Norma Gouvêa Rumjanek

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE

Aim: To evaluate the rpoB gene as a biomarker for PCR‐DGGE microbial analyses using soil DNA from the Cerrado, Brazil. 
Methods: DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. 
Results: The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. 
Conclusions, Significance and Impact of the Study: The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.

Growth characteristics and symbiotic efficiency of rhizobia isolated from cowpea nodules of the north-east region of Brazil

Abstract In Brazil, the geo-environmental conditions of the north-east region exert an effect on the rhizobia diversity. A large collection of rhizobia isolates were obtained from nodules of cowpea planted in different soils from this region of Brazil. Cowpea was selected as the scavenger for this study of rhizobia diversity because it is a known broad host-range leguminous plant and it is also widespread in the region. A total of 536 nodule isolates were obtained and later arranged in 78 different groups based on colony morphology and growth. Growth in culture media of isolate representatives of these groups revealed that 27 of them showed fast growth characteristics although the plant is characteristically nodulated by strains described as Bradyrhizobium spp. Furthermore, the analysis of the isolates from each region showed an increased proportion of fast-growing rhizobia when going from the coast to the semi-arid region. Fifty-five of the rhizobia representatives from the Brazilian north-east region induced Hup+ phenotype in cowpea nodules indicating the predominance of this phenotype. They were also inoculated into soybean, since this legume plant is nodulated by the slow-growing rhizobia species B. japonicum and B. elkanii. Twenty-five of the group representatives were not capable of nodulating soybean. Inoculation with some of them promoted the formation of pseudo-nodule structures which despite not contributing significantly to the nitrogen content of the plant are an indication that signal exchange between plant and rhizobia is occurring. The remaining isolate representatives were capable of nodulating soybean. The nodulation of soybean by the isolates obtained in soils of the semi-arid region of Brazil is quite remarkable since this crop has never been cultivated in the region before. Data on host-regulated uptake hydrogenase phenotype (Hup-hr) is discussed.

Small heat shock proteins genes are differentially expressed in distinct varieties of common bean

Plants respond to temperature stress by synthesizing a set of heat shock proteins (HSPs), which may be responsible for the acquisition of thermotolerance. In this study, the induction of small HSPs (sHSPs) in eight common bean varieties was evaluated by Northern blot analysis using the W HSP 16.9 cDNA as heterologous probe. Cowpea was used, as a positive control since this plant, as opposed to common bean, is known to grow well under high temperature regimes such as that found in the Brazilian semi-arid region. After the growth period, the plants were submitted to two h of heat shock at 40 oC. All varieties tested were able to induce sHSP mRNAs that hybridized with W HSP 16.9 probe. However, significant kinetic differences were found when comparing different varieties. SHSP mRNA levels induced after heat shock in cowpea was higher than the levels observed on the bean varieties displaying the highest expression of these proteins. Besides, the sHSP expression was also assessed at the protein accumulation level by Western-blot analysis for cowpea and both IPA 7 and Negro Argel varieties of bean plants. The revealed protein pattern confirmed that sHSPs are differentially expressed in distinct varieties of common bean according their heat stress tolerance.

Identification of differentially expressed genes by cDNA‐AFLP technique during heat stress in cowpea nodules

Legume nodules formed by diazotrophic microorganisms are active sites for biological nitrogen fixation (BNF). In tropical regions, a significant part of N supply for soybean, peanut and bean crops is derived from BNF, which is nevertheless often limited by high temperature stress. In contrast, cowpea nodules are very resistant to high temperatures. To understand the molecular bases of thermotolerance during BNF under heat stress, we have used cDNA‐amplified fragment length polymorphism experiments to identify differentially expressed transcripts from cowpea nodules subjected to heat shock treatment. The expression profiles obtained showed approximately 600 bands, 55 up‐regulated and nine corresponding to genes repressed by heat stress. Twenty transcript‐derived fragments were isolated, cloned and sequenced. The Vigna unguiculata nodule and stress response transcripts present similarities to those that encode low molecular weight heat shock proteins, wound‐induced proteins, disease resistance protein, and xylan endohydrolase isoenzyme, as well as different housekeeping genes. The differential expression of 15 genes was confirmed by using Northern blot or reverse Northern hybridization experiments.

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)).

Assessment of cowpea rhizobium diversity in Cerrado areas of northeastern Brazil

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.

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.

Diversity and adaptability of soybean and cowpea rhizobia in tropical soils

Abstract The current interest on biodiversity, allied with the use of powerful molecular biology techniques, are revealing the nodulation abilities of tropical legumes, their preferences in terms of relationships with rhizobia, the dynamics of the rhizobia population, and data are accumulating on the cowpea group of tropical rhizobia. Studies on the ecology of cowpea-nodulating rhizobia in soils from the semi-arid region of Brazil, are providing an understanding of the ecology of soybean nodulating rhizobia in Brazilian soils and are revealing a large community of native strains capable of nodulating soybeans in soils where soybeans never have been planted. Many isolates showed a limited ability to nodulate soybean roots and formed nodule-like structures, or pseudo-nodules. Recent host range analysis of known rhizobia species has generated substantial data that is rapidly changing recognized patterns of symbiotic specificity. Cowpeas, soybeans and common beans nodulate with many different species of rhizobia, and the occurrence of both slow- and fast-growing strains in the same plant species has been reported for many genera of tropical legumes. This review will focus on recent data on the diversity of tropical rhizobia and their adaptability to tropical soils.

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.