Seed size influences the promoting activity of rhizobia on plant growth, nodulation and N fixation in lima bean / O tamanho da semente influencia a atividade promotora dos rizóbios sobre o crescimento de plantas, nodulação e fixação de N em feijão-fava

ABSTRACT: This study evaluated the activity of rhizobia isolates inoculated in large (18 mm) and small (11 mm) seeds on lima bean growth, nodulation and N fixation. Selected rhizobia isolates were compared with a reference strain CIAT899 and two controls without inoculation. Large seeds contributed for highest plant growth, nodulation and N fixation than small seeds. The isolates UFPI-59, UFPI-18 and UFPI-38 promoted the highest values of shoot and root dry weight, respectively. The isolates UFPI-32 promoted the highest values of nodule number, while UFPI-59 promoted the highest values of nodule dry weight. The isolates UFPI-38 and UFPI-59 promoted the highest accumulation of N. This study showed that seed size really influences lima bean growth, nodulation and BNF. Considering rhizobia isolates, UFPI-59, UFPI-38, and UFPI-18 contributed for plant growth, promoted better nodulation and effectiveness on biological N fixation.

RESUMO: Este estudo avaliou a atividade de isolados de rizóbios inoculados em sementes pequenas (11 mm) e grandes (18 mm) sobre o crescimento, nodulação e fixação de N de feijão-fava. Isolados selecionados de rizóbios foram comparados com uma estirpe de referência CIAT899 e dois controles sem inoculação. Sementes grandes contribuíram para maior crescimento de plantas, nodulação e fixação de N do que sementes pequenas. Os isolados UFPI-59, UFPI-18 e UFPI-38 promoveram os maiores valores de massa seca de parte aérea e raízes. Os isolados UFPI-32 e UFPI-59 promoveram os maiores valores de número e massa de nódulos, respectivamente. Os isolados UFPI-38 e UFPI-59 promoveram maior acúmulo de N. Este estudo mostrou que o tamanho de sementes influencia o crescimento, nodulação e fixação de N em feijão-fava. Considerando os isolados de rizóbios, UFPI-59, UFPI-38 e UFPI-18 contribuíram para o crescimento de plantas, promoveram melhor nodulação e eficiência na fixação de N.

Influence of Molybdenum doses in inoculation and mineral fertilization in cowpea beans / Influência de doses de molibdênio na inoculação e na fertilização nitrogenada em feijão caupi

In some leguminous plants, associations with nitrogen-fixing microorganisms allow their nutrition with nitrogen (N) from the atmosphere. This process is known as Biological Nitrogen Fixation (BNF), where through nitrogenase enzymes, N2 is converted to an available form. This process can replace in part, or in total, nitrogen fertilizers. Cowpea bean is a legume species that is recognized for its high capacity to carry out BNF. In the last decades, studies have encouraged small farmers from north and northeast Brazil to use inoculants with rhizobia species since the results of researches have demonstrated that inoculation is an interesting strategy to improve cowpea production. Considering the specific function of molybdenum (Mo) in the N assimilation, different doses of Mo were tested in this study in order to find doses that could improve and enhance BNF. Therefore, this study aimed to compare nitrogen fertilization and BNF in the N assimilation by plants with different Mo doses. Inoculation was performed with the strains UFLA 03-84 and INPA 03-11B. Doses of Mo were applied in seeds and each pot contained five seeds. Thirty-five days after germination, the plants were analyzed for shoot dry matter and fresh matter, N contents and accumulation, as well as the Soil-Plant Analysis Development (SPAD) Index and nodulation in inoculated plants. The different doses of Mo and also the nodulation treatments did not show significant differences in the contents of N. Plants with N fertilization had significant higher shoot dry matter and root dry matter production, in addition to higher N foliar contents and N accumulation. Therefore, BNF was not as efficient as nitrogen fertilization in the evaluated experimental conditions using cowpea beans.

Em algumas plantas de leguminosas, as associações com microorganismos de fixação permitem a sua nutrição com N (nitrogênio) da atmosfera. Este processo é conhecido como Fixação Biológica de Nitrogênio (FBN), onde através de enzimas nitrogenase N2 é convertido na forma disponível. Este processo pode substituir em parte, ou no total, fertilizantes nitrogenados. O feijão-caupi é uma espécie de leguminosa que é reconhecida pela sua alta capacidade de FBN. Nas últimas décadas, estudos encorajaram os pequenos agricultores do norte e nordeste do Brasil a usar inoculantes com espécies de rizobio, uma vez que os resultados das pesquisas têm demonstrado que a inoculação é uma estratégia interessante para melhorar a produção dessa cultura. Considerando a função específica do molibdênio (Mo) na assimilação do N, diferentes doses de Mo foram testadas neste estudo, a fim de encontrar doses que possam melhorar e potencializar a FBN. A inoculação foi feita com as estirpes UFLA 03-84 e INPA 03-11B. As diferentes doses de Mo foram aplicadas na semente, cinco sementes por vaso. Trinta e cinco dias após a germinação, as plantas foram analisadas com relação à matéria seca aérea e radicular, teor de N e acúmulo, índice de análise de desenvolvimento solo-planta (SPAD) e nodulação nas plantas inoculadas. As diferentes doses de Mo não mostraram diferenças significativas no conteúdo de N, nem no processo de nodulação. As plantas com adubação nitrogenada tiveram mattera seca da parte aérea e de raízes superior as inoculadas, além de maiores teores foliares de N. Portanto, para feijão-caupi, a FBN não foi tão eficiente quanto o fertilizante nitrogenado nas condições experimentais avaliadas.

Effect of inoculation of Tarwi plants with Bradyrhizobium spp. strains isolated from a wild lupine, under greenhouse conditions / Efecto de la inoculación de plantas de Tarwi con cepas de Bradyrhizobium spp. aisladas de un lupino silvestre, en condiciones de invernadero

Abstract The "tarwi" or "chocho" (Lupinus mutabilis Sweet) is the unique specie of the genus Lupinus cultivated in America, appreciated for its high protein content in seeds, and the ability to fix nitrogen in symbiotic association with rhizobia. Its genetic variability is reinforced by approximately 84 wild species in Peru. The present investigation was carried out to show if the rhizobia of a wild lupino (Lupinus sp.) might be able to nodulate and promote the growth of tarwi. The root nodules were collected from an abandoned crop field of Huaraz (Ancash-Peru) at 3497 m of altitude, were isolated 8 slow-growing (6-7 days) rhizobial strains, which due to their microbiological and molecular characteristics correspond to the genus Bradyrhizobium. These strains were inoculated in tarwi seeds, and co-inoculated to the 15-day seedlings. It was applied a completely randomized design with 11 treatments (including the control strain LMRT28, N+ with nitrogen fertilization and N- without fertilization) and 5 repetitions. After 70 days of growth in greenhouse conditions, all treatments with native strains, with the exception of LSHZ-L1 and LSHZ-L2, showed reddish-colored root nodules, indicator of leghemoglobin activity. Six of the strains significantly increased the aerial length of the plants respect to the N- and the LMRT28 treatment; also, there were significant differences in relation of aerial dry weight being better in five treatments with native strains. The major foliar coverage was developed by LSHZ-L7; and the greatest number of secondary nodules was showed in LSHZ-L3, which is correlated with the dry weight of the root.

Resumen El "tarwi" o "chocho" (Lupinus mutabilis Sweet) es la única especie cultivada del género Lupinus en América, valorado por su alto contenido proteico y capacidad de fijar nitrógeno en asociación simbiótica con rizobios. Su variabilidad genética está reforzada por aproximadamente 84 especies silvestres en el Perú. La presente investigación se realizó para evidenciar si los rizobios de una especie silvestre de lupino (Lupinus sp.) fueran capaces de nodular y promover el crecimiento del tarwi. Los nódulos radiculares fueron colectados de un campo de cultivo en abandono de Huaraz (Ancash-Perú) a 3497 m de altitud; se aislaron 8 cepas rizobianas de crecimiento lento (6-7 días) que por sus características microbiológicas y moleculares corresponden al género Bradyrhizobium. Estas fueron inoculadas en semillas de tarwi y reinoculadas a plántulas de 15 días. Se aplicó diseño completamente aleatorizado con 11 tratamientos (incluyendo la cepa control LMRT28, N+ con fertilización nitrogenada, y N- sin fertilización) y 5 repeticiones. Después de 70 días en invernadero se observó que todas las cepas nativas, a excepción de LSHZ-L1 y LSHZ-L2, mostraron nódulos radiculares de coloración rojiza indicador de la actividad de la leghemoglobina. Seis de las cepas incrementaron significativamente la longitud aérea de las plantas respecto al N- y al control LMRT28; también hubo diferencias significativas en cuanto al peso seco aéreo destacando cinco cepas nativas; la mayor cobertura foliar fue desarrollada por LSHZ-L7; y la cepa LSHZ-L3 presentó significativamente mayor número de nódulos secundarios y estuvo correlacionada con el peso seco de la raíz.

Inoculação de diferentes doses de Bradyrhizobium por cobertura e seu efeito na cultura da soja / Inoculation by different doses of Bradyrhizobium by side dressing and its effect on Soybean / Inoculación de diferentes dosis de Bradyrhizobium por cubierta y su efecto en la Cultura de la soja

A soja é a cultura que mais cresceu no Brasil, com grande destaque para o agronegócio. Sendo o nutriente mais requerido, o nitrogênio, torna se necessário muitas vezes a utilização de fertilizantes nitrogenados e como uma alternativa sustentável, recomenda-se a inoculação com Bradyrhizobium realizando desse modo a fixação biológica de nitrogênio. O objetivo deste estudo foi avaliar os benefícios da inoculação de Bradyrhizobium na cultura da soja, pela pulverização em cobertura, na nodulação, no desenvolvimento das plantas e na produtividade. Foram conduzidos quatro tratamentos com design inteiramente casualizado e com quatro repetições, sendo tratamento 1 (controle) isento de pulverização, o segundo com pulverização de 500 mL ha­1, o terceiro de 1000 mL ha­1 e o quarto de 1500 mL ha­1 do inoculante com Bradyrhizobium. O número de sacas ha­1 com a inoculação de Bradyrhizobium por pulverização de 1500 mL ha­1 foi de 59 sacas. Reificouse ainda, aumentos significativos (p ≤ 0,05), sendo de 180,65 para número de nódulos, 4,44 g planta­1 para massa seca dos nódulos, 17,30 g planta­1 para massa seca das raízes e de 64,33 g planta­1 para massa seca da parte aérea em comparação com o tratamento controle, evidenciando o maior rendimento de soja para este tratamento. Conclui-se, portanto que a inoculação da soja com 1500 mL ha­1 de Bradyrhizobium em pulverização por cobertura, é a mais eficiente diante dos parâmetros testados no desenvolvimento e produção da soja.(AU)

The cultivation of soybean has presented the greatest increase in Brazil, with strong emphasis on agribusiness. Since nitrogen is the most required nutrient, nitrogen fertilizers are sometimes necessary. As a sustainable alternative, the inoculation of Bradyrhizobium is recommended, which can perform the biological fixation of nitrogen. The objective of this study was to evaluate the benefits of Bradyrhizobium inoculation in soybean crop, by spraying on its side dressing, nodulation, plant development and on productivity. Four treatments were carried out with a completely randomized design with four replications. The first treatment was left spray-free, the second was sprayed with 500 mL ha­1, the third with 1000 mL ha­1, and the fourth with 1500 mL ha­1 of the Bradyrhizobium inoculant. A total of 59 bags ha­1 were harvested with the application of the inoculation of Bradyrhizobium by spraying 1500 mL ha­1, and significant increases (p ≤ 0.05) were observed, namely 180.65 nodules, 4.44 g plant­1 for nodule dry matter, 17.30 g plant­1 for root dry matter, and 64.33 g plant­1 for shoot dry matter when compared to the control treatment, evidencing the higher soybean yield for this treatment. It can be concluded that the inoculation of soybean with 1500 mL ha­1 Bradyrhizobium in spray by side dressing is the most efficient in the tested parameters for the development and production of soybean.(AU)

La soja es la cultura que más creció en Brasil, con gran destaque para el agronegocio. Con el nutriente más requerido el nitrógeno, se hace necesario a menudo la utilización de fertilizantes nitrogenados y como una alternativa sostenible, se recomienda la inoculación con Bradyrhizobium realizando de ese modo la fijación biológica de nitrógeno. El objetivo de este estudio fue evaluar los beneficios de la inoculación de Bradyrhizobium en el cultivo de la soja, la pulverización en cobertura, la nodulación, el desarrollo de las plantas y la productividad. Se realizaron cuatro tratamientos con diseño completamente casualizado y con cuatro repeticiones, siendo tratamiento 1 (control) exento de pulverización, el segundo con pulverización de 500 mL ha-1, el tercero de 1000 mL ha-1 y el cuarto de 1500 mL ha-1 del inoculante con Bradyrhizobium. El número de sacas ha-1 con la inoculación de Bradyrhizobium por pulverización de 1500 mL ha-1 fue de 59 sacas Se observaron también aumentos significativos (p ≤ 0,05), siendo de 180,65 para número de nódulos, 4,44 g planta-1 para masa seca de los nódulos, 17,30 g planta-1 para masa seca de las raíces y de 64,33 g planta-1 para masa seca de la parte aérea en comparación con el tratamiento control, evidenciando mayor rendimiento de soja para este tratamiento. Se concluye, por lo tanto, que la inoculación de la soja con 1500 mL ha-1 de Bradyrhizobium en pulverización por cubierta es la más eficiente, frente a los parámetros probados en el desarrollo y producción de la soja.(AU)

Bradyrhizobium elkanii nod regulon: insights through genomic analysis

Abstract A successful symbiotic relationship between soybean [Glycine max (L.) Merr.] and Bradyrhizobium species requires expression of the bacterial structural nod genes that encode for the synthesis of lipochitooligosaccharide nodulation signal molecules, known as Nod factors (NFs). Bradyrhizobium diazoefficiens USDA 110 possesses a wide nodulation gene repertoire that allows NF assembly and modification, with transcription of the nodYABCSUIJnolMNOnodZ operon depending upon specific activators, i.e., products of regulatory nod genes that are responsive to signaling molecules such as flavonoid compounds exuded by host plant roots. Central to this regulatory circuit of nod gene expression are NodD proteins, members of the LysR-type regulator family. In this study, publicly available Bradyrhizobium elkanii sequenced genomes were compared with the closely related B. diazoefficiens USDA 110 reference genome to determine the similarities between those genomes, especially with regards to the nod operon and nod regulon. Bioinformatics analyses revealed a correlation between functional mechanisms and key elements that play an essential role in the regulation of nod gene expression. These analyses also revealed new genomic features that had not been clearly explored before, some of which were unique for some B. elkanii genomes.

Seeds inoculation and nitrogen fertilization for cowpea production on latosol in the western amazon / Produtividade de feijão-caupi com inoculante e fonte mineral nitrogenada em latossolo da amazônia ocidental

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.

O feijão-caupi é uma das principais alternativas alimentares para as populações das regiões norte e nordeste do Brasil, sendo consumido na forma de grãos verdes ou maduros. Apesar da importância social e da origem tropical, a espécie apresenta baixa produtividade na região amazônica, incluindo o estado de Roraima, devido à baixa qualidade agronômica e manejo incorreto do solo. O objetivo do trabalho foi caracterizar o desenvolvimento da cultura do feijão-de-corda com duas diferentes fontes de nitrogênio (N) tratadas com inoculante. O experimento foi realizado no município de Rorainópolis (RR). O delineamento foi em blocos casualizados, com quatro tratamentos e cinco repetições. Foi utilizada a cultivar de feijão-caupi 'BRS Guariba'. Os tratamentos foram: testemunha (ausência de adubação nitrogenada), inoculante Bradyrhizobium elkanii, 60 kg ha-1 de N provenientes da uréia e 60 kg ha-1 de N da fonte sulfato de amônio. Foram avaliados os teores foliares de macronutientes (N, P, K, Ca, Mg e S), peso da massa verde e seca da parte aérea, número de grãos secos por planta, produção de vagens e produção de grãos. Em relação ao acúmulo de nutrientes a uréia e sulfato de amônio foram as fontes que apresentaram desempenho superior. Em relação as características produtivas, o sulfato de amônio foi o que mais se destacou, sendo uma fonte viável de suprimento de N para o feijão-caupi na região amazônica. A nodulação com Bradyrhizobium elkanii não foi eficiente para substituir a adubação com fertilizantes nitrogenados para o feijão-caupi BRS Guariba.

Nursery growth and rhizobia symbiosis of scandent Leguminosae species native to the Amazon region / Crescimento e simbiose com rizóbios em casa de vegetação de espécies de Leguminosae escandentes nativas da região Amazônica

A great number of species and individuals of scandent legumes establishing symbiosis with nitrogen fixing bacteria occurs in the Amazon Forest. These symbiosis probably play an important role in contributing to nitrogen incorporation in this ecossystem. The objectives of this study were to evaluate the growth of eight species of scandent legumes in five nursery substrates; to compare nodulation with rhizobia strains introduced or native to these substrates; and to characterize phenotypically and genetically these rhizobia. The experiment was carried out in a completely randomized design with five replications. Five to seven months after seedling emergency, according to the legume species, growth and nodulation parameters were determined. Rhizobia identification of strains was carried out by 16S rRNA gene partial sequencing. The survival of seedlings after the transplanting varied from 93 to 98%, in Ultisol (Argissolo in Brazilian classification), collected in primary forest, and fertilized with all nutrients, except nitrogen (ULTfert); and in a clay and sand mixture, in a ratio 3:2 (CONV), respectively. Species with height superior to 30 cm, in general, grew better in substrates with higher fertility: ULTfert and Humic Gley soil (HG). Seven out of the eight species were able to nodulate. The percentage of nodulation per substrate was: SAND, washed sand with mixed inoculum of 100 rhizobia strains plus fertilization (100), HG (80), CONV (100), ULT, A-horizon of red-yellow Ultisol collected in the Ducke Forest Reserve (Manaus) (44), and ULTfert (55%). Bradyrhizobium spp. were isolated from nodules of all species and substrates. Burkolderia fungorum was isolated from Dalbergia inundata. For Dalbergia riedelli and Dalbergia inundata, this is the first report on the identification of symbiotic strains. Scandent legumes present high survival of seedlings in nursery, and develop better in substrates with higher fertility, and generally present symbiosis with [...].

Um grande número de espécies e indivíduos de leguminosas escandentes estabelecendo simbiose com bactérias fixadoras de nitrogênio ocorrem na floresta Amazônica. Estas simbioses provavelmente desempenham importante papel, contribuindo para incorporação de nitrogênio neste ecossistema. Os objetivos deste trabalho foram avaliar o crescimento de oito espécies de leguminosas escandentes em cinco substratos no viveiro; comparar a nodulação com as estirpes de rizóbio introduzidas ou nativas destes substratos; e caracterizar esses rizóbios fenotípica e geneticamente. O experimento foi conduzido em delineamento inteiramente casualizado com cinco repetições. Cinco a sete meses após a emergência das mudas parâmetros de crescimento e nodulação foram determinados. A identificação das estirpes de rizóbios foi feita pelo sequenciamento parcial do gene 16S rRNA. A sobrevivência das mudas após o transplante variou de 93 a 98%, em um Argissolo (Ultisol de acordo com a classificação da USDA), coletado em floresta primária e adubado com todos os nutrientes, exceto nitrogênio (ULTfert), e na mistura argila e areia, na proporção 3:2 (CONV), respectivamente. Espécies que apresentaram altura superior a 30 cm, no geral, cresceram melhor nos substratos de maior fertilidade: ULTfert e solo Glei Húmico (GH). Das oito espécies estudadas, sete nodularam. A porcentagem de nodulação por substrato foi: AREIA, areia lavada com inóculo misto de 100 estirpes de rizóbio mais adubação (100), GH (80), CONV (100), ULT, horizonte A de Argissolo vermelho-amarelo coletado na Reserva Florestal Ducke (Manaus) (44) e ULTfert (55%). Bradyrhizobium spp. foram isolados de nódulos de todas as espécies e substratos. Burkolderia fungorum foi isolada de Dalbergia inundata. Este é o primeiro relato de identificação de estirpes simbióticas para Dalbergia riedelli e Dalbergia inundata. Leguminosas escandentes apresentaram alta sobrevivência de mudas em casa de vegetação e desenvolveram-se [...]

Interrogantes en la tecnología de la inoculación de semillas de soja con Bradyrhizobium spp / Queries related to the technology of soybean seed inoculation with Bradyrhizobium spp

With the aim of exploiting symbiotic nitrogen fixation, soybean crops are inoculated with selected strains of Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens or Bradyrhizobium elkanii (collectively referred to as Bradyrhizobium spp.). The most common method of inoculation used is seed inoculation, whether performed immediately before sowing or using preinoculated seeds or pretreated seeds by the professional seed treatment. The methodology of inoculation should not only cover the seeds with living rhizobia, but must also optimize the chances of these rhizobia to infect the roots and nodulate. To this end, inoculated rhizobia must be in such an amount and condition that would allow them to overcome the competition exerted by the rhizobia of the allochthonous population of the soil, which are usually less effective for nitrogen fixation and thus dilute the effect of inoculation on yield. This optimization requires solving some queries related to the current knowledge of seed inoculation, which are addressed in this article. I conclude that the aspects that require further research are the adhesion and survival of rhizobia on seeds, the release of rhizobia once the seeds are deposited in the soil, and the movement of rhizobia from the vicinity of the seeds to the infection sites in the roots

Con el fin de aprovechar la fijación simbiótica de nitrógeno, el cultivo de soja se inocula con cepas seleccionadas de Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens o Bradyrhizobium elkanii (conjuntamente referidas como Bradyrhizobium spp.). El método más común de hacerlo es la inoculación en semillas, ya sea que esta se realice en el momento previo a la siembra o que se utilicen semillas preinoculadas o pretratadas mediante el tratamiento profesional de semillas. La metodología de inoculación no debe limitarse a recubrir las semillas con rizobios vivos, sino que también debe optimizar las chances de esos rizobios para infectar las raíces y nodular. Para ello los rizobios inoculados deben estar en una cantidad y un estado tales que les permitan superar la competición ejercida por los rizobios de la población alóctona del suelo, los cuales usualmente son menos eficaces para la fijación de nitrógeno y así diluyen el efecto de la inoculación sobre el rendimiento. Esta optimización requiere resolver algunos interrogantes, que son abordados en el presente artículo. Concluyo que los aspectos que requieren más investigación son la adhesión y supervivencia de los rizobios en las semillas, la liberación de los rizobios una vez que las semillas se depositan en el suelo y el movimiento de los rizobios desde las inmediaciones de las semillas hasta los sitios de infección en las raíces

Multilocus sequence analysis (MLSA) of Bradyrhizobium strains: revealing high diversity of tropical diazotrophic symbiotic bacteria

Symbiotic association of several genera of bacteria collectively called as rhizobia and plants belonging to the family Leguminosae (=Fabaceae) results in the process of biological nitrogen fixation, playing a key role in global N cycling, and also bringing relevant contributions to the agriculture. Bradyrhizobium is considered as the ancestral of all nitrogen-fixing rhizobial species, probably originated in the tropics. The genus encompasses a variety of diverse bacteria, but the diversity captured in the analysis of the 16S rRNA is often low. In this study, we analyzed twelve Bradyrhizobium strains selected from previous studies performed by our group for showing high genetic diversity in relation to the described species. In addition to the 16S rRNA, five housekeeping genes (recA, atpD, glnII, gyrB and rpoB) were analyzed in the MLSA (multilocus sequence analysis) approach. Analysis of each gene and of the concatenated housekeeping genes captured a considerably higher level of genetic diversity, with indication of putative new species. The results highlight the high genetic variability associated with Bradyrhizobium microsymbionts of a variety of legumes. In addition, the MLSA approach has proved to represent a rapid and reliable method to be employed in phylogenetic and taxonomic studies, speeding the identification of the still poorly known diversity of nitrogen-fixing rhizobia in the tropics.

Caracterización fenotípica y genotípica de doce rizobios aislados de diversas regiones geográficas de Venezuela

Phenotypic and genotypic characterization of twelve rhizobial isolates from different regions of Venezuela. Rhizobial taxonomy and systematics have progressed substantially, nevertheless, few studies have been developed on venezuelan species. This study evaluated the phenotypic and genetic variation between 12 venezuelan indigenous rhizobial isolates and 10 international referential strains, by phenotypical traits and DNA molecular markers. In this regard, a PCR-RFLP of the 16S rDNA gene, the presence of large plasmids, metabolic assays in solid media, salinity resistance, pH and temperature growth conditions, and intrinsic antibiotic resistance were assayed. In reference to the phenotypic attributes, we recognized three main groups: A group I, which comprised all the strains metabolizing between 67.5%-90% of the C and N sources. They were also acid-tolerant, as well as acid producers, capable of growing at 40ºC and in high salinity conditions (2-2.5% NaCl). With regard to the antibiotic sensitivity, this group was susceptible to a 30% of the antibiotic assayed. Strains belonging to Group II exhibited a lower salt tolerance (0.1-1.5%NaCl), as well as a lower acid tolerance, since they grew well at pH values equal or higher than 5.0. This group appeared to be resistant to all of the antibiotics assayed and only metabolized between 52.5%-82.5% of the C and N sources. Group III was represented by a single bacterial strain: it has a extremely low salt tolerance (0.1% NaCl). This strain grew at a pH equal or higher than 5.6, was susceptible to 50% of the antibiotics assayed and metabolized 72% of the C and N sources. On the basis of a PCR- RFLP of the 16S rDNA, three groups were also obtained. Members of the group A showed a close resemblance to Rhizobium tropici CIAT 899 and Sinorhizobium americanum CFN-EI 156, while Group B was closely related to Bradyrhizobium spp. Group C, was also represented by only one isolate. The Trebol isolate, was the only one strain able to form nodules and does not appear to be related to any of the referential rhizobial strains, suggesting a possible symbiotic horizontal gene transfer. Finally, in this work, there are evidences of a genetic diversity in the venezuelan rhizobial strains. A different geographical origin is perhaps an important factor affecting the diversity of the indigenous rhizobia in this study. Rev. Biol. Trop. 59 (3): 1017-1036. Epub 2011 September 01.

Rasgos fenotípicos y marcadores moleculares de ADN se utilizaron para investigar la variación fenotípica y genética entre 12 aislados rizobianos venezolanos y 10 cepas de referencia internacionales. Para ello, se realizó un PCR-RFLP del gen rDNA 16S, la presencia de plásmidos grandes, análisis metabólicos en medios sólidos, resistencia a la salinidad, condiciones del crecimiento a diferentes pH y temperaturas y la resistencia intrínseca a antibióticos. En referencia a las cualidades fenotípicas, se diferenciaron tres grupos principales, un grupo I que abarcó a todas aquellas cepas que metabolizaban entre 67.5% y 90% de las fuentes de C y de N. También eran tolerantes a la acidez y productoras de ácido, capaces de crecer a 40ºC y a altas condiciones de salinidad (NaCl 2-2.5%). Con respecto a la sensibilidad a antibióticos, este grupo era susceptible a un 30% de los antibióticos empleados. Las cepas que pertenecen al grupo II exhibieron una tolerancia salina más baja (0.1- 1.5%NaCl), así como una menor tolerancia a la acidez, puesto que crecieron bien en valores de pH iguales o mayores a 5.0. Este grupo era resistente a todos los antibióticos probados y metabolizaban solamente entre 52.5%-82.5% de las fuentes de C y de N. Una sola cepa bacteriana representó al grupo III, con una baja tolerancia salina (0.1% NaCl). Este aislado creció a un pH mayor o igual a 5.6, era susceptible a 50% de los antibióticos probados y metabolizó el 72% de las fuentes de C y de N. Al tener como base el PCR-RFLP del 16S rDNA, se diferenciaron también tres grupos. Los miembros del grupo A demostraron una estrecha relación con Rhizobium tropici CiAT 899 y Sinorhizobium americanum CFN-Ei156, mientras que el grupo B está estrechamente vinculado a Bradyrhizobium spp. El grupo C, está representado por solo un aislado. El aislado Trebol, fue la única cepa capaz de formar nódulos y no aparece relacionado con ninguna cepa de referencia, y sugiere una transferencia horizontal de genes simbióticos. Finalmente, en este trabajo se evidencia una diversidad genética en las cepas rizobianas venezolanas. El origen geográfico diverso de estas cepas, quizás sea un factor importante que influencie la diversidad de los rizobios indígenas utilizados en este estudio.

Doses de inoculante e nitrogênio na semeadura da soja em área de primeiro cultivo / Doses of inoculant and nitrogen at sowing of soybean in first cultivation area

A inoculação da cultura da soja com bactérias do gênero Bradyrhizobium, é uma prática amplamente difundida, devido aos ótimos resultados alcançados em produtividade, porém, algumas dúvidas ainda surgem em relação à necessidade de aplicação de nitrogênio mineral na semeadura. Neste sentido, este trabalho teve como objetivo avaliar a resposta da cultura da soja a doses de inoculante e nitrogênio mineral na semeadura em solo de primeiro ano de cultivo. Os experimentos foram realizados no Campo Experimental da Universidade do Estado de Mato Grosso, Campus de Alta Floresta, MT, nos anos agrícolas de 2006/2007 e 2007/2008. O delineamento experimental foi em blocos ao acaso, num esquema fatorial 2x4, com quatro repetições. Os fatores consistiram de inoculação das sementes com Bradyrhizobium japonicum (3 e 6 mL kg-1 de sementes), e adubação nitrogenada na semeadura (0, 10, 20 e 40 kg de N ha-1). As seguintes variáveis foram avaliadas: altura de plantas, altura de inserção da primeira vagem, número de vagens por planta, número de grãos por vagem, massa de 100 grãos e produtividade de grãos. Nos dois anos agrícolas não houve diferença significativa para as doses de inoculante aplicadas em primeiro ano de cultivo com soja. Para a aplicação de nitrogênio na semeadura da cultura da soja houve efeito quadrático para altura de plantas e de inserção da primeira vagem e linear crescente para número de vagens por planta no segundo ano de cultivo, porém, sem incremento significativo na produtividade.

The inoculation of soybean crop with Bradyrhizobium bacteria is a widespread practice, due to excellent results achieved in productivity, however, some doubt still arise in relation the need of application of mineral nitrogen at sowing. In that mean, this work aimed to evaluate the response of the doses at inoculant and mineral nitrogen in the sowing of crop soybean in area of first cultivation. The experiments were carried in the experimental field of the University of the Estate of Mato Grosso, Campus of Alta Floresta - MT, Brazil, in the growing seasons of 2006/2007 and 2007/2008. The experimental design was randomized blocks, in a factorial escheme 2 x 4, with four replications. The factors consisted in the inoculation of seeds with Bradyrhizobium japonicum (3 e 6 mL kg-1 of seeds), and nitrogen fertilizer at sowing (0, 10, 20 e 40 kg ha-1). The following variables were evaluated: plant height, insertion height of first pod, number of pod per plant, number of grain per pod, mass of 100 grain and grain productivity. In the two growing seasons there was no significant difference for doses of inoculants applied in first year of cultivation with soybean. To nitrogen application at sowing of soybean crop there was quadratic effect to plant height and of insertion height of first pod and growing linear to number of pod per plant in the second year of cultivation, however, without significant increase the productivity

Fixação biológica de nitrogênio e teores foliares de nutrientes na soja em função de doses de molibdênio e gesso agrícola / Biological nitrogen fixation and leaf nutrient concentration on soybean as a function of molybdenum and gypsum levels

A competitividade econômica da soja brasileira no mercado mundial se deve, em grande parte, aos benefícios da fixação biológica do nitrogênio na cultura. O trabalho foi conduzido a campo, sob sistema plantio direto, em condição de sequeiro, no Município de Maracaju-MS, com o objetivo de avaliar a nodulação, o crescimento, nutrição mineral e produtividade de grãos da soja submetida a diferentes doses de gesso agrícola e molibdênio. Foi utilizado o delineamento experimental de blocos ao acaso, com cinco repetições e esquema de parcelas subdivididas, sendo as parcelas representadas por quatro doses de gesso agrícola (0, 1.000, 2.000 e 3.000 kg ha-1) e as subparcelas, por quatro doses de molibdênio (0, 20, 40 e 60 g ha-1). Não houve efeito da interação gesso x Mo sobre a produtividade da soja. O gesso agrícola não influencia no teor foliar de N e na produtividade. O Mo proporciona incrementos na produtividade e no teor de proteínas dos grãos.

The economic competitiveness of Brazilian soybeans on the world market occurs, in large part, due to the benefits of biological nitrogen fixation in this crop. The field experiment was carried out in Maracaju, Mato Grosso do Sul State, Brazil, under no-tillage system, in rainfed condition. The aim was to evaluate nodulation, growth, mineral nutrition and grain yield of soybeans under different doses of gypsum and molybdenum. The experimental design used was a randomized block with five replicates and arranged in a split-plot squeme, with the plot represented by four doses of gypsum (0, 1.000, 2.000 and 3.000 kg ha-1) and the subplots by four doses of molybdenum (0, 20, 40 and 60 g ha-1). There were no interaction effects of Mo x gypsum for grain yield. Gypsum has no influence in the N leaf content and grain yield. Mo increases grain yield and protein levels in the grain.

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

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

Production and some properties of crude alkaline proteases of indigenous Central Amazonian rhizobia strains

Two rhizobia strains isolated from soils of the Central Amazonian floodplain produced appreciable quantities of crude alkaline protease extracts with inexpensive carbon and nitrogen sources. These protease crude extracts were optimally active at pH 9.0-11.0. The optimum temperatures were 35 ºC for Rhizobium sp. strain R-986 and 55 ºC for Bradyrhizobium sp. strain R-993. Protease activities in the crude extracts were enhanced in the presence of 5 mM metal ions, such as Na+, Ca2+, Mg2+ and Mn2+. Rhizobia proteases were strongly inhibited by PMSF, a serine-protease inhibitor. The enzymes were active in the presence of surfactants (SDS and Triton X-100) and stable in oxidizing (H2O2) and reducing agents (β-mercaptoethanol), and organic solvents (acetone, hexane, methanol, 1-propanol and toluene).

Duas estirpes de rizóbia isoladas de solos de várzea da Amazônia Central produziram grandes quantidades de proteases alcalinas extracelulares, usando fontes baratas de carbono e nitrogênio. Os extratos brutos de proteases foram ativos em pH 9,0-11,0. As temperaturas ótimas foram de 35 ºC para a enzima do Rhizobium R-986 e de 55 ºC para a do Bradyrhizobium R-993. As atividades proteolíticas aumentaram na presença de 5 mM dos íons Na+, Ca2+ , Mg2+ e Mn2+ . As proteases secretadas pelos rizóbios foram fortemente inibidas por PMSF, um inibidor de serina protease. As enzimas foram ativas na presença de surfactantes (SDS e Triton X-100), e estáveis na presença de agentes oxidantes (H2O2) e redutores (β-mercaptoetanol) e solventes orgânicos (acetona, hexano, metanol, 1-propanol e tolueno).

Nodulation and N2 fixation effectiveness of Bradyrhizobium strains in symbiosis with Adzuki Bean, Vigna angularis

In pot experiment, one isolate Knj from a Serbian soil, four strains of Bradyrhizobium japonicum and three strains of Bradyrhizobium spp. were examined for the effect on adzuki bean nodulation and effectiveness in symbiotic N2 fixation. All the tested strains produced root nodules in adzuki bean. Strains of B. japonicum showed high potential of N2 fixation, particularly 525 and 542. B. japonicum strains resulted 65-71 percent shoot dry weight and 99-138 percent total N content of uninoculated control with full N content (100 percent). No significant difference was found between the plants inoculated with Bradyrhizobium spp. strains and uninoculated control plants without N (40-42 and 42 percent shoot dry weight, respectively), which indicated symbiotic N2 fixation inactivity of the Bradyrhizobium spp. strains. Knj strain had the middle position (56 percent shoot dry weight). These data showed that B. japonicum 525 and 542 strains could be used in further investigations in order to apply them as inoculants in microbiological N fertilizers.

Partial characterization of amylases of two indigenous Central Amazonian rhizobia strains

Amylase production and partial characterization of crude enzyme preparations from two rhizobia strains (R-926 and R-991) were evaluated. For both the strains, maximal amylase activities were achieved during the early-to-mid- exponential growth phase; both were active over a pH range from 4.5 to 8.5 and temperature from 30 to 50 ºC. None of the ions studied (K+, Na+, Ca2+, Hg2+, Mg2+, Mn2+, Cu2+ and Zn2+) was required for the catalytic activity of strain R-926; amylase activity of strain R-991 was stimulated in the presence of K+, Hg2+ and Zn2+. The surfactants SDS, Triton X-100 and Tween-80 did not have a pronounced inhibitory effect on enzyme activities; SDS and Tween-80 caused the highest stimulatory effects. Amylase activities from the rhizobia strains were reduced by up to 30 percent in the presence of EDTA; amylase activity of R-926 was also inhibited by HgCl2, suggesting that Ca2+and cysteine residues could be important for activity of this strain.

A produção e parcial caracterização de extratos brutos de amilase de duas estirpes de rizóbio (R-926 e R-991) foram avaliadas. Para ambas as estirpes, as máximas atividades amilolíticas foram obtidas no início/meio da fase exponencial de crescimento. As amilases rizobiais foram ativas numa variação de pH de 4,5 a 8,5 e temperatura de 30 a 50 ºC. Nenhum dos íons testados (K+, Na+, Ca2+, Hg2+, Mg2+, Mn2+, Cu2+ e Zn2+) foi exigido para a atividade catalítica da estirpe R-926. A amilase produzida pelo R991 foi estimulada na presença de K+, Hg2+ e Zn2+. Os surfactantes SDS, Triton X-100 e Tween-80 não exerceram um pronunciado efeito inibitório sobre as atividades enzimáticas, e SDS e Tween-80 causaram os maiores efeitos estimulatórios. A atividade amilolítica rizobial foi reduzida em até 30 por cento na presença de EDTA; a amilase produzida pela estirpe R-926 também foi inibida pelo HgCl2, sugerindo, ao menos em parte, a importância de Ca2+ e resíduos de cisteína na atividade amilolítica dessa estirpe.

Coinoculação rizóbio e Bacillus subtilis em feijão-caupi e leucena: efeito sobre a nodulação, a fixação de N2 e o crescimento das plantas / Co-inoculation rhizobia and Bacilus subtilis in cowpea and Leucaena: effects on nodulation, N2 fixation and plant growth

O objetivo deste trabalho foi avaliar o efeito da associação entre o rizóbio e o Bacillus subtilis sobre a nodulação, a fixação de N2 e o crescimento do feijão-caupi (Vigna unguiculata) e da leucena (Leucaena leucocephala) cultivados em um Latossolo vermelho. Os tratamentos consistiram de: a) testemunha; b) fertilização com NPK; c) inoculação com rizóbio + PK; e d) inoculação com rizóbio + Bacillus subtilis + PK. Houve um aumento na nodulação do feijão-caupi com a coinoculação. A massa da parte aérea seca, o acúmulo de N e a leitura da clorofila em feijão-caupi foram maiores nos tratamentos fertilizados (NPK) e no tratamento coinoculado. No caso da leucena, não houve diferenças entre os tratamentos NPK, inoculado e coinoculado. As massas das raízes secas do feijão-caupi e leucena aumentaram com a coinoculação. A coinoculação rizóbio e Bacillus subtilis favoreceu o aumento na nodulação em feijão-caupi e tem potencial para aumentar a fixação biológica do N2 e o crescimento das plantas.

This research aimed to evaluate co-inoculation of Bradyrhizobium and Bacillus subtilis on nodulation, N2 fixation and growth of cowpea (Vigna unguiculata) and leucaena (Leucaena leucocephala) grown in a red Oxisol. Treatments consisted by: a) control; b) NPK; c) inoculation with Bradyrhizobium + PK; d) co-inoculation Bradyrhizobium + Bacillus subtilis + PK. There was an increase in nodulation of cowpea with co-inoculation. The shoot dry weight, N accumulation and chlorophyll were higher in both co-inoculated and fertilized (NPK) treatments. For leucaena, there were not differences between NPK, inoculated and co-inoculated treatments. The dry weight roots of cowpea and leucaena increased with co-inoculation. The co-inoculation Bradyrhizobium and Bacillus subtilis favored the increase in nodulation of cowpea and it has potential to increase biological N2 fixation and plant growth.

Caracterización de cepas de Rhizobium y Bradyrhizobium (con habilidad de nodulación) seleccionados de los cultivos de fríjol caupi (Vigna unguiculata) como potenciales bioinóculos / Characterization of Rhizobium and Bradyrhizobiu´s strains (with ability of nodulation) selected from bean (Vigna unguiculata) cultures as a potentials bioinoculants

Los suelos de Latinoamérica son ácidos y deficientes en nitrógeno; por esta razón es necesario explorar nuevas alternativas con el fin de modular el desarrollo de plantas, especialmente las leguminosas. El uso del potencial de las bacterias para inducir nodulación y fijar el nitrógeno en las plantas leguminosas se ha estudiado como una opción con potencial impacto. En este estudio se analizó la diversidad de cepas de rhizobios aislados del fríjol Vigna unguiculata (fríjol caupí) cultivado en el norte del departamento de Bolívar (Colombia). Se identificaron aislados capaces de crecer mejor en ambientes hostiles (los cuales tienen un uso potencial como bioinoculantes). Se describe además un acercamiento a la taxonomía de estas bioespecies. Se caracterizaron 52 cepas de rizobios (basados en sus características morfológicas, requerimientos de cultivo, metabólicas, resistencia a metales y antibióticos y de autenticación). De acuerdo con sus propiedades de crecimiento, el 63,5 por ciento fueron de cepas de lento, mientras que el 36,5 por ciento, de rápido crecimiento. Prevalecieron las de lento crecimiento con un 63,5 por ciento del total sobre un 36,5 por ciento de rápido crecimiento. Los aislados fueron caracterizados de acuerdo con sus patrones de asimilación de carbohidratos, y se encontraron microorganismos de los géneros Rhizobium, Bradyrhizobium y Mesorhizobium (12 cepas identificadas como potenciales bioinoculantes).

Latin America´s soils are acidic and nitrogen-deficient, for this reason is necessary to explore new alternatives in order to modulate in the development of plants, specially the leguminous plants. The use of bacteria´s potential to induce nodulation and to fix nitrogen in leguminous plants has been studied as an option with potential impact. In our study, we analyzed the diversity of rhizobial isolated of the bean Vigna unguiculata (beans cowpea) cultured in the north of Bolivar department (Colombia). We identified strains able of growing in hostile environment (which have a potential use like bioinoculants). Additionally, we described an approach to taxonomy of these biospecies. We reached the characterization of 52 rhizobial strains (based on their morphologic, requires of culture, metabolic, resistance to metals and antibiotics and authentication characteristics). According with their growing properties, 63.5 percent were slow growth strains while the 36.5 percent were of rapid growth. The isolates were characterized according to their assimilation carbohydrate´s pattern, finding microorganism as Rhizobium, Bradyrhizobium and Mesorhizobium (12 of which were identified as potential bioinoculants).

Inoculação e adubação nitrogenada sobre a nodulação e a produtividade de grãos de feijão-caupi / Inoculation and nitrogen fertilization on nodulation and grain yield of cowpea

O objetivo deste trabalho foi avaliar a influência da inoculação e da adubação nitrogenada sobre a nodulação e a produtividade do feijão-caupi. O estudo foi conduzido em uma área experimental, em Parnaíba, PI. Os tratamentos foram dispostos em delineamento em blocos ao acaso em arranjo fatorial de 5 x 2, sendo cinco doses de N (0, 20, 40, 80 e 160kg ha-1 de N) e dois tratamentos microbiológicos (com e sem inoculação). No tratamento com inoculação, foi utilizado um inoculante contendo Bradyrhizobium sp, estirpe BR2001. A avaliação da nodulação e da produtividade de grãos foi realizada aos 36 e 65 dias após a emergência das plantas, respectivamente. Houve interação significativa entre inoculação e doses de N para todas as variáveis analisadas, sendo a regressão quadrática a que melhor ajustou-se aos dados observados. O número e a massa dos nódulos apresentaram, no tratamento com inoculação, valores máximos de 29 nódulos e 118,6mg nas doses estimadas de 34,5 e 17,5kg ha-1 de N, respectivamente. Os resultados obtidos no presente estudo indicam que a produtividade de grãos do feijão-caupi aumenta com o uso da inoculação e o efeito dessa prática é favorecido quando o feijão-caupi é adubado com no máximo 20kg ha-1 de N.

The aim of this research was to evaluate the influence of inoculation and nitrogen fertilization on nodulation and grain yield of cowpea. The experiment was carried out in an experimental area in Parnaíba, PI. The treatments were disposed in a completely randomized block in factorial design of 5 x 2, consisted of five N levels (0, 20, 40, 80 and 160kg ha-1 N) and two microbiological treatments (with and without inoculation). In the treatment with inoculation, an inoculant was used containing Bradyrhizobium sp, strain BR2001. The evaluation of nodulation and grain yield was performed 36 and 65 days after plant emergency, respectively. There was significative interaction among inoculation and N levels in all variables, being the quadratic regression the one that best adjusted to data. The number and mass of nodules presented, in the treatment with inoculation, maximum values calculated of 29 nodules and 118,6mg in doses estimated of 34.5 and 17.5kg ha-1 N, respectively. The results indicate that the grain yield increase by use of inoculation and the effect of this practice is favored when the cowpea is fertilized with maximum 20kg ha-1 of N.

Primary Study on Co-inoculum of Peanut Bradyrhizobium and Molybdate and Boron / 微生物学通报

Aiming at molybdate and boron deficient acid purple soil from main peanut cultivated areas in Sichuan,and Mo and B requirement of peanut growth,the feasibility of Co-inoculum of peanut Bradyrhizobium and molybdate and boron was studied.The tolerance to molybdate and boron of the tested strains Spr2-9,Spr4-5 was inspected.The result indicated that the two tested strains could tolerate higher concentration of molybdate than that of boron.The compound inoculum of Bradyrhizobial strain and trace element Mo was developed.The optimum concentration of Mo was 0.4%.