All-Russian Research Institute of Agricultural Microbiology

Arbuscular mycorrhizal fungi (AMF) play a key role in the regenerative successions of plant communities after anthropogenic disturbances, particularly in quarries. AMF help plants with water and mineral nutrition, contributing to the restoration rate of vegetation cover. The research is aimed to study the biodiversity of AMF using molecular genetic methods at different stages of overgrowth of two quarries in the Leningrad region. Molecular genetic identification of fungi was carried out using Illumina MiSeq analysis of the ITS1 and ITS2 regions as barcodes for the identification of operational taxonomic units (OTUs) with species-level identification. An adapted and error-checked AMF genetic sequence database from NCBI was used as a reference. The study applied an optimized nucleic acid isolation technique for sandy soils. The results showed maximum AMF biodiversity at the initial stages of overgrowth – pioneer and grass stages – with minimum diversity observed at the shrub stage, where it decreased by five times. At the forest stage, the biodiversity of AMF was almost restored to the level seen at the grass stage. It has been shown that the biodiversity and species composition of AMF can vary greatly between the stages of regenerative succession and probably depends primarily on the biodiversity of grasses, with which AMF most effectively enter into symbiotic relationships. The analysis showed a reliable negative correlation between the number of AMF species and the number of woody plant species. Such studies can aid in understanding how plant-fungal symbiosis develops in regenerative successions and which AMF most effectively contribute to vegetation cover restoration.

In contrast to Rhizobium johnstonii strain 3841, R. johnstonii strain NaPi is able to form large pink nodules on the roots of pea (Pisum sativum L.) mutants in the gene Sym40. The genetic determinants underlying such efficiency have not been discovered yet. In this study, we report the complete genome sequence of the strain NaPi.

In pea (Pisum sativum L.), alleles of the Sym2 gene determine the specificity of the interaction with nodule bacteria (rhizobia). The Sym2A allele present in landraces from Afghanistan provides higher selectiveness toward rhizobia than the Sym2E allele present in European cultivars. Rhizobial strains possessing the nodX gene can interact with both Sym2A and Sym2E peas, while strains lacking nodX can interact only with Sym2E peas. Here, we studied the previously obtained introgression line A33.18 bearing Sym2A in a homozygous state in the genome of the European pea cultivar ‘Rondo’. A33.18 has proved its high selectiveness in pot experiments. Genome sequencing has shown that A33.18 possesses an 18.2 Mb region inherited from Afghanistan pea with 63 genes, including 5 receptor kinase genes, among which was the Sym2 candidate gene LykX. In a field experiment, under inoculation with the nodX+ strain TOM, over 95% of nodules of A33.18 contained TOM, as opposed to less than 8% of nodules containing TOM in the parental European cultivar ‘Rondo’. Thus, introgression of Sym2A enabled peas to interact specifically with the nodX+ strain, favoring the formation of nodules by the strain from the inoculum and protecting peas from the indigenous soil microbiota.

Introduction Soil microbiome is a major source of physiologically active microorganisms, which can be potentially mobilized by adding various nutrients. To study this process, a long-term experiment was conducted on the decomposition of oat straw and leaf litter using soil as a microbial inoculum. Methods Combined analyses of enzymatic activity and NGS data for 16S rRNA gene amplicon and full metagenome sequencing were applied to study taxonomic, carbohydrate-active enzyme (CAZy), and polysaccharide utilization loci (PULs) composition of microbial communities at different stages of decomposition between substrates. Results In straw degradation, the microbial community demonstrated higher amylase, protease, catalase, and cellulase activities, while peroxidase, invertase, and polyphenol oxidase were more active in leaf litter. Consistent with this, the metagenome analysis showed that the microbiome of straw compost was enriched in genes for metabolic pathways of simpler compounds. At the same time, there were more genes for aromatic compound degradation pathways in leaf litter compost. We identified nine metagenome-assembled genomes (MAGs) as the most promising prokaryotic decomposers due to their abnormally high quantity of PULs for their genome sizes, which were confirmed by 16S rRNA gene amplicon sequencing to constitute the bulk of the community at all stages of substrate degradation. MAGs from Bacteroidota (Chitinophaga and Ohtaekwangia) and Actinomycetota (Streptomyces) were found in both composts, while those from Bacillota (Pristimantibacillus) were specific for leaf litter. The most frequently identified PULs were specialized on xylans and pectins, but not cellulose, suggesting that PUL databases may be underrepresented in clusters for complex substrates. Discussion Our study explores microbial communities from natural ecosystems, such as soil and lignocellulosic waste, which are capable of decomposing lignocellulosic substrates. Using a comprehensive approach with chemical analyses of the substrates, amplicon, and full metagenome sequencing data, we have shown that such communities may be a source of identifying the highly effective decomposing species with novel PULs.

In this study, using the original neural network information technology, it was possible to perform an intelligent analysis of molecular genetic data of silage microflora and evaluate the influence of silage starters and preservatives on the directionality of microbiological biochemical processes in silage biomass. The output product of the intelligent analysis of fractal profiles of silage microflora was the CSIgaZ and CSImU indices, chosen to represent quantitatively, in terms of microbiological data, the intensity of silage outgassing and the amount of lactic and acetic acids in silage. According to the CSImU index it was possible to evaluate the increase of lactic acid and decrease of acetic acid in silage, which signals the slowing down of development of putrefactive microorganisms in silage, and according to the CSIgaZ index - the decrease of silage gas emissions, which indicates favourable anaerobic conditions in which microbiological biochemical processes in silage take place. The laboratory experiment on silage of hedgehog silage conducted in the molecular genetic laboratory of ‘Biotrof’ Ltd. showed that ‘Biotrof-111’ (based on Bacillus subtilis, produced by ‘Biotrof’ Ltd.) creates the best anaerobic conditions in the silaged plant biomass, where the microflora of silage bioconsolidates in the biosystem of lactic acid bacteria. LLC ‘Biotrof’) creates the best anaerobic conditions in the silage plant biomass, in which the silage microflora bioconsolidates into a biosystem of lactic acid bacteria with a minimum value of CSIgaZ index = 3.1 and a maximum value of CSImU index = 6.8, that is, in this variant of silage silage hedgehog silage microflora of silage manages to group in such a way, that simultaneously provides a relatively low intensity of silage gas emissions, accelerated formation of lactic acid and slow formation of acetic acid in silage, and thus in these conditions the processes will be completed with the best quality nutritional indicators of silage.

It is well known that individual pea (Pisum sativum L.) cultivars differ in their symbiotic responsivity. This trait is typically manifested with an increase in seed weights, due to inoculation with rhizobial bacteria and arbuscular mycorrhizal fungi. The aim of this study was to characterize alterations in the root proteome of highly responsive pea genotype k-8274 plants and low responsive genotype k-3358 ones grown in non-sterile soil, which were associated with root colonization with rhizobial bacteria and arbuscular mycorrhizal fungi (in comparison to proteome shifts caused by soil supplementation with mineral nitrogen salts). Our results clearly indicate that supplementation of the soil with mineral nitrogen-containing salts switched the root proteome of both genotypes to assimilation of the available nitrogen, whereas the processes associated with nitrogen fixation were suppressed. Surprisingly, inoculation with rhizobial bacteria had only a minor effect on the root proteomes of both genotypes. The most pronounced response was observed for the highly responsive k-8274 genotype inoculated simultaneously with rhizobial bacteria and arbuscular mycorrhizal fungi. This response involved activation of the proteins related to redox metabolism and suppression of excessive nodule formation. In turn, the low responsive genotype k-3358 demonstrated a pronounced inoculation-induced suppression of protein metabolism and enhanced diverse defense reactions in pea roots under the same soil conditions. The results of the study shed light on the molecular basis of differential symbiotic responsivity in different pea cultivars. The raw data are available in the PRIDE repository under the project accession number PXD058701 and project DOI 10.6019/PXD058701.

Amyloids are protein fibrils with a characteristic cross-β structure that is responsible for the unusual resistance of amyloids to various physical and chemical factors, as well as numerous pathogenic and functional consequences of amyloidogenesis. The greatest diversity of functional amyloids was identified in bacteria. The majority of bacterial amyloids are involved in virulence and pathogenesis either via facilitating formation of biofilms and adaptation of bacteria to colonization of a host organism or through direct regulation of toxicity. Recent studies have shown that, beside their commonly known activity, amyloids may be involved in the spatial regulation of proteome by modulating aggregation of other amyloidogenic proteins with multiple functional or pathological effects. Although the studies on the role of microbiome-produced amyloids in the development of amyloidoses in humans and animals have only been started, it is clear that humans as holobionts contain amyloids encoded not only by the host genome, but also by microorganisms that constitute the microbiome. Amyloids acquired from external sources (e.g., food) can interact with holobiont amyloids and modulate the effects of bacterial and host amyloids, thus adding another level of complexity to the holobiont-associated amyloid network. In this review, we described bacterial amyloids directly or indirectly involved in disease pathogenesis in humans and discussed the significance of bacterial amyloids in the three-component network of holobiont-associated amyloids.

Among the species of the genus Crotalaria L., Crotalaria juncea is the only cover crop cultivated for its fiber. The quality of the latter depends on nutritional conditions, the accumulation of biophilic elements in the biomass and the synthesis of proteinogenic amino acids. Thus, the purpose of the study was a qualitative and quantitative assessment of the green harvest at the stage of active flowering, before the phase of bean formation, when the entire biochemical cycle of the plant is reconfigured, and biochemical indicators are maximum. The plants were grown for 140 days (from April 22 to September 9, 2023) in protected soil conditions. The experimental design consisted of two blocks of 50 plants: in the first (control) plants were grown in a soil mixture without any additives; in the second, the crop was treated three times during the growing season with an organic suspension of humic acids (1000 ppm) obtained from sapropel (Pskov region) – UDGSS, by applying it when watering at the root. The soil substrate in both cases was typical chernozem (Kamennaya Steppe nature reserve, Voronezh region, 51°01ʹ41.6ʹʹN 40°43ʹ39.3ʹʹE) with a 20% addition of volcanic zeolite. Over the course of every 14 days, a systematic record of the dynamics of changes in the morphometric indicators of crop growth (height and dry biomass) was carried out, by removing five cultivars from each block of variants. After 140 days, the remaining five replicates per variant were subjected to biochemical analysis using inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC) using 7500 and LC 1200 instruments (Agilent Technologies, USA), according to the manufacturer's method. Treatment with UDGSS increased productivity and shoot height by 51.1% and 11.3%, respectively. The concentration of macroelements in dry biomass was in the following order: NaKCaSPMg in the control and NaKCaMgPS in the variant with UDGSS, and the export of microelements – FeMnZn BaBCuMo and FeMnBZnBaCuMo. At the same time, in total, the increase was more manifested in the amount of accumulation of microelements, due to a twofold increase in the concentration of iron ions. Among amino acids, an increase in the concentration of L-Lysine, L-Glycine, L-Glutamine and L-tyrosine was observed. The first three are structural components of biological tissues, which indirectly indicates an increase in the fiber strength of the culture.

In this article, we announce the sequence and draft analysis of the genome of endophyte Bacillus amyloliquefaciens strain Apn01R, isolated from the roots of Alhagi pseudalhagi (Bieb.) Fisch. Our results show genes that may be crucial to defy abiotic stress and repel bacterial and fungal pathogens.

We report the whole-genome sequence of the non-rhizobial endosymbiotic bacteria Tardiphaga sp. strain 709, which was isolated from the root nodule of Astragalus inopinatus Borris. on the Kamchatka Peninsula, Russia. The genome consists of one chromosome and one plasmid with a total length of 6,359,564 bp and 61.5% of GC content.

In this study, we sequence, assemble, and analyze the genome of endophyte Bacillus amyloliquefaciens Can02R isolated from the roots of the resurrection plant host, Chenopodium album. The assembly of the strain’s genome amounts to 3,965,760 bp and contains 3,989 coding sequences, among which synthetic antibiotic clusters and multidrug resistance transporters can be found.

One of the major strategies for evolution of bacteria, accompanied by pronounced changes in their genetic organization, is the formation of symbioses with eukaryotes. They provide their micropartners with nutrients and ecological niches, in which bacteria implement trophic or protective functions used by their hosts. Acquisition of the ability for symbiosis is associated with formation of specialized sym gene systems, which is often accompanied by a modification in bacterial genome structure. In nodule bacteria (rhizobia) – N2-fixing symbionts of leguminous plants, most of which belong to α-proteobacteria of order Hyphomicrobiales, the symbiogenic changes in genomes vary depending on the taxonomic position. In the evolutionarily primary rhizobia of family Bradyrhizobiaceae, which emerged directly from free-living N2-fixers, transition to symbiosis is accompanied by a significant (1.5–2 times) increase in the genome size. However, their genomes retain a unitary structure: in the majority of Bradyrhizobium strains, more than 95% of genes are located in chromosomes. In the secondary rhizobia of family Phyllobacteriaceae (Mesorhizobium, Phyllobacterium), which emerged by transfer of sym genes into soil bacteria, various stages were revealed for formation of multipartite genome harboring a significant part of genes in extrachromosomal elements (ECE) – plasmids and chromids. The most pronounced multicomponent genome structure is found in the Rhizobiaceae family (Rhizobium, Sinorhizobium, Neorhizobium), in which the total size of ECEs containing sym genes can exceed the size of chromosome. In these bacteria, transfer from tropical to temperate ecosystems was accompanied by narrowing the host specificity, however, modification of genome structure was revealed only in Sinorhizobium, in which the proportion of ECE increases up to 51% of genome. Such genomes were also revealed in associative (rhizospheric, endophytic) N2-fixers of genus Azospirillum, in which ECE comprise up to 60% of a genome. The irreversibly differentiated N2-fixing cellular forms, bacteroids formed by rhizobia are included into the temporary organelles, symbiosomes considered as precursors of N2 -fixing organelles which are revealed in some protists and are probably may be constructed for the crop plants.

The review is devoted to the application of quantitative trait loci (QTL) analysis to study the interactions of common pea (Pisum sativum L.), one of the most important grain legumes, with soil microorganisms. Pea, like other legumes, forms symbioses with nodule bacteria and arbuscular mycorrhiza fungi. The formation of symbioses leads to improved nitrogen and phosphorus nutrition of plants, resulting in increased plant resistance to abiotic and biotic stress factors, in particular, to phytopathogens. The main objective of QTL analysis is to identify genomic regions whose allelic state affects the manifestation of quantitative traits, including such traits as nitrogen fixation efficiency and pathogen resistance. The identified QTLs and molecular markers created on their basis can be used in the selection of new pea varieties with improved agronomic characteristics, such as resistance to changing environmental conditions and high efficiency of symbiotic systems. This article reviews the historical stages of the emergence of QTL analysis, the basic principles of QTL mapping, and modern approaches. The need for an integrated approach to the analysis of the characteristics of symbiosis efficiency and stability is noted, and the use of integrated phenotypic assessments for working with such traits is discussed.

Insecticides are used commonly in agricultural production to defend plants, including legumes, from insect pests. It is a known fact that insecticides can have a harmful effect on the legume–rhizobial symbiosis. In this study, the effects of systemic seed treatment insecticide Imidor Pro (imidacloprid) and foliar insecticide Faskord (alpha-cypermethrin) on the structural organization of pea (Pisum sativum L.) nodules and their transcriptomic activity were investigated. The plants were treated as recommended by the manufacturer (10 mg/mL for Imidor Pro and 50 µg/mL for Faskord) and twofold concentrations were used for both insecticides. Insecticides had no visible effect on the growth of pea plants. The nodules also showed no visible changes, except for the variant treated with twofold concentration of Imidor Pro. However, the dry weight of shoots and roots differed significantly in insecticide-treated plants compared to untreated plants in almost all treatments. The number of nodules decreased in variants with Imidor Pro treatment. At the ultrastructural level, both insecticides caused cell wall deformation, poly-β-hydroxybutyrate accumulation in bacteroids, expansion of the peribacteroid space in symbiosomes, and inclusions in vacuoles. Treatment with Faskord caused chromatin condensation in nucleus. Imidor Pro treatment caused hypertrophy of infection droplets by increasing the amount of matrix, as confirmed by immunofluorescence analysis of extensins. Transcriptome analysis revealed upregulation of expression of a number of extensin-like protein-coding genes in nodules after the Imidor Pro treatment. Overall, both insecticides caused some minor changes in the legume–rhizobial system when used at recommended doses, but Faskord, an enteric contact insecticide, has fewer negative effects on symbiotic nodules and legume plants; of these two insecticides, it is preferred in pea agricultural production.

Tobacco BY-2 cell culture is one of the most widely used models in plant biology. The main advantage of BY-2 suspension cultures is the synchronization of cell development and the appearance of polar elongation. In batch culture, BY-2 cells passed through the lag, proliferation, elongation, and stationary phases. During this process, the composition of the growth medium changed dramatically. Sucrose was rapidly eliminated; hexose first accumulated and then depleted. The medium’s pH initially decreased and then rose with aging. As a result of the crosstalk between the internal and external stimuli, cells pass through complicated systemic rearrangements, which cause metabolomic alterations. The early stages were characterized by high levels of amino acids and sterols, which could be interpreted as the result of synthetic activity. The most intense rearrangements occurred between the proliferation and active elongation stages, including repression of amino acid accumulation and up-regulation of sugar metabolism. Later stages were distinguished by higher levels of secondary metabolites, which may be a non-specific response to deteriorating conditions. Senescence was followed by some increase in fatty acids and sterols as well as amino acids, and probably led to self-destructive processes. A correlation analysis revealed relationships between metabolites’ covariation, their biochemical ratio, and the growth phase.

Background. According to modern concepts, the SWEET family may be the only family of plant sugar transporters that includes genes specifically expressed during the formation and development of plant symbiosis with fungi of arbuscular mycorrhiza (AM). The data on the key genetic markers of the development of effective AM symbiosis can contribute an active development of organic agriculture in various conditions of phosphorus (P) availability in the soil. Materials and methods. The study was performed using a highly efficient plant-microbial system “Medicago lupulina + Rhizophagus irregularis”. Results. The aim was to evaluate the effect of AM on the expression of SWEET genes in M. lupulina during key stages of host plant development (stages of leaves rosette, stooling initiation, stooling, lateral branching initiation, lateral branching and flowering). Under condition of high P level in the substrate it was shown for the first time the MlSWEET1b and MlSWEET3c genes were characterized by specific expression during mycorrhization in M. lupulina leaves. Conclusion. These genes and their orthologs can be considered as marker genes of effective symbiosis development, as a tool of biotechnology to increase agricultural productivity with using biostimulants based on AM fungi.

The aim of this research was to study the effect of plant-growth-promoting bacteria (PGPB) isolated from the drought-tolerant plants camel thorn (Alhagi pseudoalhagi (M.Bieb.) Fisch) and white pigweed (Chenopodium album L.) on wheat (Triticum aestivum L.) plants cv. Lenigradskaya 6, growing under hydroponic conditions and osmotic stress (generated by 12% polyethylene glycol-6000 (PEG)). Based on the assumption that plants create a unique microbiome that helps them overcome various stresses, we hypothesized that bacteria isolated from drought-tolerant plants may assist cultivated wheat plants in coping with drought stress. PGPB were isolated from seeds and leaves of plants and identified as Bacillus spp. (strains Cap 07D, Cap 09D, and App 11D); Paenibacillus sp. (Cap 286); and Arthrobacter sp. (Cap 03D). All bacteria produced different phytohormones such as indole acetic acid (IAA), abscisic acid (ABA), and gibberellic acid (GAS3) and were capable of stimulating wheat growth under normal and osmotic stress conditions. All PGPB reduced the malondialdehyde (MDA) content, increased the total chlorophyll content by increasing chlorophyll a, and modulated wheat hormone homeostasis and CAT and POX activities under osmotic conditions. Selected strains can be promising candidates for the mitigating of the drought stress of wheat plants.