Rhizobium Galegae Research Articles

Polymorphism of Core and Symbiotically Specialized Genes in the Polytypic Species of Nodule Bacteria

The polytypic species of nodule bacteria Rhizobium leguminosarum (includes biovars viciae and trifolii) and Neorhizobium galegae (biovars orientalis and officinalis) differ in the nucleotide polymorphism of housekeeping genes (hkg) and of symbiotically specialized genes (sym) that control the formation of N2-fixing nodules in leguminous plants. In R. leguminosarum, p-distance values for sym genes are higher than for hkg genes in strains from the same and from different biovars. In N. galegae, differences between biovars in sym genes are higher than in hkg genes while within biovars, polymorphism in sym genes is lower than in hkg genes. Coefficients of biovar differentiation for both groups of genes are higher in N. galegae than in R. leguminosarum, possibly reflecting the spatial isolation of N. galegae biovars. In these species of rhizobia, the phylogenetic congruence of sym and hkg genes is more pronounced in N. galegae than in R. leguminosarum. This difference indicates an active transfer of sym genes in R. leguminosarum populations possibly representing an important factor of the deep diversification for symbiotic traits in this rhizobia species.

Impacts of Natural Selection on Evolution of Core and Symbiotically Specialized (sym) Genes in the Polytypic Species Neorhizobium galegae

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 (N2-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 (P0 < 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 < P0 < 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.

Occurrence of the Ability to Suppress the Growth of Related Strains in Rhizobia

Abstract—Screening of 1019 strains of rhizobia Rhizobium leguminosarum and Neorhizobium galegae from the “Symbiont” collection of symbiotic rhizospheric microorganisms of the Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences, which have been isolated from root nodules of 20 leguminous species, for the ability to suppress the growth of related strains of rhizobia was carried out. Inhibition of the growth of competing bacteria by rhizobia was shown to be rather common. On average, when grown on rich media, ~10% of rhizobial strains released antibacterial substances into the medium. At the same time, when a live culture of rhizobia was applied to the lawn of bacteria, the percentage of suppression was higher. The percentage of strains synthesizing antibacterial substances correlated also, to some extent, with the level of genetic diversity of the population of legume microsymbionts. A weak signal of impaired protein synthesis was found in the study of the mechanism of action of metabolites of 20 strains of rhizobia R. leguminosarum bv. viciae from root nodules of Lathyrus palustris and Lathyrus vernus. In other cases, the nature of the antibacterial action of bacterial metabolites could not be determined using the DualRep2 system.

The Nexus between Fire and Soil Bacterial Diversity in the African Miombo Woodlands of Niassa Special Reserve, Mozambique.

(1) Background: the Miombo woodlands comprise the most important vegetation from southern Africa and are dominated by tree legumes with an ecology highly driven by fires. Here, we report on the characterization of bacterial communities from the rhizosphere of Brachystegia boehmii in different soil types from areas subjected to different regimes. (2) Methods: bacterial communities were identified through Illumina MiSeq sequencing (16S rRNA). Vigna unguiculata was used as a trap to capture nitrogen-fixing bacteria and culture-dependent methods in selective media were used to isolate plant growth promoting bacteria (PGPB). PGP traits were analysed and molecular taxonomy of the purified isolates was performed. (3) Results: Bacterial communities in the Miombo rhizosphere are highly diverse and driven by soil type and fire regime. Independent of the soil or fire regime, the functional diversity was high, and the different consortia maintained the general functions. A diverse pool of diazotrophs was isolated, and included symbiotic (e.g., Mesorhizobium sp., Neorhizobium galegae, Rhizobium sp., and Ensifer adhaerens), and non-symbiotic (e.g., Agrobacterium sp., Burkholderia sp., Cohnella sp., Microvirga sp., Pseudomonas sp., and Stenotrophomonas sp.) bacteria. Several isolates presented cumulative PGP traits. (4) Conclusions: Although the dynamics of bacterial communities from the Miombo rhizosphere is driven by fire, the maintenance of high levels of diversity and functions remain unchanged, constituting a source of promising bacteria in terms of plant-beneficial activities such as mobilization and acquisition of nutrients, mitigation of abiotic stress, and modulation of plant hormone levels.

The Biology of Canadian Weeds: 158.Galega officinalisL.

The biology of the weed goat’s-rue, Galega officinalis (Fabaceae), is reviewed. Introduced to Canada in the late 19th Century as an ornamental, it has become established at scattered localities and is spreading locally in southern Ontario. The plant is considered a noxious weed and is legally regulated in many jurisdictions due to the production of toxic alkaloids and its invasive characteristics. Primarily a weed of pastures, grasslands and perennial crops, it also displaces native vegetation in areas where it becomes established. Originally endemic in the Black Sea region, it was spread by humans through Europe and eastward to Pakistan as a herbal medicine. More recently it has been introduced widely as a forage and ornamental plant. As a folk remedy it has been used primarily to treat diabetes in humans and to enhance milk production in both humans and livestock. The plant has also been used as a forage by limiting consumption to early growth stages and the quantity ingested. Effective control has been achieved with 2,4-D, dicamba, tryclopyr, metsulfuron methyl and other herbicides. Goat’s-rue forms a highly specific nitrogen-fixing symbiosis with the soil bacterium Neorhizobium galegae symbiovar officinalis. Successful establishment of Goat’s rue in new regions depends on the co-introduction of plant and bacterium. The lack of long-distance dispersal adaptations, soil pH requirements and its symbiont dependency, reduces the ability of G. officinalis to spread into novel areas without anthropogenic activities. These constraints to establishment may facilitate management and eradication strategies.

Creation of an inducible vector system based on the rhizobia nodA gene promoter

BACKGROUND: The possibility of changing the properties of rhizobial bacteria by giving them the ability to regulate the expression of additionally introduced genes into them is an urgent task both for fundamental science and for applied agrobiology, since this will make it possible to obtain microsymbionts with desired properties. An expression construct using the rhizobia regulatory system was created in this work. The rhizobia nodD gene encodes a regulatory protein that, in the presence of plant inducers, flavonoids, activates the transcription of nod-genes involved in the early stages of the formation of legume-rhizobium symbiosis.
 MATERIALS AND METHODS: A vector construct containing the nodD gene from Rhizobium leguminosarum bv. trifoli under the regulation of its own promoter and the gfp gene under the regulation of the nodA gene promoter from the same rhizobia was obtained. Neorhizobium galegae CIAM 0702 were transformed with the vector construct.
 RESULTS: It has been shown that in recombinant strains synthetic flavonoids are capable of inducing expression of gfp gene to varying degrees.
 CONCLUSION: In the future, the results can be used to obtain rhizosphere microorganisms with a controlled synthesis of growth-stimulating and protective substances.

Post-reclamation microbial diversity and functions in hexachlorocyclohexane (HCH) contaminated soil in relation to spontaneous HCH tolerant vegetation

The toxicity, volatility and persistence of the obsolete organochlorine pesticide hexachlorocyclohexane (HCH), makes reclamation of contaminated areas a priority for the health and welfare of neighboring human communities. Microbial diversity and functions and their relation to spontaneous vegetation in post-excavation situations, are essential indicators to consider in bioaugmentation or microbe-assisted phytoremediation strategies at field scale. Our study aimed to evaluate the effects of long-term HCH contamination on soil and plant-associated microbial communities, and whether contaminated soil has the potential to act as a bacterial inoculum in post-excavation bioremediation strategies. To scrutinize the role of vegetation, the potential nitrogen fixation of free-living and symbiotic diazotrophs of the legume Lotus tenuis was assessed as a measure of nutrient cycling functions in soil under HCH contamination. Potential nitrogen fixation was generally not affected by HCH, with the exception of lower nifH gene counts in excavated contaminated rhizospheres, most probably a short-term HCH effect on early bacterial succession in this compartment. HCH shaped microbial communities in long-term contaminated bulk soil, where we identified possible HCH tolerants such as Sphingomonas and Altererythrobacter. In L. tenuis rhizosphere, microbial community composition was additionally influenced by plant growth stage. Sphingobium and Massilia were the bacterial genera characteristic for HCH contaminated rhizospheres. Long-term HCH contamination negatively affected L. tenuis growth and development. However, root-associated bacterial community composition was driven solely by plant age, with negligible HCH effect. Results showed that L. tenuis acquired possible HCH tolerant bacteria such as the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade, Sphingomonas, Massilia or Pantoea which could simultaneously offer plant growth promoting (PGP) benefits for the host. Finally, we identified an inoculum with possibly HCH tolerant, PGP bacteria transferred from the contaminated bulk soil to L. tenuis roots through the rhizosphere compartment, consisting of Mesorhizobium loti, Neorhizobium galegae, Novosphingobium lindaniclasticum, Pantoea agglomerans and Lysobacter bugurensis.

Genome Analysis of Endobacterium cerealis, a Novel Genus and Species Isolated from Zea mays Roots in North Spain.

In the present work, we analyse the genomic and phenotypic characteristics of a strain named RZME27T isolated from roots of a Zea mays plant grown in Spain. The phylogenetic analyses of 16S rRNA gene and whole genome sequences showed that the strain RZME27T clustered with the type strains of Neorhizobium galegae and Pseudorhizobium pelagicum from the family Rhizobiaceae. This family encompasses several genera establishing symbiosis with legumes, but the genes involved in nodulation and nitrogen fixation are absent in its genome. Nevertheless, genes related to plant colonization, such as those involved in motility, chemotaxis, quorum sensing, exopolysaccharide biosynthesis and hydrolytic enzymes production were found. The comparative pangenomic analyses showed that 78 protein clusters present in the strain RZME27T were not found in the type strains of its closest relatives N. galegae and P. pelagicum. The calculated average nucleotide identity (ANI) values between the strain RZME27T and the type strains of N. galegae and P. pelagicum were 75.61% and 75.1%, respectively, similar or lower than those found for other genera from family Rhizobiaceae. Several phenotypic differences were also found, highlighting the absence of the fatty acid C19:0 cyclo ω8c and propionate assimilation. These results support the definition of a novel genus and species named Endobacterium cerealis gen. nov. sp. nov. whose type strain is RZME27T.

Evolutionary Geography of Root Nodule Bacteria: Speciation Directed by the Host Plants

Compared to free-living relatives, bacterial symbionts of plants exhibit higher rates of population diversification (microevolution), as well as of speciation and formation of super-species taxa (macroevolution). Analysis of rhizobia, the N2-fixing symbionts of leguminous plants, suggests that both evolutionary modes are determined by the hosts, which have mutagenic and recombinogenic effects on their microsymbionts, apart from activating the selective processes that determine the fate of newly emerged genotypes in their populations. Migration induced by leguminous plants plays a key role in the evolution of rhizobia, including: (1) joint movement of the symbionts and their hosts into new ecological zones and (2) local circulation of the symbionts in plant‒soil systems. In the first case, allopatric speciation occurs in rhizobia due to transfer of the symbiotically specialized (sym) genes from the introduced strains into local bacteria, which are transformed into novel symbionts. In the second case, a sympatric divergence of Rhizobium populations occurs, resulting in formation of intraspecies taxa (biovars and symbiovars) contrasting in their host ranges. In the evolutionarily advanced rhizobia, Rhizobium leguminosarum and Neorhizobium galegae interacting with galegoid legumes (tribes Fabeae, Galegae, and Trifolieae), these processes are associated with diversification of the sym genes representing the accessory genome parts due to the interaction between bacteria and the hosts differing in their symbiotic affinities. In parallel, formation of new cryptic (genomic) species was revealed in local rhizobia populations, which is associated with divergence of the core genome parts. It may be caused either by genetic drift or by selection for resistance against local edaphic stresses, which in combination with disruptive selection can sufficiently enhance the rhizobia diversity and change the structures of their pangenomes.

Rhizobium deserti sp. Nov Isolated from Biological Soil Crusts Collected at Mu Us Sandy Land, China.

A novel gram-negative, aerobic, non-spore-forming, rod-shaped, and non-nitrogen-fixing bacterium, named SPY-1T, was isolated from biological soil crusts collected at Mu Us Sandy Land, China. Based on 16S rRNA sequence similarity, strain SPY-1T was most closely related to Neorhizobium alkalisoli CCTCC AB 2014138T (98.7%), Neorhizobium huautlense CGMCC 1.2538T (98.6%), Neorhizobium galegae DSM 11542T (98.4%), Rhizobium wenxiniae 166T (97.9%), and Rhizobium smilacinae CCTCC AB 2013016T (97.5%). Phylogenetic analysis based on 16S rRNA sequencing and multilocus sequence analysis of partial sequences of atpD-glnII-glnA-recA-ropD-thrC housekeeping genes both indicated that strain SPY-1T was a member of the genus Rhizobium. The draft genome of strain SPY-1T was 4.75Mb in size, and the G + C content was 60.0%. The average nucleotide identity(ANI) values to N. alkalisoli CCTCC AB 2014138T and R. smilacinae CCTCC AB 2013016T were both 84.0%. The digital DNA-DNA hybridization (dDDH) values to N. alkalisoli CCTCC AB 2014138T and R. smilacinae CCTCC AB 2013016T were 20.9% and 20.2%, respectively. The major cellular fatty acids were summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C16:0. Based on the data from chemotaxonomic, phylogenetic, and phenotypic evidence, strain SPY-1T represents a novel species in the genus Rhizobium, for which the name Rhizobium deserti sp. nov. is proposed. The type strain is SPY-1T (= ACCC 61627T = JCM 33732T).

Invasive Galega officinalis (Goat's rue) plants in Canada form a symbiotic association with strains of Neorhizobium galegae sv. officinalis originating from the Old World.

The toxic legume plant, Galega officinalis, is native to the Eastern Mediterranean and Black Sea regions. This legume is considered to be a noxious weed, and its establishment in Canada may have resulted from ornamental planting and/or field trials. In its native range, a highly specific nitrogen‐fixing symbiosis with the bacterium, Neorhizobium galegae symbiovar (sv.) officinalis, is required for normal growth. In North America, nothing is known about the bacterial symbionts of G. officinalis. Our purpose was to determine the species and symbiovar identity of symbiotic bacteria associated with invasive plants of G. officinalis at five sites in the province of Ontario, Canada. Sequence analysis of four housekeeping (16S rRNA, atpD, glnII, and recA) and two symbiosis (nodC and nifH) genes showed that all 50 bacterial isolates from root nodules of G. officinalis at the five Canadian sites were identical to strains of N. galegae sv. officinalis originating either from Europe or the Caucasus. Plant tests indicated that soils collected from four Canadian sites without a history of agriculture or presence of G. officinalis were deficient in symbiotic bacteria capable of eliciting nodules on this plant. Collectively our data support the hypothesis of anthropogenic co‐introduction of G. officinalis and its specific symbiotic bacterium into Canada from the Old World. Factors that may limit the spread of G. officinalis in new environments are discussed.

Evolution of Goat’s Rue Rhizobia (Neorhizobium galegae): Analysis of Polymorphism of the Nitrogen Fixation and Nodule Formation Genes

The goat’s rue rhizobia (Neorhizobium galegae) represent a convenient model to study the evolution and speciation of symbiotic bacteria. This rhizobial species is composed of two biovars (bv. orientalis and bv. officinalis), which form N2-fixing nodules with certain species of goat’s rue (Galega orientalis and G. officinalis). The cross-inoculation between them results in the formation of nodules unable to fix nitrogen. On the basis of the data on the whole-genome sequencing, we studied the nucleotide polymorphism of 11 N. galegae strains isolated from the North Caucasus ecosystems, where G. orientalis has higher diversity than G. officinalis. The low level of differences in the polymorphism within the group of the sym genes in comparison with the nonsymbiotic genes can be associated with the active participation of host plants in the evolution of rhizobia. The intragenic polymorphism of bv. orientalis proved to be significantly higher than that of bv. officinalis. The level of polymorphism of nonsymbiotic genes was lower than that of the symbiotic genes, which are functionally more homogeneous. The divergence of the nitrogen fixation genes (nif/fix) is more pronounced than that of the nodule formation genes (nod) in the N. galegae biovars. These facts indicate the leading role of the host-specific nitrogen fixation in the evolution of the studied rizhobial species.

Neorhizobium tomejilense sp. nov., first non-symbiotic Neorhizobium species isolated from a dryland agricultural soil in southern Spain

We describe for the first time a non-symbiotic species of the recently described genus Neorhizobium, lacking nodulation or nitrogen fixation genes. The strains were isolated from a dryland agricultural soil in southern Spain where no record of legume cultivation is available, thus we propose the name Neorhizobium tomejilense sp. nov. (type strain T17_20T, LMG 30623T and CECT 9621T). N. tomejilense exhibit a clear distinct lineage from the other Neorhizobium species, Neorhizobium galegae, Neorhizobium alkalisoli and Neorhizobium huautlense, based on polyphasic evidence. Phylogenetic marker analysis of 16S rDNA, atpD, glnII, recA, rpoB and thrC genes and genomic identity data derived from the draft genomic sequences showed that N. tomejilense strains clearly separated from the other Neorhizobium species and that N. galegae represents the closest species, with Average Nucleotide Identities (ANIb) ranging from 90% (for type strain HAMBI 540T) to just under 95.0% (for two N. galegae sv. officinalis strains). Genomes from N. galegae and N. tomejilense, however, clearly differed in important traits, such as the number of rRNA operon copies or the number of tRNAs. Phenotypic characterisation of N. tomejilense also displayed differences with the other Neorhizobium species. Whole-cell matrix-assisted laser-desorption time-of-flight mass spectrometry (WC MALDI-TOF-MS) fingerprint analysis and the dendrogram derived from the fingerprint profiles, showed a clearly distinct group formed by the three N. tomejilense isolates (T17_20T, T20_22 and T11_12) from the other Neorhizobium especies.

Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX–PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

Microbiomes associated with infective stages of root-knot and lesion nematodes in soil.

Endoparasitic root-knot (Meloidogyne spp.) and lesion (Pratylenchus spp.) nematodes cause considerable damage in agriculture. Before they invade roots to complete their life cycle, soil microbes can attach to their cuticle or surface coat and antagonize the nematode directly or by induction of host plant defenses. We investigated whether the nematode-associated microbiome in soil differs between infective stages of Meloidogyne incognita and Pratylenchus penetrans, and whether it is affected by variation in the composition of microbial communities among soils. Nematodes were incubated in suspensions of five organically and two integrated horticultural production soils, recovered by sieving and analyzed for attached bacteria and fungi after washing off loosely adhering microbes. Significant effects of the soil type and nematode species on nematode-associated fungi and bacteria were revealed as analyzed by community profiling using denaturing gradient gel electrophoresis. Attached microbes represented a small specific subset of the soil microbiome. Two organic soils had very similar bacterial and fungal community profiles, but one of them was strongly suppressive towards root-knot nematodes. They were selected for deep amplicon sequencing of bacterial 16S rRNA genes and fungal ITS. Significant differences among the microbiomes associated with the two species in both soils suggested specific surface epitopes. Among the 28 detected bacterial classes, Betaproteobacteria, Bacilli and Actinobacteria were the most abundant. The most frequently detected fungal genera were Malassezia, Aspergillus and Cladosporium. Attached microbiomes did not statistically differ between these two soils. However, Malassezia globosa and four fungal species of the family Plectosphaerellaceae, and the bacterium Neorhizobium galegae were strongly enriched on M. incognita in the suppressive soil. In conclusion, the highly specific attachment of microbes to infective stages of phytonematodes in soil suggested an ecological role of this association and might be involved in soil suppressiveness towards them.

THE FEATURES OF INTERACTION RHIZOBIUM GALEGAE WITH GALEGA ORIENTALIS

The strain of Rhizobium galegae К- 3 - active symbiote of Galega orientalis has been received by means of selection. Positive influence of this strain on formation and functioning of symbiosis during the first and second years of cultivation of Galega orientalis plants is shown. R. galegae К- 3 promotes to formation of generous amount of indeterminate nodules on the roots, increase of activity of symbiotic nitrogen fixation and increase in the yield of Galega orientalis.

THE SCIENTIFIC BASIS FOR THE CREATION OF NEW FORMS OF MICROBIAL BIOCHEMICALS

Herein we summarize the results of a comprehensive study aimed on the creation of liquid form of biological products for symbiotic and associative rhizobacteria. The objects of study were nodule bacteria from the rhizosphere of galegae (Rhizobium galegae), soybean (Bradyrhizobium japonicum) and associative rhizobacteria (Arthrobacter mysorens, Azospirillum brasilense, Agrobacterium radiobacter), as well as plants of soybean (Glycine max L.), barley (Hordeum L.), alfalfa (Medicago L.), etc. As carriers for bacteria, we used gamma-sterile peat, vermiculite and improved sterile liquid media on the basis of components of vegetable or synthetic origin, with the introduction of nutrients and stabilizers (humates, glycerine, potassium sorbate, carboxymethylcellulose, etc.). The efficacy of tested products and forms were compared in a biological survey carried out in different regions of the Russian Federation. Under the conditions of Leningrad Region the effectiveness was evaluated for the preparations based on recently used and perspective strains of alfalfa nodule bacteria Sinorhizobium meliloti. An optimized combination of stabilizers, nutrients and protective substrates provided a long-term storage and high efficiency of biological products. Nevertheless, plant nutrient media (bean broth) did not provide the required quality. Using bean medium for cultivation it is possible to obtain the desired titer of bacteria in the product, but their numbers are rapidly decreasing. Furthermore, preparations obtained with use of bean broth lose quality after 2-3 months after the beginning of storage because of extraneous microflora therein. The new forms of biochemicals designed on the basis of a synthetic nutrient medium can be stored at room temperature for at least 6 months, when sterile-packed in plastic bottles. High bacteria titer (3.6-4.2 billion CFU/ml of liquid formulation) was obtained after cultivation. It is shown that an improved liquid form has a high efficiency. Its application on leguminous plants increased grain yield by 20-40 % in soy, and by 15-25 % in peas. Productivity of cereal crops (wheat, barley) increased by 15-25 %, and the productivity of alfalfa was 20-45 % higher. A significant increase in crop quality was found to be due to higher protein content. The effectiveness of biological products considerably depended on agroecological conditions and biological features of the crops. Using different strains of nodule bacteria Sinorhizobium meliloti in the Leningrad Region allowed us to create the effective plantmicrobe systems. Thus on the sod-podzolic soil the strain A-4 was most effective providing yield increase by more than 25 %, while on sod-carbonate soil it was the strain A-5 which increased the productivity of alfalfa by 140 %.

The effect of cadmium on the efficiency of development of legume-rhizobium symbiosis

Screening of nodule bacteria (rhizobia) forming symbiotic relationships with legumes has been performed in order to isolate strains resistant to cadmium ions in a wide range of concentrations (6-132 mg/kg). The effect ofcadmium salts (6, 12, 24 mg/kg) on the legume-rhizobium symbiosis ofthe pea Pisum sativum L. with Rhizobium leguminosarum and of the fodder galega Galega orientalis Lam. with Rhizobium galegae has been studied under experimental laboratory conditions. No statistically significant differences have been revealed in the growth and biomass of plants with regard to the control in the range of concentrations given above. However, it was found that cadmium inhibited nodulation in P. sativum and stimulated it in G. orientalis.

Pseudorhizobium pelagicum gen. nov., sp. nov. isolated from a pelagic Mediterranean zone

Two novel Alphaproteobacteria strains, R1-200B4T and R2-400B4, were isolated from the Mediterranean Sea off the coast of Alicante in Spain. The phylogenetic analysis of the 16S rRNA gene showed that they are related to members of Family Rhizobiaceae. The 16S rRNA gene sequence of strain R1-200B4T presents 97.0% and 95.6% similarity with respect to the type strains of the type species from genera Neorhizobium and Rhizobium, Neorhizobium galegae HAMBI 540T and Rhizobium leguminosarum USDA 2370T, respectively. The remaining genera of family Rhizobiaceae showed similarities lower than 95%. The recA and atpD gene sequences of strain R1-200B4T showed, respectively, 90% and 88.6% similarity with respect to N. galegae HAMBI 540T and 87% and 86% with respect to R. leguminosarum USDA 2370T. The calculated ANI values between the genomes of the strain R1-200B4T and those of N. galegae HAMBI 540T and R. leguminosarum 3841 are 75.9% and 74.0%, respectively. The major fatty acids are those from summed feature 8 (C18:1 ω6c/C18:1 ω7c) and the C16:0. Catalase and oxidase were positive. Nitrate reduction and aesculin hydrolysis were positive. Production of β-galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Growth was observed in presence of 7% NaCl. Therefore, based on the phylogenetic, chemotaxonomic and phenotypic data obtained in this study, we propose to classify the strains isolated in this study in a new genus named Pseudorhizobium gen. nov. and a new species named Pseudorhizobium pelagicum sp. nov. with the type strain R1-200B4T (=LMG 28314T=CECT 8629T).

Genomic features separating ten strains of Neorhizobium galegae with different symbiotic phenotypes.

BackgroundThe symbiotic phenotype of Neorhizobium galegae, with strains specifically fixing nitrogen with either Galega orientalis or G. officinalis, has made it a target in research on determinants of host specificity in nitrogen fixation. The genomic differences between representative strains of the two symbiovars are, however, relatively small. This introduced a need for a dataset representing a larger bacterial population in order to make better conclusions on characteristics typical for a subset of the species. In this study, we produced draft genomes of eight strains of N. galegae having different symbiotic phenotypes, both with regard to host specificity and nitrogen fixation efficiency. These genomes were analysed together with the previously published complete genomes of N. galegae strains HAMBI 540T and HAMBI 1141.ResultsThe results showed that the presence of an additional rpoN sigma factor gene in the symbiosis gene region is a characteristic specific to symbiovar orientalis, required for nitrogen fixation. Also the nifQ gene was shown to be crucial for functional symbiosis in both symbiovars. Genome-wide analyses identified additional genes characteristic of strains of the same symbiovar and of strains having similar plant growth promoting properties on Galega orientalis. Many of these genes are involved in transcriptional regulation or in metabolic functions.ConclusionsThe results of this study confirm that the only symbiosis-related gene that is present in one symbiovar of N. galegae but not in the other is an rpoN gene. The specific function of this gene remains to be determined, however. New genes that were identified as specific for strains of one symbiovar may be involved in determining host specificity, while others are defined as potential determinant genes for differences in efficiency of nitrogen fixation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1576-3) contains supplementary material, which is available to authorized users.