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Albizia adianthifolia (Schumach) W. F. Wright, a N-fixing legume tree, has a wide distribution in Africa, in Ghana occurring in high rainfall forests and in seasonally droughted forests, and is associated in the Ghanaian forest zone with dry, infertile sites. We hypothesised that A. adianthifolia hosted different rhizobial strains in different forest types, and that these different strains would show different growth responses to moisture stress and different motility and mortality in droughted soil. Three isolates, extracted from seedlings of A. adianthifolia growing in three forest types differing in seasonal drought, were identified as Bradyrhizobium elkanii and exposed to varying levels of osmotic stress. Growth responses varied between the three strains, one of which displayed clear signs of drought tolerance. A novel approach using soil leaching columns was used to test the effects of soil pore water (in terms of neck diameter) on both the survival and movement of wet and dry forest rhizobial isolates through soil columns. The responses of the isolates were significantly different, the pore neck diameter, marginally insignificant and the drought treatment insignificant. Thus the dry forest isolate survived better in all treatments, and showed less response to the treatments, than the isolate from the wet forest. The results offer preliminary evidence that Bradyrhizobium elkanii strains from A. adianthifolia in Ghana have evolved in response to local differences in seasonal water availability. These differences could assist the selection of A. adianthifolia provenances for agroforestry or land rehabilitation.

Genetic diversity and distribution of indigenous soybean-nodulating bradyrhizobia in Japan were investigated based on restriction fragment length polymorphism analysis of PCR product (PCR-RFLP) analysis of the 16S−23S rDNA internal transcribed spacer (ITS) region using Bradyrhizobium USDA strains as reference strains. Soil samples were collected from five field sites in Hokkaido, Fukushima, Kyoto, Miyazaki and Okinawa in Japan. A total of 300 isolates were derived from three Rj-genotype soybean cultivars, Akishirome (non-Rj), CNS (Rj2Rj3) and Fukuyutaka (Rj4), and five field site combinations. The PCR products of the ITS region were digested with HaeIII, HhaI, MspI and XspI. Electrophoresed patterns were analyzed for phylogenetic relationship using Bradyrhizobium reference strains. Results revealed 22 RFLP patterns and 11 clusters. The RFLP patterns of the seven clusters were similar or identical to Bradyrhizobium japonicum USDA 6, 38, 110, 115, 123 and Bradyrhizobium elkanii USDA 76 and 94. Four minor clusters were independent from the clusters of the reference strains. The isolation ratio revealed the major clusters at each field site. These results suggested that major clusters of indigenous bradyrhizobia might be in the order Bj123, Bj38, Bj110, Bj6 and Be76 from the northern to southern regions in Japan.

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.

International audience; The distribution of rhcRST genes encoding the type III secretion system (T3SS) in a collection of Bradyrhizobium strains was characterized by PCR and Southern blot hybridization. The polymorphism of the corresponding sequences amplified by PCR was characterized by RFLP and sequencing together with those available in the databank. Genomic group I is characterized by the presence of Bradyrhizobium elkanii strains and group II by the presence of B. japonicum and B. liaoningense strains. Highly conserved T3SS-like genes were detected by PCR in all Bradyrhizobium strains isolated from soybean belonging to genomic group II, and in none of the strains belonging to genomic group I. These data were confirmed by Southern blot hybridization that further indicated the presence of sequences showing similarity to the rhcRST sequence in B. elkanii strains. The high level of conservation of rhcRST among Bradyrhizobia of genomic group II and sharing the same host-plant suggests that T3SS-like genes might have undergone horizontal genetic transfer within this genomic group. When considering the three Rhizobiaceae genera, a clear congruence was recorded between the rhcRST, rRNA gene and ITS sequences in bacteria harbouring sequences encoding T3SS, suggesting a relatively ancient emergence of the T3SS in these genera.

Biosolubilization of rock phosphate (RP) using a Penicillium spp ., an Aspergillus spp ., Pleurotus ostreatus , Bradyrhizobium elkanii SEMIA 5019 and their fungal–rhizobial biofilms was investigated. Eppawala Rock Phosphate (ERP, total P concentration 17.6%), a RP from a deposit in Sri Lanka was used. Penicillium spp.– B elkanii SEMIA 5019 biofilm released the highest amount of P from the ERP with the highest P release-to-P uptake ratio. The P release of Penicillium spp. alone was significantly lower than that of its biofilm. Similarly, P. ostreatus – B. elkanii SEMIA 5019 biofilm showed a higher P release than P. ostreatus alone. However, P. ostreatus alone or its biofilm showed lower P release-to-P uptake ratios indicating relatively higher P uptake compared to the P release. The Aspergillus spp ., showed a moderate P release. Large bradyrhizobial cell clusters attached to the mycelial mat of Penicillium spp. and P. ostreatus were observed under light microscope after 12 and 25 days of incubation, respectively. The present study, identified an effective method of fungal–rhizobial biofilm mediated solubilization of RP.

Os efeitos do herbicida Roundup, formulado à base de glifosato, foram avaliados sobre três estirpes de Bradyrhizobium elkanii (BR 29, INPA 80A e INPA 553A), e uma de B. japonicum (BR 86), e sobre três espécies de fungos micorrízicos arbusculares (FMA) (Gigaspora margarita, Glomus etunicatum e Scutellospora heterogama), em meios de cultivo com concentrações crescentes do herbicida (0 a 454 µmol L-1); foram também avaliados os efeitos sobre a nodulação e micorrização da soja, em casa de vegetação, em solo que recebeu, antes da semeadura, doses do herbicida equivalentes a 1,25 até 10 L ha-1. O Roundup mostrou-se inibitório ao crescimento de Bradyrhizobium spp. e aos fungos em meio de cultura, e esse efeito foi crescente com o aumento das concentrações aplicadas, tendo variado em razão das espécies ou estirpes avaliadas. No entanto, a inibição in vitro só ocorreu em concentrações muito superiores à dose recomendada para aplicações no campo. As estirpes BR 29, INPA 553A e INPA 80A mostraram-se mais tolerantes ao glifosato, em relação à estirpe BR 86. O efeito do herbicida sobre a germinação e o crescimento dos tubos germinativos dos esporos dos FMA foi diferenciado, tendo sido observada inibição decrescente de G. etunicatum para S. heterogama e G. margarita. A aplicação do herbicida ao solo, antes da semeadura, até a dose equivalente a 10 L ha-1 não influenciou na nodulação e na colonização micorrízica da soja. The effects of the Roundup herbicide on three strains of Bradyrhizobium elkanii (BR 29, INPA 80A and INPA 553A), one of B. japonicum (BR 86), and on three species of arbuscular mycorrhizal fungi (AMF) (Gigaspora margarita,Glomus etunicatum and Scutellospora heterogama), were evaluated in culture media containing increasing concentrations of the herbicide (0_454 µM); evaluations were also made on the effects on nodulation and mycorrhiza colonization of soybean grown in a soil treated with Roundup doses equivalent to 1.25 to 10 L ha-1 before sowing. The herbicide inhibited growth of Bradyrhizobium spp. and AMF in culture medium. These effects were directly related to increasing concentrations, and varied depending on the strain and species evaluated. However, in vitro inhibition occurred only when concentrations were greater than that recommended for use in the field. Strains BR 29, INPA 553A and INPA 80A showed to be more resistant to glyphosate, when compared to BR 86. Herbicide inhibition on germination and growth of AMF spore germ tubes decreased from G. etunicatum to S. heterogama and G. margarita. Soil application of Roundup before sowing up to a dose equivalent to 10 L ha-1 had no effect on nodulation and mycorrhiza colonization of soybean.

Soybean bradyrhizobia, Bradyrhizobium japonicum and Bradyrhizobium elkanii differ in various traits such as DNA fingerprint, rhizobitoxine production, indole-3-acetic acid production and uptake of hydrogenase. In this communication, we investigated whether the differences between both species extend to host preference in multistrain environments. Nodule occupancy of B.japonicum and B. elkanii significantly depended on host plants. B. japonicum and B. elkanii preferentially nodulated Glycine max and M. atropurpureum, respectively. Both bacterial species were shown to nodulate G. soja with similar efficiency. There was a significant divergence of DNA sequences in and around nodulation genes between the B. japonicum and B. elkanii field isolates. However, flavonoid and lipo-chitin nodulation signals were not involved in the difference in host preference of B. japonicum and B. elkanii. The ecological implications of host preference are discussed.

In order to contribute for the optimization of biological nitrogen fixation (BNF) associated with cowpea in Cerrado areas in the Northeast region of Brazil, this work aimed to analyze the diversity of rhizobial populations in eight areas of Cerrado, during a soybean and rice-cowpea rotation. Morphological traits (mucous production and colony morphology), genotypic analyzes (ARDRA 16S) and intrinsic resistance to antibiotics were determined for a collection of isolates captured using cowpea as a host-plant. The morphological data showed a inverse correlation (p < 0.05) between the number of legume (soybean and cowpea) crops, according to the history of each area, and rhizobium diversity, estimated by the Shannon-Weaver index. ARDRA data showed that native Cerrado areas were exclusively colonized by Bradyrhizobium elkanii, corroborating previous data. In the areas where legumes were grown, we observed two distinct situations: where soybean only were grown, a high proportion of B. japonicum was found, and where soybean and cowpea were grown, we observed more B. elkanii. The analysis of antibiotic resistance revealed five different profiles. High percentage of antibiotic resistant Bradyrhizobium spp. isolates were found in the areas cultivated for a long time, whereas the native area and areas with a few crops had fewer resistant strain. There was an inverse relationship between intrinsic antibiotic resistance and rhizobial diversity, while the last decreases as more legume crops are introduced into the area, the former increases, suggesting that the presence of legumes may provide ecological conditions to select specific rhizobium groups, which acquire competitiveness traits and become successfully established.

A soja pode ser considerada uma alternativa viável para cultivo em solos hidromórficos. O sucesso da cultura, porém, depende do funcionamento do processo de fixação biológica do N2. Sabe-se que a fixação biológica de N2 é, geralmente, prejudicada pela inundação do solo, dependendo do genótipo do macrossimbionte. Não se conhece, no entanto, o comportamento, em solo inundado, das diferentes estirpes de Bradyrhizobium elkanii e Bradyrhizobium japonicum recomendadas para a soja. O presente trabalho teve como objetivo avaliar a eficiência das estirpes recomendadas para a cultura da soja, em solo submetido à inundação, em comparação com a adubação nitrogenada. O experimento em casa de vegetação foi realizado em vasos com solo, nos quais se fez a semeadura de soja da variedade CEP 20. Os tratamentos consistiram na aplicação de dois níveis de umidade ao solo – 100 % da capacidade de campo e inundado com cerca de 1 cm de lâmina de água – e de duas formas de fornecimento de N – inoculação com as estirpes de rizóbio SEMIA 587, SEMIA 5019, SEMIA 5079 ou SEMIA 5080 e aplicação de 100 mg kg-1 de N mineral. A inundação do solo foi aplicada 14 dias após a emergência, tendo sido mantida por 30 dias. Foram avaliados o número de nódulos, a massa seca de nódulos e da parte aérea das plantas de soja, aos 46 e aos 75 dias da emergência. A quantidade de N na parte aérea foi avaliada aos 75 dias da emergência. Nas duas avaliações, o número e a massa de nódulos foram reduzidos com a inundação do solo. Não houve diferenças na massa seca da parte aérea das plantas inoculadas com as estirpes, as quais apresentaram rendimentos inferiores aos do tratamento com adubação nitrogenada. Na segunda avaliação, porém, o tratamento com adubação nitrogenada apresentou menor rendimento de massa seca no solo que havia sido inundado, em comparação com o mesmo tratamento em capacidade de campo. A quantidade de N acumulada na parte aérea também foi reduzida no solo submetido à inundação. A partir dos resultados obtidos, pôde-se concluir que a inundação do solo prejudicou o processo de fixação biológica de N2 em plantas de soja. Todavia, após a remoção do excesso de umidade, houve recuperação da nodulação e da fixação biológica de N2 em soja inoculada com Bradyrhizobium. As plantas adubadas com fertilizante nitrogenado mineral também foram influenciadas pela inundação. Soybean can be a viable alternative crop for lowlands. However, soybean relies on biological nitrogen fixation, a process that is normally affected by soil flooding, depending on the macrosymbiont genotype. Moreover, the behavior of different bradyrhizobia strains in flooded soil is yet unknown. The present research aimed to evaluate the efficiency of Bradyrhizobium elkanii and Bradyrhizobium japonicum strains recommended for soybean in comparison to mineral nitrogen fertilization, in flooded soil. The experiment was carried out in a greenhouse, where soybean cultivar CEP 20 was sown in pots with soil. The treatments applied were either inoculation with one of the Bradyrhizobia strains: SEMIA 587, SEMIA 5019, SEMIA 5079 or SEMIA 5080, or the application of 100 mg kg-1 mineral nitrogen at two soil moisture levels - 100% of field capacity or flooded with a 1 cm-water layer on the soil surface. The soil was flooded 14 days after plant emergence and it was maintained under such conditions for 30 days. The nodule number and dry matter, and shoot dry matter were evaluated at 46 and 75 days after plant emergence. The amount of accumulated nitrogen in the soybean shoot was determined 75 days after plant emergence. Soil flooding reduced the nodule number and dry mass in both evaluation periods. There was no difference in the soybean shoot dry matter among the plants inoculated with distinct bradyrhizobia strains, which accumulated less dry matter than the treatment with mineral nitrogen. However, in the second evaluation period the treatment with mineral nitrogen accumulated less dry matter in the flooded soil as compared to the same treatment in soil at field capacity. The amount of nitrogen accumulated in the shoot was also lower in the flooded soil. Results show that the process of nitrogen fixation is affected by soil flooding, hobut, when waterlogging cease nodulation and biological nitrogen fixation is recovered. Plants fertilized with mineral nitrogen are also affected by soil flooding.

Four decades of soybean [Glycine max (L.) Merr.] cultivation in South Africa has resulted in the establishment of populations of bradyrhizobia against which the recently introduced inoculant strain CB 1809 must compete. Serological and DNA fingerprinting methods were used to study the diversity of nodule isolates from soils at Bergville, Koedoeskop and Morgenzon. Dominant serogroups included Bradyrhizobium elkanii serotype 76 at Bergville (67%), Bradyrhizobium japonicum serotype 123 at Morgenzon (81%) and B. japonicum serotype 135 at Koedoeskop (100%). Their origin is unknown as they do not correspond in serotype to strains used in previous inoculants. A small percentage of isolates from Bergville (13%) and Morgenzon (16%) were serologically homologous to strain WB 1 (serotype 31/76), applied for two decades before CB 1809 (serotype 122). Nitrogen-fixing effectiveness of CB 1809 was superior to 60% of the isolates tested from Bergville and Morgenzon, but similar to 73% of the Koedoeskop isolates. Seed and liquid-in-furrow application methods increased CB 1809 nodule occupancy at least three-fold above background levels at Bergville (pH 5.16) and Morgenzon (pH 6.33). Inoculation did not, however, increase CB 1809 nodule occupancy at Koedoeskop (pH 7.76), possibly because alkaline soil conditions favoured the serotype 135 population predominant at this site.

Bacteria were isolated from root-nodules collected from indigenous legumes at 38 separate locations in the Gascoyne and Pilbara regions of Western Australia. Authentication of cultures resulted in 31 being ascribed status as root-nodule bacteria based upon their nodulation of at least one of eight indigenous legume species. The authenticated isolates originated from eight legume genera from 19 sites. Isolates were characterised on the basis of their growth and physiology; 20 isolates were fast-growing and 11 were slow-growing (visible growth within 3 and 7d, respectively). Fast-growers were isolated from Acacia, Isotropis, Lotus and Swainsona, whilst slow-growers were from Muelleranthus, Rhynchosia and Tephrosia. Indigofera produced one fast-growing isolate and seven slow-growing isolates. Three indigenous legumes (Swainsona formosa, Swainsona maccullochiana and Swainsona pterostylis) nodulated with fast-growing isolates and four species (Acacia saligna, Indigofera brevidens, Kennedia coccinea and Kennedia prorepens) nodulated with both fast- and slow-growing isolates. Swainsona kingii did not form nodules with any isolates. Fast-growing isolates were predominantly acid-sensitive, alkaline- and salt-tolerant. All slow-growing isolates grew well at pH 9.0 whilst more than half grew at pH 5.0, but all were salt-sensitive. All isolates were able to grow at 37deg;C. The fast-growing isolates utilised disaccharides, whereas the slow-growing isolates did not. Symbiotic interactions of the isolates were assessed on three annual, one biennial and nine perennial exotic legume species that have agricultural use, or potential use, in southern Australia. Argyrolobium uniflorum, Chamaecytisus proliferus, Macroptilium atropurpureum, Ononis natrix, Phaseolus vulgaris and Sutherlandia microphylla nodulated with one or more of the authenticated isolates. Hedysarum coronarium, Medicago sativa, Ornithopus sativus, Ornithopus compressus, Trifolium burchellianum, Trifolium polymorphum and Trifolium uniflorum did not form nodules. Investigation of the 31 authenticated isolates by polymerase chain reaction with three primers resulted in the RPO1 primer distinguishing 20 separate banding patterns, while ERIC and PucFor primers distinguished 26 separate banding patterns. Sequencing the 16S rRNA gene for four fast- and two slow-growing isolates produced the following phylogenetic associations; WSM1701 and WSM1715 (isolated from Lotus cruentus and S. pterostylis, respectively) displayed 99% homology with Sinorhizobium meliloti, WSM1707 and WSM1721 (isolated from Sinorhizobium leeana and Indigofera sp., respectively) displayed 99% homology with Sinorhizobium terangae, WSM1704 (isolated from Tephrosia gardneri) shared 99% sequence homology with Bradyrhizobium elkanii, and WSM1743 (isolated from Indigofera sp.) displayed 99% homology with Bradyrhizobium japonicum.

To quantify the contribution of biological nitrogen fixation (BNF) to legume crops using the 15N natural abundance technique, it is necessary to determine the 15N abundance of the N derived from BNF—the B value. In this study, we used a technique to determine B whereby both legume and non-N2-fixing reference plants were grown under the same conditions in two similar soils, one artificially labelled with 15N, and the other not. The proportion of N derived from BNF (%Ndfa) was determined from the plants grown in the 15N-labelled soil and it was assumed that the %Ndfa values of the legumes grown in the two soils were the same, hence the B value of the legumes could be calculated. The legumes used were velvet bean (Mucuna pruriens), sunnhemp (Crotalaria juncea), groundnut (Arachis hypogaea) and soybean (Glycine max) inoculated, or not, with different strains of rhizobium. The values of %Ndfa were all over 89%, and all the legumes grown in unlabelled soil showed negative δ15N values even though the plant-available N in this soil was found to be approximately +6.0‰. The B values for the shoot tissue (Bs) were calculated and ranged from approximately −1.4‰ for inoculated sunnhemp and groundnut to −2.4 and −4.5‰ for soybean inoculated with Bradyrhizobium japonicum strain CPAC 7 and Bradyrhizobium elkanii strain 29W, respectively. The B (Bwp) values for the whole plants including roots, nodules and the original seed N were still significantly different between the soybean plants inoculated with CPAC 7 (−1.33‰) and 29W (−2.25‰). In a parallel experiment conducted in monoxenic culture using the same soybean variety and Bradyrhizobium strains, the plants accumulated less N from BNF and the values were less negative, but still significantly different for soybean inoculated with the two different Bradyrhizobium strains. The results suggest that the technique utilized in this study to determine B with legume plants grown in soil in the open air, yields B values that are more appropriate for use under field conditions.

Nitrogen-fixing nodules are formed as a result of a series of interactions between rhizobia and leguminous plants. 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 and nodulation competitiveness by inhibiting ethylene biosynthesis in host roots. In addition, the gene for 1-aminocyclopropane-1-carboxylate (ACC) deaminase was recently found in some rhizobia, such as Mesorhizobium loti, Bradyrhizobium japonicum and Rhizobium sp. ACC deaminase also facilitates symbiosis by decreasing ethylene levels in host roots. The cumulative evidence reveals general strategies by which rhizobia produce an inhibitor and an enzyme to decrease ethylene levels in host roots and thereby enhance nodulation. In this review, we compare these strategies and discuss how they function and have evolved in terms of genetics, biochemistry, and ecology. These rhizobial strategies might be utilized as tools in agriculture and biotechnology.

One hundred isolates were trapped by soybean (Glycine max) plants inoculated with a soil from the Cerrados, the main producing area in Brazil. The soil was originally void of rhizobia able to nodulate soybean, and 15 years before received inoculant containing Bradyrhizobium elkanii strains SEMIA 587 and SEMIA 5019; the area has been annually cropped with soybean since then, but with no further inoculation for the past 7 years. Enormous diversity was observed among the isolates, with thirteen serologically distinct groups, twelve protein and seven lipopolysaccharide profiles; no more than five isolates shared similar characteristics. An unexpected feature was that 48% of the isolates showed multiple reactions with the antisera to the serogroups established in the soils. Also 40% of the isolates reacted with the antiserum to B. japonicum strain SEMIA 566, that has never been introduced into the soil, probably due to dispersion from other cropping areas, associated with its high saprophytic competence; 13% of the isolates did not react with any of the antisera. Nodulation and N2 fixation capacity also varied considerably among the isolates. Although one third of the isolates were fast growers with an acid reaction in vitro, and many formed pseudo-nodules on common bean (Phaseolus vulgaris), they shared several properties with the Bradyrhizobium inoculant strains. A high level of genetic diversity was confirmed when the DNAs were amplified with BOX and RPO1 primers, and several isolates were positioned in far different clusters in the analysis of interspersed repetitive or nif-directed sequences. Moreover, serological properties showed higher correlation with BOX than with RPO1 products. The high diversity could be attributed both to lateral transfer of genetic material between inoculant and indigenous strains and to genomic rearrangements during the adaptation to the Cerrados, and may play an important role as a biological buffer, avoiding the dominance of a particular strain.

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.

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.

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 alpha-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.

Phenolic acids, low molecular weight phenolics, are precursors of a variety of antimicrobial compounds, root signalling molecules, and phytoalexins that play an important role in plant defence responses. In agro ecosystem, a large amount of litter is turned over during the cropping season, fallow period and land preparation. This releases a flush of phenolic acids, amounts of which exceed very much the quantities released in root exudation. In rhizobial inoculation of legumes, these phenolic acids, depending on the concentration, may affect the persistence of rhizobia in the soil and their symbiotic efficiency, in terms of N2 fixation. The present study evaluates the effects of different concentrations of four phenolic acids (protocatechuic, p-coumaric, ferulic and vanillic) on population size of four rhizobial strains (Bradyrhizobium elkanii SEMIA 5019, B. japonicum TAL 102 and TAL 620, and Azorhizobium caulinodans ORS 571). Culture media with different concentrations of phenolic acids in the presence or absence of manitol were used to evaluate rhizobial population size on day 6. Rhizobial total proteins were extracted and electrophoresed on polyacrylamide gels. Further, the effects of phenolic acid-affected rhizobia on N2 fixing capacity were also investigated by inoculating two of those strains to soybean. Phenolic acid-treated B. elkanii SEMIA 5019 and B. japonicum TAL 102 were inoculated to soybean, and plant growth, N accumulation and nodule dry weight were assessed in a pot experiment. The population size of TAL 102 was induced when the culture medium was supplied with different phenolic acids as the sole carbon source. In many cases, the presence of manitol in the medium masked the differential effects of phenolic acids on the rhizobial population size. All four phenolic acids used in our study suppressed the population size of TAL 620. Strain ORS 571 showed low population size at low concentrations followed by a growth recovery at high phenolic acid concentrations. Strain SEMIA 5019 treated with 0.03 mM ferulic acid produced the highest increase in shoot growth of soybean, (ca. 65%). Treating strain SEMIA 5019 with 9 mM protocatechuic acid produced the largest decrease in nodule dry weight (ca. 50%) without any significant changes in shoot N accumulation. P-coumaric acid, even at 0.12 mM, could stimulate the N2 fixing activity of SEMIA 5019, whereas the same concentration reduced the effectiveness of TAL102 in a soybean-rhizobium symbiosis. Phenolic acid interactions with rhizobia led to biochemical, and hence physiological changes, resulting in an alteration in their symbiotic ability. Different leguminous plants secrete different phenolic compounds other than phenolic acids during root exudation. Further studies should therefore be conducted to evaluate the effects of those compounds on the symbiosis. It is concluded from this study that the effect of phenolic acids is concentration and structure dependant, and strain-specific. The effect will also be pH dependant. Thus, phenolic acids are possible agents for modifying the legume-rhizobial symbiosis.

TWenty-six isolates were obtained from nodules of various legume plants (Glycine max, Vigna sinensis, Arachis hypogaea, Desmanthus virgatus, Acacia mangium, Centrosema pascuorum, Pterocarpus indicus, Xylia xylocarpa, and Sesbania rostrata) in Thailand. After confirming their nodulation and nitrogen-fixing abilities, they were identified by 16S rRNA gene analysis as Bradyrhizobium japonicum, Bradyrhizobium elkanii, Rhizobium leguminosarum, Rhizobium gallicum, and Rhizobium galegae. Using these local isolates, the distribution of the activities of both NAD+-dependent (DME: EC 1.1.1.39) and NADP+-dependent (TME: EC 1.1.1.40) malic enzymes was surveyed. The malic enzyme activities were present in all the isolated rhizobia and in other 17 local Bradyrhizobium strains in Thailand. In almost all the rhizobia, the DME activity predominated whereas the TME activity predominated only in the Rhizobium gallicum strains that were major symbionts of Sesbania rostrata. Southern hybridization analysis was perfor...

Major differences in evolutionary relationships of the 16S rRNA gene and the nitrogenase alpha-subunit gene (nifD) were observed among 38 strains of Bradyrhizobium sp. nodule bacteria from North America, Central America, Asia and Australia. Two lineages were evident in the 16S rRNA phylogeny representing strains related to Bradyrhizobium japonicum (29 isolates) or Bradyrhizobium elkanii (9 isolates). Both clades were distributed across most or all of the geographic regions sampled. By contrast, in the nifD tree almost all isolates were placed into one of three groups each exclusively composed of taxa from a single geographic region (North Temperate, Central America or Australia). Isolates that were closely related or identical in gene sequence at one locus often had divergent sequences at the other locus and a partition homogeneity test indicated that the 16S rRNA and nifD phylogenies were significantly incongruent. No evidence for any gene duplication of nifD was found by Southern hybridization analysis on a subset of the strains, so unrecognized paralogy is not likely to be responsible for the discrepancy between 16S rRNA and nifD tree topologies. These results are consistent with a model whereby geographic areas were initially colonized by several diverse 16S rRNA lineages, with subsequent horizontal gene transfer of nifD leading to increased nifD sequence homogeneity within each regional population.

Natural genetic diversity and symbiotic status were studied among 41 root nodule bacteria from ecologically diverse sites in Kenya having no history of growing domesticated legumes, but where species of Acacia and Sesbania sesban grow naturally. Isolates originated either from nodules of field plants (trees and shrubs) or from trap host plants grown in field-collected soil. The host plants were Acacia mellifera , Acacia nilotica , Acacia nubica , Acacia polyacantha , Acacia seyal , Acacia tortilis , Acacia xanthophloea , and Faidherbia (Syn. Acacia ) albida , Prosopis juliflora , S. sesban , Macroptilium atropurpureum , Phaseolus vulgaris and Vigna unguiculata . Two molecular methods were used to characterize these strains; PCR–RFLP of the 16S rRNA gene (12 distinct genotypes) and sequence analysis of a 230 bp fragment of this gene (10 genotypes) with the former showing better discriminatory power. The two methods were in good agreement in delineating five bacterial genera: Agrobacterium , Bradyrhizobium , Mesorhizobium , Rhizobium and Sinorhizobium . Bradyrhizobium strains were all alkali-producing, exhibiting very slow to intermediate growth; Mesorhizobium strains were acid-producing with intermediate to fast growth; Rhizobium and Sinorhizobium were acid-producing with fast to very fast growth, and Agrobacterium strains were acid-producing and showed very fast growth. PCR–RFLP analysis further revealed strains corresponding to the species Bradyrhizobium elkanii , Bradyrhizobium japonicum , Rhizobium leguminosarum and Rhizobium tropici IIB, as well as others characterized to genus level only. Apparent diversity varied from site to site and was greatly influenced by host of isolation. For example, five rhizobial genotypes (four genera) were found using five different trap host species in a single Acacia tortilis woodland site, thus reaffirming occurrence of a heterogeneous population regardless of their nodulating ability with the native legume species. Bradyrhizobium spp. ( F. albida ), known for their preference of F. albida in tropical African soils, were abundant and widespread mainly because F. albida was the most frequently used trap host. Twenty Kenyan and eight reference strains were tested on tree species that are a priority for Kenyan use; these showed considerable diversity in symbiotic characters. Whereas Acacia species and F. albida showed variation in symbiotic effectiveness with test strains, S. sesban nodulated most effectively only with its homologous strains. Agrobacterium -allied strains failed to nodulate any of the host species tested. The high diversity (rhizobial genotypes, host affinities and their symbiotic effectiveness) found in the Kenyan sites will require rigorous selection for host×strain×site combinations in order to obtain effective and competitive inoculants for legume trees and crops in rehabilitation and agroforestry programmes.

The objective of this study was to demonstrate that municipal and industrial wastewater sludges could be used as a sole raw material to sustain growth of rhizobia. Growth of two different groups of rhizobium (fast growing: Sinorhizobium meliloti, Rhizobium leguminosarum bv viciae; and slow growing: Bradyrhizobium japonicum and Bradyrhizobium elkanii) was tested on primary, secondary and mixed sludges obtained from different wastewater treatment plants. The results obtained in Erlenmeyer flasks indicated that slow- and fast-growing rhizobia grew well in sludge. Generally, the number of cells of rhizobia exceeds 1 × 109 cfu/mL in 72 h. The composition of sludges varies with the sludge type and origin. The sludge composition affected the generation time, cell yield and nodulation index. Higher solids concentration tended to give higher generation time. The high sludge metals concentration did not affect the growth kinetics of rhizobia. However, primary sludge could inhibit cell growth. Acid, alkaline and oxidative pre-treatments increased the primary sludge biodegradability and consequently the cell count of S. meliloti. Pre-treatment of pulp and paper sludge with NaOH enhanced the bacterial cell concentration to a maximum 1 × 1010 cfu/mL. Sludge pre-treatment decreased the generation time and reduced the process time.

We investigated the horizontal transfer of nodulation (nod) genes to a Bradyrhizobium elkanii strain, lacking common nod genes as a recipient, in soils and microcosms using selection systems of antibiotic resistance and legume nodulation. We observed the horizontal transfer of nod genes at 4C in Nakazawa soil where peculiar strains (HRS strains) of B. japonicum harboring high copy numbers of insertion sequences dominated. In microcosms containing HRS strains as donors, we detected a similar horizontal transfer from B. japonicum HRS strain NK5 to the B. elkanii recipient more efficiently at 4C, which was verified by examining hybridization, nodulation and Nod factor production. These traits were, however, gradually lost during successive cultures. Plasmid analysis indicated that this event was not due to the simple transfer of plasmid carrying common nod genes. These results suggest the potential for horizontal transfer of nod genes among bradyrhizobia and other bacterial populations in soil environments.

Summary A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus erinaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S–23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S–23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorhizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrhizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrhizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.

Cowpea (Vigna unguiculata (L.) Walp.), a plant broadly cultivated for human consumption and animal feed, is among the most nutritious grain legumes. Most of the areas where cowpea is grown are drought-prone, and there is a need to address this issue, with water scarcity becoming a major concern in agriculture. Cowpea is known to form mutualistic associations with nitrogen-fixing (NF) bacteria and arbuscular mycorrhizal (AM) fungi. These beneficial soil microorganisms have the capacity to benefit plants by reducing the effects of environmental stresses, including drought. Our aim was to study the effect of inoculation with Bradyrhizobium elkanii and Rhizophagus irregularis on the growth and grain yield of cowpea under water-deficit conditions. Under moderate water deficit, grain yield was increased by 63%, 55% and 84% in plants inoculated with B. elkanii, R. irregularis and B. elkanii + R. irregularis, respectively. Under severe water deficit, inoculation with B. elkanii and B. elkanii + R. irregularis resulted in grain-yield enhancement of 45% and 42%, respectively. The use of cowpea inoculated with NF bacteria and AM fungi has great potential for sustainable agricultural production under drought conditions.

Root clusters are bunches of hairy rootlets that enhance nutrient uptake among many plants. Since first being reported in 1974, the involvement of rhizobacteria in their formation has received conflicting support. Attempts to identify specific causative organisms have failed and their role has remained speculative. We set up a gnotobiotic experiment using two root-clustered species, Viminaria juncea (Fabaceae) and Hakea laurina (Proteaceae), and inoculated them with two plant-growth-promoting rhizobacteria (PGPR), Bradyrhizobium elkanii and Bacillus mageratium, that produce indole-3-acetic-acid (IAA). Plants were suspended in water culture with four combinations of nitrogen and phosphorus. Clusters only developed in the presence of PGPR in two treatments, were greatly enhanced in another four, suppressed in five, and unaffected in five. Nitrogen amendment was associated with a higher density of clusters. Bradyrhizobium promoted cluster formation in Hakea, whereas Bacillus promoted cluster formation in Viminaria and suppressed it in Hakea. Greater root cluster numbers were due either to a larger root system induced by PGPR (indirect resource effect) and/or to more clusters per unit length of parent root (direct morphogenetic effect). The results are interpreted in terms of greater IAA production by Bradyrhizobium than Bacillus and greater sensitivity of Viminaria to IAA than Hakea.

The nodulation of Erythrophleum fordii has been recorded recently, but its microsymbionts have never been studied. To investigate the diversity and biogeography of rhizobia associated with this leguminous evergreen tree, root nodules were collected from the southern subtropical region of China. A total of 166 bacterial isolates were obtained from the nodules and characterized. In a PCR-based restriction fragment length polymorphism (RFLP) analysis of ribosomal intergenic sequences, the isolates were classified into 22 types within the genus Bradyrhizobium . Sequence analysis of 16S rRNA, ribosomal intergenic spacer (IGS), and the housekeeping genes recA and glnII classified the isolates into four groups: the Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi groups, comprising the dominant symbionts, Bradyrhizobium yuanmingense , and an unclassified group comprising the minor symbionts. The nodC and nifH phylogenetic trees defined five or six lineages among the isolates, which was largely consistent with the definition of genomic species. The phylogenetic results and evolutionary analysis demonstrated that mutation and vertical transmission of genes were the principal processes for the divergent evolution of Bradyrhizobium species associated with E. fordii , while lateral transfer and recombination of housekeeping and symbiotic genes were rare. The distribution of the dominant rhizobial populations was affected by soil pH and effective phosphorus. This is the first report to characterize E. fordii rhizobia.

The genus Bradyrhizobium has been considered to be a taxonomically difficult group. In this study, phylogenetics and evolutionary genetics analyses were used to investigate divergence levels among Bradyrhizobium strains nodulating soybeans in China. Eleven genospecies were identified by sequence analysis of three phylogenetic and taxonomic markers ( SMc00019 , thrA , and truA ). This was also supported by analyses of eight genes outside the symbiosis island (“off-island” genes; SMc00019 , thrA , truA , fabB , glyA , phyR , exoN , and hsfA ). However, seven genes inside the symbiosis island (“island” genes; nifA , nifH , nodC , nodV , fixA , trpD , and rhcC2 ) showed contrasting lower levels of nucleotide diversity and recombination rates than did off-island genes. Island genes had significantly incongruent gene phylogenies compared to the species tree. Four phylogenetic clusters were observed in island genes, and the epidemic cluster IV (harbored by Bradyrhizobium japonicum , Bradyrhizobium diazoefficiens , Bradyrhizobium huanghuaihaiense , Bradyrhizobium liaoningense , Bradyrhizobium daqingense , Bradyrhizobium sp. I, Bradyrhizobium sp. III, and Bradyrhizobium sp. IV) was not found in Bradyrhizobium yuanmingense , Bradyrhizobium sp. II, or Bradyrhizobium elkanii . The gene flow level of island genes among genospecies is discussed in the context of the divergence level of off-island genes.

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.

We cloned and sequenced a cluster of genes involved in the biosynthesis of rhizobitoxine, a nodulation enhancer produced by Bradyrhizobium elkanii . The nucleotide sequence of the cloned 28.4-kb DNA region encompassing rtxA showed that several open reading frames (ORFs) were located downstream of rtxA . A large-deletion mutant of B. elkanii , USDA94Δ rtx ::Ω1, which lacks rtxA , ORF1 ( rtxC ), ORF2, and ORF3, did not produce rhizobitoxine, dihydrorhizobitoxine, or serinol. The broad-host-range cosmid pLAFR1, which contains rtxA and these ORFs, complemented rhizobitoxine production in USDA94Δ rtx ::Ω1. Further complementation experiments involving cosmid derivatives obtained by random mutagenesis with a kanamycin cassette revealed that at least rtxA and rtxC are necessary for rhizobitoxine production. Insertional mutagenesis of the N-terminal and C-terminal regions of rtxA indicated that rtxA is responsible for two crucial steps, serinol formation and dihydrorhizobitoxine biosynthesis. An insertional mutant of rtxC produced serinol and dihydrorhizobitoxine but no rhizobitoxine. Moreover, the rtxC product was highly homologous to the fatty acid desaturase of Pseudomonas syringae and included the copper-binding signature and eight histidine residues conserved in membrane-bound desaturase. This result suggested that rtxC encodes dihydrorhizobitoxine desaturase for the final step of rhizobitoxine production. In light of results from DNA sequence comparison, gene disruption experiments, and dihydrorhizobitoxine production from various substrates, we discuss the biosynthetic pathway of rhizobitoxine and its evolutionary significance in bradyrhizobia.

The symbiotic effectiveness of 30 fast-growing rhizobial strains (doubling times of 85–225 min and acid reaction in yeast mannitol medium) isolated from soybean nodules in Brazil and of Sinorhizobium fredii reference strains was evaluated under greenhouse and field conditions. Most Brazilian fast-growing strains were genetically related to the Rhizobium tropici-Rhizobium genomic species Q-Agrobacterium spp. branch and five to the Bradyrhizobium japonicum and B. elkanii species. Under axenic conditions, some of the fast-growing strains fixed as much N2 as the B. japonicum/B. elkanii strains carried in Brazilian commercial inocula. However, in a co-inoculation experiment, very few strains were able to compete against B. elkanii strain SEMIA 5019. Although isolated from acid soils (pH 3.0–5.1), the competitiveness of Brazilian fast growers and of S. fredii reference strains against B. japonicum/B. elkanii was low under acid conditions (pH 5.1 and pH 5.4), but increased when the pH was raised to 6.8 and 7.9. Therefore, as the great majority of Brazilian soils are acidic and show a very high population of naturalized B. japonicum/B. elkanii, the low competitiveness of S. fredii and of the Brazilian rhizobial strains investigated in this study, under the given conditions, limits, at this time, their recommendation for use in commercial inocula.

Enzyme electrophoresis and rRNA sequencing were used to analyze relationships of Bradyrhizobium sp. nodule bacteria from four papilionoid legumes ( Clitoria javitensis, Erythrina costaricensis, Rhynchosia pyramidalis , and Desmodium axillare ) growing on Barro Colorado Island (BCI), Panama. Bacteria with identical multilocus allele profiles were commonly found in association with two or more legume genera. Among the 16 multilocus genotypes (electrophoretic types [ETs]) detected, six ETs formed a closely related cluster that included isolates from all four legume taxa. Bacteria from two other BCI legumes ( Platypodium and Machaerium ) sampled in a previous study were also identical to certain ETs in this group. Isolates from different legume genera that had the same ET had identical nucleotide sequences for both a 5′ portion of the 23S rRNA and the nearly full-length 16S rRNA genes. These results suggest that Bradyrhizobium genotypes with low host specificity may be prevalent in this tropical forest. Parsimony analysis of 16S rRNA sequence variation indicated that most isolates were related to Bradyrhizobium japonicum USDA 110, although one ET sampled from C. javitensis had a 16S rRNA gene highly similar to that of Bradyrhizobium elkanii USDA 76. However, this isolate displayed a mosaic structure within the 5′ 23S rRNA region: one 84-bp segment was identical to that of BCI isolate Pe1-3 (a close relative of B. japonicum USDA 110, based on 16S rRNA data), while an adjacent 288-bp segment matched that of B. elkanii USDA 76. This mosaic structure is one of the first observations suggesting recombination in nature between Bradyrhizobium isolates related to B. japonicum versus B. elkanii .

The 16S rDNA sequence of Bradyrhizobium liaoningense was determined and analysed together with sequences of other Bradyrhizobium species and related taxa. In addition, DNA-DNA hybridizations were performed between representative strains of the three Bradyrhizobium species. Bradyrhizobium liaoningense is genotypically highly related to Bradyrhizobium japonicum, whereas Bradyrhizobium elkanii is more distantly related to these two species. The fact that Afipia, Agromonas, Blastobacter, Nitrobacter and Rhodopseudomonas are phylogenetically more closely related to Bradyrhizobium japonicum than to Bradyrhizobium elkanii is discussed.

The effects of growth phase, reductions in the water activity (a(w)) of the growth medium and mild desiccation on the composition and the degree of unsaturation of cellular fatty acids (CFA) of Sinorhizobium meliloti, Bradyrhizobium elkanii and Bradyrhizobium japonicum were studied. During the course of growth, an interchange of cis-vaccenic with lactobacillic acid and a slight increase in palmitic acid were observed while other fatty acids remained constant. The degree of unsaturation was significantly higher in the exponential phase of growth. Reductions in the a(w) of the medium led to an increase in lag phase, a reduction in growth rate and maximal optical densities (OD) in stationary phase cells. A decrease in the degree of unsaturation of CFA was also observed as the a(w) was reduced from 0.999 to 0.969 and after desiccation to 83.5% relative humidity (R.H.). The changes in the degree of unsaturation of CFA observed after growth at reduced a(w) may be one of the pre-adaptation steps to endure more severe desiccation.

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.

Application of 1-aminoocyclopropane-1-carboxylic acid, an ethylene precursor, decreased nodulation of Macroptilium atropurpureum by Bradyrhizobium elkanii. B. elkanii produces rhizobitoxine, an ethylene synthesis inhibitor. Elimination of rhizobitoxine production in B. elkanii increased ethylene evolution and decreased nodulation and competitiveness on M. atropurpureum . These results suggest that rhizobitoxine enhances nodulation and competitiveness of B. elkanii on M. atropurpureum .

In order to identify soybean cultivars with higher biological N2 fixation capacities, North American and Brazilian soybean [Glycine max (L.) Merrill] cultivars, belonging to maturity groups VI–VIII, were evaluated for nodulation parameters and N2 fixation rates. The symbiotic performance of 152 cultivars was evaluated in pots containing 4 kg soil with an established population of the three Bradyrhizobium elkanii strains [29w (SEMIA 5019):SEMIA 566 : SEMIA 587, 22%:36%:34%] which are established in most Brazilian soils cultivated with soybean. Differences were verified among cultivars, with some accumulating up to twice as much nodule dry weight and N in tissues as others. The variability among cultivars was also confirmed when six of them were used in a field experiment, resulting in differences in nodulation, yield and total N accumulated in grains. The analysis of nodule occupancy in 12 cultivars grown either under sterile conditions and receiving a double inoculum and N-free nutrient solution, or in pots containing soil with an established population of bradyrhizobia, showed the preference of cultivars for specific strains.

We isolated 11 low-indole-3-acetic acid (IAA)-producing mutants of Bradyrhizobium elkanii by Tn5 mutagenesis. The amount of IAA produced by each mutant was 2.2-13.6% of that of the wild-type. It was found by resting cell reactions that the biosynthetic step to convert indole-3-pyruvic acid to indole-3-acetaldehyde was blocked in all the mutants.

Although Bradyrhizobium elkanii is a mutualistic symbiont of legumes, it synthesizes a phytotoxin, rhizobitoxine, that causes chlorosis on a variety of legume hosts, giving a pathogenic character to these interactions. No positive role for rhizobitoxine has been previously demonstrated. Interestingly, rhizobitoxine inhibits the rate-limiting step for ethylene biosynthesis, a plant hormone known to inhibit or down-regulate nodule development. We hypothesized that rhizobitoxine plays a positive role in nodule development through its inhibition of ethylene biosynthesis. To test this hypothesis, host plants of B. elkanii were screened for a differential nodulation response to the wild-type and rhizobitoxine mutant strains. In Vigna radiata (mungbean), the rhizobitoxine mutant strains induced many aborted nodules arrested at all stages of pre-emergent and post-emergent development and formed significantly fewer mature nodules than the wild type. Experiments revealed that nodulation of mungbean plants is sensitive to exogenous ethylene, and that the ethylene inhibitors aminoethoxyvinylglycine and Co2+ were able to partially restore a wild-type nodulation pattern to the rhizobitoxine mutants. This is the first demonstration of a nodulation phenotype of the rhizobitoxine mutants and suggests that rhizobitoxine plays a positive and necessary role in Rhizobium-legume symbiosis through its inhibition of ethylene biosynthesis.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.

To elucidate the phylogenetic relationships between Thai soybean bradyrhizobia and USDA strains of Bradyrhizobium, restriction fragment length polymorphism (RFLP) analysis using the nifDK gene probe and sequencing of the partial 16S rRNA gene were performed. In our previous work, Thai isolates of Bradyrhizobium sp. (Glycine max) were separated clearly from Bradyrhizobium japonicum and Bradyrhizobium elkanii based on the RFLP analysis using the nodDYABC gene probe. RFLP analysis using the nifDK gene probe divided 14 Thai isolates and eight USDA strains of B. japonicum into different groups, respectively, but categorized into the same cluster. All of seven strains within these Thai isolates had the same sequence of the partial 16S rRNA gene, and it was an intermediate sequence between those of B. japonicum USDA 110 and B. elkanii USDA 76T. Furthermore, three USDA strains of B. japonicum, USDA 6 (B. japonicum ATCC 10324T), USDA 115 and USDA 129, had the same partial 16S rRNA gene sequence that seven Thai isolates had. These results suggest that Thai isolates of Bradyrhizobium sp. (Glycine max) are genetically distinct from USDA strains of B. japonicum and B. elkanii, but also indicate a close relationship between Thai isolates and USDA strains of B. japonicum.Key words: Bradyrhizobium, 16S rRNA gene, nif gene, soybean, Thailand.

Two hundred and thirteen isolates of soybean bradyrhizobia indigenous to six field sites in Japan were characterized using hybridization probes RSα, RSβ, nifDK and hupSL from Bradyrhizobium japonicum and indole-3-acetic acid production to clarify diversity and endemism of their population structures. Significant diversities and site-dependent variations were observed in terms of Bradyrhizobium species, hup genotype and fingerprints with repeated sequences RSα and RSβ. The fingerprints at one site with no history of soybean cultivation were less diverse than those at other sites. Even within the populations of B. japonicum hup− isolates, which were commonly indigenous to the six field sites, several RSα copies clustering around nif genes were highly conserved. The results suggest that soybean bradyrhizobia may be diversified in individual fields as associated with host plants and local soil conditions.

A group of Bradyrhizobium strains isolated from soybean plants in Thailand did not correspond to any known DNA homology groups of Bradyrhizobium japonicum and Bradyrhizobium elkanii reported by Hollis et al. (J. Gen. Microbiol., 123, 215–222, 1981). To clarify the phenotypic characteristics of the group, serological properties and intrinsic antibiotic resistance (IAR) profile of 94 Thai strains were compared with those of USDA and Japanese strains. Indirect ELISA tests for each Thai strain were performed agaiIl.st polyclonal antisera prepared against 15 USDA standard serotype strains of B. japonicum and B. elkanii. Among the 94 Thai strains tested, 36 which were previously identified as B. elkanii, with the exception of one strain, were strongly responsive to an antiserum prepared against USDA 31. The remaining 58 strains, with the exception of two strains, showed multiple cross reactions which were peculiar to the Thai strains. These serological reaction patterns did not correspond to any known ...

Rhizobitoxine is synthesized by the legume symbiont Bradyrhizobium elkanii and the plant pathogen Burkholderia andropogonis . Rhizobitoxine competitively inhibited 1-aminocyclopropane-1-carboxylate (ACC) synthase bLE-ACS2 from the tomato, a key enzyme in the pathway of ethylene biosynthesis. Based on this inhibition of ACC synthase, we have developed a new assay for rhizobitoxine.

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.

Eleven isolates of slow-growing oligotrophic bacteria from grassland soil were found to be closely related by partial 16S rRNA sequence similarity and many common taxonomic traits. Analysis of full 16S rRNA gene sequences of four representative isolates and Agromonas oligotrophica S58 indicated that they were more closely related to Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium, (similarity values: 98.1–98.8%) than other strains such as Bradyrhizobium elkanii, Nitrobacter spp., Rhodopseudomonas palustris, and Afipia spp. This result was supported by analysis of phenotypic traits and DNA-DNA hybridization analysis. No strain showed hybridization to nodD1YABC of B. japonicum, and only strain G14130 exhibited hybridization to nifDK- and hupSL-specific DNA. These latter genotypes are involved in the phenotypes of nodulation and nitrogen fixation under microaerobic conditions. These results suggest that the isolates possess a unique phylogenetic position since they are closely related to B. japonicum though they do not have characteristics of symbiotic nitrogen fixation.

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.

Symbiotic efficiency of soybean Glycine max L. cv. Doko nodulated by BR-33 (CB 1809) strain of Bradyrhizobium japonicum or BR-29 (29W) strain of B. elkanii was investigated under two light intensities: full sunlight or 30% of full sunlight. Plants were harvested during pod-filling and at maturity. Soybeans cultivated under full sunlight and inoculated with strain BR-33 showed higher shoot dry mass and nitrogen accumulation, nitrogenase activity, ureide-N in the xylem sap and grain yield than plants inoculated with strain BR-29, confirming the superior symbiotic effectiveness of strain BR-33. In contrast, soybean submitted to shading treatment showed no significant differences regardless of the strain used as inoculant. When submitted to shading, plants inoculated with BR-29 strain showed an increase in most of the variables observed while plants inoculated with strain BR-33 were not significantly affected by the level of shading. The results suggest that strain efficiency used as an inoculant for soybean can be different in plants cultivated either in the greenhouse or in the field. This can help to explain why the potential of hydrogenase activity to improve symbiotic effectiveness is still a matter of controversy when comparing results from different experiments.

Several genes that restrict nodulation with specific Bradyrhizobiumstrains are known in Glycine max (soybean), and a similar system of nodulation restriction has recently been discovered in the related North American legume Amphicarpaea bracteata. We analyzed how nodulation-restrictive genotypes of each plant interacted with Bradyrhizobium strains sampled from the other host species. Ten bacterial isolates from A. bracteata that nodulated differentially with genotypes of their homologous host legume showed uniform responses to two soybean isogenic lines that differed at the Rj4 locus controlling nodulation restriction: all isolates formed nodules of normal size and morphology on both isolines. However, little or no nitrogen fixation occurred in any of these symbioses. A. bracteata genotypes that displayed broad vs. restricted symbiotic phenotypes toward naturally-associated bradyrhizobia were also tested with two bacterial isolates from soybean (USDA 76 and USDA 123). Both isolates formed nodules and fixed nitrogen in association with both A. bracteata genotypes. However, symbiotic effectiveness (as measured by acetylene reduction assays) was normal only for the combination of USDA 76 with the restrictive A. bracteata genotype. Overall, these results indicate that plant genes that restrict nodulation by certain naturally-associated bradyrhizobia do not confer comparable specificity when plants interact with bacteria from another related legume species.

Bacterial strains isolated from root nodules on Acacia mangium at various locations in Indonesia were characterized by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA and by an intergenic repetitive extragenic palindromic (REP) sequence. The RFLP-16S rDNA analysis used four restriction endonucleases classified into seven Sau3AI, eight TaqI, nine HinfI, and nine MspI groups. Analysis of the amplified REP-PCR products showed intra-specific variations in the rhizobial genomic DNA. From the data obtained, 10 of the 15 strains isolated from A. mangium were classified as Bradyrhizobium. These isolates clustered with B. elkanii, and were predominant in the A. mangium nodulation systems. Partial 16S rDNA sequence analysis of some representative strains demonstrated that the RFLP clusters were stable and phylogenetically relevant.