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Soybean [Glycine max (L.) Merr.] is one of the most important and oldest crops. Due to its ability to form symbiotic interactions with nitrogen-fixing bacteria, it is a valuable source of nitrogen for agriculture and proteins for humans and livestock. In Europe, for instance, in Poland, the soybean cultivation area is still not large but is gradually increasing due to climate change. The lack of indigenous soybean microsymbionts in Polish soils forces the application of commercial strains to establish effective symbioses. Fast and reliable identification methods are needed to study the persistence, competitiveness, and dispersal of bradyrhizobia introduced as inocula. Our study aimed to apply real-time PCR coupled with high-resolution melting curve (HRM) analysis to detect and differentiate bacterial strains occupying soybean nodules. HRM-PCR was performed on crude extracts from nodules using primers specific for recA, a highly conserved nonsymbiotic gene. By comparing them with the reference strains, we were able to identify and assign Bradyrhiobium strains that had been introduced into field locations in Poland. In conclusion, HRM analysis was proven to be a fast and accurate method for identifying soybean microsymbionts and might be successfully used for identifying other legume-nodulating bacteria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bradyrhizobium is thought to be the largest and most diverse rhizobial genus, but this is not reflected in the number of described species. Although it was one of the first rhizobial genera recognised, its taxonomy remains complex. Various contemporary studies are showing that genome sequence information may simplify taxonomic decisions. Therefore, the growing availability of genomes for Bradyrhizobium will likely aid in the delineation and characterization of new species. In this study, we addressed two aims: first, we reviewed the availability and quality of available genomic resources for Bradyrhizobium. This was achieved by comparing genome sequences in terms of sequencing technologies used and estimated level of completeness for inclusion in genome-based phylogenetic analyses. Secondly, we utilized these genomes to investigate the taxonomic standing of Bradyrhizobium in light of its diverse lifestyles. Although genome sequences differed in terms of their quality and completeness, our data indicate that the use of these genome sequences is adequate for taxonomic purposes. By using these resources, we inferred a fully resolved, well-supported phylogeny. It separated Bradyrhizobium into seven lineages, three of which corresponded to the so-called supergroups known for the genus. Wide distribution of key lifestyle traits such as nodulation, nitrogen fixation and photosynthesis revealed that these traits have complicated evolutionary histories. We present the first robust Bradyrhizobium species phylogeny based on genome sequence information for investigating the evolution of this important assemblage of bacteria. Furthermore, this study provides the basis for using genome sequence information as a resource to make important taxonomic decisions, particularly at the species and genus levels. [ABSTRACT FROM AUTHOR]

Bradyrhizobium species are symbiotic partners of soybean plants. However, some Bradyrhizobium bacteria do not form functional nodules on the roots of Rj4 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 Rj4 and other Rj genotype soybeans. Strains in groups (i), (ii), and (iii) are known to be incompatible with Rj4 soybean genotypes. MMY3-5 and MMY3-7 produced functional nodules when associated with Rj4 and other Rj genotype soybean cultivars, except Hill (Rj4) 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 Rj4 soybean cultivars were > 0.5, demonstrating incompatibility between these bacterial strains and the Rj4 genotype. Interestingly, the I/T ratios of MMY6-1 and MMY6-2 were higher than that of Is-34 in almost all Rj4 soybean cultivars. Thus, the nodule-forming abilities of the B. elkanii strains MMY6-1 and MMY6-2 were strongly suppressed in Rj4 soybean cultivars; these strains may therefore be useful to identify the Rj4 genotype in soybean cultivars. [ABSTRACT FROM AUTHOR]

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.

Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean (Glycine max cv. BARC2) and unable to nodulate either plant. This incompatibility is due to the presence of a functional type III secretion system (T3SS) that translocates effector protein into host cells. We previously identified five genes in B. elkanii that are responsible for its incompatibility with KPS1 plants. Among them, a novel gene designated as innB exhibited some characteristics associated with the T3SS and was found to be responsible for the restriction of nodulation on KPS1. In the present study, we further characterized innB by analysis of gene expression, protein secretion, and symbiotic phenotypes. The innB gene was found to encode a hypothetical protein that is highly conserved among T3SS-harboring rhizobia. Similar to other rhizobial T3SS-associated genes, the expression of innB was dependent on plant flavonoids and a transcriptional regulator TtsI. The InnB protein was secreted via the T3SS and was not essential for secretion of other nodulation outer proteins. In addition, T3SS-dependent translocation of InnB into nodule cells was confirmed by an adenylate cyclase assay. According to inoculation tests using several Vigna species, InnB promoted nodulation of at least one V. mungo cultivar. These results indicate that innB encodes a novel type III effector controlling symbiosis with Vigna species.

The cowpea is an important food crop in the North and Northeast regions of Brazil, where the beans are consumed either green or ripe. Yet, considering its socio-economic importance and its tropical origin, cowpea yields are low in those regions, due to inadequate cultivation practices and incorrect soil management. Therefore, the objective of this study was to study the development of cowpea crop inoculated and fertilized with two different nitrogen (N) sources. The experiment was conducted in the municipality of Rorainópolis (RR). The experimental design was randomized blocks with four treatments and five replications. The plant material was 'BRS Guariba' cowpea cultivar, and the treatments were: (i) control (no nitrogen fertilization), (ii) seeds inoculated with Bradyrhizobium elkanii, (iii) urea as N source (60 kg N ha-1), and (iv) ammonium sulphate as N source (60 kg N ha-1). The following crop traits were evaluated: foliar macronutrients (N, P, K, Ca, Mg and S), green and dry weight of shoots, dry beans per plant, pod yield and bean yield. Urea and ammonium sulphate promoted high accumulation of nutrients in leaves. Ammonium sulphate also stood out regarding productivity traits, thus proving to be a viable N source for cowpea in the Amazon region. The nodulation with Bradyrhizobium elkanii wasn't efficient to replace the fertilization with nitrogen fertilizers for cowpea BRS Guariba.

The establishment of a root nodule symbiosis between a leguminous plant and a rhizobium requires complex molecular interactions between the two partners. Compatible interactions lead to the formation of nitrogen-fixing nodules, however, some legumes exhibit incompatibility with specific rhizobial strains and restrict nodulation by the strains. Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean cultivars carrying the Rj4 allele. Here, we explored genetic loci in USDA61 that determine incompatibility with V. radiata KPS1. We identified five novel B. elkanii genes that contribute to this incompatibility. Four of these genes also control incompatibility with soybean cultivars carrying the Rj4 allele, suggesting that a common mechanism underlies nodulation restriction in both legumes. The fifth gene encodes a hypothetical protein that contains a tts box in its promoter region. The tts box is conserved in genes encoding the type III secretion system (T3SS), which is known for its delivery of virulence effectors by pathogenic bacteria. These findings revealed both common and unique genes that are involved in the incompatibility of B. elkanii with mung bean and soybean. Of particular interest is the novel T3SS-related gene, which causes incompatibility specifically with mung bean cv. KPS1.The establishment of a root nodule symbiosis between a leguminous plant and a rhizobium requires complex molecular interactions between the two partners. Compatible interactions lead to the formation of nitrogen-fixing nodules, however, some legumes exhibit incompatibility with specific rhizobial strains and restrict nodulation by the strains. Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean cultivars carrying the Rj4 allele. Here, we explored genetic loci in USDA61 that determine incompatibility with V. radiata KPS1. We identified five novel B. elkanii genes that contribute to this incompatibility. Four of these genes also control incompatibility with soybean cultivars carrying the Rj4 allele, suggesting that a common mechanism underlies nodulation restriction in both legumes. The fifth gene encodes a hypothetical protein that contains a tts box in its promoter region. The tts box is conserved in genes encoding the type III secretion system (T3SS), which is known for its delivery of virulence effectors by pathogenic bacteria. These findings revealed both common and unique genes that are involved in the incompatibility of B. elkanii with mung bean and soybean. Of particular interest is the novel T3SS-related gene, which causes incompatibility specifically with mung bean cv. KPS1. [ABSTRACT FROM AUTHOR]

Nitrogen (N) and phosphorus (P) are nutrients that frequently limit plant performance and crop yield. Currently, awareness of the importance of plant-microbe interactions in plant nutrient acquisition and growth promotion persist, particularly under stressful conditions, such as those resulting from P scarcity. The aim of the present study was to evaluate the effects of P starvation on the influence of arbuscular mycorrhiza (AM) symbiosis on N 2 -fixing capacities, plant growth, nutrient uptake and photosynthetic performance of soybean plants at flowering and grain filling stages. To this end, soybean ( Glycine max ) plants were nodulated with Bradyrhizobium elkanii and inoculated or not with the AM fungus Glomus macrocarpum . The +AM and −AM plants were grown under low P conditions (50 μM), and a control treatment with −AM plants under sufficient P concentration (500 μM) was also performed (−AM + P). Plants were grown until the beginning of the flowering and another set till the grain filling stage. The results showed that mycorrhization improved soybean plant performance under P scarcity compared with non-AM associated plants under the same conditions. AM positively influenced growth and plant nodulation at both flowering and grain-filling stages, without significant changes in plant P content. AM symbiosis increased nitrogenase activity, P content in nodules and leaf N content, the maximum carboxylation rate of Rubisco and reduced the metabolic limitation of photosynthesis under P starvation, which indicated the AM stimulus to symbiotic N 2 fixation (SNF) and photosynthesis. Under these conditions, mycorrhizal symbiosis could not completely meet soybean P demand compared with well-nourished soybean plants, which produced higher plant and nodules biomass. In conclusion, AM association in nodulated soybean plants is likely an important strategy for maintaining SNF functioning, conferring increased plant growth and better N status under P scarcity conditions and significantly attenuating the negative effects of low Pi in nodulated-soybean plants. [ABSTRACT FROM AUTHOR]

Aims: 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. Methods: 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. Results: 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. Conclusions: 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. [ABSTRACT FROM AUTHOR]

Controlled-release phosphate carriers based on hydrocalumite-like Layered Double Hydroxides (Ca-Al-LDHs) have been synthesized to serve as a P-source for Bradyrhizobium elkanii growth. [Ca-Al]-LDHs loaded with different phosphate contents were prepared by structural reconstruction (SR) and ion exchange (IE) and characterized by powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), adsorption experiments, solid-state nuclear magnetic resonance (NMR) and thermal analysis (TG/DTG). [Ca-Al-PO 4 3 − ]-LDHs comprising similar crystalline phases were formed regardless the phosphate uptake route. However, phosphate loadings were found to be higher in [Ca-Al-PO 4 3 − ]-LDH obtained by IE rather than by SR. This was related to differences in specific surface area between precursors used in each route. [Ca-Al]-LDH IE-33.10 mM significantly increased the growth of Bradyrhizobium elkanii , a nitrogen-fixing symbiotic bacterium widely used in crop (mainly soybean) plantations. Once [Ca-Al-PO 4 3 − ]-LDHs are eco-friendly materials, they have enormous potential to be used in leguminous crops and raise agriculture production. [ABSTRACT FROM AUTHOR]

A expansão da cultura da soja evidenciou uma alta especificidade hospedeira, requerendo a pesquisa de novas estirpes que apresentassem capacidade de nodular a soja e bom potencial de competição com a população de rizóbios naturalizada nos solos. O objetivo deste trabalho foi avaliar a especificidade hospedeira de variantes isolados de estirpes de Bradyrhizobium spp quanto à nodulação e eficiência relativa de fixação de N2 atmosférico em soja (cvs peking e clark), caupi e guandu. O experimento foi realizado sob condições controladas em câmara de crescimento por meio de testes de variantes de B. japonicum e B. elkanii e suas respectivas estirpes originais quanto à habilidade de nodular soja, caupi e guandu. A colheita foi realizada aos 35 dias, sendo avaliada a nodulação (número, peso dos nódulos secos), produção de matéria seca na parte aérea, eficiência relativa de fixação de N2 atmosférico. Os variantes e estirpes de Bradyrhizobium spp nodularam Glycine max (cultivares BR-16, Clark e Peking), Vigna unguiculata e Cajanus cajan, contudo, apenas para Glycine max a interação rizóbio-leguminosa demonstrou eficiência simbiótica significativa.The expansion of the soybean crop has evidenced a high host specificity, indicating the requirement of new strains with capacity of nodulating soybean and competition capacity with the natural soil rhizobial population. The purpose of this study was to evaluate the host specificity of mutants isolated from Bradyrhizobium spp strains in relation to nodulation and relative N2 fixation effectiveness in soybean (cultivars Peking and Clark), cowpea and pigeon pea. The experiment was carried out under controlled conditions in a growth chamber, where B. japonicum and B. elkanii mutants and the respective original strains were tested for their nodulation ability on soybean, cowpea and pigeon pea. The crop was harvested 35 days after planting and the nodulation (number, dry nodule weight), shoot dry matter production and relative N2 fixation effectiveness were evaluated. The mutants and strains of Bradyrhizobium spp nodulate Glycine max (cultivars BR-16, Clark and Peking), Vigna unguiculata and Cajanus cajan, however the interaction rhizobia-leguminous demonstrated significant symbiotic effectiveness for Glycine max only.

In commercial plantations of soybean in both the Southern and the Cerrado regions, contributions from biological nitrogen fixation (BNF) are generally proportionately high. When using the 15N natural abundance technique to quantify BNF inputs, it is essential to determine, with accuracy, the 15N abundance of the N derived from BNF (the 'B' value). This study aimed to determine the effect of four recommended strains of Bradyrhizobium spp. (two B. japonicum and two B. elkanii) on the 'B' value of soybean grown in pots in an open field using an equation based on the determination of δ15N natural abundance in a non-labelled soil, and estimate of the contribution of BNF derived from the use of 15N-isotope dilution in soils enriched with 15N. To evaluate N2 fixation by soybean, three non-N2-fixing reference crops were grown under the same conditions. Regardless of Bradyrhizobium strain, no differences were observed in dry matter, nodule weight and total N between labelled and non-labelled soil. The N2 fixation of the soybeans grown in the two soil conditions were similar. The mean 'B' values of the soybeans inoculated with the B. japonicum strains were -1.84 ‰ and -0.50 ‰, while those inoculated with B. elkanii were -3.67 ‰ and -1.0 ‰, for the shoot tissue and the whole plant, respectively. Finally, the 'B' value for the soybean crop varied considerably in function of the inoculated Bradyrhizobium strain, being most important when only the shoot tissue was utilised to estimate the proportion of N in the plant derived from N2 fixation.Em plantações comerciais de soja na região Sul e do Cerrado, as contribuições da fixação biológica de Nitrogênio (FBN) são geralmente elevadas. Quando usamos a técnica da abundância natural de 15N para quantificar a FBN, é essencial determinar com exatidão a abundância de 15N do N derivado da FBN (valor 'B'). Este trabalho buscou determinar o efeito das quatro estirpes de Bradyrhizobium spp. (duas B. japonicum, duas B. elkanii) sobre o valor 'B' de soja crescida em vasos em ambiente aberto usando uma equação na determinação da abundância natural de 15N em um solo não enriquecido com 15N, e estimativas da contribuição da FBN derivado do uso da técnica de diluição isotópica de 15N em solo enriquecido com 15N. Para avaliar a fixação de N2 pela soja três plantas referenciam foram crescidas nas mesmas condições. Independente da estirpe de Bradyrhizobium, não foi observada diferença para matéria seca, massa de nódulos e N total entre solo marcado e não marcado. A fixação de N2 em soja crescida nas duas condições de marcação do solo foi semelhante. Os valores médios de 'B' para plantas de soja inoculadas com estirpes de B. japonicum foram, em média, de -1,84 ‰ e -0,50 ‰ enquanto as inoculadas com B. elkanii apresentaram médias de -3,67 ‰ e -1,00 ‰, para parte aérea e planta inteira, respectivamente. Finalmente, o valor 'B' para a cultura da soja variou consideravelmente em função da estirpe de Bradyrhizobium inoculada, sendo mais importante quando se utiliza somente a parte aérea da planta para estimar a proporção do N da planta derivado da fixação de N2.

O objetivo deste trabalho foi avaliar as relações filogenéticas de estirpes de Bradyrhizobium e a contribuição destas estirpes para a fixação biológica de nitrogênio em caupi, em solos do Cerrado. Na avaliação da relação filogenética, o gene 16S rDNA de cada uma das estirpes foi amplificado e seqüenciado, e para a análise da eficiência simbiótica, determinou-se: N total, matéria seca das plantas, massa de nódulos e redução de acetileno, em casa de vegetação, e ocupação nodular, em experimento de campo. A maioria das estirpes estudadas pertence a B. elkanii e, pelo menos dez das estirpes, independentemente da espécie, apresentaram bom desempenho quanto à fixação biológica de N2. As estirpes BR3262, BR3280 (caracterizadas como B. elkanii) e BR3267, BR3287 e BR3288 (Bradyrhizobium sp.) mostram-se como inoculantes potenciais para o caupi, em razão do bom desempenho tanto na eficiência simbiótica quanto na ocupação nodular.The obejctive of this study was to evaluate the phylogenetic relationships of Bradyrhizobium strains, and the contribution of these strains to cowpea biological nitrogen fixation in Cerrado soils. To elucidate the phylogenetic relationships among strains, their 16S rDNA gene was extracted, amplified and sequenced. In order to evaluate the symbiotic efficiency, total N, plant dry matter, nodular mass and acetylene reduction in a greenhouse experiment, and nodular occupation, in a field experiment, were determined. Most part of the strains belonge to B. elkanii, and at least ten of the analyzed strains, independently of the species, presented high capacity to fix N2. The strains BR3262, BR3280 (characterized as B. elkanii), BR3267, BR3287 and BR3288 (Bradyrhizobium sp.) exhibit a potential as inoculant for cowpea, due to their high biological nitrogen fixation.

As culturas da soja [Glycine max (L.) Merrill] e do feijoeiro (Phaseolus vulgaris L.) são de grande importância econômica e social para o Brasil e ambas podem ter seu requerimento de nitrogênio suprido pela simbiose com bactérias da ordem Rhizobiales. Para garantir a maximização do processo biológico, deve-se proceder à inoculação de estirpes de rizóbio eficientes e competitivas, recomendadas pela pesquisa. No Brasil, foram comercializados, na safra 2001/2002, 14 milhões de doses de inoculantes, dos quais 99 % para as culturas da soja e do feijoeiro. Neste trabalho, determinou-se a posição taxonômica das estirpes utilizadas em inoculantes comerciais para as duas culturas, pelo seqüenciamento da região do DNA que codifica o gene 16S rRNA, que é suficientemente variável, mas carrega as informações necessárias para permitir a análise filogenética de bactérias. O seqüenciamento permitiu definir que duas das estirpes recomendadas para a cultura da soja, SEMIA 587 e SEMIA 5019 (= 29 w), pertencem à espécie Bradyrhizobium elkanii e as duas outras, SEMIA 5079 (=CPAC 15) e SEMIA 5080 (= CPAC 7), à espécie B. japonicum. Determinou-se, ainda, que a estirpe SEMIA 4080 (=PRF 81), recomendada para o cultura do feijoeiro, pertence à espécie Rhizobium tropici. As seqüências obtidas foram depositadas no banco mundial de genes do National Center for Biotechnology Information.Soybean [Glycine max (L.) Merrill] and common bean (Phaseolus vulgaris L.) crops are of economical and social importance in Brazil; their requirement for nitrogen can be supplied by the symbiosis with bacteria belonging to the order Rhizobiales. However, to guarantee the maximization of the biological nitrogen fixation, seeds must be inoculated with efficient and competitive strains of rhizobia recommended by research. In 2001/2002, 14 million doses of inoculant were sold in Brazil, 99 % of these for soybean and common bean crops. In this study the taxonomic position of the strains used in commercial inoculants for both crops was evaluated by sequencing the DNA region that carries the information for the 16S rRNA gene. Although variable, it codes enough information to allow a phylogenetic analysis of the bacteria. Sequencing determined that two of the strains recommended for the soybean crop, SEMIA 587 and SEMIA 5019 (= 29 w), belong to the Bradyrhizobium elkanii, while the two other, SEMIA 5079 (=CPAC 15) and SEMIA 5080 (=CPAC 7), belong to the B. japonicum species. Strain SEMIA 4080 (=PRF 81), recommended for common bean crop, was identified as member of the species Rhizobium tropici. The sequences were included in the GenBank database of the National Center for Biotechnology Information.

Background and aims: Bradyrhizobium japonicum and Bradyrhizobium elkanii dominated soybean nodules in temperate and subtropical regions in Nepal, respectively, in our previous study. The aims of this study were to reveal the effects of temperature on the nodulation dominancy of B. japonicum and B. elkanii and to clarify the relationship between the effects of temperature and the climate-dependent distribution of Bradyrhizobium species. Methods: A laboratory competition experiment was conducted between B. japonicum and B. elkanii strains isolated from the same temperate location in Nepal. A mixture of each strain was inoculated into sterilized vermiculite with or without soybean seeds, and inoculated samples were incubated at 33/27 (day/night) and 23/17 °C. Relative populations in the non-rhizosphere, rhizosphere, and nodules were determined by competitive PCR using specific primers for each strain at 0, 1, 2, and 4 weeks after inoculation. Results: Both separately inoculated B. japonicum and B. elkanii strains formed nodules at both temperatures. Under competitive conditions, B. japonicum strains dominated at low temperature; however, at high temperature, both strains achieved co-nodulation in 1 week, with B. elkanii dominating after 2 weeks. The relative populations of both strains were similar in the non-rhizosphere and rhizosphere at low temperature, but B. elkanii strains dominated in these regions at high temperature. Conclusions: The domination of B. japonicum strains in nodules at the low temperature appeared to be due to preferential infection, while the domination of B. elkanii strains at high temperature appeared to be due to the higher population of B. elkanii in the non-rhizosphere and rhizosphere, in addition to its domination in nodules after co-nodulation. The effects of temperature on the competition between B. japonicum and B. elkanii strains were remarkable and corresponded with the distribution of bradyrhizobial species in Nepal. [ABSTRACT FROM AUTHOR]

The article discusses role of Bradyrhizobium elkanii (B. elkanii), species of nitrogen-fixing bacterium and related strains in degradation of methoxychlor in soil and surface water environment. Topics discussed include degradation of methoxychlor by B. elkanii (USDA94) species, identification of microbial degraded pesticides and isolation of two strains, Bradyrhizobium sp. 4-1 and 17-4. It also mentions that bradyrhizobium consists of large number of cytochrome P450 genes.

Microbial conversion is an important technology for the refinement of renewable resources. Here, we describe the biotechnological conversion of glycerol to 2-amino-1,3-propanediol (serinol), a relevant intermediate in several chemical syntheses processes. Either the dihydroxyacetone phosphate aminotransferase/dihydrorhizobitoxine synthase (RtxA) of Bradyrhizobium elkanii USD94 or only the N-terminal domain (RtxA513) comprising the first reaction, respectively, was expressed in recombinant Escherichia coli. Serinol contents of up to 3.3 g/l were achieved in batch cultures. We could further clarify that glutamic acid is the preferred cosubstrate for the transamination of dihydroxyacetone phosphate to serinolphosphate, which is the essential step in serinol synthesis. An in vivo detoxification of serinol employing wax ester synthase/acyl-CoA:diacyl-glycerol acyl transferase from Acinetobacter baylyi ADP1 was not accomplished. This study paves the way for biotechnological production of serinol from glycerol derived from the biodiesel industry. [ABSTRACT FROM AUTHOR]

Cloning and sequencing of a 47.1-kb chromosomal DNA region revealed the presence of a type III secretion system (T3SS) in Bradyrhizobium elkanii USDA61. The identified genes are likely to encode the transcriptional activator TtsI, core components of the secretion apparatus and secreted proteins. Several ORFs within the cluster are not conserved in other rhizobia. Nine tts box motifs, a promoter element of TtsI-regulated genes, were found; six of them upstream of annotated genes. For functional analyses, the rhcC2 and rhcJ genes were disrupted. These mutations had a cultivar-specific effect on nodulation. Vigna radiata cv. KPS1 developed nodules if infected with the mutant strains but not with the wild type. In contrast, V. radiata cv. CN36 was nodulated by all strains. Nodulation of rj1 soybean depended on the T3SS. A comparison of the protein patterns from supernatants of the wild type and rhcJ mutant by two-dimensional gel electrophoresis revealed proteins that are secreted only in the wild-type background. These results show that B. elkanii encodes a functional T3SS that is involved in the interaction with host legumes. [ABSTRACT FROM AUTHOR]

Previously, restriction fragment length polymorphism analysis using the nodD1YABC gene probe showed the genetic diversity of common nodD1ABC gene regions of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and the Thai soybean Bradyrhizobium. The nodD1 sequences of representative strains of the 3 groups differed phylogenetically, suggesting that responses of NodD1 proteins of the 3 Bradyrhizobium groups to diverse flavonoids may differ. To confirm this hypothesis, 6 representative strains were chosen from the 3 Bradyrhizobium groups. Six reporter strains were constructed, all carrying the pZB32 plasmid, which contains a nod box and the nodY-lacZ fusion of B. japonicum USDA 110. Differences in nodY-lacZ expression among the strains in response to 37 flavonoid compounds at various concentrations were evaluated. Of those compounds, prunetin (4′,5-dihydroxy-7-methoxyisoflavone) and esculetin (6,7-dihydroxycoumarin) were identified as Bradyrhizobium group-specific nod gene inducers. Esculetin showed nod gene induction activities unique to Thai Bradyrhizobium strains. The levels of nodY-lacZ induction among B. japonicum and Thai Bradyrhizobium strains increased with increasing concentration of prunetin, whereas, those in B. elkanii strains did not. [ABSTRACT FROM AUTHOR]

We examined the genetic basis and transfer for production of rhizobitoxine, an inhibitor of ethylene biosynthesis in plants, directed by the rtx genes of Bradyrhizobium elkanii. Comparison with genome sequences of Bradyrhizobium japonicum and Xanthomonas oryzae suggests that the rtx genes extend from the previously identified rtxAC genes through four additional genes rtxDEFG. Reverse transcription-PCR analysis showed that the rtxACDEFG genes are expressed as an operon. Mutational analysis indicated that rtxDEG mutants reduced rhizobitoxine biosynthesis, while the rtxA gene is essential for its synthesis. Introduction of the rtxACDEFG into Agrobacterium tumefaciens resulted in strong expression of rtxACDEFG and production of RtxA protein, but no rhizobitoxine was detectable. Addition of O-acetylhomoserine, a precursor of rhizobitoxine, to the Agrobacterium derivative, however, fostered production of rhizobitoxine in culture. The diluted culture supernatant inhibited the activities of β-cystathionase and 1-aminocyclopropane-1-carboxylate synthase, indicating that A. tumefaciens carrying rtxACDEFG genes excreted biologically active rhizobitoxine. [ABSTRACT FROM AUTHOR]

Abstract: Bradyrhizobium elkanii produces rhizobitoxine, an enol-ether amino acid, which has been regarded as a phytotoxin because it causes chlorosis in soybeans. However, recent studies have revealed that rhizobitoxine plays a positive role in establishing symbiosis between B. elkanii and host legumes: rhizobitoxine enhances the nodulation process by inhibiting ACC (1-aminocyclopropane-1-carboxylate) synthase in the ethylene biosynthesis of host roots. B. elkanii rtxA and rtxC genes are required for rhizobitoxine production. In particular, rtxC gene is involved in the desaturation of dihydrorhizobitoxine into rhizobitoxine. A legume with a mutated ethylene receptor gene produced markedly higher numbers of rhizobial infection threads and nodule primordia. Thus, endogenous ethylene in legume roots negatively regulates the formation of nodule primordia, which is overcome by rhiozbitoxine. Although a plant pathogen Burkholderia andropogonis has been known to produce rhizobitoxine, the genome sequence of Xanthomonas oryzae showed the existence of a putative rhizobitoxine transposon in the genome. The cumulative evidence suggests that rhizobitoxine-producing bacteria modulate plant–microbe interactions via ethylene in the rhizosphere and phyllosphere environments. In addition, rhizobitoxine-producing capability might be utilized as tools in agriculture and biotechnology. [Copyright &y& Elsevier]

Regression analysis of results from a mathematical competition model showed that rhizobitoxine production by Bradyrhizobium elkanii USDA94 gave this strain a nodulation competitiveness about 10 times greater than that of a non-rhizobitoxine-producing mutant strain on Macroptilium atropurpureum (Siratro). Rhizobitoxine enhancement of competitive nodulation occurred at a late stage in the time-course of nodulation. All other known rhizobial factors that affect nodulation competitiveness act in the rhizosphere and during the initial interaction with legumes. This unique late action of rhizobitoxine could prove advantageous in inoculant production, because inoculum often fails to nodulate in the latter stages of nodulation kinetics. [Copyright &y& Elsevier]

Many undomesticated legumes harbor nodule bacteria related to the soybean symbiont Bradyrhizobium elkanii, but little is known about their phylogenetic relationships or geographic distribution. Sequences of ribosomal genes (16S rRNA and partial 23S rRNA) and the nitrogenase α-subunit gene (nifD) were analyzed in 22 isolates of this group sampled from diverse legumes in Korea, Japan, the USA, Mexico, Costa Rica and Panama. Some strains from Asia and North America shared identical sequences for both ribosomal genes. However, pairs of strains with closely related nifD sequences were almost never found in different regions. The major exceptions involved North American isolates B. elkanii USDA 76 and USDA 94, which had nifD sequences highly similar to certain Korean strains. However, 16S rRNA sequences of USDA 76 and USDA 94 were closely related to Central American rather than Asian bradyrhizobia, implying that these strains are genetic mosaics combining sequences from distinct ancestral areas. Several other conflicts between rRNA and nifD tree topologies indicated that the genealogical histories of these loci have been influenced by recurrent lateral gene transfer events. [Copyright &y& Elsevier]

Indole-3-acetaldehyde (IAAId) was detected in the culture supernatant of Bradyrhizobium elkanii. Deuteriumlabelled L-tryptophan (Trp) was incorporated into IAAId and indole-3-acetic acid (IAA), suggesting that B. elkanii produces IAA via IAAId from Trp. In B. elkanii cell suspension, indole-3-pyruvic acid (IPyA) was converted to IAAId, and exogenously added IAAId was rapidly converted to IAA. Furthermore, the activity of indolepyruvate decarboxylase (IPDC), which catalyzes the decarboxylation of IPyA to produce IAAId and is a key enzyme for IPyA pathway, was detected in B. elkanii cell-free extract. The IPDC activity depended on Mg2+ and thiamine pyrophosphate, cofactors of decarboxylation. This mounting evidence strongly suggests that IAA synthesis occurs via IPyA pathway (Trp → IPyA →p IAAId → IAA) in B. elkanii. [ABSTRACT FROM AUTHOR]

In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N2 fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N2 fixation and nodule efficiency (mg of N mg–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N2 fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N2 fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N2 fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N2 fixation to soybean nutrition. [ABSTRACT FROM AUTHOR]

Several years of research have shown that there is a high genetic and physiological variability among Bradyrhizobium japonicum strains, culminating in a subdivision into two bacterial genotypes, and the description of the new species B. elkanii. In Brazil, large-scale soybean inoculation started in 1960 and today 15 million doses of inoculants are sold per year for an estimated area of 12 million ha. Efforts have been made to find strains able to fix high amounts of N2 under Brazilian soil conditions, but few laboratories cover basic studies on N2 fixation, such as strain classification into the two Bradyrhizobium species. In this study several characteristics of 40 soybean Bradyrhizobium strains, including 4 reference strains of B. japonicum (genotype I) species, 3 of B. elkanii (genotype II) and 1 of a mixed genotype were evaluated. The parameters analysed in vitro were: colony morphology, serological grouping, intrinsic resistance to antibiotics, synthesis of indole acetic acid, expression of hydrogenase activity and growth in a medium enriched with asparagine. In vivo, analyses performed included the nodulation of Rj 4 soybean cultivar Hill and the detection of symptoms caused by rhizobitoxine. These evaluations allowed a phenotypic grouping which positioned most of the strains utilized in Brazilian inoculants and studies, as well as some new strains isolated from the Cerrado region, within the species B. elkanii. However, environmental stresses and adaptation of Bradyrhizobium strains to the soil caused a large physiological and genetic variability in some isolates from the Cerrado soils in relation to the putative parental strain introduced 15 years ago, placing these isolates in an intermediate position between the two Bradyrhizobium species. [ABSTRACT FROM AUTHOR]

The genome of Brahyrhizobium japonicum and B. elkanii contains multiple copies of the repeated DNA sequence RSα. A collection of 18 B. japonicum, 4 B. elkanii and 72 other bacterial strains was screened by polymerase chain reaction (PCR) using a pair of primers specific for RSα. Only strains of B. japonicum and B. elkanii gave the predicted amplification product. Restriction analysis of PCR products obtained from different strains of B. japonicum showed that the RSα sequence was generally conserved. The usefulness of RSα as a specific probe for Brahyrhizobium strains capable of nodulating soybean was also demonstrated. [ABSTRACT FROM AUTHOR]

Bradyrhizobium elkanii USDA61 possesses a functional type III secretion system (T3SS) that controls host-specific symbioses with legumes. Here, we demonstrated that B. elkanii T3SS is essential for the nodulation of several southern Asiatic Vigna mungo cultivars. Strikingly, inactivation of either Nod factor synthesis or T3SS in B. elkanii abolished nodulation of the V. mungo plants. Among the effectors, NopL was identified as a key determinant for T3SS-dependent symbiosis. Mutations of other effector genes, such as innB, nopP2, and bel2-5, also impacted symbiotic effectiveness, depending on host genotypes. The nopL deletion mutant formed no nodules on V. mungo, but infection thread formation was still maintained, thereby suggesting its pivotal role in nodule organogenesis. Phylogenetic analyses revealed that NopL was exclusively conserved among Bradyrhizobium and Sinorhizobium (Ensifer) species and showed a different phylogenetic lineage from T3SS. These findings suggest that V. mungo evolved a unique symbiotic signaling cascade that requires both NFs and T3Es (NopL). [ABSTRACT FROM AUTHOR]