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Background Red clover ( Trifolium pratense L.) is widely cultivated as forage crop. Due to the ability to accumulate potentially toxic elements from the soil, red clover is proposed as a bioindicator plant, but this potential is still being explored. Rhizobium leguminosarum biovar trifolii forms endosymbiosis with red clover which provides nitrogen to the plant, but also produces different plant growth-promoting compounds contributing to alleviation of different stress factors. The aim of this study was to characterize the native rhizobial strains associated with red clover plants and to evaluate in vitro the capacity of nitrogen fixation, plant-growth promotion potential and tolerance to nickel (Ni) as well as the effect of selected strains on elemental composition and growth of red clover in soil with elevated Ni concentration. Results More than 60% of tested strains nodulated red clover when re-inoculated in in vitro culture and induced significant increase in height, shoot dry weight and shoot nitrogen content up to 140.53%, 1026.27% and 196.85%, respectively, over the non-inoculated plants. The strains were characterized as Rhizobium sp. Plant growth-promoting traits such as indole-3-acetic acid (22.18–135.32 µg/mL in the presence of 2 g/L L-tryptophan) and 1-aminocyclopropane-1-carboxylate deaminase production and phosphate solubilization were detected in some strains. Inoculation of red clover in soil with increased Ni concentration showed that inoculated treatments had increased shoot dry weight (up to 16.13%) and nitrogen content (up to 16.21% in shoots and 18.62% in roots) compared to non-treated plants. Inoculation increased Ni, Cr and Pb concentrations (up to 52.33, 145.88 and 218.03%, respectively) compared to the control. Generally, red clover plants accumulated more metals in their roots than in shoots, and shoot concentrations were mainly within common range for plants. Conclusion Inoculation of red clover with effective rhizobial strains showed potential for plant growth improvement and phytostabilization of Ni in metal-burdened soils.

Legume-rhizobium symbiosis provides a sustainable nitrogen source for agriculture. Nitrogen fixation efficiency depends on both legume and rhizobium genotypes, but the implications of their interactions for plant performance in environments with many competing rhizobium strains remain unclear. Here, we let 399 Rhizobium leguminosarum complex sv. viciae strains compete for nodulation of 212 faba bean genotypes. We find that the strains can be categorised by their nodule occupancy profiles into groups that show distinct competitive interactions and plant growth-promoting effects. Further, we show that the diversity of strains occupying root nodules affects plant growth and is under plant genetic control. These insights provide a basis for re-designing rhizobium inoculation and plant breeding strategies to enhance symbiotic nitrogen fixation in agriculture. Optimising plant-rhizobium interactions can enhance nitrogen fixation, improving sustainability in agriculture. This study identified four groups of faba bean symbionts that show growth-promoting characteristics and whose diversity across root nodules is under plant genetic control.

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.

Most species in the Leguminosae (legume family) can fix atmospheric nitrogen (N₂) via symbiotic bacteria (rhizobia) in root nodules. Here, the literature on legume-rhizobia symbioses in field soils was reviewed and genotypically characterised rhizobia related to the taxonomy of the legumes from which they were isolated. The Leguminosae was divided into three sub-families, the Caesalpinioideae, Mimosoideae and Papilionoideae. Bradyrhizobium spp. were the exclusive rhizobial symbionts of species in the Caesalpinioideae, but data are limited. Generally, a range of rhizobia genera nodulated legume species across the two Mimosoideae tribes Ingeae and Mimoseae, but Mimosa spp. show specificity towards Burkholderia in central and southern Brazil, Rhizobium/Ensifer in central Mexico and Cupriavidus in southern Uruguay. These specific symbioses are likely to be at least in part related to the relative occurrence of the potential symbionts in soils of the different regions. Generally, Papilionoideae species were promiscuous in relation to rhizobial symbionts, but specificity for rhizobial genus appears to hold at the tribe level for the Fabeae (Rhizobium), the genus level for Cytisus (Bradyrhizobium), Lupinus (Bradyrhizobium) and the New Zealand native Sophora spp. (Mesorhizobium) and species level for Cicer arietinum (Mesorhizobium), Listia bainesii (Methylobacterium) and Listia angolensis (Microvirga). Specificity for rhizobial species/symbiovar appears to hold for Galega officinalis (Neorhizobium galegeae sv. officinalis), Galega orientalis (Neorhizobium galegeae sv. orientalis), Hedysarum coronarium (Rhizobium sullae), Medicago laciniata (Ensifer meliloti sv. medicaginis), Medicago rigiduloides (Ensifer meliloti sv. rigiduloides) and Trifolium ambiguum (Rhizobium leguminosarum sv. trifolii). Lateral gene transfer of specific symbiosis genes within rhizobial genera is an important mechanism allowing legumes to form symbioses with rhizobia adapted to particular soils. Strain-specific legume rhizobia symbioses can develop in particular habitats.

Chickpea ( Cicer arietinum L. ) is a vital legume crop worldwide, valued for its high nutritional content and significant contribution to food security and soil fertility through biological nitrogen fixation. Despite its importance, chickpea yields remain suboptimal in many regions, including Ethiopia, primarily due to constraints such as poor soil fertility and inadequate use of effective rhizobia inoculants. This study aimed to isolate and characterize native Rhizobium strains from chickpea root nodules collected from fields in the Woldia region and to assess their potential to promote plant growth. A total of 41 bacterial isolates were obtained, of which 12 were presumptively identified as Rhizobium based on growth characteristics on Congo red and bromothymol blue media. These isolates were further characterized morphologically and biochemically. Five biochemically promising isolates were selected for evaluation in a controlled 45-day greenhouse experiment under sterile conditions. Inoculation with these isolates significantly enhanced seed germination and early seedling growth compared to uninoculated controls. The symbiotic effectiveness of the isolates ranged from 74.3% to 121.9%, with isolates WUSFDG-23, WUSFMC-31, and WUSFMC-23 demonstrating high effectiveness, isolate WUSFDG-23 markedly increased nodulation and biomass accumulation. This study highlights the potential of native Rhizobium isolates from Woldia chickpea fields, especially WUSFDG-23, as effective bio-inoculants to promote sustainable chickpea production and reduce dependence on chemical fertilizers.

The Type VI secretion systems (T6SSs) allow bacteria to translocate effector proteins to other bacteria or to eukaryotic cells. However, little is known about the role of T6SS in endosymbiotic bacteria. In this work we describe the T6SS of Rhizobium etli Mim1, a bacteria able to effectively nodulate common beans. Structural genes and those encoding possible effectors have been identified in a 28-gene DNA region of R. etli Mim1 pRetMIM1f plasmid. Immunodetection of Hcp protein, a conserved key structural component of T6SS systems, indicates that this secretion system is active at high cell densities, in the presence of root exudates, and in bean nodules. Rhizobium etli mutants affected in T6SS structural genes produced plants with lower dry weight and smaller nodules than the wild-type strain, indicating for the first time that the T6SS plays a positive role in Rhizobium-legume symbiosis.

Background Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean ( Phaseolus vulgaris ) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. Results Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. Conclusions Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.

Fabeae legumes such as pea and faba bean form symbiotic nodules with a large diversity of soil Rhizobium leguminosarum symbiovar viciae (Rlv) bacteria. However, bacteria competitive to form root nodules (CFN) are generally not the most efficient to fix dinitrogen, resulting in a decrease in legume crop yields. Here, we investigate differential selection by host plants on the diversity of Rlv. A large collection of Rlv was collected by nodule trapping with pea and faba bean from soils at five European sites. Representative genomes were sequenced. In parallel, diversity and abundance of Rlv were estimated directly in these soils using metabarcoding. The CFN of isolates was measured with both legume hosts. Pea/faba bean CFN were associated to Rlv genomic regions. Variations of bacterial pea and/or faba bean CFN explained the differential abundance of Rlv genotypes in pea and faba bean nodules. No evidence was found for genetic association between CFN and variations in the core genome, but variations in specific regions of the nod locus, as well as in other plasmid loci, were associated with differences in CFN. These findings shed light on the genetic control of CFN in Rlv and emphasise the importance of host plants in controlling Rhizobium diversity.

One of the largest contributions to biologically available nitrogen comes from the reduction of N₂ to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N₂ fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N₂ fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N₂ fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis.

By analyzing successive lifestyle stages of a model Rhizobium–legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N₂-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N₂. Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N₂ fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium–legume symbiosis.