17, Vaf-18(T) and Vaf-43 had notable amounts of C16:0 (4.8-6.0 %), C16:0 3-OH (6.4-6.6 %), C16:1 ω5c (8.8-9.0 %), C17:0 cyclo (13.5-13.9 %), C18:1 ω7c (43.4-45.4 %), C19:0 cyclo ω8c (10.5-12.6 %) and Summed Feature (SF) 3 (6.4-8.0 %). The DNA-DNA relatedness between the strains Vaf-18(T) and B. lathyri LMG 26379(T) was 24.0 %. On the basis of genotypic and phenotypic analysis a new species Bosea vaviloviae sp. nov. (type strain RCAM 02129(T) = LMG 28367(T) = Vaf-18(T)) is proposed.

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. foundin the soil population, 12 were present in the root nodulepopulation, 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 nodiles. 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 evolutioin in the course of symbiosis may be associated with development of highly polymorphic virulent:subpopulations subjected to directional selection in the "plant-soil" system.

By using the method which has been developed for isolation and studying of microorganism strains, associative to particular plant species, the strain Pseudomonas fluorescens P10 has been isolated and its features of colonization of cabbage plant organs has been studied. In this study, different colonizing abilities of the strain are shown depending on the site of its isolation during germination and development of plants from inoculated seeds Brassica capitata var. alba Lizg. The ability of the strain to exist in epiphytic and endophytic forms and interaction with cabbage plants has been determined. It has been shown that this method can be used for strain activation during passage through plants.

Sixteen bacterial strains were isolated from root nodules of Vavilovia formosa plants originated from the North Ossetian State Natural Reserve (Caucasus, Russia). Phylogenetic analysis of these strains was performed using partial 16S rRNA gene and internally transcribed spacer (ITS) sequences. The results showed that the isolates belong to three families of root nodule bacteria. Twelve of them were related to the genus Rhizobium (family Rhizobiaceae) but four strains can be most probably identified as Phyllobacterium-related (family Phyllobacteriaceae), Bosea- and Rhodopseudomonas-related (family Bradyrhizobiaceae). Amplified fragment length polymorphism clustering was congruent with ITS phylogeny but displayed more variability for Rhizobium isolates, which formed a single group at the level of 30 % similarity. We expect that the isolates obtained can belong to new taxa at genus, species or subspecies levels. The results of PCR amplification of the nodulation genes nodC and nodX showed their presence in all Rhizobium isolates and one Rhodopseudomonas-related isolate. The nodC gene sequences of V. formosa isolates were closely related to those of the species Rhizobium leguminosarum bv. viciae but formed separate clusters and did not intermingle with any reference strains. The presence of the nodX gene, which is necessary for nodulation of Afghan peas (Pisum sativum L.) originated from the Middle East, allows the speculation that these wild-type pea cultivars may be the closest existing relatives of V. formosa. Thus, the studies of genetic diversity and symbiotic genes of V. formosa microsymbionts provide the primary information about their phylogeny and contribute to the conservation of this relict leguminous species.

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.

Legume plants can obtain combined nitrogen for their growth in an efficient way through symbiosis with specific bacteria. The symbiosis between Rhizobium galegae and its host plant Galega is an interesting case where the plant species G. orientalis and G. officinalis form effective, nitrogen-fixing, symbioses only with the appropriate rhizobial counterpart, R. galegae bv. orientalis and R. galegae bv. officinalis, respectively. The symbiotic properties of nitrogen-fixing rhizobia are well studied, but more information is needed on the properties of the host plants. The Caucasus region in Eurasia has been identified as the gene centre (centre of origin) of G. orientalis, although both G. orientalis and G. officinalis can be found in this region. In this study, the diversity of these two Galega species in Caucasus was investigated to test the hypothesis that in this region G. orientalis is more diverse than G. officinalis. The amplified fragment length polymorphism fingerprinting performed here showed that the populations of G. orientalis and R. galegae bv. orientalis are more diverse than those of G. officinalis and R. galegae bv. officinalis, respectively. These results support the centre of origin status of Caucasus for G. orientalis at a genetic level. Analysis of the symbiosis-related plant genes NORK and Nfr5 reveals remarkable diversity within the Nfr5 sequence, although no evidence of adaptive evolution could be found., (© 2011 Blackwell Publishing Ltd.)