Lindete Míria Vieira Martins

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.

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.

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.

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.

Field performance of new cowpea cultivars inoculated with efficient nitrogen-fixing rhizobial strains in the Brazilian Semiarid

The objective of this work was to evaluate the contribution of efficient nitrogen-fixing rhizobial strains to grain yield of new cowpea cultivars, indicated for cultivation in the Brazilian Semiarid region, in the sub-medium of the Sao Francisco River Valley. Two experiments were set up at the irrigated perimeters of Mandacaru (Juazeiro, state of Bahia) and Bebedouro (Petrolina, state of Pernambuco). The treatments consisted of single inoculation of five rhizobial strains - BR 3267, BR 3262, INPA 03-11B, UFLA 03-84 (Bradyrhizobiumsp.), and BR 3299T(Microvirga vignae) -, besides a treatment with nitrogen and a control without inoculation or N application. The following cowpea cultivars were evaluated: BRS Pujante, BRS Tapaihum, BRS Carijo, and BRS Acaua. A randomized complete block design, with four replicates, was used. Inoculated plants showed similar grain yield to the one observed with plants fertilized with 80 kg ha-1 N. The cultivars BRS Tapaihum and BRS Pujante stood out in grain yield and protein contents when inoculated, showing their potential for cultivation in the sub-medium of the Sao Francisco River Valley.

Symbiotic and agronomic efficiency of new cowpea rhizobia from Brazilian Semi-Arid

Cowpea is a very important crop to Brazilian Semi-Arid mainly small family-based farmers. Rhizobia inoculation is a practice, easy to use, and cheap technology that increases cowpea productivity. The aim of this study was to evaluate the efficiency of two new rhizobia isolates in greenhouse and field as well as classify them taxonomically. To bacterial identification the 16S rRNA gene of ESA 17 and ESA 18 isolates were sequenced. The greenhouse test was conducted with pots containing 3 L of soil and the bacterial isolates evaluated were ESA 17, ESA 18,BR 3267 or BR 3262 strains. A field experiment was implemented on a Vertisol in Juazeiro, Bahia State, to evaluate the cowpea growth and productivity. In this experiment, the peat-based inoculants with ESA 17, ESA 18, BR 3267 or UFLA 3-84 were used in 2 cowpea cultivars. Both bacteria were identified as Bradyrhizobium, but related to different species. ESA 17 was related to B. japonicumand ESA 18 was closer to B. pachyrhizi. At greenhouse, both isolates increased cowpea nitrogen content in the shoots due to the presence of very efficient nodules. In the field, the isolate ESA 18 inoculated at BRS Pujante cultivar induced higher production than observed for the absolute control, and for BR 17 Gurgueia cultivar, he ESA 17 and BR 3267 stood out both by inducing high production and grain protein content. The results indicate that both isolates can be evaluated in network experiments aiming at official recommendation for new bacteria to cowpea inoculant in Brazil.