Maria Cristina Prata Neves

Nitrogenase activity on the roots of tropical forage grasses

Abstract Nitrogen fixation in the rhizospheres of field grown tropical forage grasses was studied by the acetylene reduction method. Values varied considerably between sites but indicate the possible economic importance of several of the species studied. Maximal nitrogenase activity measured (nmoles C 2 H 4 g −1 dry roots h −1 ) was 754 for Pennisetum purpureum , 750 for Brachiaria mutica , 341 for Digitaria decumbens , 299 for Panicum maximum , 283 for Paspalum notatum , 269 for Cynodon dactylon , 41 for Melinis minutiflora and 29 for Hyparrhenia rufa . Nitrogenase activity varied considerably with season and was maximal during active vegetative growth of two of the grasses. Significant differences between Paspalum notatum ecotypes and cultivars. in Azotohacter paspali occurrence and nitrogen fixation, indicate the possibility of plant breeding to enhance nitrogen fixation in grass rhizospherc associations. Other research lines of agronomic importance are fertilizer effects. In intact soil plant cores with the Paspalum system 10 parts/10 6 NH 4 + J-N inhibited nitrogenase activity within 2 h and 10 parts/10 6 NO − 3 -N within 4 h. but after 1 week these effects were negligible. In the field, nitrogenase activity on roots of P. purpureum and D. decumbens , assayed 2 weeks after top dressings of 20 kg N ha −1 as NH 4 NO 3 . was not affected even after eight such dressings.

Quantification of the contribution of N2 fixation to field-grown grain legumes-a strategy for the practical application of the 15N isotope dilution technique.

Abstract Four experiments were performed at two sites in Brazil to evaluate the use of the 15 N isotope dilution technique for the quantification of N 2 fixation to inoculated and non-inoculated soybean, cowpea and groundnut using three independent control crops in each experiment. The data showed that the 15 N enrichment of the N derived from the soil by the different control crops was different even though slow-release forms of labelled N had been applied. These results illustrate the principal problem associated with the use of the isotope dilution technique in that its application theoretically requires that all test (“N 2 -fixing”) and control crops accumulate N from the soil with the same 15 N enrichment. It was also shown that the three control crops behaved differently at the different sites, so that the idea that “appropriate” controls can be universally recommended for use with any particular legume crop is fallacious. The use of several control crops, in combination with slow-release labelled fertilizer, was shown to be a successful strategy for the application of this technique to quantify the contribution of N 2 fixation to field-grown grain legumes.

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.

The response of field grownPhaseolus vulgaris to Rhizobium inoculation and the quantification of N2 fixation using15N

SummaryA field experiment was performed to assess the effects of Rhizobium inoculation and nitrogen fertilizer (100 kg N ha−1) on four cultivars of Phaseolus beans; Carioca, Negro Argel, Venezuela 350 and Rio Tibagi. In the inoculated treatment 2.5 kg N ha−1 of15N labelled fertilizer was added in order to apply the isotope dilution technique to quantify the contribution of N2 fixation to the nutrition of these cultivars.Nodulation of all cultivars in the uninoculated treatments was poor, but the cultivars Carioca and Negro Argel were well nodulated when inoculated. Even when inoculated, nodulation of the cultivars Venezuela 350 and Rio Tibagi was poor and these cultivars showed little response to inoculation in terms of nitrogen accumulation or grain yield. The estimates of the contribution of N2 fixation estimated using the isotope dilution technique, for the Carioca and Negro Argel cultivars, amounted to 31.7 and 18.4 kg N ha−1 respectively. These two cultivars produced 991 and 883 kg ha−1 of grain, respectively, when inoculated and 663 and 620 kg ha−1 with the addition of 100 kg N ha−1 of N fertilizer. The response to nitrogen was particularly poor due to high leaching losses in the very sandy soil at the experimental site.The Venezuela 350 and Rio Tibagi cultivars only responded to N fertilizer and not to inoculation with Rhizobium which stresses the great importance of selecting plant cultivars for nitrogen fixation in the field.

The physiology of nitrogen fixation in tropical grain legumes

In the tropics, improving protein production depends on increasing agricultural productivity while maintaining low production costs. A major factor limiting productivity is nitrogen fertilizer and, in this context, grain legumes are a foremost alternative because of their high protein content and ability to obtain fixed nitrogen via biological fixation. Nitrogen fixation in the legume/Rhizobium symbiotic system is the result of a complex integration of physiological functions between the host plant and the endophyte. It depends on an exchange of specific metabolities: the bacteroids have an obligatory requirement for carbon substrates as their source of energy and carbon skeletons for nitrogen fixation and assimilation, and the plant receives the nitrogenous products exported from nodules which are required for protein synthesis. Maximizing biological nitrogen fixation to provide nitrogen for high and reliable yields of grain legumes clearly requires an understanding of the functioning of the symbiotic sy...

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.

Cultivar and Rhizobium strain effect on nitrogen fixation and transport inPhaseolus vulgaris L.

The effects of Rhizobium strain and its interaction with plant cultivar were examined in glasshouse-grownPhaseolus vulgaris in two experiments where the physiological attributes defining the symbiotic efficiency were determined.Strains of Rhizobium significantly affected nodulation, rates of N accumulation, partitioning of N within the mature shoot and remobilizaton of the N stored in the vegetative organs to the seeds.The most efficient symbiosis (strain CO5 with Negro Argel), in comparison with the least efficient symbiosis (strain 127 K-17 with Venezuela-350) showed higher rates of C2H2 reduction from flowering to mid pod fill stage, evolved less hydrogen from nodules and showed higher rates of N transport as well as higher percentages of ureide-N in the xylem sap. At maturity, the best cultivar/strain association exceeded the total N accumulated in the seed and the harvest index of the poorest symbiosis in 88% and 20%, respectively. The other symbiotic combinations were intermediate in all characteristics.Nitrogen accumulation in plant shoot showed highly significant correlation with acetylene reduction rates, nodule relative efficiency, total N transport in the xylem sap and percentage of N transported as ureides.

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.

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.

Relative efficiency, ureide transport and harvest index in soybeans inoculated with isogenic HUP mutants of Bradyrhizobium japonicum

SummaryDifferences between isogenic uptake hydrogenase (HUP) mutants of Bradyrhizobium japonicum in terms of nodule efficiency, N2 fixation and N incorporation into various plant parts were studied in a monoxenic greenhouse experiment in order to confirm previous results with soybeans and beans inoculated with various HUP+ and HUP− strains. The HUP+ revertant PJ17-1 of a HUP− mutant (PJ17) of strain USDA DES 122 showed a completely restored relative efficiency (100% versus 78±2% for the HUP− mutant), higher nodule efficiency (N2 fixed per g nodules), higher ureide-N transport rates, higher N contents in pods and higher N harvest indices. All these observations confirm previous experiments with HUP+ and HUP− strains.