Root nodule bacteria were isolated from nodules on Mimosa pudica L. growing in neutral-alkaline soils from the Distrito Federal in central Brazil. The 16S rRNA gene sequence analysis of 10 strains placed them into the genus Rhizobium with the closest neighbouring species (each with 99 % similarity) being Rhizobium grahamii, Rhizobium cauense, Rhizobium mesoamericanum and Rhizobium tibeticum. This high similarity, however, was not confirmed by multi-locus sequence analysis (MLSA) using three housekeeping genes (recA, glnII and rpoB), which revealed R. mesoamericanum CCGE 501T to be the closest type strain (92 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [with majority being C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c)], DNA G+C content (57.6 mol%), and carbon compound utilization patterns supported the placement of the novel strains in the genus Rhizobium. Results of average nucleotide identity (ANI) differentiated the novel strains from the closest species of the genus Rhizobium, R. mesoamericanum, R. grahamii and R. tibeticum with 89.0, 88.1 and 87.8 % similarity, respectively. The symbiotic genes essential for nodulation (nodC) and nitrogen fixation (nifH) were most similar (99-100 %) to those of R. mesoamericanum, another Mimosa-nodulating species. Based on the current data, these 10 strains represent a novel species of the genus Rhizobium for which the name Rhizobium altiplani sp. nov. is proposed. The type strain is BR 10423T (=HAMBI 3664T).

Resumo -Os fungos endofíticos conhecidos como DSEF ou "dark septate" podem ser promotores de crescimento vegetal. Com o objetivo de aperfeiçoar uma metodologia para o isolamento, armazenamento e determinação da colonização de raízes de arroz por fungos "dark septate" desenvolveu-se um estudo a partir da obtenção destes microrganismos em plantas de Oryza glumaepatula coletadas em ambientes amazônicos. As raízes das plantas de doze amostras de O. glumaepatula foram seccionadas, desinfestadas e dispostas em placas com o meio ágar-malte adicionado de cloranfenicol, sulfametazol e trimetropina. Os isolados obtidos foram avaliados quanto às características de "dark septate" e armazenados em água destilada com e sem a adição de glicerol. Cinco isolados fúngicos considerados "dark septate" foram reinoculados em plantas de O. glumaepatula e em O. sativa e, também, caracterizados molecularmente através da amplificação e sequenciamento da região ITS1-5.8S-ITS2. O meio de cultura ágar-malte foi eficiente para a obtenção de "dark septate", obtendo-se mais de 50 isolados. O armazenamento em água destilada e em água destilada adicionada de glicerol mostrou-se eficiente para a manutenção das culturas fúngicas por pelo menos 12 meses. Os cinco isolados fúngicos reinoculados foram capazes de colonizar tanto raízes O. glumaepatula quanto O. sativa, não desencadeando sintomas de patogenicidade. A análise molecular mostrou que estes fungos apresentam baixa similaridade entre eles e não possuem similaridade com fungos "dark septate" já descritos, indicando que a metodologia empregada permitiu a obtenção de espécies fúngicas diversas e que a espécie O. glumaepatula abriga alta diversidade deste grupo de fungos. Palavras-Chave -Amazônia. DSEF. Oryza glumaepatula. Oryza sativa.Abstract -Endophyte fungi known as DSEF can be plant-growth-promoting. Aiming to improve a methodology for the isolation, storage and determine the rice roots colonization by "dark septate", a study was developed based on the achievement of these microorganisms in Oryza glumaepatula plants collected in Amazon region. The plant roots of twelve O. glumaepatula samples were sectioned, disinfected, and placed in plates with agar-malte medium added of chloramphenicol, sulphametazole and trimetropin. The isolates were evaluated in relation of dark septate characteristics and stored in distilled water, and in distilled water added of glycerol. Five isolates considered dark septate were re-inoculated in O. glumaepatula and O. sativa, and also analyzed through amplification and sequencing of ITS1-5.8S-ITS2 region. The agar-malte medium was efficient to isolate dark septate, and more than 50 isolates were obtained. The distilled water and also distilled water added of glycerol were efficient to maintain the fungus culture at least 12 months. The five isolates re-inoculated were able to colonize both O. glumaepatula and O. sativa, without pathogenic symptoms observed. The molecular approach reveled that these fungus are not similar to each other and also to others dark septa...

Root nodule bacteria were isolated from Centrolobium paraense Tul. grown in soils from the Amazon region, State of Roraima (Brazil). 16S rRNA gene sequence analysis of seven strains (BR 10247 T , BR 10296, BR 10297, BR 10298, BR 10299, BR 10300 and BR 10301) placed them in the genus Bradyrhizobium with the closest neighbours being the type strains of Bradyrhizobium paxllaeri (98.8 % similarity), Bradyrhizobium icense (98.8 %), Bradyrhizobium lablabi (98.7 %), Bradyrhizobium jicamae (98.6 %), Bradyrhizobium elkanii (98.6 %), Bradyrhizobium pachyrhizi (98.6 %) and Bradyrhizobium retamae (98.3 %). This high similarity, however, was not confirmed by the intergenic transcribed spacer (ITS) 16S-23S rRNA region sequence analysis nor by multi-locus sequence analysis. Phylogenetic analyses of five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed Bradyrhizobium iriomotense EK05 T (5LMG 24129 T ) to be the most closely related type strain (95.7 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [major components being C 16 : 0 and summed feature 8 (18 : 1v6c/18 : 1v7c)], DNA G+C content, slow growth rate and carbon compound utilization patterns, supported the placement of the novel strains in the genus Bradyrhizobium. Results of DNA-DNA relatedness studies and physiological data (especially carbon source utilization) differentiated the strains from the closest recognized species of the genus Bradyrhizobium. Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) placed the novel species in a new branch within the genus Bradyrhizobium. Based on the current data, these seven strains represent a novel species for which the name Bradyrhizobium neotropicale sp. nov. is proposed. The type strain is BR 10247 T (5HAMBI 3599 T ).

The objective of this work was to isolate and characterize rhizobia from nodules of Centrolobium paraense and to evaluate their symbiotic efficiency. Soil samples collected from four sites of the Roraima Cerrado, Brazil, were used to cultivate C. paraense in order to obtain nodules. Isolates (178) were obtained from 334 nodules after cultivation on medium 79. Twenty-five isolates belonging to six morphological groups were authenticated using Vigna unguiculata and they were characterized by 16S rRNA. Isolates identified as Bradyrhizobium were further characterized using rpoB gene sequencing. A greenhouse experiment was carried out with C. paraense to test the 18 authenticated isolates. Approximately 90% of the isolates grew slowly in medium 79. The 16S rRNA analysis showed that 14 authenticated isolates belong to the genus Bradyrhizobium, and rpoB indicated they constitute different groups compared to previously described species. Only four of the 11 fast-growing isolates nodulated V. unguiculata, two of which belong to Rhizobium, and two to Pleomorphomonas, which was not previously reported as a nodulating genus. The Bradyrhizobium isolates ERR 326, ERR 399, and ERR 435 had the highest symbiotic efficiency on C. paraense and showed a contribution similar to the nitrogen treatment. Centrolobium paraense is able to nodulate with different rhizobium species, some of which have not yet been described.

Bacterias diazotróficas aisladas de arroz silvestre Oryza glumaepatula (Poaceae) en la Amazonia brasileña. The association of wild grasses with diazotrophic bacteria in Brazilian biomes is poorly understood. The isolation and characterization of bacteria associated with wild grasses can contribute to understand the diazotrophic ecology as well as to identify bacteria with biotechnological applications. In this study, we isolated and characterized diazotrophic bacterial isolates from Oryza glumaepatula collected in Cerrado and Forest areas of the Amazon in Roraima State, Brazil. Healthy O. glumepatula plants were collected at five sampling sites at Forest and seven at Cerrado, respectively. The plants were collected at the Cerrado areas in September 2008 while the Forest plants were collected in June/2008 and April/2009. The plants and the soil adhering to the roots were transferred to pots and grown for 35 days in greenhouse conditions. During the harvest, the shoots and the roots were crushed separately in a saline solution; the suspension was diluted serially and inoculated in Petri dishes containing Dyg's medium. All distinct bacterial colonies were purified in the same medium. The diazotrophic capacity of each bacterium in microaerophilic conditions was assessed in semisolid BMGM medium. In addition, the pellicles forming bacterial isolates were also evaluated by PCR amplification for nifH gene. The diversity of nifH + bacteria was analyzed by Box-PCR fingerprinting. For selected strains, the growth promoting capacity of O. sativa as a model plant was also evaluated. A total of 992 bacterial isolates were obtained. Fiftyone bacteria were able to form pellicles in the semisolid medium and 38 also positively amplified the 360bp nifH gene fragment. Among the 38 nifH + isolates, 24 were obtained from the shoots, while 14 originated from the roots. The Box-PCR profiles showed that the bacterial isolates obtained in this study presented a low similarity with the reference strains belonging to the Herbaspirillum, Azospirillum and Burkholderia genus. The growthpromoting ability was confirmed for at least five isolates. For these bacteria, the root and shoot growing results showed higher increases when compared to those observed in plants inoculated with the evaluated reference strains. These results indicate that O. glumaepatula is colonized by a high diverse diazotrophic community in