Seeds enriched with phosphorus and molybdenum improve the contribution of biological nitrogen fixation to common bean as estimated by 15n isotope dilution

Seeds with a high concentration of P or Mo can improve the growth and N accumulation of the common bean (Phaseolus vulgaris L.), but the effect of enriched seeds on biological N2 fixation has not been established yet. This study aimed to evaluate the effect of seeds enriched with P and Mo on growth and biological N2 fixation of the common bean by the 15N isotope dilution technique. An experiment was carried out in pots in a 2 x 3 x 2 x 2 factorial design in randomized blocks with four replications, comprising two levels of soil applied P (0 and 80 mg kg-1), three N sources (without N, inoculated with rhizobia, and mineral N), two seed P concentrations (low and high), and two seed Mo concentrations (low and high). Non-nodulating bean and sorghum were used as non-fixing crops. The substrate was 5.0 kg of a Red Latosol (Oxisol) previously enriched with 15N and mixed with 5.0 kg of sand. Plants were harvested 41 days after emergence. Seeds with high P concentration increased the growth and N in shoots, particularly in inoculated plants at lower applied P levels. Inoculated plants raised from high P seeds showed improved nodulation at both soil P levels. Higher soil P levels increased the percentage of N derived from the atmosphere (%Ndfa) in bean leaves. Inoculation with the selected strains increased the %Ndfa. High seed P increased the %Ndfa in inoculated plants at lower soil P levels. High seed Mo increased the %Ndfa at lower soil P levels in plants that did not receive inoculation or mineral N. It is concluded that high seed P concentration increases the growth, N accumulation and the contribution of the biological N2 fixation in the common bean, particularly in inoculated plants grown at lower soil P availability.

Growth, nodulation and nitrogen fixation of cowpea in soils amended with composted tannery sludge

Tannery wastes generation is increasing every year and a suitable method for tannery sludge management is necessary in order to decrease this environmental problem. The composting is recognized as a suitable method for sludge recycling.. The effect of tannery sludge compost (TSC) rates on growth, nodulation and N fixation of cowpea was investigated. Sandy and clayey soils were amended with TSC at rates of 0, 7.5, 15, 30, and 60 t ha-1. The shoot dry weight of cowpea plants 45 days after emergence (DAE) was greater in the TSC-amended than in the unamended soil. In the sandy soil, nodule dry weight increased with TSC application 45 DAE. In the clayey soil, 45 DAE, nodule dry weight decreased with TSC amendment levels greater than 7.5 t ha-1 compared to the unamended control. The application of TSC increased N accumulation in the cowpea plants. The results suggest that cowpea responds differently to TSC depending on the amendment rate and initial soil type.

Growth and yield of common bean cultivars at two soil phosphorus levels under biological nitrogen fixation

The genotypic differences on growth and yield of common bean (Phaseolus vulgaris L.) in response to P supply were evaluated in a field experiment under biological N2 fixation. Eight cultivars were grown at two levels of applied P (12 and 50 kg ha-1 of P -- P1 and P2 respectively), in randomized block design in factorial arrangement. Vegetative biomass was sampled at three ontogenetic stages. The effects of genotype and phosphorus were significant for most traits, but not the genotype ´ phosphorus interaction. The cultivars presented different patterns of biomass production and nutrient accumulation, particularly on root system. At P1, P accumulation persisted after the beginning of pod filling, and P translocation from roots to shoots was lower. The nodule senescence observed after flowering might have reduced N2 fixation during pod filling. The responses of vegetative growth to the higher P supply did not reflect with the same magnitude on yield, which increased only 6% at P2; hence the harvest index was lower at P2. The cultivars with highest yields also presented lower grain P concentrations. A sub-optimal supply of N could have limited the expression of the yield potential of cultivars, reducing the genotypic variability of responses to P levels.

Effect of water stress on nitrogen fixation and nodule structure of common bean

The aim of this work was to investigate the effect of water stress on N2 fixation and nodule structure of two common bean (Phaseolus vulgaris L.) cultivars Carioca and EMGOPA-201. Plants were harvested after five and eight days of water stress. Carioca had lower nodule dry weight on both water stress periods; shoot dry weight was lower at five days water stress and did not differ from control after eight days stress. Both cultivars had lower nitrogenase activity than control after five and eight days water stress. For both cultivars, after eight days stress bacteroid membranes were damaged. Carioca presented more pronounced damage to infected tissue, with host cell vacuolation and loss of the peribacteroid membrane at five days after stress; at eight days after stress, there was degradation of cytoplasm host cells and senescence of bacteroids, with their release into intercellular spaces. Intensity of immunogold-labeling of intercellular cortical glycoprotein with the monoclonal antibodies MAC 236/265 was different for both cultivars.

Nitrogen fixation by groundnut and velvet bean and residual benefit to a subsequent maize crop

Chemical fertilisers are rarely avaiable to poor farmers, for whom the nitrogen (N) is often the most limiting element for cereal grain production. The objective of this study was to quantify the contribution of biological nitrogen fixation (BNF) to groundnut (Arachis hypogaea) and velvet bean (Mucuna pruriens) crops using the 15N natural abundance (delta15N) technique and to determine their residual effect and that of a natural fallow, on growth and N accumulation by two rustic maize varieties. The contribution of BNF calculated from delta15N data was 40.9, 59.6 and 30.9 kg ha-1, for groundnut, velvet bean and the natural fallow, respectively. The only legume grain harvested was from the groundnut, which yielded approximately 1.000 kg ha-1. The subsequent maize varieties ("Sol de Manhã" and "Caiana Sobralha") yielded between 1.958 and 2.971 kg ha-1, and were higher after velvet bean for both maize varieties and "Sol da Manhã" groundnut, followed by "Caiana" after groundnut and, finally, the natural fallow. For a small-holder producer the most attractive system is the groundnut followed by maize, as, in this treatment, both groundnut and maize grain harvest are possible. However, a simple N balance calculation indicated that the groundnut-maize sequence would, in the long term, deplete soil N reserves, while the velvet bean-maize sequence would lead to a build up of soil nitrogen.

Genetic characterization and nitrogen fixation capacity of Rhizobium strains on common bean

This study aimed to genetically characterize four new Rhizobium strains, and to evaluate their nodulation and fixation capacity compared to commercial strains and to native rhizobia population of a Brazilian Rhodic Hapludox. Two experiments were carried out in randomized blocks design, under greenhouse conditions, in 2007. In the first experiment, the nodulation and nitrogen fixation capacity of new strains were evaluated, in comparison to the commercial strains CIAT-899 and PRF-81 and to native soil population. It was carried out in plastic tubes filled with vermiculite. DNA extractions and PCR sequencing of the intergenic space were made from the isolated pure colonies, in order to genetically characterize the strains and the native rhizobia population. In the second experiment, the nodulation and productivity of common beans Perola cultivar were determined, with the use of evaluated strains, alone or in mixture with PRF-81 strain. It was carried out in pots filled with soil. The native soil population was identified as Rhizobium sp. and was inefficient in nitrogen fixation. Three different Rhizobium species were found among the four new strains. The LBMP-4BR and LBMP-12BR new strains are among the ones with greatest nodulation and fixation capacity and exhibit differential responses when mixed to PRF-81.

Diversity and nitrogen fixation efficiency of rhizobia isolated from nodules of Centrolobium paraense

The objective of this work was to isolate and characterize rhizobia from nodules of Centrolobium paraense and to evaluate their symbiotic efficiency. Soil samples collected from four sites of the Roraima Cerrado, Brazil, were used to cultivate C. paraense in order to obtain nodules. Isolates (178) were obtained from 334 nodules after cultivation on medium 79. Twenty-five isolates belonging to six morphological groups were authenticated using Vigna unguiculata and they were characterized by 16S rRNA. Isolates identified as Bradyrhizobium were further characterized using rpoB gene sequencing. A greenhouse experiment was carried out with C. paraense to test the 18 authenticated isolates. Approximately 90% of the isolates grew slowly in medium 79. The 16S rRNA analysis showed that 14 authenticated isolates belong to the genus Bradyrhizobium, and rpoB indicated they constitute different groups compared to previously described species. Only four of the 11 fast-growing isolates nodulated V. unguiculata, two of which belong to Rhizobium, and two to Pleomorphomonas, which was not previously reported as a nodulating genus. The Bradyrhizobium isolates ERR 326, ERR 399, and ERR 435 had the highest symbiotic efficiency on C. paraense and showed a contribution similar to the nitrogen treatment. Centrolobium paraense is able to nodulate with different rhizobium species, some of which have not yet been described.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

Inoculation with Azospirillum, associated with nitrogen fertilization in maize

The biological nitrogen fixation is an alternative to supply the nitrogen needed for maize. The objective of this study was to evaluate the development and yield of maize in response to inoculation with Azospirillum associated with nitrogen fertilization. We conducted two field experiments in the summer harvest, the first in the 2000/2001 crop year in the region of Marechal Cândido Rondon, under conventional tillage, and second in the 2002/2003 agricultural year in the region of Cascavel, under no tillage. The experimental design in both experiments was a randomized complete block, with four replications, 2x2x2 factorial, with two levels of nitrogen at sowing (zero and 20 kg ha-1), two levels of inoculum (zero and 200 g ha-1) and two levels of nitrogen in topdressing (zero and 100 kg ha-1). There was evaluated the height of ear insertion, total plant height, leaf N content, shoot dry biomass and grain yield. The height of ear insertion and total plant height were not influenced by the factors under study. Nitrogen fertilization at sowing increased the leaf N content, causing the opposite effect when combined with inoculation. Inoculation with Azospirillum in the absence of nitrogen, provide productivity increases of 15.4% and 7.4% for 2000/2001 and 2002/2003 crops, respectively. The inoculation provided productivity similar to that obtained with 100 kg ha-1 in topdressing in crop 2000/2001, while in association with the topdressing, reduced productivity and shoot dry biomass in crop 2002/2003.

Performance of pineapple slips inoculated with diazotrophic phosphate-solubilizing bacteria and rock phosphate

Besides fixing N2, some diazotrophic bacteria or diazotrophs, also synthesize organic acids and are able to solubilize rock phosphates, increasing the availability of P for plants. The application of these bacteria to pineapple leaf axils in combination with rock phosphate could increase N and P availability for the crop, due to the bacterial activity of biological nitrogen fixation and phosphate solubilization. The objectives of this study were: (i) to select and characterize diazotrophs able to solubilize phosphates in vitro and (ii) evaluate the initial performance of the pineapple cultivars Imperial and Pérola in response to inoculation with selected bacteria in combination with rock phosphate. The experiments were conducted at Universidade Estadual do Norte Fluminense Darcy Ribeiro, in 2009. In the treatments with bacteria the leaf contents of N, P and K were higher than those of the controls, followed by an increase in plant growth. These results indicate that the combined application of diazotrophic phosphate-solubilizing bacteria Burkholderia together with Araxá rock phosphate can be used to improve the initial performance of pineapple slips.

Diversity and capacity to promote maize growth of bacteria isolated from the Amazon region

ABSTRACT Maize plants can establish beneficial associations with plant growth-promoting bacteria. However, few studies have been conducted on the characterization and inoculation of these bacteria in the Amazon region. This study aimed to characterize endophytic bacteria isolated from maize in the Amazon region and to assess their capacity to promote plant growth. Fifty-five bacterial isolates were obtained from maize grown in two types of ecosystems, i.e., a cerrado (savanna) and a forest area. The isolates were characterized by the presence of the nifH gene, their ability to synthesize indole-3-acetic acid (IAA) and solubilize calcium phosphate (CaHPO4), and 16S rRNA partial gene sequencing. Twenty-four bacteria contained the nifH gene, of which seven were isolated from maize plants cultivated in a cerrado area and seventeen from a forest area. Fourteen samples showed the capacity to synthesize IAA and only four solubilized calcium phosphate. The following genera were found among these isolates: Pseudomonas; Acinetobacter; Enterobacter; Pantoea; Burkholderia and Bacillus. In addition, eight isolates with plant growth-promoting capacity were selected for a glasshouse experiment involving the inoculation of two maize genotypes (a hybrid and a variety) grown in pots containing soil. Inoculation promoted the development of the maize plants but no significant interaction between maize cultivar and bacterial inoculation was found. A high diversity of endophytic bacteria is present in the Amazon region and these bacteria have potential to promote the development of maize plants.

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

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.

Exopolysaccharides produced by the symbiotic nitrogen-fixing bacteria of leguminosae

The process of biological nitrogen fixation (BNF), performed by symbiotic nitrogen fixing bacteria with legume species, commonly known as α and β rhizobia, provides high sustainability for the ecosystems. Its management as a biotechnology is well succeeded for improving crop yields. A remarkable example of this success is the inoculation of Brazilian soybeans with Bradyrhizobium strains. Rhizobia produce a wide diversity of chemical structures of exopolysaccharides (EPS). Although the role of EPS is relatively well studied in the process of BNF, their economic and environmental potential is not yet explored. These EPS are mostly species-specific heteropolysaccharides, which can vary according to the composition of sugars, their linkages in a single subunit, the repeating unit size and the degree of polymerization. Studies have showed that the EPS produced by rhizobia play an important role in the invasion process, infection threads formation, bacteroid and nodule development and plant defense response. These EPS also confer protection to these bacteria when exposed to environmental stresses. In general, strains of rhizobia that produce greater amounts of EPS are more tolerant to adverse conditions when compared with strains that produce less. Moreover, it is known that the EPS produced by microorganisms are widely used in various industrial activities. These compounds, also called biopolymers, provide a valid alternative for the commonly used in food industry through the development of products with identical properties or with better rheological characteristics, which can be used for new applications. The microbial EPS are also able to increase the adhesion of soil particles favoring the mechanical stability of aggregates, increasing levels of water retention and air flows in this environment. Due to the importance of EPS, in this review we discuss the role of these compounds in the process of BNF, in the adaptation of rhizobia to environmental stresses and in the process of soil aggregation. The possible applications of these biopolymers in industry are also discussed.

Effect of 15n-labeled hairy vetch and nitrogen fertilization on maize nutrition and yield under no-tillage¹

This study evaluated the effect of hairy vetch (Vicia villosa Roth) as cover crop on maize nutrition and yield under no tillage using isotope techniques. For this purpose, three experiments were carried out: 1) quantification of biological nitrogen fixation (BNF) in hairy vetch; 2) estimation of the N release rate from hairy vetch residues on the soil surface; 3) quantification of 15N recovery by maize from labeled hairy vetch under three rates of mineral N fertilization. This two-year field experiment was conducted on a sandy Acrisol (FAO soil classification) or Argissolo Vermelho distrófico arênico (Brazilian Soil Classification), at a mean annual temperature of 18 ºC and mean annual rainfall of 1686 mm. The experiment was arranged in a double split-plot factorial design with three replications. Two levels of hairy vetch residue (50 and 100 % of the aboveground biomass production) were distributed on the surface of the main plots (5 x 12 m). Maize in the sub-plots (5 x 4 m) was fertilized with three N rates (0, 60, and 120 kg ha-1 N), with urea as N source. The hairy vetch-derived N recovered by maize was evaluated in microplots (1.8 x 2.2 m). The BFN of hairy vetch was on average 72.4 %, which represents an annual input of 130 kg ha-1 of atmospheric N. The N release from hairy vetch residues was fast, with a release of about 90 % of total N within the first four weeks after cover crop management and soil residue application. The recovery of hairy vetch 15N by maize was low, with an average of 12.3 % at harvest. Although hairy vetch was not directly the main source of maize N nutrition, the crop yield reached 8.2 Mg ha-1, without mineral fertilization. There was an apparent synergism between hairy vetch residue application and the mineral N fertilization rate of 60 kg ha-1, confirming the benefits of the combination of organic and inorganic N sources for maize under no tillage.

Symbiotic capability of calopo rhizobia from an agrisoil with different crops in Pernambuco

Biological nitrogen fixation by rhizobium-legume symbiosis represents one of the most important nitrogen sources for plants and depends strongly on the symbiotic efficiency of the rhizobium strain. This study evaluated the symbiotic capacity of rhizobial isolates from calopo (CALOPOGONIUM MUCUNOIDES) taken from an agrisoil under BRACHIARIA DECUMBENS pasture, sabiá (MIMOSA CAESALPINIIFOLIA) plantations and Atlantic Forest areas of the Dry Forest Zone of Pernambuco. A total of 1,575 isolates were obtained from 398 groups. A single random isolate of each group was authenticated, in randomized blocks with two replications. Each plant was inoculated with 1 mL of a bacterial broth, containing an estimated population of 10(8) rhizobial cells mL-1. Forty-five days after inoculation, the plants were harvested, separated into shoots, roots and nodules, oven-dried to constant mass, and weighed. Next, the symbiotic capability was tested with 1.5 kg of an autoclaved sand:vermiculite (1:1) mixture in polyethylene bags. The treatments consisted of 122 authenticated isolates, selected based on the shoot dry matter, five uninoculated controls (treated with 0, 50, 100, 150, or 200 kg ha-1 N) and a control inoculated with SEMIA 6152 (=BR1602), a strain of BRADYRHIZOBIUM JAPONICUM The test was performed as described above. The shoot dry matter of the plants inoculated with the most effective isolates did not differ from that of plants treated with 150 kg ha-1 N. Shoot dry matter was positively correlated with all other variables. The proportion of effective isolates was highest among isolates from SABIÁ forests. There was great variation in nodule dry weight, as well as in N contents and total N.