Elkanii Strains Research Articles

Developing a genomic-based strategy to confirm microbial identity in bio-inputs containing multiple strains: an easy, fast, and low-cost multiplex PCR applied to inoculants carrying soybean Bradyrhizobium

Brazil stands out in research, industrial development, and farmers' use of microbial inoculants, with an emphasis on getting benefits from the biological nitrogen fixation process with the soybean crop. Nowadays, about 140 million doses of inoculants are commercialized annually for the soybean in the country, and strain identification is achieved by rep-PCR, an effective but time-consuming method. Aiming to develop an easy, low-cost, and low-time-consuming method, we used a complete genome-based approach based on the unequivocal identification of unique genes present in the genomes of each of the four Bradyrhizobium strains used in commercial inoculants: Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019, Bradyrhizobium japonicum SEMIA 5079, and Bradyrhizobium diazoefficiens SEMIA 5080. The unique pairs of primers able to amplify genomic regions of different sizes allowed the identification of the four strains in a simple multiplex polymerase chain reaction (PCR). Validation was confirmed by using single colonies, multiple cultures, and commercial inoculants. The number of labor hours of a technician was 3.08 times higher, and the final cost was 3.25 times higher in the rep-PCR than in the multiplex PCR. Most importantly, the results for multiplex PCR were obtained on the same day, in contrast with 15 days in the traditional methodology. The genomic approach developed can be easily applied to a variety of microbial inoculants worldwide, in addition to studies of ecology and evaluation of the competitiveness of the strains.Graphical

High efficient broad-spectrum Bradyrhizobium elkanii Y63-1

Soybean (Glycine max), the primary source of high-quality plant protein, plays a crucial role as a grain and oil crop in China. Harnessing the full potential of symbiotic nitrogen fixation in soybean production holds immense significance for agriculture and ecology alike. Zhongdou 63, a newly developed early-maturing summer soybean cultivar in 2021, exhibits remarkable traits such as high yield, superior quality, multi-resistance, and wide adaptability. In this study, eight distinct rhizobia strains from diverse regions were meticulously screened to identify highly effective strains specifically suited for Zhongdou 63. The aboveground biomass, plant height, chlorophyll content, root length, nodule number, and nodule dry weight of Zhongdou 63 were measured and the data were subjected to statistical analysis. The results demonstrated that Y63-1 is a predominant strain of Zhongdou 63. Subsequently, we conducted further investigations on the broad-spectrum nodulation characteristics of Y63-1. Ten representative soybean cultivars were individually inoculated with Y63-1 and subsequently analyzed for nodule numbers and nodule dry weight in their symbiotic systems with rhizobia. The findings revealed that Y63-1 effectively formed nodules with all ten soybean varieties tested. In summary, our current study identified highly efficient broad-spectrum Bradyrhizobium elkanii strain Y63-1 as the predominant strain in Zhongdou 63 and provided a theoretical foundation for enhancing yield potential not only in Zhongdou 63 but also in other varieties through inoculation with highly efficient rhizobia in production.

Analysis of the importance of the inoculation technique in brazilian soybean crop

Soybean (Glycine max) is the most important aleuro-oleaginous cultivated in the world, being undoubtedly the crop that had most advanced in Brazilian agriculture, due to a series of joint factors such as the increase in productivity, producer efficiency, new technologies, especially in the southern and central regions of Brazil. For the 2020/2021 harvest, Brazilian soybean production is expected to break a new record, reaching 131 million tons. The use of bacteria that promote plant development helps to reduce cultivation costs, being an alternative to increase soybean crop yield. In the Brazilian agricultural scenario, soybean is able to maintain high yields in commercial crops inoculated with Bradyrhizobium japonicum and B. elkanii strains through the symbiosis between leguminous plants and N2 fixing bacteria. Thus, the objective of this revisional work is to investigate the inoculation technique in Brazilian soybean production and analyze its advances in the last decade. Inoculation is one of the most important practices in soybean cultivation, so the producer must inoculate the soybean to fix N2 and thus help in the production of nitrogen compounds for the plant, achieving greater productivity. Finally, the scientific studies analyzed show that the inoculation technique in Brazilian soybean farming has evolved a lot in the last years, significantly contributing to the increased production and productivity of the soybean crop.

Isolation and characterization of Bradyrhizobium elkanii as a root nodule symbiont of red sword bean Canavalia gladiata var. gladiata

ABSTRACT Bacterial strains were isolated from root nodules of red sword bean (Canavalia gladiata var. gladiata) cultivated in Shizuoka, Japan, in order to elucidate the taxonomy of the symbionts. Of the 52 bacterial isolates, 28 strains were identified as the genus Bradyrhizobium and 10 as Rhizobium, based on the nucleotide sequences of 16S rRNA genes. Nine Bradyrhizobium isolates, which were phylogenetically selected from the 28 strains, exhibited nucleotide sequences of ITS regions that were 99.9% or 100% identical with known B. elkanii strains. These nine strains shared more than 70% similarity with B. elkanii USDA 76 T or USDA 94 in DNA-DNA hybridization analysis, indicating that the strains are B. elkanii. In a nodule formation experiment using red sword bean seeds treated with mercury chloride solution, all of the plants which were inoculated with each of the B. elkanii strains (the isolate TI06 or MI08, or the type strain USDA 76 T) formed round-type root nodules (234–664 nodules per plant), while no nodules were observed in control plants that were cultivated without inoculating bacterial strains. The bacterial strains, which were isolated from the obtained nodules, exhibited nucleotide sequences of the ITS regions that were identical to those of the corresponding inoculated strains. The root nodules formed in the experiment exhibited acetylene-reducing activity, suggesting the nitrogen-fixation activity of the nodules. We thus conclude that B. elkanii is a root nodule symbiont of red sword bean.

Growth and Competitive Infection Behaviors of Bradyrhizobium japonicum and Bradyrhizobium elkanii at Different Temperatures

Growth and competitive infection behaviors of two sets of Bradyrhizobium spp. strains were examined at different temperatures to explain strain-specific soybean nodulation under local climate conditions. Each set consisted of three strains—B. japonicum Hh 16-9 (Bj11-1), B. japonicum Hh 16-25 (Bj11-2), and B. elkanii Hk 16-7 (BeL7); and B. japonicum Kh 16-43 (Bj10J-2), B. japonicum Kh 16-64 (Bj10J-4), and B. elkanii Kh 16-7 (BeL7)—which were isolated from the soybean nodules cultivated in Fukagawa and Miyazaki soils, respectively. The growth of each strain was evaluated in Yeast Mannitol (YM) liquid medium at 15, 20, 25, 30, and 35 °C with shaking at 125 rpm for one week while measuring their OD660 daily. In the competitive infection experiment, each set of the strains was inoculated in sterilized vermiculite followed by sowing surface-sterilized soybean seeds, and they were cultivated at 20/18 °C and 30/28 °C in a 16/8 h (day/night) cycle in a phytotron for three weeks, then nodule compositions were determined based on the partial 16S-23R rRNA internal transcribes spacer (ITS) gene sequence of DNA extracted from the nodules. The optimum growth temperatures were at 15–20 °C for all B. japonicum strains, while they were at 25–35 °C for all B. elkanii strains. In the competitive experiment with the Fukagawa strains, Bj11-1 and BeL7 dominated in the nodules at the low and high temperatures, respectively. In the Miyazaki strains, BjS10J-2 and BeL7 dominated at the low and high temperatures, respectively. It can be assumed that temperature of soil affects rhizobia growth in rhizospheres and could be a reason for the different competitive properties of B. japonicum and B. elkanii strains at different temperatures. In addition, competitive infection was suggested between the B. japonicum strains.

Searching for Myanmar indigenous Bradyrhizobium type C strains that best identify Rj 4 genotypes in soybean

ABSTRACT Bradyrhizobium species are symbiotic partners of soybean plants. However, some Bradyrhizobium bacteria do not form functional nodules on the roots of Rj 4 genotype soybean cultivars. Our objective was to identify the strains of Bradyrhizobium (i.e., type C strains) that are least competent to form nodules on the roots of this plant genotype. We checked (i) previously isolated type C strains of Myanmar Bradyrhizobium elkanii (MMY6-1, MMY6-2, and MMY6-5), (ii) previously isolated type C strains of Myanmarese Bradyrhizobium spp. (MMY3-5 and MMY3-7), and (iii) strain Is-34 of B. japonicum, for nodule formation when associated with Rj 4 and other Rj genotype soybeans. Strains in groups (i), (ii), and (iii) are known to be incompatible with Rj 4 soybean genotypes. MMY3-5 and MMY3-7 produced functional nodules when associated with Rj 4 and other Rj genotype soybean cultivars, except Hill (Rj 4) cultivar. The ratios of ineffective nodule numbers/total nodule numbers (I/T ratios) for MMY6-1, MMY6-2, MMY6-5, and Is-34 in association with Rj 4 soybean cultivars were > 0.5, demonstrating incompatibility between these bacterial strains and the Rj 4 genotype. Interestingly, the I/T ratios of MMY6-1 and MMY6-2 were higher than that of Is-34 in almost all Rj 4 soybean cultivars. Thus, the nodule-forming abilities of the B. elkanii strains MMY6-1 and MMY6-2 were strongly suppressed in Rj 4 soybean cultivars; these strains may therefore be useful to identify the Rj 4 genotype in soybean cultivars.

Isolation and characterization of rhizobia from nodules of Clitoria ternatea in Thailand.

Rhizobia were isolated from the root nodules of Clitoria ternatea in Thailand. The phylogeny of the isolates was investigated using 16S rDNA and the internal transcribed spacer (ITS) region from 16S to 23S rDNA. The phylogenetic tree of the 16S rDNA showed that ten of the eleven isolates belonged to Bradyrhizobium elkanii, and one belonged to Bradyrhizobium japonicum. The topology of the ITS tree was similar to that of 16S rDNA. The acetylene reduction activity was higher for the nodules inoculated with the isolated B. elkanii strains than for those inoculated with B. japonicum strains. When C. ternatea plants were inoculated with various Bradyrhizobium USDA strains isolated from Glycine max, C. ternatea formed many effective nodules with B. elkanii, especially USDA61. However, acetylene reduction activity per plant and the growth were higher in C. ternatea inoculated with our isolates. From these data we propose that effective rhizobia inoculant were identified for C. ternatea cultivation.

Genetic diversity of indigenous soybean-nodulating Bradyrhizobium elkanii from southern Japan and Nueva Ecija, Philippines

Understanding the factors that influence the diversity of soybean-nodulating rhizobia is important before doing inoculation. Since studies about this topic in tropical regions are limited, this could lay the groundwork for related research particularly on Bradyrhizobium elkanii. To determine the genetic diversity of B. elkanii in different regions, we conducted Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and sequence analysis of 16S rRNA gene, internal transcribed spacer (ITS) region and rpoB gene. Also, sequence analysis of symbiotic nifD and nodD1 genes was conducted. Analysis of the rpoB gene revealed a higher genetic diversity than the ITS region, and possible endemic B. elkanii strains were observed. Meanwhile, no variation was detected among the strains in both nifD and nodD1 phylogenies. Through rpoB gene analysis, variations in the ITS-rpoB type of B. elkanii strains were distinguished and differentiated with that of the closest reference strains. We identified potential soybean inoculants which possess symbiotic efficiency regardless of the Rj genotypes used, suggesting broad host-range of the strains. We show how the genetic diversity of soybean-nodulating B. elkanii strains in subtropical and tropical regions might be influenced by temperature and soil pH and, provided some insights between the symbiotic genes and Rj genotypes.

Incompatible Nodulation of <i>Bradyrhizobium elkanii</i> Strains BLY3-8 and BLY6-1 with <i>Rj</i>3 Gene-Harboring Soybean Cultivars

Bradyrhizobia are known symbiotic partners of soybean. However, some soybean cultivars restrict nodulation by some Bradyrhizobium bacterial strains. These restrictions are related to compatibility between the Rj genes of soybean cultivars and nodulation types of inoculated bacteria. The objective of this study was to determine nodulation incompatibility of Type B strains with Rj3 soybean cultivars. Newly isolated B. elkanii strains BLY3-8 and BLY6-1 from Myanmar and specific strain Bradyrhizobium elkanii USDA33, which are incompatible with Rj3 soybean cultivars, and B. japonicum USDA110 were used as inoculants to check compatibility or incompatibility with Rj3 soybean cultivars. Nitrogen fixation activity was measured by the acetylene reduction method. Ethylene concentration (reduction of acetylene) was determined by flame ionization gas chromatography. According to the inoculation test results, USDA110 was compatible with all soybean cultivars because it formed effective nodules (Figure S1 in Appendix) and possessed nitrogenase activity. Similarly, B. elkanii strains BLY3-8, BLY6-1, and USDA33 were highly compatible with non-Rj and Rj4-gene harboring soybean cultivars because they had the ability to form functional nodules and possessed nitrogenase activity. Inversely, BLY3-8, BLY6-1, and USDA33 were incompatible with Rj3 soybean cultivars because they produced ineffective nodules. Consequently, the ratio of ineffective nodule number to total nodule number was >0.5. Therefore, nodule formation by the newly isolated B. elkanii strains BLY3-8 and BLY6-1 was restricted by the Rj3 soybean cultivars potentially making them useful as specific strains to detect the Rj3 gene in soybean cultivars.

Rj4, a Gene Controlling Nodulation Specificity in Soybeans, Encodes a Thaumatin-Like Protein But Not the One Previously Reported.

Rj4 is a dominant gene in soybeans (Glycine max) that restricts nodulation by many strains of Bradyrhizobium elkanii. The soybean-B. elkanii symbiosis has a low nitrogen-fixation efficiency, but B. elkanii strains are highly competitive for nodulation; thus, cultivars harboring an Rj4 allele are considered favorable. Cloning the Rj4 gene is the first step in understanding the molecular basis of Rj4-mediated nodulation restriction and facilitates the development of molecular tools for genetic improvement of nitrogen fixation in soybeans. We finely mapped the Rj4 locus within a small genomic region on soybean chromosome 1, and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We demonstrated that Rj4 encodes a thaumatin-like protein, for which a corresponding allele is not present in the surveyed rj4 genotypes, including the reference genome Williams 82. Our conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (previously annotated as Glyma01g37060). Instead, we provide convincing evidence that Rj4 is Glyma.01g165800-D, a duplicated and unique version of Glyma.01g165800, that has evolved the ability to control symbiotic specificity.

Phylogeny and nitrogen fixation potential of Bradyrhizobium species isolated from the legume cover crop Pueraria phaseoloides (Roxb.) Benth. in Eastern Cameroon

Pueraria is an herbaceous, perennial legume crop originating in Asia. Pueraria phaseoloides (Roxb.) Benth. (tropical kudzu) is frequently introduced into production systems and is used as green manure, a cover crop and a forage plant, making it important economically. We used P. phaseoloides as a trap crop to study and characterize soil rhizobia in Eastern Cameroon. Bacteria were isolated from fresh nodules collected from field-grown P. phaseoloides roots. The 16S-23S rRNA internal transcribed spacer (ITS) sequences from 30 bacterial isolates were amplified by polymerase chain reaction (PCR) and the reaction products were sequenced. Phylogenetic analysis revealed that all isolates were ascribed to the genus Bradyrhizobium and were grouped into three clusters of Bradyrhizobium sp. strains, one cluster of B. yuanmingense strains, and one cluster of B. elkanii strains. Acetylene reduction assay (ARA) results indicated that the B. yuanmingense strains had significantly higher nitrogen fixation potential and that they could be used as inoculants to enhance nitrogen fixation in Pueraria grown in Eastern Cameroon.

Emulsification Properties of Bioemulsifiers Produced by Wild-Type and Mutant Bradyrhizobium elkanii Strains

The use of biosurfactant is a promising alternative over the chemical surfactant as they are better biodegradable and do not pollute the environment. To date, there is little information on the biosurfactant producing microorganisms belonging to different Rhizobium and Bradyrhizobium species. In this study, the emulsifying properties of both culture media and a substantially cell-free medium from bacterial cell cultures in which B. elkanii SEMIA 587 or mutant strains cultivated in defined media supplemented with sucrose were determined by measuring the emulsifying activity, which determined the ability of biosurfactant in forming oil-water emulsion, and emulsifying index (E24), which determined the capacity of surfactant in forming emulsions on different hydrophobic substrates (soybean oil, sunflower oil or diesel oil). The performance of strains as bio emulsifier were very contrasting as shown by their specific emulsification indexes with different substrates, beginning at 24 hours. Regarding emulsifying index for cells, best result of E24 was obtained by using sunflower oil for wild type and mutants with exception for 587::TnphoA31 that witch got better results in soybean oil. However, analysing using cell-free medium have shown higher E24 values mostly in soybean oil except for 587::TnphoA31 and 587::TnphoA50 which have performed better in diesel oil. This finding indicates a probable production of bioemulsificants that adhere to the cell wall of this bacterium and are extracellular. The same E24 value (79.17%) was observed in the case of soybean oil with culture medium from both the wild-type and the mutant strain 587::TnphoA50. The emulsification capacity was very sensitive to the temperature, pH and NaCl concentration changes. These results demonstrate that the Bradyrhizobium strains could be attractive for use in biosurfactants applications, these products is great due to growing demand for biodegradable and environmentally friendly analogues for synthetic chemicals.

Fine mapping of the Rj4 locus, a gene controlling nodulation specificity in soybean

Leguminous plants have the ability to make their own nitrogen fertilizer by forming a root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This biological process plays a critical role in sustainable agriculture because it reduces the need for external nitrogen input. One remarkable property of legume–rhizobial symbiosis is its high level of specificity, which occurs at both inter- and intra-species levels and takes place at multiple phases of the interaction, ranging from initial bacterial infection and nodulation to late nodule development associated with nitrogen fixation. Knowledge of the molecular mechanisms controlling symbiotic specificity will facilitate the development of new crop varieties with improved agronomic potential for nitrogen-fixing symbiosis. In this report, we describe fine mapping of the Rj4 locus, a gene controlling nodulation specificity in soybean (Glycine max). The Rj4 allele prevents the host plant from nodulation with many strains of Bradyrhizobium elkanii, which are frequently present in soils of the southeastern USA. Since B. elkanii strains are poor symbiotic partners of soybean, cultivars containing an Rj4 allele are considered favorable. We have delimited the Rj4 locus within a 57-kb genomic region on soybean chromosome 1. The data reported here will facilitate positional cloning of the Rj4 gene and the development of genetic markers for marker-assisted selection in soybean.

Characterization and Phylogenetic Analysis of Soybean Rhizobial Strains from Java and Sumatra

Twenty-seven and twenty-four soybean rhizobial isolates from Java and Sumatra, respectively,were characterized. Based on cross-inoculation, eight isolates from Java and nine from Sumatra could be grouped as soybean specific rhizobial species , while 19 isolates from Java and 15 from Sumatra were promiscuous. ARDRA of intergenic spacer region of 16S-23S rDNA showed that the isolates from Java were different from those from Sumatra. Six soybean specific isolates from Java and one from Sumatra were in the same cluster with the reference strain, Bradyrhizobium japonicum USDA 110, thus could be classified as B. japonicum. One soybean specific isolate from Java’s has a distinct position, while the other soybean specific isolate from Java was placed in another group dominated by isolates from Sumatra. The nineteen promiscuous isolates from Java were clustered in a different group. This group, together with the isolate with distinct position and the other group that were dominated by isolates from Sumatra, were distinct from B. japonicum USDA 110. Therefore it is tempting to speculate that they represent indigenous soybean rhizobial strains. Based on complete sequencing of the amplified 16S rDNA of 21 selected isolates, these isolates could be divided into three groups consisting of twelve Bradyrhizobium elkanii, eight Bradyrizobium japonicum and one Sinorhizobium fredii. Most of the B. elkanii strains were isolated from acid soils at Sitiung, West-Sumatra, while only two isolates were obtained from Java. Four isolates from Java, two isolates from Sitiung, and two isolates from Bukit Tinggi were identified as B. japonicum. One isolate from Java with a distinct position on the ARDRA was identified as S. fredii.

Expression and Functional Roles ofBradyrhizobium japonicumGenes Involved in the Utilization of Inorganic and Organic Sulfur Compounds in Free-Living and Symbiotic Conditions

Strains of Bradyrhizobium spp. form nitrogen-fixing symbioses with many legumes, including soybean. Although inorganic sulfur is preferred by bacteria in laboratory conditions, sulfur in agricultural soil is mainly present as sulfonates and sulfur esters. Here, we show that Bradyrhizobium japonicum and B. elkanii strains were able to utilize sulfate, cysteine, sulfonates, and sulfur-ester compounds as sole sulfur sources for growth. Expression and functional analysis revealed that two sets of gene clusters (bll6449 to bll6455 or bll7007 to bll7011) are important for utilization of sulfonates sulfur source. The bll6451 or bll7010 genes are also expressed in the symbiotic nodules. However, B. japonicum mutants defective in either of the sulfonate utilization operons were not affected for symbiosis with soybean, indicating the functional redundancy or availability of other sulfur sources in planta. In accordance, B. japonicum bacteroids possessed significant sulfatase activity. These results indicate that strains of Bradyrhizobium spp. likely use organosulfur compounds for growth and survival in soils, as well as for legume nodulation and nitrogen fixation.

Diversity and Biogeography of Rhizobia Isolated from Root Nodules of Glycine max Grown in Hebei Province, China

A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S-23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline-saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.

Application of representational difference analysis to identify genomic differences between Bradyrhizobium elkanii and B. Japonicum species

Bradyrhizobium elkanii is successfully used in the formulation of commercial inoculants and, together with B. japonicum, it fully supplies the plant nitrogen demands. Despite the similarity between B. japonicum and B. elkanii species, several works demonstrated genetic and physiological differences between them. In this work Representational Difference Analysis (RDA) was used for genomic comparison between B. elkanii SEMIA 587, a crop inoculant strain, and B. japonicum USDA 110, a reference strain. Two hundred sequences were obtained. From these, 46 sequences belonged exclusively to the genome of B. elkanii strain, and 154 showed similarity to sequences from B. japonicum genome. From the 46 sequences with no similarity to sequences from B. japonicum, 39 showed no similarity to sequences in public databases and seven showed similarity to sequences of genes coding for known proteins. These seven sequences were divided in three groups: similar to sequences from other Bradyrhizobium strains, similar to sequences from other nitrogen-fixing bacteria, and similar to sequences from non nitrogen-fixing bacteria. These new sequences could be used as DNA markers in order to investigate the rates of genetic material gain and loss in natural Bradyrhizobium strains.

Genetic variability of soybean bradyrhizobia populations under different soil managements

Due to their ecologic and economic importance, bradyrhizobia have been extensively studied in recent years. Since 1992, Bradyrhizobium elkanii SEMIA 587 and SEMIA 5019 and Bradyrhizobium japonicum SEMIA 5079 and SEMIA 5080 have been widely used in most Brazilian soybean fields. The objective of this work was to estimate the genetic variability of bradyrhizobial isolates recovered from soils under rhizobial inoculation and different soil managements. Only 25% of the isolates demonstrated high similarities to the original strains, and a strong correlation was obtained between the bradyrhizobial genetic variability and soil management. A high level of genetic diversity was observed both within isolates (H = 5.46) as well as among the different soil practices. Soil under no-tillage presented a higher bradyrhizobia diversity compared with bradyrhizobia isolated from soil under conventional tillage. Serological characterization also indicated that B. elkanii strains SEMIA 587 and SEMIA 5019 were more competitive and presented a higher nodular occupancy capacity than strains belonging to B. japonicum species in Southern Brazilian soils.

15N natural abundance of biologically fixed N2 in soybean is controlled more by the Bradyrhizobium strain than by the variety of the host plant

The 15 N natural abundance technique is one of those most easily applied ‘on farm’ to evaluate the contribution of biological N 2 fixation (BNF) to legume crops. When proportional BNF inputs are high, the accuracy of this technique is highly dependent on an accurate estimate of the 15 N abundance of the N derived from N 2 fixation (the ‘ B ’ value). The objective of this study was to determine the influence of soybean variety on ‘ B ’ value. Plants of five soybean varieties were inoculated separately with two Bradyrhizobium strains (one Bradyrhizobium japonicum and one Bradyrhizobium elkanii ) grown in pots of soil virtually free of bradyrhizobia capable of nodulating soybean. The proportion of N derived from BNF (%Ndfa) was estimated in separate pots where a small quantity of enriched 15 N ammonium sulphate was added. The %Ndfa was then used with the 15 N natural abundance data of the nodulated soybean and non-N 2 -fixing reference plants, to determine the ‘ B ’ value for each soybean variety/ Bradyrhizobium association. The varieties nodulated by the B. japonicum strain showed significantly greater N content and %Ndfa than those nodulated by the B. elkanii strain, and in all cases the ‘ B ’ value of the shoot tissue (‘ B s ’) was higher. The differences in ‘ B s ’ values between varieties nodulated by the same Bradyrhizobium strain were insignificant, indicating that this parameter is influenced much more by the Bradyrhizobium strain than by the variety of the host plant.

Application of representational difference analysis to identify genomic differences between Bradyrhizobium elkanii and B. Japonicum species

Bradyrhizobium elkanii is successfully used in the formulation of commercial inoculants and, together with B. japonicum, it fully supplies the plant nitrogen demands. Despite the similarity between B. japonicum and B. elkanii species, several works demonstrated genetic and physiological differences between them. In this work Representational Difference Analysis (RDA) was used for genomic comparison between B. elkanii SEMIA 587, a crop inoculant strain, and B. japonicum USDA 110, a reference strain. Two hundred sequences were obtained. From these, 46 sequences belonged exclusively to the genome of B. elkanii strain, and 154 showed similarity to sequences from B. japonicum genome. From the 46 sequences with no similarity to sequences from B. japonicum, 39 showed no similarity to sequences in public databases and seven showed similarity to sequences of genes coding for known proteins. These seven sequences were divided in three groups: similar to sequences from other Bradyrhizobium strains, similar to sequences from other nitrogen-fixing bacteria, and similar to sequences from non nitrogen-fixing bacteria. These new sequences could be used as DNA markers in order to investigate the rates of genetic material gain and loss in natural Bradyrhizobium strains.