Milton A. T. Vargas

Environmental factors affecting N2 fixation in grain legumes in the tropics, with an emphasis on Brazil

Abstract Biological nitrogen (N 2 ) fixation is key to sustainable agricultural systems in tropical soils, which are frequently deficient in N. However, high temperature, drought and soil acidity constrain legume root-nodule formation and function in the tropics. In most cases, the microsymbiont is the more affected partner, with plants growing on mineral N usually less sensitive to these stresses. High temperature and moisture deficiency are major causes of nodulation failure, affecting all stages of the symbiosis and limiting rhizobial growth and survival in soil. They may also contribute to undesirable changes in rhizobia, including plasmid deletions, genomic rearrangements and reduced diversity. Acidity affects several steps in the development of the symbiosis, including the exchange of molecular signals between the legume and the microsymbiont. Liming is effective in overcoming soil acidity and aluminium toxicity, but in Brazil few soils are limed to near neutral pH. Selection of rhizobial inoculant strains that are genetically stable under the often harsh soil conditions of this region is essential, but that task is impaired by a lack of knowledge of tolerance in the microsymbiont. However, good results have been obtained by selecting naturally occurring rhizobia from acid tropical soils affected by water stress and high temperatures. In Brazil, increases in grain yields of common bean and soybean have resulted from inoculation with such stress-tolerant strains. Appropriate soil management practices, e.g. no-till, are other approaches that may decrease soil temperatures and preserve moisture, thereby increasing N 2 fixation.

Response of field-grown bean (Phaseolus vulgaris L.) to Rhizobium inoculation and nitrogen fertilization in two Cerrados soils

Abstract Most soils sown with field beans (Phaseolus vulgaris L.) contain indigenous rhizobia which might interfere with the establishment of inoculated strains. As a consequence, the benefits of bean inoculation are usually questioned, and the use of N fertilizer is gradually becoming a common practice. The present study had the objective of evaluating the effectiveness of inoculation and N fertilization in field soil with (site 1) and without (site 2) a previous bean-cropping history. At site 1, which had a rhizobial population of 7×102 cells g–1 soil, inoculation had no effect on nodulation or yield, whereas at site 2 (<10 cells g–1 soil) inoculation increased nodulation, nodule occupancy by the inoculated strain and grain yield. N fertilizer decreased nodulation at both sites, but increased grain yield at site 1 but not at site 2, indicating that the response to inoculation and N fertilization depends on the cropping history. When bean was cultivated for the first time, indigenous populations of rhizobia were low and high yields were accomplished solely with seed inoculation, with no further response to N fertilizer. In contrast, previous cultivation of bean increases soil rhizobia, preventing nodule formation by inoculated strains, and N fertilizer may be necessary for maximum yields. A significant interaction effect between N fertilizer and inoculation was detected for serogroup distribution only at site 2, with N fertilizer decreasing nodule occupancy by the inoculated strain and increasing the occurrence of indigenous strains. Consequently, although no benefits were obtained by the combination of inoculation and N fertilizer, this practice may be feasible with the selection of appropriate N-tolerant strains from the indigenous rhizobial population.

Nitrogen fixation capacity and nodule occupancy by Bradyrhizobium japonicum and B. elkanii strains

Abstract In a previous study soybean Bradyrhizobium strains, used in Brazilian studies and inoculants over the last 30 years, and strains adapted to the Brazilian Cerrados, a region frequently submitted to environmental and nutritional stresses, were analyzed for 32 morphological and physiological parameters in vivo and in vitro. A cluster analysis allowed the subdivision of these strains into species Bradyrhizobium japonicum, Bradyrhizobium elkanii and a mixed genotype. In this study, the bacteria were analyzed for nodulation, N2 fixation capacity, nodule occupancy and the ability to increase yield. The goal was to find a relationship between the strain groups and the symbiotic performance. Two strains of Brazilian B. japonicum showed higher rates of N2 fixation and nodule efficiency (mg of N mg–1 of nodules) under axenic conditions. These strains also showed greater yield increases in field experiments when compared to B. elkanii strains. However, no differences were detected between B. japonicum and B. elkanii strains when comparing nodule occupancy capacity. The adapted strains belonging to the serogroup B. elkanii SEMIA 566, most clustered in a mixed genotype, were more competitive than the parental strain, and some showed a higher capacity of N2 fixation. Some of the adapted strains, such as S-370 and S-372, have shown similar N2 fixation rates and nodulation competitiveness to two Brazilian strains of B. japonicum. This similarity demonstrates the possibility of enhancing N2 fixing ability, after local adaptation, even within B. elkanii species. Differences in the DNA profiles were also detected between the parental SEMIA 566 and the adapted strains by analyses with the ERIC and REP-PCR techniques. Consequently, genetic, morphological and physiological changes can be a result of adaptation of rhizobia to the soil. This variability can be used to select strains capable of increasing the contribution of N2 fixation to soybean nutrition.

Soybean response to starter nitrogen and Bradyrhizobium inoculation on a Cerrado oxisol under no-tillage and conventional tillage systems

SUMMARY In Brazil, Bradyrhizobium inoculation has successfully replaced the use of N fertilizer on soybean [Glycine max (L) Merr.] crops. However, with the expansion of no-tillage cropping systems in the Cerrados region, the idea that it is necessary to use small N rates at the sowing to overcome problems related with N immobilization has become widespread, mainly when soybean is cultivated after a non-legume crop. In this study we examined soybean response to small rates of N fertilizer under no-tillage (NT) and conventional tillage (CT) systems. Four experiments (a completely randomized block with five replicates) were carried out in a red yellow oxisol, during the periods of 1998/1999 and 1999/ 2000, under NT and CT. The treatments consisted of four urea rates (0, 20, 30 and 40 kg ha-1 N). All treatments were inoculated with Bradyrhizobium japonicum strains SEMIA 5080 and SEMIA 5079, in the proportion 1 kg of peat inoculant (1,5 x 10 9 cells g -1 ) per 50 kg of seeds. In both experiments, soybean was cultivated after corn and the N fertilizer was band applied at sowing. In all experiments, N rates promoted reductions of up to 50 % in the nodule number at 15 days after the emergence. Regardless of the management system, these reductions disappeared at the flowering stage and there was no effect of N rates on either the number and dry weight of nodules or on soybean yields. Therefore, in the Brazilian Cerrados, when an efficient symbiosis is established, it is not necessary to apply starter N rates on soybean, even when cultivated under notillage systems.

Random amplified polymorphic DNA analysis of effective Rhizobium sp. associated with beans cultivated in brazilian cerrado soils

Efficient bean nodulating Rhizobium strains, isolated from different Brazilian cerrado soils, were characterized by RAPD. This study showed great genetic heterogeneity among R. tropici and R. leguminosarum bv. phaseoli strains and allowed the constitution of genetic clusters, besides indicating the most suitable primers for this characterization. The groups of genetically distinct strains can be used in competitiveness studies to select appropriate Rhizobium strains for bean inoculation in cerrado soils.

The Brazilian Experience with the Soybean (Glycine max) and Common Bean (Phaseolus vulgaris) Symbioses

Soybean and common bean are cultivated on more than 17 million hectares in Brazil. Soybean is the main cash crop and relies exclusively on BNF to supply the plant N demands. However, to maintain yields of more productive cultivars, a search for efficient, competitive and stress tolerant strains continues. Common bean is the most important legume used for food and inoculation with an efficient, competitive Rhizobium tropici strain increases in nodulation, nodule occupancy and yield. An adequate soil management is essential to guarantee the benefits of BNF with both crops.

Changes in electrophoretic profiles of lipopolysaccharides from competitive strains of Bradyrhizobium spp. induced by soybean roots

The soybean Bradyrhizobium strain Semia 566 was introduced into soils of the Cerrados (Brazilian edaphic savannas) in the late 1960s. Then, nodule occupancy by this strain was not greater than 2%. Recently, this serogroup has been found in approximately 60% of nodules formed on soybeans cultivated in the Cerrados, replacing the strains 29W and Semia 587, the Brazilian commercial inoculant for soybean. Although some re‐isolates of Semia 566, adapted to Cerrado soils, were more competitive than 29W under both field and aseptic conditions, they did not differ from the parental strain, based on their lipopolysaccharide (LPS) electrophoretic profile. The only exceptions were the isolates 4A‐5 and CPAC‐15 which presented an additional polysaccharidic band of low molecular weight or higher mobility. On the other hand, this same band may be induced and intensified in LPS extracted from competitive strains (29W, 220, 204, 370, 372, 516, 122 and CPAC‐15) after bacterial contact with soybean roots for 6 or 12 h. In addition, a 29W Tn5 mutant with a phenotype of delayed nodulation showed a delayed induction of this polysaccharidic band. Conversely, the LPS of less competitive strains was not modified or showed a weak intensification of this band. As this band alteration was correlated with the concurrent elevation of dominance in nodules, it may be suggested that LPS plays a role in the competitive ability of rhizobia strains for nodulation.

Brazilian Trials to Evaluate the Effects of Soybean Reinoculation

Although Brazilian soils are very poor on N, the nitrogen fixation process is able to fully supply the N plant demands. Reinoculation with selected strains promotes increases in nodulation, nodule occupancy and yield.

Selection of Efficient and Competitive Strains of Bradyrhizobium for the Soybean Crop in Brazil

Variant strains showing higher capacity of N 2 fixation and competitiveness can be obtained under laboratory conditions, searching for individual colonies with specific properties.

Selection of Rhizobium Strains for the Common Bean Crop

The good symbiotic performance of strain PRF 81 encourages the identification of new competitive, efficient and genetically stable Rhizobium tropici strains for the bean crop in tropical regions.