Gustavo Ribeiro Xavier

Microvirga vignae sp. nov., a root nodule symbiotic bacterium isolated from cowpea grown in semi-arid Brazil

16S rRNA gene sequence analysis of eight strains (BR 3299(T), BR 3296, BR 10192, BR 10193, BR 10194, BR 10195, BR 10196 and BR 10197) isolated from nodules of cowpea collected from a semi-arid region of Brazil showed 97 % similarity to sequences of recently described rhizobial species of the genus Microvirga. Phylogenetic analyses of four housekeeping genes (gyrB, recA, dnaK and rpoB), DNA-DNA relatedness and AFLP further indicated that these strains belong to a novel species within the genus Microvirga. Our data support the hypothesis that genes related to nitrogen fixation were obtained via horizontal gene transfer, as sequences of nifH genes were very similar to those found in members of the genera Rhizobium and Mesorhizobium, which are not immediate relatives of the genus Microvirga, as shown by 16S rRNA gene sequence analysis. Phenotypic traits, such as host range and carbon utilization, differentiate the novel strains from the most closely related species, Microvirga lotononidis, Microvirga zambiensis and Microvirga lupini. Therefore, these symbiotic nitrogen-fixing bacteria are proposed to be representatives of a novel species, for which the name Microvirga vignae sp. nov. is suggested. The type strain is BR3299(T) ( = HAMBI 3457(T)).

Edaphic factors as determinants for the distribution of intrinsic antibiotic resistance in a cowpea rhizobia population

Abstract A large collection of cowpea rhizobia strains was obtained from soil samples collected from either a semi-arid or a tropical rain forest area located at about the same latitude in the north-eastern region of Brazil and evaluated for their intrinsic antibiotic resistance to eight commercial antibiotics. The aim of this study was to correlate antibiotic resistance of native rhizobia strains to edaphic-climatic factors as a way to establish suitable inoculants for specific areas. A large diversity regarding intrinsic antibiotic resistance was found, and 17 clusters were identified as varying from sensitive to gradually resistant up to 500 μg·ml–1 of the antibiotics tested. Clustering analysis did not show any pattern related to the geographic region where isolates have been obtained. On the other hand, an increase in the antibiotic-resistant rhizobia population was associated with an increase in soil P and Al contents. lsolates which were sensitive to spectinomycin, ampicillin, streptomycin, chloramphenicol and tetracycline were present at higher rates in soils devoid of Al. Rhizobia bacteria producing mucus type I (fluid and capable of spreading over the solid media) were found preferentially in soil with Al concentrations up to 36 mg·kg–1, diminishing quickly at higher levels.

Bradyrhizobium manausense sp. nov., isolated from effective nodules of Vigna unguiculata grown in Brazilian Amazonian rainforest soils

Root nodule bacteria were trapped within cowpea (Vigna unguiculata) in soils with different cultivation histories collected from the Amazonian rainforest in northern Brazil. Analysis of the 16S rRNA gene sequences of six strains (BR 3351(T), BR 3307, BR 3310, BR 3315, BR 3323 BR and BR 3361) isolated from cowpea nodules showed that they formed a distinct group within the genus Bradyrhizobium, which was separate from previously identified type strains. Phylogenetic analyses of three housekeeping genes (glnII, recA and rpoB) revealed that Bradyrhizobium huanghuaihaiense CCBAU 23303(T) was the most closely related type strain (96% sequence similarity or lower). Chemotaxonomic data, including fatty acid profiles (predominant fatty acids being C16 : 0 and summed feature 8), the slow growth rate and carbon compound utilization patterns supported the assignment of the strains to the genus Bradyrhizobium. The results of DNA-DNA hybridizations, antibiotic resistance and physiological tests differentiated these novel strains from the most closely related species of the genus Bradyrhizobium with validly published names. Symbiosis-related genes for nodulation (nodC) and nitrogen fixation (nifH) grouped the novel strains of the genus Bradyrhizobium together with Bradyrhizobium iriomotense strain EK05(T), with 94% and 96% sequence similarity, respectively. Based on these data, these six strains represent a novel species for which the name Brabyrhizobium manausense sp. nov. (BR 3351(T) = HAMBI 3596(T)), is proposed.

Contribuição de estirpes de rizóbio para o desenvolvimento e produtividade de grãos de feijão-caupi em Roraima

The study aimed to evaluate the contribution of biological nitrogen fixation (BNF), promoted by rhizobium strains, to development and grain yield of cowpea in Roraima. In the years 2005 and 2006 experiments were performed in cerrado and mata alterada areas, where were tested the strains INPA 03-11B, UFLA 3-84, BR3267 (recommended to the cowpea), the strains BR3299 and BR3262, two mineral nitrogen doses (50 and 80 kg ha-1 of N) and a control. The analyzed variables were: nodulation and cowpea plants dry matter production, and the grain yield in the harvest. It was observed, in the mean, that BR3262 strain provided a number and nodule dry mass significantly larger than the control, while among the recommended strains, this only occurred in a sporadic form with INPA 03-11B and BR3267. Besides, it was also observed that soil rhizobium population was determinant to plants nodulation in the experiments. Comparatively to the other strains, BR3262 together with BR3267, provided superior effectiveness in BNF to plant dry mass production. In relation to grain yield, the strain BR3267 and INPA 03-11B presented better resulted compared to UFLA 3-84, however, just the strains BR3262 provided grain yield (in the mean about 1700 kg h-1), equal to 50 kg ha-1 N dose and superior to the control in three of the four experiments performed, showing to be most suitable for cowpea inoculation in Roraima.

Polymers as carriers for rhizobial inoculant formulations

O objetivo deste trabalho foi avaliar a eficiencia de misturas polimericas de carboximetilcelulose (CMC) e amido, como veiculos de inoculante para rizobios, quanto a sua capacidade de manter celulas rizobianas viaveis e promover a nodulacao em feijao-caupi (Vigna unguiculata). O delineamento experimental foi completamente casualizado, com tres repeticoes. Quarenta diferentes composicoes polimericas de carboximetilcelulose (CMC) e amido, compatibilizadas ou nao com proporcoes de MgO ou ZnO, foram inicialmente avaliadas quanto a sua capacidade de manter celulas rizobianas viaveis pelo periodo de um mes. Posteriormente, veiculos de inoculantes selecionados foram avaliados quanto a capacidade de manter celulas rizobianas viaveis pelo periodo de 165 dias, e seu desempenho como veiculos de inoculantes foi comparado com os de inoculantes turfosos, em casa de vegetacao. Celulas rizobianas sobreviveram melhor em misturas com 50-60% de CMC. Os agentes compatibilizantes nao aumentaram a sobrevivencia das celulas rizobianas apos 30 dias de estocagem. A nodulacao do feijao-caupi com o uso das misturas polimericas nao diferiu estatisticamente da nodulacao com o uso da turfa. As misturas de CMC/amido, quando compatibilizadas com MgO (1%), sao veiculos eficientes para inoculantes rizobianos por ate 165 dias de armazenamento.

Biodiversity of rhizobia associated with cowpea cultivars in soils of the lower half of the São Francisco River Valley

The biodiversity of rhizobium in soils of the Sao Francisco Valley is unknown and can be studied using cowpea as trap plants. The objective of this study was to verify the diversity of diazotrophic bacteria that nodulate cowpea in soils of the lower half of the Sao Francisco River Valley by morphological and genotypic characterization. Seven soil samples (A1, A2, A3, A4, C1, C2 and MC) were collected to capture bacteria associated to five cowpea cultivars (IPA 206, BRS Pujante, BRS Marataoa, Canapu Roxo, and Sempre Verde), in a 5x7 factorial design with three replications. Thirty days after plant emergence, the nodules were collected and the bacteria isolated and analyzed in relation to their growth characteristics in YMA medium. The 581 isolates were grouped in 49 morphologic groups. Of this total, 62.3 % formed colonies in up to three days, 33.4 % grew from the 6 th day on, and 4.3 % began to grow 4 to 5 days after incubation. Regarding the formation of acids and alkalis, 63 % acidified the medium, 12 % made it alkaline and 25 % maintained the medium at neutral pH. The highest diversity was observed in the A3 sample and in isolates associated with the cultivars Canapu Roxo and BRS Pujante. Thirty-eight representative isolates were chosen for the genotypic characterization, clustered in four groups based on the restriction analysis of 16s rDNA. This grouping was strongly correlated with the sampling site; 13 rhizobium isolates had an electrophoretic profile distinct from the standard rhizobium strains used in this study.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.

Variabilidade genética em acessos de caupi analisada por meio de marcadores RAPD

The knowledge on genetic variability and the relationship among different cowpea accesses is important to maximize resource use represented by available cowpea genotypes. The objective of this work was to determine the genetic variability among 45 cowpea (Vigna unguiculata (L.) Walp.) accesses from Brazil, USA and Niger, characterized by RAPD (Random Amplified Polymorphic DNA) markers. Eight polymorphic primers were identified, comprehending a total of 48 informative bands. Based on the obtained polymorphic profiles, four major clusters were formed. Clustering was mainly influenced by the genotype origin. Most accesses from Brazilian landraces belong to just one cluster, suggesting a limited genetic basis. It is worth noting that none of the genotypes from Niger considered as possessing superior agronomical traits, such as high productivity, was present in this cluster. RAPD shows to be an efficient tool, capable of assisting cowpea genotype selection adapted to Brazilian edaphoclimatic conditions, aiming at increasing productivity and improving other desirable characteristics to meet the needs of specific regional demands.

Characterization of indigenous rhizobia from Caatinga

The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100% similarity, according to ARDRA results.

Cowpea Nodules Harbor Non-rhizobial Bacterial Communities that Are Shaped by Soil Type Rather than Plant Genotype

Many studies have been pointing to a high diversity of bacteria associated to legume root nodules. Even though most of these bacteria do not form nodules with legumes themselves, it was shown that they might enter infection threads when co-inoculated with rhizobial strains. The aim of this work was to describe the diversity of bacterial communities associated with cowpea (Vigna unguiculata L. Walp) root nodules using 16S rRNA gene amplicon sequencing, regarding the factors plant genotype and soil type. As expected, Bradyrhizobium was the most abundant genus of the detected genera. Furthermore, we found a high bacterial diversity associated to cowpea nodules; OTUs related to the genera Enterobacter, Chryseobacterium, Sphingobacterium, and unclassified Enterobacteriacea were the most abundant. The presence of these groups was significantly influenced by the soil type and, to a lesser extent, plant genotype. Interestingly, OTUs assigned to Chryseobacterium were highly abundant, particularly in samples obtained from an Ultisol soil. We confirmed their presence in root nodules and assessed their diversity using a target isolation approach. Though their functional role still needs to be addressed, we postulate that Chryseobacterium strains might help cowpea plant to cope with salt stress in semi-arid regions.