Rhizobial Strains Research Articles

Frankia [NiFe] uptake hydrogenases and genome reduction: different lineages of loss.

Uptake hydrogenase (Hup) recycles H2 formed by nitrogenase during nitrogen fixation, thereby preserving energy. Among root nodule bacteria, most rhizobial strains examined are Hup-, while only one Hup- Frankia inoculum had been identified. Previous analyses had led to the identification of two different [NiFe] hydrogenase syntons. We analysed the distribution of different types of [NiFe] hydrogenase in the genomes of different Frankia species. Our results show that Frankia strains can contain four different [NiFe] hydrogenase syntons representing groups 1f, 1h, 2a and 3b according to Søndergaard et al. (2016); no more than three types were found in any individual genome. The phylogeny of the structural proteins of groups 1f, 1h and 2a follows Frankia phylogeny; the phylogeny of the accessory proteins does not consistently. An analysis of different [NiFe] hydrogenase types in Actinomycetia shows that under the most parsimonious assumption, all four types were present in the ancestral Frankia strain. Based on Hup activities analysed and the losses of syntons in different lineages of genome reduction, we can conclude that groups 1f and 2a are involved in recycling H2 formed by nitrogenase while group 1h and group 3b are not.

Atypical rhizobia trigger nodulation and pathogenesis on the same legume hosts

The emergence of commensalism and mutualism often derives from ancestral parasitism. However, in the case of rhizobium-legume interactions, bacterial strains displaying both pathogenic and nodulation features on a single host have not been described yet. Here, we isolated such a bacterium from Medicago nodules. On the same plant genotypes, the T4 strain can induce ineffective nodules in a highly competitive way and behave as a harsh parasite triggering plant death. The T4 strain presents this dual ability on multiple legume species of the Inverted Repeat-Lacking Clade, the output of the interaction relying on the developmental stage of the plant. Genomic and phenotypic clustering analysis show that T4 belongs to the nonsymbiotic Ensifer adhaerens group and clusters together with T173, another strain harboring this dual ability. In this work, we identify a bacterial clade that includes rhizobial strains displaying both pathogenic and nodulating abilities on a single legume host.

Assessing the Phytoextraction of Cd, Pb, and Zn from a Slag-Contaminated Soil by Legume Species Inoculated with Rhizobial Strains

Assessing the Phytoextraction of Cd, Pb, and Zn from a Slag-Contaminated Soil by Legume Species Inoculated with Rhizobial Strains

Rhizobial variation, more than plant variation, mediates plant symbiotic and fitness responses to herbicide stress.

Symbiotic mutualisms provide critical ecosystem services throughout the world. Anthropogenic stressors, however, may disrupt mutualistic interactions and impact ecosystem health. The plant-rhizobia symbiosis promotes plant growth and contributes to the nitrogen (N) cycle. While off-target herbicide exposure is recognized as a significant stressor impacting wild plants, we lack knowledge about how it affects the symbiotic relationship between plants and rhizobia. Moreover, we do not know whether the impact of herbicide exposure on symbiotic traits or plant fitness might be ameliorated by plant or rhizobial genetic variation. To address these gaps, we conducted a greenhouse study where we grew 17 full-sibling genetic families of red clover (Trifolium pratense) either alone (uninoculated) or in symbiosis with one of two genetic strains of rhizobia (Rhizobium leguminosarum) and exposed them to a concentration of the herbicide dicamba that simulated "drift" (i.e., off-target atmospheric movement) or a control solution. We recorded responses in immediate vegetative injury, key features of the plant-rhizobia mutualism (nodule number, nodule size, and N fixation), mutualism outcomes, and plant fitness (biomass). In general, we found that rhizobial variation more than plant variation determined outcomes of mutualism and plant fitness in response to herbicide exposure. Herbicide damage response depended on plant family, but also whether plants were inoculated with rhizobia and if so, with which strain. Rhizobial strain variation determined nodule number and size, but this was herbicide treatment-dependent. In contrast, strain and herbicide treatment independently impacted symbiotic N fixation. And while herbicide exposure significantly reduced plant fitness, this effect depended on inoculation state. Furthermore, the differential fitness benefits that the two rhizobial strains provided plants seemed to diminish under herbicidal conditions. Altogether, these findings suggest that exposure to low levels of herbicide impact key components of the plant-rhizobia mutualism as well as plant fitness, but genetic variation in the partners determines the magnitude and/or direction of these effects. In particular, our results highlight a strong role of rhizobial strain identity in driving both symbiotic and plant growth responses to herbicide stress.

Influence of Enhanced Synthesis of Exopolysaccharides in Rhizobium ruizarguesonis and Overproduction of Plant Receptor to these Compounds on Colonizing Activity of Rhizobia in Legume and Non-Legume Plants and Plant Resistance to Phytopathogenic Fungi.

Rhizobial exopolysaccharides (EPS) may provide stabilization of membranes against external factors, as well as improved surface adhesion, but their role in interaction with legume and non-legume plants is still far from understanding. In this work, the transcriptional regulator RosR of Rhizobium ruizarguesonis, which regulates the synthesis of EPS, was overproduced in a pHC60 plasmid and expressed in the RCAM 1026 strain. This resulted in an improved production of EPS by this recombinant strain. Comparative analysis of the inoculation of pea Pisum sativum plants with R. ruizarguesonis pHC60-rosR and strain carrying the empty plasmid revealed an essential increase in the number of nodules, root length and biomass in plants inoculated with this EPS-overproducing strain. It demonstrates that the enhanced EPS synthesis by rhizobia may stimulate plant root colonization and subsequent nodule formation in pea plants. The influence of enhanced EPS synthesis in rhizobia on colonizing activity was also estimated in non-legume plant tomato Solanum lycopersicum. Our findings shown the increased colonization of the root surface and stimulation of the shoot biomass of inoculated plants. Inoculation of pea and tomato with EPS-overproducing rhizobial strain essentially increased plant resistance to phytopathogenic fungi Fusarium culmorum and F. oxysporum in both legume and non-legume plants, demonstrating a significant biocontrol effect of this recombinant strain. Furthermore, we have identified the PsLYK10 gene that encodes a putative EPS receptor in P. sativum, although no significant effect of PsLYK10 overexpression on nodulation in legume (pea P. sativum) and colonization of roots of non-legume plants by rhizobia was found compared to enhanced production of EPS by rhizobia.

Potential of novel Rhizobium strains as biofertilizer for improved groundnut (Arachis hypogaea L.) cultivation in Eastern India

ABSTRACT Comprehensive assessments were conducted on eight fast growing, novel Rhizobium isolates, short listed from a preliminary selection of 68 isolates obtained from root nodules of groundnut plants grown in diverse agro-climatic regions of eastern India. Additionally, two conventional rhizobia strains, viz. IGR6 and GN2, (collected from the Nodule Research Center, BCKV), were utilized for comparative analysis. Cluster analysis, based on carbon utilization and biochemical parameters, grouped all the test isolates with an average similarity level of 73%. The ability of the isolates to utilize a wide range of carbon sources, including hexoses, pentoses, and disaccharides, confirms their fast-growing nature. Further analysis through 16S rDNA extraction and nucleotide sequencing revealed that the promising fresh isolates exhibited 95–99% similarity to known Rhizobium species in public databases. When evaluated in pot culture experiments using new alluvial soil from the Gangetic Plains in eastern India, the native fresh isolate NRA1(PP355674) outperformed other isolates and conventional strains in terms of nodulation efficiency, plant dry matter production, and plant nitrogen uptake. This study highlights the significant potential of native Rhizobium isolates to surpass conventionally used strains in biological nitrogen fixation, supporting the development of sustainable, environmentally-friendly, and cost-effective agricultural practices.

Isolation and characterization of mung bean (Vigna radiata L.) rhizobia in Myanmar

AbstractWe collected soil samples from six major mung bean cropping regions in Myanmar: Sagaing, Mandalay, Nay Pyi Taw, and Magway in the tropical savanna climate zone and Bago and Yangon in the tropical monsoon climate zone. All fields grew mung bean for at least 5 years and had no history of rhizobial inoculation. Mung bean ‘Yezin-11’, a popular cultivar in Myanmar, was inoculated with soil suspensions. From the nodules formed on the roots, we isolated 55 rhizobial strains. Identification of the isolates revealed the dominant species of indigenous rhizobia in each region. We identified 53 Bradyrhizobium strains and 2 Ensifer strains. Bradyrhizobium yuanmingense was dominant in the tropical savanna zone and Bradyrhizobium sp. (B. liaoningense or B. diversitatis) and B. centrosematis were dominant in the tropical monsoon zone. Principal component analysis indicates that the dominance of B. yuanmingense in the tropical savanna zone might be due to high concentration of NO3-N and P2O5 in the soil. It also indicates that the dominance of B. centrosematis in the tropical monsoon zone might be caused by drastically low pH and high concentration of NH4 in the soil. Bradyrhizobium centrosematis YGN-M9, B. yuanmingense SGG-M3, and Bradyrhizobium sp. BGO-M5 significantly increased nodulation (nodule number and nodule dry weight), acetylene reduction activity, and shoot dry weight, respectively, relative to Ensifer terangae MDY-M6. Co-inoculation with these three strains increased nodulation significantly compared with single inoculation of BGO-M5. The characterization of mung bean rhizobia and selection of microbial inoculant candidates will be useful for the development of microbial inoculants in Myanmar.

Rhizobia inoculation’s impact on the biomass and moisture content of leguminous Bambara groundnut (Vigna subterranean L. Verdc)

Bambara groundnut (BG) (Vigna subterranean L. verdc) is a highly nutritious underutilized leguminous crop of African origin with high economic importance. Its production rate (300,000 metric tonnes/year) does not meet the current demand (800,000 metric tonnes/year). Despite it’s useful properties, it has remained neglected, with poor market and low yields because research about improving BG has been neglected. rhizobia have proved to be useful in promoting the growth of legumes, yet, there is unavailable information about the role of indigenous BG-symbiont rhizobia inoculant (which are better affordable alternatives to chemical fertilizers) for increasing BG yield and this needs to be explored, hence this study. This experiment studied the role six rhizobial strains in improving the biomass and plant vigour of two selected varieties of BG (TVSU 1248, TVSU 631).These strains were applied as inoculant (at concentrations of > 0.85 × 1010 cfu/ml) on field compared alongside commercial USDA110 strains and Urea (rates of 60 kg/ha and 20 kg/ha) treated plants and analysed for biomass of shoot, root, shaft and seed, and moisture etc. Rhizobia significantly influenced the shoot biomass (≥ 19.26 g), (79.81 ± 1.81 g and 63.15 ± 3.21 g), root biomass (≥ 0.63 g), pod biomass (≥ 155.3 g), seed biomass (≥ 103.96 g), shaff biomass (≥ 52.29 g) and moisture content of the plants, with differences in varietal response. Indigenous BG-symbiotic strains are able to significantly influence plant vigour and biomass, promoting the growth and health of BG. Rhizobia, proved to be useful for enhancing the plant biomass, vigour and growth and should be recommended for improving the production of BG for higher yield and productivity.

Hot Spots of Site-Specific Integration into the Sinorhizobium meliloti Chromosome.

The diversity of phage-related sequences (PRSs) and their site-specific integration into the genomes of nonpathogenic, agriculturally valuable, nitrogen-fixing root nodule bacteria, such as Sinorhizobium meliloti, were evaluated in this study. A total of 314 PRSs, ranging in size from 3.24 kb to 88.98 kb, were identified in the genomes of 27 S. meliloti strains. The amount of genetic information foreign to S. meliloti accumulated in all identified PRSs was 6.30 Mb. However, more than 53% of this information was contained in prophages (Phs) and genomic islands (GIs) integrated into genes encoding tRNAs (tRNA genes) located on the chromosomes of the rhizobial strains studied. It was found that phiLM21-like Phs were predominantly abundant in the genomes of S. meliloti strains of distant geographical origin, whereas RR1-A- and 16-3-like Phs were much less common. In addition, GIs predominantly contained fragments of phages infecting bacteria of distant taxa, while rhizobiophage-like sequences were unique. A site-specific integration analysis revealed that not all tRNA genes in S. meliloti are integration sites, but among those in which integration occurred, there were "hot spots" of integration into which either Phs or GIs were predominantly inserted. For the first time, it is shown that at these integration "hot spots", not only is the homology of attP and attB strictly preserved, but integrases in PRSs similar to those of phages infecting the Proteobacteria genera Azospirillum or Pseudomonas are also present. The data presented greatly expand the understanding of the fate of phage-related sequences in host bacterial genomes and also raise new questions about the role of phages in bacterial-phage coevolution.

Study of Plant Growth Promoting Abilities of Rhizobium Strains Isolated from Mungbean Root Nodules

Background: Plant Growth Promoting Rhizobacteria (PGPR) are a type of bacteria that use biofertilizers and other plant growth-promoting agents to increase plant output and growth. In the last several decades, there has been a global surge in the usage of beneficial soil microorganisms like PGPR for safe and sustainable agriculture due to the detrimental effects of artificial fertilizers on the environment and their rising prices. Plant-beneficial rhizobacteria have the potential to reduce the world's reliance on dangerous agricultural chemicals that upset agro-ecosystems. Today a large part of yield is lost due to the various stress mechanisms of plants. It could be classified into biotic and abiotic stress. These are generally a group of microorganisms that is found either in the rhizosphere or in the root nodules of plants. PGPR has beneficial impact, they colonize in the plant roots and improve crop yields and agricultural productivity and also produce some useful growth promoting growth hormones viz., IAA, Auxins, Cytokinin and Gibberellins. Methods: The bacterial isolates were screened for plant growth promoting traits such as phosphate solubilization, IAA production and siderophore production was tested qualitatively by using pikovskayas media, TSB (Tryptone Soy Broth) agar and Chrome Azurol Sulphate (CAS) blue agar respectively. Results: In present study out of the eight rhizobial isolates, five exhibited the phosphate solubilization zones, three shows negative result. However out of eight isolates five isolates produced IAA and three showed negative result. Among all isolates seven isolates were able to produce siderophore, while one showed negative result.

Phylogenetic diversity of Rhizobium species recovered from nodules of common beans (Phaseolus vulgaris L.) in fields in Uganda: R. phaseoli, R. etli, and R. hidalgonense.

A total of 75 bacterial isolates were obtained from nodules of beans cultivated across 10 sites in six agro-ecological zones in Uganda. Using recA gene sequence analysis, 66 isolates were identified as members of the genus Rhizobium, while nine were related to Agrobacterium species. In the recA gene tree, most Rhizobium strains were classified into five recognized species. Phylogenetic analysis based on six concatenated sequences (recA-rpoB-dnaK-glnII-gyrB-atpD) placed 32 representative strains into five distinct Rhizobium species, consistent with the species groups observed in the recA gene tree: R. phaseoli, R. etli, R. hidalgonense, R. ecuadorense, and R. sophoriradicis, with the first three being the predominant. The rhizobial strains grouped into three nodC subclades within the symbiovar phaseoli clade, encompassing strains from distinct phylogenetic groups. This pattern reflects the conservation of symbiotic genes, likely acquired through horizontal gene transfer among diverse rhizobial species. The 32 representative strains formed symbiotic relationships with host beans, while the Agrobacterium strains did not form nodules and lacked symbiotic genes. Multivariate analysis revealed that species distribution was influenced by the environmental factors of the sampling sites, emphasizing the need to consider these factors in future effectiveness studies to identify effective nitrogen-fixing strains for specific locations.

Identification and functional analysis of recent IS transposition events in rhizobia

Rhizobia are alpha- and beta- Proteobacteria that, through the establishment of symbiotic interactions with leguminous plants, are able to fix atmospheric nitrogen as ammonium. The successful establishment of a symbiotic interaction is highly dependent on the availability of nitrogen sources in the soil, and on the specific rhizobia strain. Insertion sequences (ISs) are simple transposable genetic elements that can move to different locations within the host genome and are known to play an important evolutionary role, contributing to genome plasticity by acting as recombination hot-spots, and disrupting coding and regulatory sequences. Disruption of coding sequences may have occurred either in a common ancestor of the species or more recently. By means of ISComapare, we identified Differentially Located ISs (DLISs) in nearly related rhizobial strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium. Our results revealed that recent IS transposition could have a role in adaptation by enabling the activation and inactivation of genes that could dynamically affect the competition and survival of rhizobia in the rhizosphere.Graphical

Differential symbiotic compatibilities between rhizobium strains and cultivated and wild soybeans revealed by anatomical and transcriptome analyses.

Various species of rhizobium establish compatible symbiotic relationships with soybean (Glycine max) leading to the formation of nitrogen-fixing nodules in roots. The formation of functional nodules is mediated through complex developmental and transcriptional reprogramming that involves the activity of thousands of plant genes. However, host transcriptome that differentiate between functional or non-functional nodules remain largely unexplored. In this study, we investigated differential compatibilities between rhizobium strains (Bradyrhizobium diazoefficiens USDA110 Bradyrhizobium sp. strain LVM105) and cultivated and wild soybeans. The nodulation assays revealed that both USDA110 and LVM105 strains effectively nodulate G. soja but only USDA110 can form symbiotic relationships with Williams 82. LVM105 formed pseudonodules on Williams 82 that consist of a central nodule-like mass that are devoid of any rhizobia. RNA-seq data revealed that USDA110 and LVM105 induce distinct transcriptome programing in functional mature nodules formed on G. soja roots, where genes involved in nucleosome assembly, DNA replication, regulation of cell cycle, and defense responses play key roles. Transcriptome comparison also suggested that activation of genes associated with cell wall biogenesis and organization and defense responses together with downregulation of genes involved in the biosynthesis of isoprenoids and antioxidant stress are associated with the formation of non-functional nodules on Williams 82 roots. Moreover, our analysis implies that increased activity of genes involved in oxygen binding, amino acid transport, and nitrate transport differentiates between fully-developed nodules in cultivated versus wild soybeans.

Phenanthrene-Degrading and Nickel-Resistant Neorhizobium Strain Isolated from Hydrocarbon-Contaminated Rhizosphere of Medicago sativa L.

Pollutant degradation and heavy-metal resistance may be important features of the rhizobia, making them promising agents for environment cleanup biotechnology. The degradation of phenanthrene, a three-ring polycyclic aromatic hydrocarbon (PAH), by the rhizobial strain Rsf11 isolated from the oil-polluted rhizosphere of alfalfa and the influence of nickel ions on this process were studied. On the basis of whole-genome and polyphasic taxonomy, the bacterium Rsf11 represent a novel species of the genus Neorhizobium, so the name Neorhizobium phenanthreniclasticum sp. nov. was proposed. Analysis of phenanthrene degradation by the Rsf1 strain revealed 1-hydroxy-2-naphthoic acid as the key intermediate and the activity of two enzymes apparently involved in PAH degradation. It was also shown that the nickel resistance of Rsf11 was connected with the extracellular adsorption of metal by EPS. The joint presence of phenanthrene and nickel in the medium reduced the degradation of PAH by the microorganism, apparently due to the inhibition of microbial growth but not due to the inhibition of the activity of the PAH degradation enzymes. Genes potentially involved in PAH catabolism and nickel resistance were discovered in the microorganism studied. N. phenanthreniclasticum strain Rsf11 can be considered as a promising candidate for use in the bioremediation of mixed PAH-heavy-metal contamination.

Microvirga sesbaniae sp. nov. and Microvirga yunnanensis sp. nov., Pink-Pigmented Bacteria Isolated from Root Nodules of Sesbania cannabina (Retz.) Poir.

Four pigment-producing rhizobial strains nodulating Sesbania cannabina (Retz.) Poir. formed a unique group in genus Microvirga in the phylogeny of a 16S rRNA gene and five housekeeping genes (gyrB, recA, dnaK, glnA, and atpD) in a genome analysis, phenotypic characteristics analysis, and chemotaxonomic analysis. These four strains shared as high as 99.3% similarity with Microvirga tunisiensis LmiM8T in the 16S rRNA gene sequence and, in an MLSA, were subdivided into two clusters, ANI (genome average nucleotide) and dDDH (digital DNA-DNA hybridization) which shared sequence similarities lower than the species thresholds with each other and with the reference strains for related Microvirga species. The polar lipids elucidated that phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin were the main components for strain SWF67558T and for strain HBU65207T, with the exception of PC. SWF67558T and HBU65207T strains had similar predominant cellular fatty acids, including C16:0, C18:0, summed feature 2, and summed feature8, but with different contents. In addition, all the four novel strains produced pink-pigment, and the main coloring material extract from strain SWF67558T was identified as zeaxanthin, which presented antioxidant ability and reduction power. With all the phylogenetic and phenotypic divergency, we proposed these pink-pigmented symbiotic bacteria as two novel species, named Microvirga sesbaniae sp. nov. and Microvirga yunnanensis sp. nov., with SWF67558T (=KCTC82331T=GDMCC1.2024T) and HBU65207T (=KCTC92125T=GDMCC1.2023T) as the type strains, respectively.

Developed Rhizobium Strains Enhance Soil Fertility and Yield of Legume Crops in Haryana, India.

Three strains of Gram-negative bacterium,Rhizobium, were developed by gamma (γ)-irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants-chickpea, field-pea, and lentil-in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control-uninoculated crops and crops grown with indigenous or commercial crop-specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field-pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson's correlation coefficientsrevealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro- and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility.

Cereal–Legume Intercropping: Which Partners Are Preferred in Northwestern Europe?

To increase Europe’s self-sufficiency for protein sources, boosting plant protein production is a prerequisite. Yield variability is one of the main problems regarding the cultivation of protein crops. In this light, cereal–legume intercropping can offer a solution, as well-balanced intercropping systems are less prone to yield variations. Therefore, in this study the effects of (i) species/genotype combination, (ii) intercropping sowing densities and (iii) fertilizer regime were evaluated under Belgian (Northwestern European) conditions over three years (i.e., the 2020–2021, 2021–2022 and 2022–2023 seasons). Regarding the species combinations, winter barley x winter pea, winter wheat x winter faba bean and winter triticale x winter faba bean, it was observed that the best-performing combination varied from year to year depending on the prevailing weather conditions. A reduced sowing density (i.e., 130 seeds/m2 for the cereal partner and 20 seeds/m2 in the case of faba bean or 40 seeds/m2 in the case of pea) was sufficient to achieve competitive yields under the prevailing conditions. Inoculation with commercial Rhizobium strains did not result in an increased yield. Fertilization with one or two nitrogen fractions significantly increased the total yield thanks to a yield increase in the cereal partner; however, as a consequence, the proportion of legumes in the mixture decreased. In conclusion, it can be stated that with the investigated cereal–legume combinations, a competitive yield and qualitative protein yield can be achieved with a reduced fertilizer input.

Harnessing the Efficacy of a Rhizobium Strain in Individual and Consortium Mode to Promote Sustainable Lentil Production and Biofortification under Diverse Soil Conditions

Harnessing the Efficacy of a Rhizobium Strain in Individual and Consortium Mode to Promote Sustainable Lentil Production and Biofortification under Diverse Soil Conditions

Genetic Characterization of Rhizobium spp. Strains in an Organic Field Pea (Pisum sativum L.) Field in Lithuania.

Biological nitrogen fixation in legume plants depends on the diversity of rhizobia present in the soil. Rhizobial strains exhibit specificity towards host plants and vary in their capacity to fix nitrogen. The increasing interest in rhizobia diversity has prompted studies of their phylogenetic relations. Molecular identification of Rhizobium is quite complex, requiring multiple gene markers to be analysed to distinguish strains at the species level or to predict their host plant. In this research, 50 rhizobia isolates were obtained from the root nodules of five different Pisum sativum L. genotypes ("Bagoo", "Respect", "Astronaute", "Lina DS", and "Egle DS"). All genotypes were growing in the same field, where ecological farming practices were applied, and no commercial rhizobia inoculants were used. The influence of rhizobial isolates on pea root nodulation and dry biomass accumulation was determined. 16S rRNA gene, two housekeeping genes recA and atpD, and symbiotic gene nodC were analysed to characterize rhizobia population. The phylogenetic analysis of 16S rRNA gene sequences showed that 46 isolates were linked to Rhizobium leguminosarum; species complex 1 isolate was identified as Rhizobium nepotum, and the remaining 3 isolates belonged to Rahnella spp., Paenarthrobacter spp., and Peribacillus spp. genera. RecA and atpD gene analysis showed that the 46 isolates identified as R. leguminosarum clustered into three genospecies groups (B), (E) and (K). Isolates that had the highest influence on plant dry biomass accumulation clustered into the (B) group. NodC gene phylogenetic analysis clustered 46 R. leguminosarum isolates into 10 groups, and all isolates were assigned to the R. leguminosarum sv. viciae.

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria

BackgroundMercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg.ResultsOur analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg.ConclusionsMer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.