Soybean [Glycine max (L.) Merr.] holds critical significance globally as a primary source of protein and oil [1] , contributing to a production exceeding 175 million tons across approximately 80 million hectares [2] . In Mexico, soybean cultivation is primarily practiced in rainfed production systems, encompassing an average of 164 thousand hectares over the last three years during both autumn-winter and spring-summer cycles. However, the average yield per hectare in Mexico remains modest, not surpassing 1.55 tons [3] [4] . Enhancing soybean productivity involves strategic interventions such as organic fertilization and the use of bioinoculants containing specific strains of the Bradyrhizobium genus [5] [6] . These measures have demonstrated effectiveness in increasing grain yield, both in rainfed and irrigated production systems [5] . The identification and selection of strains displaying high affinity and adaptation to soybean plants are crucial steps in optimizing productivity [7] . Molecular markers, particularly Multilocus Sequence Analysis (MLSA), have emerged as valuable tools for bacterial identification, enabling taxonomic assignments at the species level within the Bradyrhizobium genus.

Studies by Delamuta et al. [8] utilized MLSA, analyzing the 16S rRNA gene along with five housekeeping genes (recA, atpD, glnII, gyrB, and rpoB) to reliably identify strains of the Bradyrhizobium genus. This methodology proved effective in phylogenetic and taxonomic analyses of nitrogen-fixing rhizobia [8] . Similarly, Li et al. [9] , employed a polyphasic approach, combining the 16S rRNA gene and concatenated MLSA genes (glnII, recA, dnaK) alongside digital DNA-DNA hybridization (dDDH) in characterizing rhizobia isolates from peanut nodules (Arachis hypogaea). Their findings resulted in the identification of three new species: Bradyrhizobium nanningense sp. nov., Bradyrhizobium guangzhouense sp. nov., and Bradyrhizobium zhanjiangense sp. nov. These studies underscore the utility of MLSA in advancing our understanding of nitrogen-fixing rhizobia and optimizing symbiotic relationships for improved crop outcomes.

The MLSA method, leveraging the analysis of housekeeping genes, has proven to be a rapid and reliable approach for identifying strains at the species level within the Bradyrhizobium genus. This method facilitates the construction of phylogenetic and taxonomic trees, providing in-depth insights into the diversity of nitrogen-fixing rhizobia [10] [11] . Notable studies, such as the one conducted in Brazil by Kruschewsky et al. [12] , utilized MLSA to analyze the diversity of rhizobium strains isolated from root nodules of different Inga Mill. species. The study employed various gene sequences, including dnaK, recA, rpoB, gyrB, glnII, 16S rRNA, internal transcribed spacer regions (ITS), and symbiosis-associated genes nodC and nifH. MLSA combined with the ITS region yielded the best genetic structure, revealing four main groups of Bradyrhizobium strains.

Recent applications of MLSA have extended to the identification and characterization of new Bradyrhizobium species isolated from nodules of forage legumes in Australia and South Africa [13] . Researchers have adopted a polyphasic approach in the identification of bacterial isolates, integrating MLSA with other techniques such as PCR-RFLP, phenotypic characteristics, fatty acid analysis, 16S rRNA gene sequencing, and whole-genome sequencing [10] [11] [14] [15] .

The study of bacterial isolates from soybean root nodules, particularly those capable of symbiotic nitrogen fixation, is a recurrent theme. Common molecular tools include the 16S rRNA gene, MLSA genes (atpD, recA, glnII, rpoB, dnaK), and nifH genes associated with biological nitrogen fixation [16] . In Brazil, researchers have established a database of MLSA sequences for the Bradyrhizobium genus, facilitating species-level studies [17] . In light of this, the primary objective of the current study is to employ MLSA for the molecular identification of strains isolated from root nodules of commercial soybean varieties cultivated in the southern region of the state of Tamaulipas, Mexico.

1) Soil sample and root nodules collection. Sampling locations: The bacterial isolates were obtained from soils in soybean-producing plots located in Altamira and El Mante municipalities, Tamaulipas, Mexico. Additionally, a plot without agricultural use in Reynosa, Tamaulipas, Mexico, was included (refer to Table 1). The soil sampling procedure was carried out as shown followers. A composite soil sample was created by combining five 2.5 kg subsamples. These were taken radially within one-hectare plots at a depth of 40 cm. Subsamples included one from the center and four from the plot edges. The composite sample underwent physical-chemical soil analysis following the Mexican standard NOM-021-RECNAT (2000) [18] .

2) Root nodule collection. Nodules were collected from the commercial varieties Huasteca-200, Huasteca-300, Huasteca-400, Tamesi, and Vernal, adapted for production in the state of Tamaulipas, Mexico. The greenhouse experiment for nodule collection was established as follows. In the spring of 2019, soybean materials were planted in triplicate in soil collected from sterilized plastic pots. Each experimental unit comprised 12 seeds per pot per repetition. To prevent cross-contamination, seeds were washed with 1% sodium hypochlorite and

were collected at the beginning of the plant’s flowering stage (R1 reproductive rinsed with sterile deionized water before planting. nodules stage), characterized by increased development and biological activity of atmospheric nitrogen fixation. Morphologically, nodules exhibited a pinkish-reddish color [19] .

3) Preprocessing of nodules and isolate acquisition. Nodules were disinfected using a 0.1% sodium hypochlorite solution [19] . Subsequently, they were macerated individually in 1.5 mL Eppendorf tubes with a drop of sterile deionized water per nodule. A 50 µL aliquot of the resulting macerate, diluted to 1 × 10⁻⁵, was evenly spread onto Extract Yeast-Mannitol-Agar (ELMA) modified with soil extract. The medium was supplemented with Bromothymol Blue (ABT) (0.5 g/100mL ethanol) as a pH indicator and dispensed into Petri dishes in triplicate [20] . Strain selection was based on the medium’s coloration. Yellow indicated fast-growing rhizobia (1 - 3 days), typically genus Rhizobium, while blue indicated slow-growing rhizobia (5 - 10 days), generally genus Bradyrhizobium. Purity was confirmed on yeast extract-mannitol agar (YEMA) medium. Gram staining was performed to exclude Gram-positive motile bacilli. Out of the 287 isolated strains, 81 were chosen based on macroscopic and microscopic characteristics, including color, appearance, shape, and texture [21] . Additionally, the infectivity rate of each strain in greenhouse conditions was assessed by the number of nodules formed in the roots. Interestingly, the CE soil exhibited a higher abundance of strains with characteristics typical of the Bradyrhizobium genus.

4) Multilocus Sequence Analysis (MLSA). Pure bacterial cultures were grown in Extract Yeast-Mannitol-Agar (ELMA) broth at a density of 10⁹ cells per mL. For each 15 µL of bacterial culture, 3 µL of 0.01% Tween was added and incubated at 65˚C for 20 minutes. The MLSA housekeeping genes (recA, glnII, dnaK, gyrB, and atpD) were amplified according to the protocol outlined in Table 2. Each amplification reaction consisted of a 14 µL mixture, including 7 µL of PCR Master Mix 2× (Promega™, USA), 2 µL of nuclease-free water (Promega™, USA), 1 µL of each primer pair, and 3 µL of template DNA. Amplifications were performed using a thermal cycler (C1000 Touch BIO-RAD, USA) under the conditions specified in Table 1. Amplicons were visualized through 1% agarose gel electrophoresis (Promega™, USA) and confirmed using the DNA Ladder Marker 100 bp (Promega™, USA) on a BIO-RAD Molecular Imegen^®-GEL DOC™ XR image documenter (Bio-Rad Laboratories Inc.). The amplicons of each gene were purified using the commercial ExoSAP-IT^® kit (AFFYMETRIX; Cleveland, USA). Sequencing reactions were carried out at Eurofins MWG from Operon USA (Thermo Fisher Scientific).

5) Bioinformatic analysis and diversity. The MLSA gene sequences were concatenated and subjected to taxonomic analysis using the database for the taxonomic and phylogenetic identification of the genus Bradyrhizobium

(http://mlsa.cnpso.embrapa.br/). The platform employs sequence analysis multilocus for taxonomic assignment, following the methodology outlined by Azevedo et al. [17] . Phylogenetic analysis was conducted using the MEGA Ver 6.0

(Molecular Evolutionary Genetics Analysis) program. The sequences of the 81 strains were aligned with four reference sequences (Bj_USDA110, Bj_USDA6, Bradyrhizobium_sp., and Rhizobium_sp.) using MUSCLE [26] . Phylogenetic trees were constructed using the Kimura 2-parameter method for calculating evolutionary distances [27] . The maximum likelihood method was applied with resampling of 10,000 replicates for robustness [28] .

The conclusion highlights the efficacy of MLSA as a valuable tool for investigating taxonomic relationships among bacterial species in the genus Bradyrhizobium by concatenating housekeeping genes, this method provided a high level of resolution for interspecific differences and divergence, enabling precise identification. In the context of this study, it revealed a significant presence of the species Bradyrhizobium japonicum USDA110 in soils of soybean-producing plots. Importantly, the analysis also showcased diversity at the strain level, with no clear association observed between identified species and the soil of origin. This underscores the richness and complexity of rhizobial populations in the studied agricultural soils. In practical terms, understanding the diversity and prevalence of specific Bradyrhizobium species, such as Bradyrhizobium japonicum USDA110, has implications for soybean cultivation. The absence of a clear association between species and soil collection suggests that various factors, including soybean varieties and local soil conditions, contribute to the diversity of rhizobial populations. This complexity should be considered in agricultural practices, especially in regions where soybean is a vital crop.

Expresses gratitude to CONAHCYT (Consejo Nacional de Humanidades, Ciencias y Tecnologías) for the support provided through the project TAMPS-2013-C45-201410. Additionally, Cecilia Vazquez Rodriguez expresses thanks to CONACHYT and IPN (Instituto Politécnico Nacional) for the scholarship awarded for her Master of Science training in Genomic Biotechnology. This acknowledgment recognizes the financial and educational support that has contributed to the successful completion of the research and academic endeavors.

The authors declare no conflicts of interest regarding the publication of this paper.

Vázquez Rodríguez, C., Vital López, L., García Olivares, J.G. and Gill Langarica, H.R. (2024) Identification of Rhizobia Isolated from Nodules of Mexican Commercial Soybean Varieties. American Journal of Plant Sciences, 15, 29-45. https://doi.org/10.4236/ajps.2024.151003