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Project titleAnalysis of genetic and evolutionary potential of the soil microbiome to improve the plant productivity and the soil fertility

AffiliationFederal State Budget Scientific Institution All-Russia Research Institute for Agricultural Microbiology,

KeywordsSoil, rhizosphere, microbiome, evolution, adaptation, gene, genome, metagenome, soil-microbe interactions

Annotation
Soil’s microbial communities (microbiomes) are crucial to its fertility as they determine the productivity of all components of the biocenosis, especially plants. Soil microbiome is the first-hand source of endophytic microflora, which determines plant nutrition, resistance to pathogens and abiotic stresses. New symbiotic microorganisms, evolving through the coevolution of plants and microflora, effectively cope with the aforementioned tasks and are included in various microbial preparations, widely used in the adaptive agriculture. The genetic potential of a soil microbiome has been long understood by means of its taxonomic and functional diversity, acquired through experiments. In this project, we propose to look at the issue of biodiversity from a wider perspective and explore its genetic potential as a capacity of variability, primarily comprising the causes, mechanisms and guidelines of the adaptive diversification of a microbial community. This approach will not only let to understand the evolutionary nature of metagenomic diversity, but will allow to route it in a desired direction, which opens tremendous opportunities for soil monitoring and control over fertility, and to improve the productivity and the environmental safety of agriculture. Modern technologies of molecular genetic analysis of microbiomes, based on the high-throughput sequencing methods and advanced computer bioinformatic analysis, make for such projects, the object of which is one of the most complex living natural objects - the soil microbiome - enormous in both volume and diversity. While developing the idea of this project and its general research methodology, we assumed that the main guiding factor of evolutionary processes in the microbiome of soil were plants. In the rhizosphere, under the influence of various exudates and signals produced by a plant, soil microbiome undergoes the restructuring directed towards the optimum conditions for the plant to grow. It is in the rhizosphere - as in a powerful "evolutionary reactor" - where symbiotically active forms of microorganism, which interact with a plant from the level of a relatively non-specific association of microorganisms to the highly specialized intracellular symbiosis that is the nitrogen-fixing nodule, evolve. The intensity of mutation and recombination, as well as the symbiosis-specific forms of selection, dramatically increases in the rhizosphere and endophytic communities. This guides the evolution of the soil microbiome to improve the environmental efficiency of plant-targeted interactions. According to this logic, we will develop a project, wherein the influence of a plant will be analyzed from the «distant interactions» in soil, representing the influence of unspecific environmental effects, and in the rhizosphere, where the interactions of soil microorganisms and plants - though not much specific - are nevertheless clearly expressed, and finally in the internal environment of plants, where the coevolutionary interactions are the strongest and, in fact, reveal a host controlled«construction" of microsymbionts. In each of the three niches a study on the mechanisms of variability of soil microbiome will be performed at three levels of organization of genetic material - gene, demonstrating the evolution of groups of genes involved in the adaptive capacity of microorganisms to plants; genome - demonstrating the evolution of genomes from specialized forms to symbiotically specialized; and finally metagenomic describing the overall structure of adaptive changes microbiome. We will study the mechanisms of variability of soil microbiome at three different organization levels of genetic information - genetic, genomic and metagenomic - within each of the three niches. The first level demonstrates the evolution of groups of genes, underlying the adaptive potential of microorganisms in respect of a plant. The second one represents the evolution of individual genomes from unspecific forms to those of well-specialized symbionts. Finally, the metagenomic level describes adaptive variations of the microbiome as a whole. Our main biosystem models, listed below, are the of one of a kind gems of astonishing interest: 1. The stationary 100-year experiment in Timiryazev Agricultural Academy, comprised of more than 200 plots, that have been under the same agricultural practices (e.g. fertilization at the background of growing several major crops) for a century; 2. A representative set of the major soil types of Russia (40 samples), collected in various regions of the country in compliance with the standards of soil science. These samples will be used to analyse the widest variations in the soil microbiome’s structure due to the diversity of their soil type and regional origin, which form the background for the evolutionary interactions of plants and the microbiome of soil; 3. Unique expeditionary material of a relict legume species Vavilovia formosa, collected in 2013, which was the first exemplar extracted with nodules, allowing the extraction of unexpectedly wide range of probable symbionts, representing virtually all possible evolutionary stages of symbiosis, from the alleged ancestors of rhizobia to modern deep specialized forms; 4. Expedition material collected during 2012-2013 within nonarable fields of Ternopil and Leningrad regions, where a rare sort of experiment, aimed to create a pool of nodules and rhizosphere soil samples from several plant species, took place. A study of the collection, will allow to analyze the divergent evolution of key symbiotic genes of rhizobia in unprecedented detail; Our research methods, listed below, will provide the most technologically sophisticated approaches in both data mining and further analysis: 1. The high-throughput sequencing, which will be used to analyze the structure of soil metagenomes and to acquire 6 complete bacterial genomes; 2. Modern chromatography–mass spectrometry methods, which will provide a detailed transcript for the chemical structures of the plant root exudates, that guide the evolutionary processes in the microbiome of soil; 3. The most modern bioinformatics methods of genome and metagenome analysis, the analysis of evolutionary diversification of the symbiosis-significant genes, which will be confirmed by means of molecular modeling and docking of the corresponding gene products. The project will provide a comprehensive description of the mechanisms of the plant influence on genetic diversity of soil microorganisms in three niches, characterized by varying degrees of distance from the plant (i.e. soil, rhizosphere and plant tissue), demonstrating the corresponding extent of evolutionary influence - from vanishing to strong - at three levels of organization of genetic material in the soil microbiome - genetic, genomic and metagenomic. The aforesaid creates a ground for a large-scale research project on the genetic potential of the microbiome of Russian soil. The project will allow to: A. give a detailed description of environmental-genetic potential of major soil types, as well as to describe the main trends and reveal the mechanisms of evolution of the microbiome, which will make for new effective methods of monitoring and increasing the productivity of agricultural soils ; B. examine the impact of plants on the evolutionary diversification of soil microbiome and use the results to develop a methodology of designing highly productive, yet environmentally-friendly, agrophytocenosis, based on the adaptive interaction potential of crop plants with beneficial soil microflora.

Expected results
The main results to be obtained in the course of this project are the products of integration of the latest technologies in genetic, chemical and bioinformatic analysis of complex biological systems in fundamental agricultural science. This project has no analogues in the world, not only in agriculture but also in biological science due to its scaled and integrated approaches and a range of unique experimental models. For the first time in a single project the detailed analysis of the mechanisms of diversification of soil microbiome controlled by plants will be performed in various soil and plant-associated habitats (soil-rhizosphere-plant tissue), and in three hierarchical levels of genetic material organization (genes, genomes metagenome). Each of the experimental models by itself may serve as the basis for further interesting projects: 100 year-old TAA experiment; collection of typical Russian soils; relict legume vavilovia and its extremely interesting symbionts; collection of joined samples for studying the diversification of soil microorganisms under plant control. This subjects when joined in the frame of a single project would help to reconstruct the global evolutionary processes occurring in soil microbiomes under plant government, beginning the soil monitoring approaches as well as designing of agronomically valuable plant-microbe systems. The gross yield of the project is going to be fruitful: the taxonomic structure of about 300 soil microbiomes will be discovered as well as 6 bacterial genomes will be completely sequenced using high-throughput sequencing; applying of gas chromato-mass spectrometry will allow the detailed analysis of the root exudates of 4 agricultural plants species (2 lines of each) as well as the investigation of their impact on soil microorganisms; using the example of a bacterial genes cluster governing the adaptation of bacteria to plant the large-scale picture of the soil microbiome evolutionary variation will be given. Finally, all the results will be analyzed by the mathematical model of the plant-driven soil microbiome evolutionary dynamics. This project will provide the results of both fundamental and applyed value, involving the development of a new high-tech methodology to design environment-friendly high-productive agrophytocenosises based on the use of the adaptive potential of interaction between crop plants and beneficial soil microflora. The socially important results of the project will be: the preparation of the qualyfying papers by laboratory staff (1 doctoral thesis, 1 PhD thesis, 3 masters theses).

Annotation of the results obtained in 2016
All the research performed in the 2016 is very closely connected to the work from the previous years of our project. It was aimed on the research of evolutionary processes in the soil microbiome on the three levels of its organization – genetic, genomic and metagenomics, and in the three niches – soil, rhizosphere and endosphere, which demonstrate progressive increasing plant influence on soil microbial community and the speed of its evolution. The project is aimed on the research of Russian soil microbiomes, one of the results is going to be a creation of specialized databases, allowing services lacking in current available databases. One of the essential problems is that it is impossible to search among Next Generation Sequencing libraries of 16s rRNA gene the one which is most similar to the user library (similarly to BLAST in GeneBank). There are several reasons for this: (i) different primers were used for the amplicon libraries construction; (ii) necessity to constantly align arrays in case of adding new data or searching while the OTU-picking algorithm library analysis is used, which requires high resource capacity for this type of calculations. That’s why future operations with databases will require creation of principally new search algorithms, one of which was created as a result of working on this stage of the project. Within the framework of the project study on regularities of rhizobia genome evolution was continued, and for this reason a new collection of more than 100 R.leguminosarum bv. trifolii strains, extracted from the clover nodules from three distinct regions (Ternopolskaya region, Leningradskaya region and northern Karelia) was made. For the analysis of this collection, universal primers on symbiotic genes of clover rhizobia were constructed, including primers on nodA, nodX and nodD, which were used for the screening of acquired collection, during this screening amplified fragments of this genes were sequenced for all of the strains. Maximum polymorphism of all genes (nucleotide diversity, pi) was detected in the population from Leningradskaya region, which is characteristic of its relatively low diversity of host-plant species (Trifolium pratense, T. repens, T. hybridum) and limited by a fertility of sod-podzolic soil. Minimum polymorphism was characteristic of the population from Ukraine Ternopol region, where a wide range of plant-hosts (more than 20 clover species) grow on a rich chernozem soil. The population from the north Karelia, growing in a poor forest soil with a lack of naturally growing plant-hosts (local populations of T. pratense and T. repens can be found only as ruderal species alongside buildings and roads), is characteristic by intermediate polymorphism levels of nod-genes. 9 strains were selected to perform a full genome sequencing. At this moment the first stage of analysis is completed, which consists of contigs assembly and an annotation. According to the data obtained, genomes from Ternopol demonstrate more compact structure of symbiotic genes than those from Leningrad adea. Moreover, intergenic spaces between symbiotic genes are smaller in Ternopol strains than in Leningrad strains. For the research on dynamics of rhizobia symbiotic genes nucleotide polymorphism controlled by a plant-host, the expedition to Pskov region was held, where at the two sites, 25 km apart, plants of Melilotus alba and Medicago lupulina, 30 pieces of each at each site, were collected. Apart from plant material at both sites and for both species nodules collection (100 pieces for each) and rhizosphere soil collection were made. Biological material was combined into pools (plant, nodule and soil), 6 pools for every site (3 for each plant species), 12 pools total. For the analysis of plant diversity gene coding for symbiotic receptor Nfr5 was cloned and sequenced (30 clones for each species at each site, in total of 120 nucleotide sequences). Analysis of plant receptor diversity demonstrated unusually low genetic polymorphism for both plant species, which in turn affects rhizobia (Sinorhizobium meliloti, S. medicae) diversity. For the analysis of rhizobia diversity in nodule and soil pools, degenerate nested-primers for nodABCDE genes were constructed. As a result of deep sequencing, 32 libraries were obtained (4 genes nodABCDE for two sites, for two plant species and two pools – soil and nodule), with average output at least 1500 nucleotide sequences for each library. To date nodABC gene libraries are already analyzed. Trend, which was described earlier, is confirmed (higher plant diversity leads to higher rhizobia diversity), although two cases (nodA and nodB genes from Medicago isolates) demonstrate the opposite. Our findings will be used for the verification of “evolutionary moulding” hypothesis, which states that main selection factor, which drives the evolution of symbiotic systems, is inconsistency between diversity levels of genes coding for signal-receptor complex components in plants and bacteria. In the progress of the project, legume receptor and its docking with Nod-factor were simulated. Special task of this stage was to try mapping detected amino acid polymorphism on receptor surface. As a model for this work 300 nucleotide sequences of Nfr5 gene (100 sequences for each of 3 plants) and 100 nucleotide sequences of k1 gene for Vicia were selected. Modelling of Nfr5 protein 3D structures was performed on SWISS-MODEL server for all Vicia haplotypes. As a homolog template for modelling process crystallized with ligands CERK1 was used, pdb: 4EBZ. Based on obtained models, mapping of population-related polymorphism on receptor surface was attempted for the first time. With the help of Shrödinger package we analyzed possible binding sites of receptor and ligand for all the Nfr5 structures of Vicia plant. Site clusterisation showed that more preferable binding positions are grooves between LysM domains. However, LysM domains have chitin-binding function, which is also noted in known crystals. Important result is the observation of docking into Groove23 zone between LysM2 and LysM3 domains. This zone demonstrates big inter-domain area and high concentration of population polymorphism inside Nfr5 gene. Besides, this zone has two tunnels on the protein surface. Two most potential bindings appear to fall into these tunnels with fatty acid chains. Besides, there are a lot of polymorphism around these tunnels, and almost no polymorphism around binding sites with fatty acid ends. Thus, in this work represents a first attempt to map population-related polymorphism in plant receptor genes and connect it to Nod-factor docking. Next step of the project was dedicated to the evolution and adaptation of rhizobial fix-cluster. To achieve this, DNA from soil, rhizosphere, rhizoplane, and nodules of legume plant Vicia was extracted by standard methods. All samples were amplified using universal primers for fix-genes and sequenced on GS Junior sequencer. After that sequencing results were processed by cluster analysis on the basis of OUT-picking results, major representatives cluster analysis and its taxonomical attribution. For quantitative analysis real-time PCR method with universal primers was used. During the analysis of obtained results two patterns were detected: (1) with consequential transfer from soil to a plant (soil – rhizosphere – rhizoplane – endosphere) microbial community becomes more saturated with fix-genes, reaching its maximum in the endosphere of legume plant (nodules); (2) this process correlates with decreasing of fix-genes taxonomical diversity, reaching its minimum of almost one taxon in nodules. Interpretation of these trends leads us to the conclusion that fix-genes have significant function during plant associated niche colonization, which increases the more niche is close to a plant. The question about the meaning of fix-genes in the process of rhizobia adaptation to soil niches requires further research. To clarify the biochemical mechanisms of rhizosphere effect, the compositions of rye and wheat root exudates was analyzed, and later model experiment was conducted to study impact of separate root exudates components and their mixtures and native exudates on soil microbial community. Major components of sugars, organic acids and amino acids were selected on the basis of rye and wheat exudate spectra and added to the soil samples. After 5 days of exposition, DNA was extracted from the samples and analyzed with Next Generation sequencing of 16s rRNA gene libraries. According to the results of cluster analysis (principal component analysis), communities formed in the soils with added sugars and amino acids had almost no difference between each other. Most significant result is achieved by adding organic acids, which is consistent with the previous results demonstrating their crucial role in the forming of useful microflora of plant root zone.

Publications

1. Andronov E.E., Ivanova E.A., Pershina E.V., Orlova O.V., Kruglov Y.V., Belimov A.A., Tikhonovich I.A. Анализ показателей почвенного микробиома в процессах, связанных с почвообразованием, трансформацией органического вещества и тонкой регуляцией вегетационных процессов. Бюллетень Почвенного института им. В.В. Докучаева., № 80. С. 83-94. (year - 2015)

2. Chirak E.R., Kopat V.V., Kimeklis A.K., Safronova V.I., Belimov A.A., Chirak E.L., Tupikin A.E., Andronov E.E., Provorov N.A. Структурно-функциональная организация плазмидных регионов симбиотических генов у Rhizobium leguminosarum. Микробиология, Т. 85. № 6. С. 693-702. (year - 2016) https://doi.org/10.7868/S0026365616060070

3. Ivanova E.A., Pershina E.V., Kutovaya O.V., Andronov E.E. Сравнительный анализ микробных сообществ контрастных почвенных типов в условиях различных фитоценозов. Экология, - (year - 2017)

4. Pershina E., Valkonen J., Kurki P., Ivanova E., Chirak E., Korvigo I., Provorov N., Andronov E. Comparative analysis of prokaryotic communities associated with organic and conventional farming systems PLoS ONE, 10(12): e0145072 (year - 2015) https://doi.org/10.1371/journal.pone.0145072

5. Provorov N.A. Мережковский и происхождение эукариотической клетки: 111 лет теории симбиогенеза. Сельскохоз. биология, Т. 51, № 5, с. 746-758 (year - 2016) https://doi.org/10.15389/agrobiology.2016.5.746rus

6. Provorov N.A., Onishchuk O.P., Kurchak O.N. Габитус и продуктивность люцерны (Medicago sativa L.) в зависимости от инокуляции штаммами Sinorhizobium meliloti, различающимися по солеустойчивости. Сельскохоз. биология, Т. 51, № 3, с. 343-350 (year - 2016) https://doi.org/10.15389/agrobiology.2016.3.343rus

7. Safronova V., Belimov A., Andronov E., Popova J., Tikhomirova N.Y., Orlova O., Verkhozina A., Chimitov D., Tikhonovich I. Method for obtaining root nodules of the Baikal relict legumes in laboratory pot experiments. International Journal of Environmental Studies., - (year - 2017)

8. Safronova V., Belimov A., Sazanova A., Kuznetsova I., Popova J., Andronov E., Verkhozina A., Tikhonovich I. Does the Miocene-Pliocene relict legume Oxytropis triphylla form nitrogen-fixing nodules with a combination of bacterial strains? International Journal of Environmental Studies., - (year - 2017)

9. Safronova V., Kuznetsova I., Sazanova A., Belimov A., Andronov E., Chirak E., Osledkin Y., Onishchuk O., Kurchak O., Shaposhnikov A., Willems A., Tikhonovich I. Microvirga ossetica sp. nov. a new species of rhizobia isolated from root nodules of the legume species Vicia alpestris Steven originated from North Ossetia region in the Caucasus. Intern. J. System. Evol. Microbiol., - (year - 2017)

10. Shaposhnikov A.I., Morgunov A.I., Akin B., Makarova N.M., Belimov A.A., Tikhonovich I.A. Сравнительные характеристики корневых систем и корневой экссудации у синтетического, примитивного и современного сортов пшеницы. Сельскохоз. биология, Т. 51, № 1, с. 68-78 (year - 2016) https://doi.org/10.15389/agrobiology.2016.1.68rus

11. Skopina M.Y., Vasilieva A.A., Pershina E.V., Pinevich A.V. Разнообразие малочисленности: феномен разреженной бактериальной биосферы. Микробиология, Т. 85. № 3. С. 248-260 (year - 2016)

12. Sviridova O.V., Vorobyov N.I., Provorov N.A., Orlova O.V., Rusakova I.V. Andronov E.E., Pischik V.N., Popov A.A., Kruglov Y.V. Выравнивание почвенных условий для развития растений при деструкции растительных остатков микробными препаратами. Сельскохоз. биология, Т. 51, № 5, с. 664-672. (year - 2016) https://doi.org/10.15389/agrobiology.2016.5.664rus

13. Zverev A.O., Pershina E.V., Provorov N.A., Andronov E.E., Serikova E.N. Метагеномная характеристика ризосферного эффекта при выращивании злаковых культур в черноземной и дерново-подзолистой почве. Сельскохозяйственная биология, Т. 51, № 5, с. 654-663 (year - 2016) https://doi.org/10.15389/agrobiology.2016.5.654rus

14. Kruglov Y.V. Микробное сообщество почвы: физиологическое разнообразие и методы исследования (обзор). Сельскохоз. биология, Т. 51, №1, с. 46-59 (year - 2016) https://doi.org/10.15389/agrobiology.2016.1.46rus

15. Pischik V.N., Vorobyov N.I., Provorov N.A., Khomyakov Y.V. Механизмы адаптации растений и микроорганизмов в микробно-растительных системах к тяжелым металлам. Микробиология, Т. 85, № 3, С. 231-247 (year - 2016)

16. Provorov N.A., Andronov E.E. Эволюция клубеньковых бактерий: реконструкция процессов видообразования, обусловленных перестройками генома в системе симбиоза. Микробиология, Т. 85. № 2. С. 195-206 (year - 2016) https://doi.org/10.7868/S0026365616020166

17. Provorov N.A., Tikhonovich I.A. (eds.). Генетические основы эволюции бактерий – симбионтов растений Информ-Навигатор, С.-Петербург, 240 с. (year - 2016)

Annotation of the results obtained in 2014
The fundamental problem addressed by the project, involves a comprehensive analysis of phylogenetic patterns and of ecological and genetic mechanisms of evolution of soil microbial communities, which is influenced by two groups of factors that are determined by physical and chemical properties of the soil, and by biological characteristics of plants growing on it. There-fore, the evolutionary process involves studying in microbiome, are different in strength to the plant niches (soil – rhizosphere – plant tissues and organs), analyzed at three hierarchical levels of the organization of microbial hereditary material (genes – genomes – metagenomes). The first stage of the work was the creation of a representative collection of Russian soils and metagenomic analysis of microbial communities inhabiting them under the influence of edaphic factors associated with the particular type of soil and with the native plant communities. Out of the 67 soil sample, DNA was isolated on the basis of which we created the amplicon li-braries of the taxonomically significant 16S rRNA gene of bacteria and archaea using the univer-sal primers (high throughput sequencing was performed using the device GS Junior). Grouping of the obtained sequences into Operational Taxonomic Unites (OTUs) was per-formed according to the 97% identity level. Taxonomic identification of sequences was conduct-ed using a database RDPII, for the statistical evaluation of the results of the cluster analysis method was used jackknife. The correlation of the soil microbiome with agrochemical parameters of soil is determined through a statistical Mantel test (total analysis included 271673 sequences). Taxonomic analysis revealed representatives of 42 bacterial and 2 archaeal phyla among which representatives of 10 phyla were dominated including 9 bacterial: Actinobacteria (33,5%), Proteobacteria (28,4%), Acidobacteria (8,3%), Verrucomicrobia (7,7 %), Bacteroidetes (4,2%), Chloroflexi (3,0%), Gemmatimonadetes (2,3%), Firmicutes (2,1%), Planctomycetes (2,0%) and one archaeal - Crenarchaeota (2,6%). When analyzing the correlation of soil microbiome with agrochemical parameters of soil, significant correlation values were obtained for the values of soil pH, potassium and phosphorus, as well as for the total salt content. Differences in taxonomic structure of microbiome was confirmed by the cluster analysis: separation of clusters is mainly due to the type of soil. It turned out that the type of plant community plays, though subordinate, but still a signifi-cant role in the formation of a specific metagenomic pattern: soil microbiome employed herba-ceous vegetation, characterized by a generally more high diversity than phytocenoses microbial communities with domination of tree and shrub vegetation. In the next stage of the work was carried out the metagenomic analysis of soil samples from the 102-year-old soil experiment at the Moscow Agricultural Academy, in which factors of vary-ing composition of the soil microbiome were mineral (NPK) and organic (manure) fertilizers, liming the soil, its mechanical tillage, as well as growing crops (rye, potatoes, barley, clover, flax). During the taxonomic analysis we found more than 37000 OTUs. Analysis revealed microbiome substantially linear negative dependence between the amount of OTUs in soil samples and the number of samples in which this OTU occurs. Common to all 132 samples were only 2 OTU identified as non-classified species from genus Kaistobacter (Proteobacteria, Alphaproteo-bacteria, Sphingomonadales, Sphingomonadaceae) and family Bradyrhizobiaceae (Proteobacte-ria, Alphaproteobacteria, Rhizobiales). Analysis of the data showed that the composition of the microbial community depends primarily on factors of farming that affect the physical and chem-ical properties of the soil, which is consistent with the dominant influence of soil type on the composition of its microbiome. Plants have a statistically significant but a limited effect on the microbiota, the indirectly influenced by the factors of farming. The system which allows us to address the plant as a dominant factor in evolution of associ-ated microbial community was established on the basis of the relic legume Vavilovia formosa, which, unlike its sister members of the tribe Fabeae (Pisum, Vicia, Lathyrus, Lens) is not re-stricted to symbiosis with only an evolutionarily advanced and specialized symbiotic strains of Rhizobium leguminosarum bv. viciae, but also with representatives of the family Bradyrhizobi-aceae (Rhodopseudomonas, Bosea, Tardiphaga), including the non-epacialized form of symbiotic nodule bacteria (Bradyrhizobium), close to the ancestral soil diazotrophes. Full genome se-quencing 8 taxonomically diverse strains of V. formosa nodules confirmed their taxonomic di-versity and showed that many slow-growing symbionts are different from the typical symbionts from legume tribe Fabeae availability and allelic status of key symbiotic genes, including the nod-genes for synthesis of lipo-oligosaccharide Nod-factors. These findings are consistent with the hypothesis that the main direction of the evolution of legume-Rhizobium symbiosis was the restriction specificity of the interaction partners, which can be seen when comparing not only the representatives of different tribes and subfamilies, but also from the same legume tribe (Fabeae). In next year we shall reconstruct the molecular mechanisms of evolution of symbiotic specificity, which may be associated with an improve in its environmental performance. The research tasks have been associated with the study of the evolution of microbial genes controlling interaction with plants in a symbiotic specialized and limited taxonomic groups nod-ule bacteria (Rhizobium, Sinorhizobium), especially in the vetch/pea cross-inoculation groups in which the plants are related taxonomically to the previously studied Vavilovia (belong to the tribe Fabeae). For this purpose were used nodule and rhizospheric populations of rhizobia isolat-ed from the Vicia and Lathurus from the Leningrad region for the preparation of amplicon librar-ies were constructed nested-primers for each of the common or species-specific nod-genes (nodABCD, nodEF). In the next years of the project we shall analyze these libraries in order to study the effect of host plant on the evolution of symbiotic (sym) genes associated with it micro-bial population, including the determination of the type and direction of natural selection, in-duced during the circulation of the microbial population in the "plant-soil" systems. The task of the final step of the work was to create software that allows to study the features of the evolution of nucleotide sequences in the protein-coding sym gene based dN/dS statistics and to identify the driving/stabilizing selection, as well as the specificity of the identified geno-types in relation to different types of plants. To solve this problem was created by the software open source in a high level language for technical computing - MATLAB. The generated soft-ware provides several indices of diversity, revealing different evolutionary significant population characteristics: (1) Pi - diversity, based on a simple count of the weighted number of mismatches of nucleotide sequences; (2) the function Pi_Haplo.m, which calculates the amount and enrich-ment (representation) of haplotypes forming the sample under consideration; (3) Shannon index, which takes into account both the total abundance of genotypes and evenness of their distribution in frequency; (4) Simpson diversity index, which shows how the population is evenness in genotypic composition. The created software allows the calculation of dN/dS statistics in two ways: based on phylogenetic information and without it. Calculation of dN/dS statistics without phylogenetic information is based on pairwise comparison of all sequences, taking into account the established model of nucleotide substitutions. Accelerated algorithm for calculating the dN/dS statistics, built for our type of sampling was carried out as a function of dndsNod.m. The parameters of the function takes as input a sequence of indices codons matrix amounts of non-synonymous substitutions and synonymous between two codons and vector quantities of synonymous and non-synonymous mutations. As additional parameters you can use the mode of sliding window, accounting Grantham matrix and the assumption that all the sequences in the sample came from the most widespread haplotype. Matrix is a parameter calculation function dN/dS statistics are calculated separately (function dndsPrepare.m) depending on which model of nucleotide substitutions researcher wants to apply. In future work, created software will be used for the analysis of sequenced libraries nod-gene, with the aim to reveal the ratio of driving and stabilizing selection affecting the different domains of the sym-genes depending on the origin of the amplicon libraries (soil, nodules) or on the plant species, which hosted the microbial populations.

Publications

1. Andronov E., Onishchuk O., Kurchak O., Provorov N. Изменение популяционной структуры ризобий клевера (Rhizobium leguminosarum bv. trifolii) при переходе из почвы в клубеньковую нишу Микробиология, T. 83. № 4. С. 500-508 (year - 2014) https://doi.org/10.7868/S0026365614030033

2. Kruglov Yu.V., Lisina T.O. Bacillus megaterium 501rif introduced into soil: factors affecting the rate of survival, sporulation and decomposition of the herbicide prometryn. Selskokhozyaistvennaya Biologiya - Agricultural Biology (Сельскохозяйственная биология), № 5, p. 107-112 (year - 2014) https://doi.org/10.15389/agrobiology.2014.5.107eng

3. Kurchak O.N., Provorov N.A., Onishchuk O.P., Vorobyov N.I., Roumiantseva M.L., Simarov B.V. Influence of salt stress on the genetically polymorphic system of Sinorhizobium meliloti – Medicago truncatula Russian Journal of Genetics, V. 50, N 7, p. 677-685. (year - 2014) https://doi.org/10.1134/S1022795414060064

4. Белимов А., Додд Ш., Сафронова В., Шапошников А. Abscisic acid metabolizing rhizobacteria decrease ABA concentrations in planta and alter plant growth Plant Physiol Biochem., V. 74, N 1, p. 84-91 (year - 2014)

5. Сафронова В., Кимеклис А., Чижевская Е., Белимов А., Тихонович И. Genetic diversity of rhizobia isolated from nodules of the relic species Vavilovia formosa (Stev.) Fed. Antonie van Leeuwenhoek, V. 105, N 3, p. 389-399 (year - 2014) https://doi.org/10.1007/s10482-013-0089-9

6. Provorov N., Shtark O. Направленная эволюция грибов и растений в системах симбиоза Микология и фитопатология, Т. 48. Вып. 3. С. 151-160 (year - 2014)

7. Provorov N., Tikhonovich I. Эволюция бактериального генома в надвидовых системах: на пути к реконструкции процессов симбиогенеза Генетика, - (year - 2015)

8. Provorov N., Tikhonovich I. Генетические и молекулярные основы симбиотических адаптаций Успехи совр. биологии, Т. 134. № 3. C. 211-226 (year - 2014)

9. Provorov N., Tikhonovich I., Vorobyov N. Симбиогенез и синтетическая теория эволюции: третий синтез Генетика, - (year - 2014)

10. Provorov N.A., Onishchuk O.P., Yurgel S.N., Kurchak O.N., Chizhevskaya E.P., Vorobyov N.I., Zatovskaya T.V., Simarov B.V. Конструирование высокоэффективных симбиотических штаммов бактерий: эволюционные модели и генетические подходы. Генетика, Т. 50. № 11. С. 1273-1285 (year - 2014) https://doi.org/10.7868/S0016675814110113

11. Provorov N.A., Vorobyov N.I. Эволюция полезных для растений признаков у азотфиксирующих бактерий: моделирование и конструирование систем межвидового альтруизма Прикладная биохимия и микробиология, - (year - 2015)

Annotation of the results obtained in 2015
The overall objective of this project is to study the evolutionary and adaptive po-tential of soil microbial communities in three niches - soil, rhizosphere and inside plant tissues and cells at three levels of the organization of the genetic material of bacteria - the gene, genomic and metagenomic. The works of the second year of the project is a logical continuation of the first year, which have focused on the most general regulari-ties characterizing the formation of the soil microbiome - the influence of soil type and nature of the plant community. In the course of this work we created and investigated the collection of different types of soil samples from different regions under different plant communities. Apart from this collection, we investigated soil samples taken in a century-long stationary experiment at Timiryazev Agricultural Academy. In all these ex-periments, it was shown that the plant is a powerful factor in the formation of the taxo-nomic structure of the soil microbiota. During the second year of the project studies the impact of the plant was investigated at a more precision level rhizosphere community. We performed the pot experiments using two contrasting soil types that have the great-est importance to agriculture of Russia - Chernozem and sod-podzolic soil, 0.5 tons of which was delivered to ARRIAM from Voronezh and Pskov regions. For this experiment we obtained from the collection of VIR the seeds of winter wheat and rye (6 varieties of each), which were grown in each type of soil. During the 42-day experiment, which was finalized at the stage of stem elongation were selected 104 samples of rhizosphere soil and control (each variant in 4 replications). In the course of this experiment we estimated a range of parameters including the taxonomic structure of the rhizosphere microbiome (high-throughout sequencing libraries of 16S rRNA gene), the genotype of the plant (genomic AFLP-fingerprinting), the elemental composition of the rhizosphere soil (17 items emission spectroscopy with inductively coupled plasma) and rhizosphere metabolome structure reflecting the secretion activity of the roots (mass spectrometry). The data allowed us to characterize the processes of succession of soil microbial communities in their transition from soil into rhizosphere, which is influenced by the plant-determined eco-chemical conditions of the adaptive zone and can be described at the level of individual taxa and the overall structure of microbiome, estimated using different diversity indices. It is shown that in the rhizosphere increased frequency of genus Dyadobacter, a typical representative of that initially identified from the surface of cereal (maize), as well as the appearance of Pedobacter, Chitinophaga, Caulobacter, Rhizobium and Luteolibacter, which is not found in the sod-podzolic soil in the absence of plants. Applications developed by the authors earlier in statistical and bioinformatics approach will allow us to characterize the effect of plants on soil formation as a microbiome plant species (rye, wheat) and certain varieties, some of which exhibit similar effects even in contrasting soil types. We also developed the fractal mathematical model, the main object for which are variations in the number of groups of microbes involved in decomposition of complex organic soil components. It allows us to analyze the phenomena of succession of soil microbes, and in the future – we shall pass from the description of the taxonomic structure microbiome to dissecting its functional structure. Analysis of the impact of plants on endosymbiotic microbial communities, at the level of genomes of individual organisms we studied the symbionts of relic legume Vavilovia formosa, which is endemic to the Caucasus and, apparently, is the nearest kin of common ancestor for the tribe Fabaea. We were able to take the first GTR-plants Vavilovia with nodules, from which we isolated from an extremely wide range microsymbionts presented not only strains R. leguminosarum bv. viciae (symbionts re-lated to the vetch isolates), but also bacteria, close to the evolutionary ancestors of rhi-zobia (Rhodopseudomonas) - Bosea, Tardiphaga, Microvirga, Phyllobacterium. This project attempts to describe the key stages in the evolution of rhizobia, the patterns of which can be obtained in the analysis of complete genomes of these organisms. To this end, in 2015 the second phase was completed genome sequencing in microsymbionts of Vavilovia - paired-sequencing, designed to facilitate the assembly of complete genomes. Since in 2015 an entirely new device PacBio RSII was appeared, which can completely replace the time-consuming and represents a very significant difficulties in many respects paired-sequencing procedure, we used this opportunity, which brought the expected results - the five genomes assembled into contigs 4 the size of which corresponds approximately to an entire chromosome (5 Mb). Comparison of these results with paired-sequencing shows the correctness of our choice. The very first results of the analysis regions sequenced genomes containing symbiotic genes revealed that the genome of strain Tardiphaga Vaf07 present cluster homologues symbiotic genes rhizobia fixNOPQ (encode cytochrome with a high oxygen affinity) and its duplication with a high degree of divergence (30 % of the sequence). Phylogenetic analysis showed that the ancestral copy of the fix-gene has an obvious kinship with the genes from closest relatives of Tardiphaga - Rhodopseudomonas, and the second copy of the fix-cluster shows a high similarity with chromosomal copies of this cluster are close to the evolutionarily advanced forms of Rhizobium and Sinorhizobium. Thus, it is probable that in the evolutionary history of rhizobia we are dealing with a classical processes of duplication-divergence (paralogization) and horizontal gene transfer, which provided the adaptation of bacteria to microaerophilic niches in nodules and soil (the latter is very important in the migraation of rhizobia from the tropics to temperate latitudes, characterized by complexly structured soils). The second evolutionary evidence for the later stages of the formation of the sym-biotic genome that we have traced a typical symbionts Vavilovia isolate R. leguminosarum bv. viciae Vaf12, in a symbiotic gene cluster which revealed nodX, characteristic of the Afghan pea rhizobia. Analysis of the data allows us to assume, that one of the basic regularities of the evolution of rhizobial genome is to increase the compactness of the localization of the sym genes (nif-, nod- and fix-regulons) located on symbiotic plasmids that provides an increased mobility of these genes in populations and intensify the evolution of the whole symbiotic system. In the third part of the project we studied the effect of legumes on nucleotide poly-morphism of rhizobia nodABCD genes encoding for the key signaling molecule - Nod-factor. Deep sequencing of libraries obtained from soil and nodule rhizobia pools isolated from three legumes (vetch, lentil, clover) demonstrated clearly the impact of the plant on the evolution of individual genes of rhizobia - all nodule libraries showed the significantly higher levels of nucleotide polymorphism than soil libraries. The role of plants as "evolutionary drivers” of rhizobia was confirmed in a study of dN/dS statistics demonstrating the ratio of nonsynonymous and synonymous mutations in the nod-genes. In this study it was shown that in general the selection of a symbiotic gene is of the stabilizing nature, but some (and perhaps the most evolutionary prospective) codons are influenced by the directed selection, both types of selection are more pronounced in nodule libraries compared with the soil ones. Since the evolution of rhizobia depends on plants, the evolution of the legume host in this project we analysed vetch, lentil and clover screened for 100 alleles of symbiotic receptor gene NFR5, which product interacts directly with the rhizobial lipo-chito-oligosaccharide Nod-factor. Analysis of nucleotide polymorphism of the receptor gene has shown that it is directly proportional to gene polymorphism symbiotic rhizobia, which indicates the close co-evolutionary communication systems interacting gene plants and microsymbionts. For more detailed study of the physicochemical nature of this connection we started computer molecular modeling of plant receptors and their interaction with Nod-factor (molecular docking). It has been shown that receptors NFR5 from vetch and lentil interact with the model Nod-factor of R. leguminosarum bv. viciae, and which is particularly interesting, with its associated chito-oligosaccharide part, but not with the unsaturated fatty acid residue. This is consistent with the hypothesis that Nod-factor interacts with the dimeric receptor complex NFR5 + K3, and second component interacts with the fatty-acid residues. So, during this phase of the project we identified a broad range of evolutionarily interactions of plants and micro-organisms at different levels of the organization of genetic material and in different niches, from variations in the taxonomic composition of microbial communities in the rhizosphere associations to modify the structure of genomes and control nucleotide polymorphism in specific genes in intracellular symbiosis. There is no doubt that the understanding of these mechanisms may serve in the future for a number of practice-oriented research, ranging from plant breeding and micro-organisms to complex solutions to ecologically friendly agriculture. In addition, the results determine the continuation of research initiated outlined in the plan for 2016. In 2016 studies will be conducted, which will create a complete picture of the evo-lution of the soil microbial community, taking place under the direct influence of host plants and gives them retaliatory co-evolutionary processes. The data will allow us for the first time to establish a relationship between the processes of adaptive evolution (succession) in soil microbiome (changes its taxonomic and functional structures influ-enced by eco-chemical factors of root zone) and the progressive evolution of symbiotically active components of microbiome, registered at the level of the overall structure of the genome, and of the sequences of individual genes (the second part of the work will be presented in the form of monograph). The immediate prospect of this research is to characterize the evolution of microbial-plant continuum, which has an integral system of heredity, which can serve as an object of bioengineering research, aimed at creating a sustainable farming systems and crop production.

Publications

1. Andronov E., Igolkina F., Kimemlis A., Vorobyov N., Provorov N. Характеристика естественного отбора в популяциях клубеньковых бактерий (Rhizobium leguminosarum), взаимодействующих с различными видами растений-хозяев Генетика, Т. 51, № 10, с. 1108–1116 (year - 2015) https://doi.org/10.7868/S0016675815100021

2. Ivanova E., Kutovaya J., Thakahava A., Cherbov T., Persgina T., Andronov E., Kogut B. Структура микробного сообщества агрегатов чернозема типичного в условиях контрастных вариантов сельскохозяйственного использования Почвоведение, № 11, с. 1367–1382 (year - 2015) https://doi.org/10.7868/S0032180X15110088

3. Kimeklis A., Safronova V., Kuznetsova I., Sazanova A., Belimov A., Pinaev A., Chizhevskaya E., Puhaev A., Popov K., Andronov E., Provorov N. Филогенетический анализ штаммов рода Rhizobium, выделенных из клубеньков Vavilovia formosa (Stev.) Fed. Сельскохозяйственная биология, № 5. С. 655-664 (year - 2015) https://doi.org/10.15389/agrobiology.2015.3.655rus

4. Kovrigo I., Pershina E., Ivanova E., Matuk N., Chirak E., Provorov N., Andronov E. Оценка длительного воздействия агротехнических приемов и сельскохозяйственных культур на почвенные микробные сообщества Микробиология, Т. 85, № 2 (year - 2016)

5. Onishchuk O., Kurchak O., Chizhevskaya E., Provorov N., Simarov B. Популяционный полиморфизм клубеньковых бактерий люцерны (Sinorhizobium meliloti) по генам симбиотической эффективности и конкурентоспособности Сельскохозяйственная биология, № 3. С. 339-344 (year - 2015) https://doi.org/10.15389/agrobiology.2015.3.339rus

6. Pershina E., Ivanova E., Nagieva A., Chirak E., Andronov E., Sergaliev N. Сравнительный анализ микробиомов природных и антропогенно-нарушенных почв Северо-Западного Казахстана Почвоведение, №4 (year - 2016)

7. Vorobyov N., Provorov N. Quorum Sensing и нодуляционная конкурентоспособность ризобий при инфицировании бобовых растений. Сельскохозяйственная биология, № 3. С. 298-304 (year - 2015) https://doi.org/10.15389/agrobiology.2015.3.298rus

8. Белимов А.А., Додд В., Сафронова В.И., Шапошников А.И., Азарова Т.С., Макарова Н.М., Дэвис В., Тихонович И.А. Rhizobacteria that produce auxins and contain 1-amino-cyclopropane-1-carboxylic acid deaminase decrease amino acid concentrations in the rhizosphere and improve growth and yield of well-watered and water-limited potato (Solanum tuberosum). Annals of Applied Biology, Annals of Applied Biology (year - 2015) https://doi.org/10.1111/aab.12203

9. Сафронова В.И., Кузнецова И.Г., Сазанова А.И., Кимеклис А.К., Белимов А.А., Андронов Е.Е., Тихонович И.А. Extra slow-growing Tardiphaga strains isolated from nodules of Vavilovia formosa (Stev.) Fed. Archives of Microbiology, V. 197, N 7, P. 889-898 (year - 2015) https://doi.org/10.1007/s10482-015-0383-9

10. Сафронова В.И., Кузнецова И.Г., Сазанова Ф.И., Кимеклис А.К., Белимов А.А., Андронов Е.Е., Пинаев А.Г., Чижевская Е.П., Тихонович И.А. Bosea vaviloviae sp. nov. a new species of slow-growing rhizobia isolated from nodules of the relict species Vavilovia Formosa (Stev.) Fed. Antonie van Leeuwenhoek, V. 107. N 2. P. 911–920. (year - 2015) https://doi.org/10.1007/s00203-015-1122-3

11. Provorov N., Tikhonovich I., Vorobyov N. Симбиогенез как модель для реконструкции ранних этапов эволюции генома Генетика, Т. 52. № 2 (year - 2016)

12. Provorov N.A. Адаптивная макроэволюция бобово-ризобиального симбиоза. Сельскохозяйственная биология, № 3. С. 323-331 (year - 2015) https://doi.org/10.15389/agrobiology.2015.3.298rus