Rhizobium etli CFN42 and Sinorhizobium meliloti 1021 bioinformatic transcriptional regulatory networks from culture and symbiosis.

Rhizobium etli CFN42 proteome-transcriptome mixed data of exponential growth and nitrogen-fixing bacteroids, as well as Sinorhizobium meliloti 1021 transcriptome data of growth and nitrogen-fixing bacteroids, were integrated into transcriptional regulatory networks (TRNs). The one-step construction network consisted of a matrix-clustering analysis of matrices of the gene profile and all matrices of the transcription factors (TFs) of their genome. The networks were constructed with the prediction of regulatory network application of the RhizoBindingSites database (http://rhizobindingsites.ccg.unam.mx/). The deduced free-living Rhizobium etli network contained 1,146 genes, including 380 TFs and 12 sigma factors. In addition, the bacteroid R. etli CFN42 network contained 884 genes, where 364 were TFs, and 12 were sigma factors, whereas the deduced free-living Sinorhizobium meliloti 1021 network contained 643 genes, where 259 were TFs and seven were sigma factors, and the bacteroid Sinorhizobium meliloti 1021 network contained 357 genes, where 210 were TFs and six were sigma factors. The similarity of these deduced condition-dependent networks and the biological E. coli and B. subtilis independent condition networks segregates from the random Erdös-Rényi networks. Deduced networks showed a low average clustering coefficient. They were not scale-free, showing a gradually diminishing hierarchy of TFs in contrast to the hierarchy role of the sigma factor rpoD in the E. coli K12 network. For rhizobia networks, partitioning the genome in the chromosome, chromids, and plasmids, where essential genes are distributed, and the symbiotic ability that is mostly coded in plasmids, may alter the structure of these deduced condition-dependent networks. It provides potential TF gen-target relationship data for constructing regulons, which are the basic units of a TRN.

NIN-like proteins (NLPs) as crucial nitrate sensors: an overview of their roles in nitrogen signaling, symbiosis, abiotic stress, and beyond.

Nitrogen is an essential macronutrient critical for plant growth and productivity. Plants have the capacity to uptake inorganic nitrate and ammonium, with nitrate playing a crucial role as a signaling molecule in various cellular processes. The availability of nitrate and the signaling pathways involved finely tune the processes of nitrate uptake and assimilation. NIN-like proteins (NLPs), a group of transcription factors belonging to the RWP-RK gene family, act as major nitrate sensors and are implicated in the primary nitrate response (PNR) within the nucleus of both non-leguminous and leguminous plants through their RWP-RK domains. In leguminous plants, NLPs are indispensable for the initiation and development of nitrogen-fixing nodules in symbiosis with rhizobia. Moreover, NLPs play pivotal roles in plant responses to abiotic stresses, including drought and cold. Recent studies have identified NLP homologs in oomycete pathogens, suggesting their potential involvement in pathogenesis and virulence. This review article delves into the conservation of RWP-RK genes, examining their significance and implications across different plant species. The focus lies on the role of NLPs as nitrate sensors, investigating their involvement in various processes, including rhizobial symbiosis in both leguminous and non-leguminous plants. Additionally, the multifaceted functions of NLPs in abiotic stress responses, developmental processes, and interactions with plant pathogens are explored. By comprehensively analyzing the role of NLPs in nitrate signaling and their broader implications for plant growth and development, this review sheds light on the intricate mechanisms underlying nitrogen sensing and signaling in various plant lineages.

Comparative genomics of the carmine cochineal symbiont Candidatus Dactylopiibacterium carminicum reveals possible protection to the host against viruses via CRISPR/Cas.

We present new genomes from the bacterial symbiont Candidatus Dactylopiibacterium carminicum obtained from non-domesticated carmine cochineals belonging to the scale insect Dactylopius (Hemiptera: Coccoidea: Dactylopiidae). As Dactylopiibacterium has not yet been cultured in the laboratory, metagenomes and metatranscriptomics have been key in revealing putative symbiont functions. Dactylopiibacterium is a nitrogen-fixing beta-proteobacterium that may be vertically transmitted and shows differential gene expression inside the cochineal depending on the tissue colonized. Here we found that all cochineal species tested had Dactylopiibacterium carminicum which has a highly conserved genome. All Dactylopiibacterium genomes analyzed had genes involved in nitrogen fixation and plant polymer degradation. Dactylopiibacterium genomes resemble those from free-living plant bacteria, some found as endophytes. Notably, we found here a new putative novel function where the bacteria may protect the insect from viruses, since all Dactylopiibacterium genomes contain CRISPRs with a spacer matching nucleopolyhedrovirus that affects insects.

Nitrogen-fixing bacteria boost floral attractiveness in a tropical legume species during nutrient limitation.

PREMISE: Legumes establish mutualistic interactions with pollinators and nitrogen (N)-fixing bacteria that are critical for plant reproduction and ecosystem functioning. However, we know little about how N-fixing bacteria and soil nutrient availability affect plant attractiveness to pollinators. METHODS: In a two-factorial greenhouse experiment to assess the impact of N-fixing bacteria and soil types on floral traits and attractiveness to pollinators in Chamaecrista latistipula (Fabaceae), plants were inoculated with N-fixing bacteria (NF+) or not (NF-) and grown in N-rich organic soil (+N organic soil) or N-poor sand soil (-N sand soil). We counted buds and flowers and measured plant size during the experiment. We also measured leaf, petal, and anther reflectance with a spectrophotometer and analyzed reflectance curves. Using the bee hexagon model, we estimated chromatic contrasts, a crucial visual cues for attracting bees that are nearby and more distant. RESULTS: NF+ plants in -N sand soil had a high floral display and color contrasts. On the other hand, NF- plants and/or plants in +N organic soil had severely reduced floral display and color contrasts, decreasing floral attractiveness to bee pollinators. CONCLUSIONS: Our findings indicate that the N-fixing bacteria positively impact pollination, particularly when nutrients are limited. This study provides insights into the dynamics of plant-pollinator interactions and underscores the significant influence of root symbionts on key floral traits within tropical ecosystems. These results contribute to understanding the mechanisms governing mutualisms and their consequences for plant fitness and ecological dynamics.

Competitive fitness and stability of ammonium-excreting Azotobacter vinelandii strains in the soil.

Non-symbiotic N2-fixation would greatly increase the versatility of N-biofertilizers for sustainable agriculture. Genetic modification of diazotrophic bacteria has successfully enhanced NH4+ release. In this study, we compared the competitive fitness of A. vinelandii mutant strains, which allowed us to analyze the burden of NH4+ release under a broad dynamic range. Long-term competition assays under regular culture conditions confirmed a large burden for NH4+ release, exclusion by the wt strain, phenotypic instability, and loss of the ability to release NH4+. In contrast, co-inoculation in mild autoclaved soil showed a much longer co-existence with the wt strain and a stable NH4+ release phenotype. All genetically modified strains increased the N content and changed its chemical speciation in the soil. This study contributes one step forward towards bridging a knowledge gap between molecular biology laboratory research and the incorporation of N from the air into the soil in a molecular species suitable for plant nutrition, a crucial requirement for developing improved bacterial inoculants for economic and environmentally sustainable agriculture. KEY POINTS: • Genetic engineering for NH4+ excretion imposes a fitness burden on the culture medium • Large phenotypic instability for NH4+-excreting bacteria in culture medium • Lower fitness burden and phenotypic instability for NH4+-excreting bacteria in soil.

Guidelines for the description of rhizobial symbiovars.

Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.

Yield, nutrition, and leaf gas exchange of lettuce plants in a hydroponic system in response to Bacillus subtilis inoculation.

Inoculation with Bacillus subtilis is a promising approach to increase plant yield and nutrient acquisition. In this context, this study aimed to estimate the B. subtilis concentration that increases yield, gas exchange, and nutrition of lettuce plants in a hydroponic system. The research was carried out in a greenhouse in Ilha Solteira, Brazil. A randomized block design with five replications was adopted. The treatments consisted of B. subtilis concentrations in nutrient solution [0 mL "non-inoculated", 7.8 × 103, 15.6 × 103, 31.2 × 103, and 62.4 × 103 colony forming units (CFU) mL-1 of nutrient solution]. There was an increase of 20% and 19% in number of leaves and 22% and 25% in shoot fresh mass with B. subtilis concentrations of 15.6 × 103 and 31.2 × 103 CFU mL-1 as compared to the non-inoculated plants, respectively. Also, B. subtilis concentration at 31.2 × 103 CFU mL-1 increased net photosynthesis rate by 95%, intercellular CO2 concentration by 30%, and water use efficiency by 67% as compared to the non-inoculated treatments. The concentration of 7.8 × 103 CFU mL-1 improved shoot accumulation of Ca, Mg, and S by 109%, 74%, and 69%, when compared with non-inoculated plants, respectively. Inoculation with B. subtilis at 15.6 × 103 CFU mL-1 provided the highest fresh leaves yield while inoculation at 15.6 × 103 and 31.2 × 103 CFU mL-1 increased shoot fresh mass and number of leaves. Concentrations of 7.8 × 103 and 15.6 × 103 increased shoot K accumulation. The concentrations of 7.8 × 103, 15.6 × 103, and 31.2 × 103 CFU mL-1 increased shoot N accumulation in hydroponic lettuce plants.

Crop rotation and inoculation increase soil bradyrhizobia population, soybean grain yields, and profitability.

Crop rotation and rhizobial inoculation are strategies to increase yield by means of organic matter addition and modulation of microbial diversity. However, the extent to which these agricultural practices change soil Bradyrhizobium populations, soybean grain yield, and economic benefits to farmers is unclear. Thus, this study aimed to evaluate the interaction between crop rotation and inoculation of soybean (Glycine max) cultivated in two contrasting soils (clayey and sandy soil) on biological nitrogen fixation components, grain yields, and profits. Field experiments with a three-year crop rotation system were carried out to compare effects of inoculation and crop rotations on soil chemical attributes, bradyrhizobia most probable number (MPN) and diversity, soybean nodulation, grain yield, and economic indicators of inoculation in different crop rotations. The crop rotation did not affect the soil MPN cells of bradyrhizobia, but the inoculation and the soil sampling time did, ranging from 3.61-4.42 to 4.40-4.82 in the sandy soil, while in the clayey soil they were from 5.19-6.34 to 6.61-7.14 in Log10 per g of soil with higher population after harvest of summer crops. In the clayey soil, crop rotation influenced soybean nodulation. The grain yield of inoculated soybean in the clayey soil was higher than that in the sandy soil. Soybean inoculation with Bradyrhizobium spp. increased the profitability of agricultural production systems by up to 45% in clayey soil and up to 7% in sandy soil.

The outstanding diversity of rhizobia microsymbionts of common bean (Phaseolus vulgaris L.) in Mato Grosso do Sul, central-western Brazil, revealing new Rhizobium species.

Common bean is considered a legume of great socioeconomic importance, capable of establishing symbioses with a wide variety of rhizobial species. However, the legume has also been recognized for its low efficiency in fixing atmospheric nitrogen. Brazil is a hotspot of biodiversity, and in a previous study, we identified 13 strains isolated from common bean (Phaseolus vulgaris) nodules in three biomes of Mato Grosso do Sul state, central-western Brazil, that might represent new phylogenetic groups, deserving further polyphasic characterization. The phylogenetic tree of the 16S rRNA gene split the 13 strains into two large clades, seven in the R. etli and six in the R. tropici clade. The MLSA with four housekeeping genes (glnII, gyrB, recA, and rpoA) confirmed the phylogenetic allocation. Genomic comparisons indicated eight strains in five putative new species and the remaining five as R. phaseoli. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparing the putative new species and the closest neighbors ranged from 81.84 to 92.50% and 24.0 to 50.7%, respectively. Other phenotypic, genotypic, and symbiotic features were evaluated. Interestingly, some strains of both R. etli and R. tropici clades lost their nodulation capacity. The data support the description of the new species Rhizobium cerradonense sp. nov. (CNPSo 3464T), Rhizobium atlanticum sp. nov. (CNPSo 3490T), Rhizobium aureum sp. nov. (CNPSo 3968T), Rhizobium pantanalense sp. nov. (CNPSo 4039T), and Rhizobium centroccidentale sp. nov. (CNPSo 4062T).

Genomes of two type strains of the Rhizobium tropici group: R. calliandrae CCGE524T and R. mayense CCGE526T.

The genome sequences of two nitrogen-fixing type strains of the Rhizobium tropici group were obtained: Rhizobium calliandrae CCGE524T and R. mayense CCGE526T. Genomic analyses confirmed their taxonomic position and identified three complete sequences of the repABC genes, indicative of three plasmids, one of them carrying symbiotic genes.

CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

Studies Using Mutant Strains of Azospirillum brasilense Reveal That Atmospheric Nitrogen Fixation and Auxin Production Are Light Dependent Processes.

As the use of microbial inoculants in agriculture rises, it becomes important to understand how the environment may influence microbial ability to promote plant growth. This work examines whether there are light dependencies in the biological functions of Azospirillum brasilense, a commercialized prolific grass-root colonizer. Though classically defined as non-phototrophic, A. brasilense possesses photoreceptors that could perceive light conducted through its host's roots. Here, we examined the light dependency of atmospheric biological nitrogen fixation (BNF) and auxin biosynthesis along with supporting processes including ATP biosynthesis, and iron and manganese uptake. Functional mutants of A. brasilense were studied in light and dark environments: HM053 (high BNF and auxin production), ipdC (capable of BNF, deficient in auxin production), and FP10 (capable of auxin production, deficient in BNF). HM053 exhibited the highest rate of nitrogenase activity with the greatest light dependency comparing iterations in light and dark environments. The ipdC mutant showed similar behavior with relatively lower nitrogenase activity observed, while FP10 did not show a light dependency. Auxin biosynthesis showed strong light dependencies in HM053 and FP10 strains, but not for ipdC. Ferrous iron is involved in BNF, and a light dependency was observed for microbial 59Fe2+ uptake in HM053 and ipdC, but not FP10. Surprisingly, a light dependency for 52Mn2+ uptake was only observed in ipdC. Finally, ATP biosynthesis was sensitive to light across all three mutants favoring blue light over red light compared to darkness with observed ATP levels in descending order for HM053 > ipdC > FP10.

Screening Digitaria eriantha cv. Suvernola Endophytic Bacteria for Maize Growth Promotion.

The search for sustainable agriculture has increased interest in using endophytic bacteria to reduce fertilizer use and increase stress resilience. Stress-adapted plants are a potential source of these bacteria. Some species of these plants have not yet been evaluated for this, such as pangolão grass, from which we considered endophytic bacteria as potential plant growth promoters. Bacteria from the root, colm, leaves, and rhizospheric soil were isolated, and 132 strains were evaluated for their in vitro biological nitrogen fixation, IAA and siderophores production, and phosphate solubilization. Each mechanism was also assessed under low N availability, water stress, and low-solubility Fe and P sources in maize greenhouse experiments. All strains synthesized IAA; 63 grew on N-free media, 114 synthesized siderophores, and 46 solubilized P, while 19 presented all four mechanisms. Overall, these strains had better performance than commercial inoculant in all experiments. Still, in vitro responses were not good predictors of in vivo effects, which indicates that the former should not be used for strain selection, since this could lead to not testing strains with good plant growth promotion potential. Their heterologous growth promotion in maize reinforces the potential of stress-adapted plant species as potential sources of strains for inoculants.

Plant Growth-Promoting Rhizobacteria for Sustainable Agricultural Production.

Rhizosheric bacteria with several abilities related to plant growth and health have been denominated Plant Growth-Promoting Rhizobacteria (PGPR). PGPR promote plant growth through several modes of action, be it directly or indirectly. The benefits provided by these bacteria can include increased nutrient availability, phytohormone production, shoot and root development, protection against several phytopathogens, and reduced diseases. Additionally, PGPR can help plants to withstand abiotic stresses such as salinity and drought and produce enzymes that detoxify plants from heavy metals. PGPR have become an important strategy in sustainable agriculture due to the possibility of reducing synthetic fertilizers and pesticides, promoting plant growth and health, and enhancing soil quality. There are many studies related to PGPR in the literature. However, this review highlights the studies that used PGPR for sustainable production in a practical way, making it possible to reduce the use of fertilizers such as phosphorus and nitrogen and fungicides, and to improve nutrient uptake. This review addresses topics such as unconventional fertilizers, seed microbiome for rhizospheric colonization, rhizospheric microorganisms, nitrogen fixation for reducing chemical fertilizers, phosphorus solubilizing and mineralizing, and siderophore and phytohormone production for reducing the use of fungicides and pesticides for sustainable agriculture.

Competitiveness and Phylogenetic Relationship of Rhizobial Strains with Different Symbiotic Efficiency in Trifolium repens: Conversion of Parasitic into Non-Parasitic Rhizobia by Natural Symbiotic Gene Transfer.

In Uruguayan soils, populations of native and naturalized rhizobia nodulate white clover. These populations include efficient rhizobia but also parasitic strains, which compete for nodule occupancy and hinder optimal nitrogen fixation by the grassland. Nodulation competitiveness assays using gusA-tagged strains proved a high nodule occupancy by the inoculant strain U204, but this was lower than the strains with intermediate efficiencies, U268 and U1116. Clover biomass production only decreased when the parasitic strain UP3 was in a 99:1 ratio with U204, but not when UP3 was at equal or lower numbers than U204. Based on phylogenetic analyses, strains with different efficiencies did not cluster together, and U1116 grouped with the parasitic strains. Our results suggest symbiotic gene transfer from an effective strain to U1116, thereby improving its symbiotic efficiency. Genome sequencing of U268 and U204 strains allowed us to assign them to species Rhizobium redzepovicii, the first report of this species nodulating clover, and Rhizobium leguminosarun, respectively. We also report the presence of hrrP- and sapA-like genes in the genomes of WSM597, U204, and U268 strains, which are related to symbiotic efficiency in rhizobia. Interestingly, we report here chromosomally located hrrP-like genes.

Contribution of Biofertilizers to Pulse Crops: From Single-Strain Inoculants to New Technologies Based on Microbiomes Strategies.

Pulses provide distinct health benefits due to their low fat content and high protein and fiber contents. Their grain production reaches approximately 93,210 × 103 tons per year. Pulses benefit from the symbiosis with atmospheric N2-fixing bacteria, which increases productivity and reduces the need for N fertilizers, thus contributing to mitigation of environmental impact mitigation. Additionally, the root region harbors a rich microbial community with multiple traits related to plant growth promotion, such as nutrient increase and tolerance enhancement to abiotic or biotic stresses. We reviewed the eight most common pulses accounting for almost 90% of world production: common beans, chickpeas, peas, cowpeas, mung beans, lentils, broad beans, and pigeon peas. We focused on updated information considering both single-rhizobial inoculation and co-inoculation with plant growth-promoting rhizobacteria. We found approximately 80 microbial taxa with PGPR traits, mainly Bacillus sp., B. subtilis, Pseudomonas sp., P. fluorescens, and arbuscular mycorrhizal fungi, and that contributed to improve plant growth and yield under different conditions. In addition, new data on root, nodule, rhizosphere, and seed microbiomes point to strategies that can be used to design new generations of biofertilizers, highlighting the importance of microorganisms for productive pulse systems.

Not just passengers, but co-pilots! Non-rhizobial nodule-associated bacteria promote cowpea growth and symbiosis with (brady)rhizobia.

AIMS: To isolate and characterize non-rhizobial nodule-associated bacteria (NAB) from cowpea root-nodules regarding their performance of plant-growth-promoting mechanisms and their ability to enhance cowpea growth and symbiosis when co-inoculated with bradyrhizobia. METHODS AND RESULTS: Sixteen NAB were isolated, identified, and in vitro evaluated for plant growth promotion traits. The ability to promote cowpea growth was analyzed when co-inoculated with Bradyrhizobium pachyrhizi BR 3262 in sterile and non-sterile substrates. The 16S rRNA gene sequences analysis revealed that NAB belonged to the genera Chryseobacterium (4), Bacillus (3), Microbacterium (3), Agrobacterium (1), Escherichia (1), Delftia (1), Pelomonas (1), Sphingomonas (1), and Staphylococcus (1). All strains produced different amounts of auxin siderophores and formed biofilms. Twelve out of the 16 strains carried the nifH, a gene associated with nitrogen fixation. Co-inoculation of NAB (ESA 424 and ESA 29) with Bradyrhizobium pachyrhizi BR 3262 significantly promoted cowpea growth, especially after simultaneous inoculation with the three strains. CONCLUSIONS: NAB are efficient cowpea growth promoters and can improve the efficiency of the symbiosis between cowpea and the N2-fixing microsymbiont B. pachyrhizi BR 3262, mainly under a specific triple microbial association.

Diversity of rhizobia, symbiotic effectiveness, and potential of inoculation in Acacia mearnsii seedling production.

Black wattle (Acacia mearnsii) is a forest species of significant economic importance in southern Brazil; as a legume, it forms symbiotic associations with rhizobia, fixing atmospheric nitrogen. Nonetheless, little is known about native rhizobia in soils where the species is cultivated. Therefore, this study aimed to evaluate the diversity and symbiotic efficiency of rhizobia nodulating A. mearnsii in commercial planting areas and validate the efficiency of a potential strain in promoting seedling development. To this end, nodules were collected from four A. mearnsii commercial plantations located in Rio Grande do Sul State, southern Brazil. A total of 80 rhizobia isolates were obtained from black wattle nodules, and thirteen clusters were obtained by rep-PCR. Higher genetic diversity was found within the rhizobial populations from the Duas Figueiras (H' = 2.224) and Seival (H' = 2.112) plantations. Twelve isolates were evaluated belonging to the genus Bradyrhizobium, especially to the species Bradyrhizobium guangdongense. The principal component analysis indicated an association between rhizobia diversity and the content of clay, Ca, Mg, and K. Isolates and reference strains (SEMIA 6163 and 6164) induced nodulation and fixed N via symbiosis with black wattle plants after 60 days of germination. The isolates DF2.4, DF2.3, DF3.3, SEMIA 6164, SEMIA 6163, CA4.3, OV3.4, and OV1.4 showed shoot nitrogen accumulation values similar to the N + control treatment. In the second experiment (under nursery conditions), inoculation with the reference strain SEMIA 6164 generally improved the growth of A. mearnsii seedlings, reinforcing its efficiency even under production conditions.

Polyphasic characterization of rhizobial isolates obtained from different common bean-producing regions

In Brazil, the common bean is a crop with significant social and economic importance. The prospecting of N2 fixing bacteria is crucial since biological nitrogen fixation (BNF) is an eco-friendly technique. This work aimed to obtain and characterize rhizobium isolates based on morpho-physiological, molecular, and symbiotic efficiency parameters, using the strains SEMIA 4077, SEMIA 4080, and SEMIA 4088 as references. The characteristics of the isolates and colonies, their tolerance to salinity and temperature, as well as their utilization of carbon sources, served as the basis for the morpho-physiological characterization. BOX-PCR, REP-PCR, and ERIC-PCR markers were used for genotypic characterization. Assessment of the symbiotic efficiency was carried out in a greenhouse, determining the number of nodules (NN), nodule dry weight (NDW), shoot dry weight (SDW), and total-N (Total-N) accumulation in the shoot. Among the isolates, those exhibiting: neutral culture medium pH, fast growth, colony diameter <2 mm, opaque transparency, homogeneous appearance, and cream color were predominant. Compared to temperature, salinity was the most restrictive factor to the growth of the isolates. Most of the isolates grew on sucrose (88.43%) and mannitol (87.28%). Genotypic analysis revealed that 90% of the isolates clustered in the same group as the reference strain SEMIA 4080. The TaMsG2R1 and BaDeG4R2 isolates showed higher Total-N in the shoot than the reference strains and should be evaluated in future studies under field conditions.