High Biological Nitrogen Fixation Research Articles

Selection of multi-purpose native rhizobia from a new red clover cultivar developed in Brazil

Red clover is an important forage legume in the livestock sector in southern Brazil, but regional climatic conditions compromise its management. Therefore, UFRGS and EMBRAPA have developed the cultivar URS BRS Mesclador adapted to the southern region of Brazil, but efficient rhizobia for it is still missing. This study aimed to select symbiotic rhizobia for the URS BRS Mesclador and evaluate their efficiency in biological nitrogen fixation in red clover and growth promotion in the rye. Seven rhizobia isolates from red clover nodules of the new cultivar and nine strains from the SEMIA Rhizobia Culture Collection were evaluated for their ability to promote plant growth. Certain strains exhibited notable abilities among the studied rhizobia, such as Rhizobium changzhiense Tp46 producing indole compounds (5.33 μg ml−1), Semia 2076 with a phosphate solubilization index of 2.4, and R. leguminosarum Tp57 and Tp34, SEMIA 2038, and SEMIA 276 with high siderophore production index. Isolates Tp46 and Tp34 demonstrated potential by displaying high biological nitrogen fixation efficiency in red clover and promoting rye growth. These promising results warrant further investigation for field studies with a long-term goal of developing inoculants for red clover cultivation in Brazil.

Contrasting potential for biological N2 fixation at three polluted central European Sphagnum peat bogs: combining the 15N2-tracer and natural-abundance isotope approaches

Abstract. Availability of reactive nitrogen (Nr) is a key control on carbon (C) sequestration in wetlands. To complement the metabolic demands of Sphagnum in pristine rain-fed bogs, diazotrophs supply additional Nr via biological nitrogen fixation (BNF). As breaking the triple bond of atmospheric N2 is energy-intensive, it is reasonable to assume that increasing inputs of pollutant Nr will lead to BNF downregulation. However, recent studies have also documented measurable BNF rates in Sphagnum-dominated bogs in polluted regions, indicating the adaptation of N2 fixers to changing N deposition. Our aim was to quantify BNF in high-elevation peatlands located in industrialized central Europe. A 15N2-tracer experiment was combined with a natural-abundance N-isotope study at three Sphagnum-dominated peat bogs in the northern Czech Republic in an attempt to assess the roles of individual BNF drivers. High short-term BNF rates (8.2 ± 4.6 g N m2 d−1) were observed at Malé mechové jezírko, which receives ∼ 17 kg Nr ha−1 yr−1. The remaining two peat bogs, whose recent atmospheric Nr inputs differed from Malé mechové jezírko by only 1–2 kg ha−1 yr−1 (Uhlír̆ská and Brumiště), showed zero BNF. The following parameters were investigated to elucidate the BNF difference: the NH4+-N / NO3--N ratio, temperature, wetness, Sphagnum species, organic-N availability, possible P limitation, possible molybdenum (Mo) limitation, SO42- deposition, and pH. At Malé mechové jezírko and Uhlír̆ská, the same moss species (S. girgensohnii) was used for the 15N2 experiment; therefore, the host identity could not explain the difference in BNF at these sites. Temperature and moisture were also identical in all incubations and could not explain the between-site differences in BNF. The N : P stoichiometry in peat and bog water indicated that Brumiště may have lacked BNF due to P limitation, whereas non-detectable BNF at Uhlír̆ská may have been related to the 70-fold higher SO42- concentration in bog water. Across the sites, the mean natural-abundance δ15N values increased in the following order: atmospheric deposition (−5.3 ± 0.3 ‰) < Sphagnum (−4.3 ± 0.1 ‰) < bog water (−3.9 ± 0.4 ‰) < atmospheric N2 (0.0 ‰). Only at Brumiště was N in Sphagnum significantly isotopically heavier than in atmospheric deposition, possibly indicating a longer-term BNF effect. Collectively, our data highlight spatial heterogeneity in BNF rates under high Nr inputs as well as the importance of environmental parameters other than atmospheric Nr pollution in regulating BNF.

Studies Using Mutant Strains of Azospirillum brasilense Reveal That Atmospheric Nitrogen Fixation and Auxin Production Are Light Dependent Processes.

As the use of microbial inoculants in agriculture rises, it becomes important to understand how the environment may influence microbial ability to promote plant growth. This work examines whether there are light dependencies in the biological functions of Azospirillum brasilense, a commercialized prolific grass-root colonizer. Though classically defined as non-phototrophic, A. brasilense possesses photoreceptors that could perceive light conducted through its host's roots. Here, we examined the light dependency of atmospheric biological nitrogen fixation (BNF) and auxin biosynthesis along with supporting processes including ATP biosynthesis, and iron and manganese uptake. Functional mutants of A. brasilense were studied in light and dark environments: HM053 (high BNF and auxin production), ipdC (capable of BNF, deficient in auxin production), and FP10 (capable of auxin production, deficient in BNF). HM053 exhibited the highest rate of nitrogenase activity with the greatest light dependency comparing iterations in light and dark environments. The ipdC mutant showed similar behavior with relatively lower nitrogenase activity observed, while FP10 did not show a light dependency. Auxin biosynthesis showed strong light dependencies in HM053 and FP10 strains, but not for ipdC. Ferrous iron is involved in BNF, and a light dependency was observed for microbial 59Fe2+ uptake in HM053 and ipdC, but not FP10. Surprisingly, a light dependency for 52Mn2+ uptake was only observed in ipdC. Finally, ATP biosynthesis was sensitive to light across all three mutants favoring blue light over red light compared to darkness with observed ATP levels in descending order for HM053 > ipdC > FP10.

Genes Associated with Biological Nitrogen Fixation Efficiency Identified Using RNA Sequencing in Red Clover (Trifolium pratense L.).

Commonly studied in the context of legume-rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant-bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover's root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.

Bradyrhizobium strains from Brazilian tropical soils promote increases in nodulation, growth and nitrogen fixation in mung bean

Mung bean [Vigna radiata (L.) Wilczek] is a legume of Asian origin cultivated in several countries, including Brazil, where its commercial cultivation has expanded in recent years. It has a promiscuous N2-fixing association with rhizobia, but nodulation in Brazilian soils has been highly variable. To achieve satisfactory yields with low production costs, studies are being carried out to select rhizobia from soils in Brazil that result in efficient symbiotic associations with mung bean. Previous studies indicate that only Bradyrhizobium strains were able to nodulate mung bean in Brazilian soils. The objective of this study was to evaluate the effect of 31 Bradyrhizobium strains and increasing levels of N fertilizer on nodulation, plant growth and N2 fixation. Bradyrhizobium strains resulted in increases of up to 71, 79, 43, 66, 40 and 55%, respectively, in nodule number and nodule, root and shoot biomasses, accumulated N and biological nitrogen fixation (BNF) contribution compared to the uninoculated control, however, there was no increase in grain yield. Compared to the control with N fertilizer, inoculated plants had lower biomass and accumulated N, despite the high BNF contribution. In general, strains of Bradyrhizobium japonicum superclade, especially those close to B. yuanmingense B071, showed greater efficiency compared to those of the superclade of B. elkanii. The application of N fertilizer increased root and shoot biomasses, however, significantly reduced nodulation. Bradyrhizobium strains isolated from Brazilian tropical soils showed potential for mung bean inoculation, however, the BNF contribution was not sufficient to meet the plant's N demand.

Selection and characterization of Spanish Trifolium-nodulating rhizobia for pasture inoculation

Identification of elite nitrogen-fixing rhizobia strains is a continuous and never ending effort, since new legume species can be cultivated in different agro systems or are introduced into new areas. This current study reports on the taxonomic affiliation and symbiotic proficiency of nine strains of Trifolium-nodulating rhizobia isolated from different pasture areas in Spain, as well as three Rhizobium leguminosarum bv. trifolii reference strains, on eleven Trifolium species. Based on 16S rRNA gene sequences the strains belonged to the R. leguminosarum species complex. Additional phylogenetic analyses of the housekeeping genes recA, atpD and rpoB showed the strains were closely related to the species R. leguminosarum, R. laguerreae, R. indicum, R. ruizarguesonis or R. acidisoli. In addition, three strains had no clear affiliation and could represent putative new species, although two of the reference strains were positioned close to R. ruizarguesonis. nodC gene phylogeny allowed the discrimination between strains isolated from annual or perennial Trifolium species and placed all of them in the symbiovar trifolii. Neither geographic origin nor host-plant species could be correlated with the taxonomic affiliation of the strains and a high degree of phenotypic diversity was found among this set of strains. The strong interaction of plant species with the rhizobial strains found for biological nitrogen fixation (BNF) was noteworthy, and allowed the identification of rhizobial strains with a maximum proficiency for certain trefoil species. Several strains showed high BNF potential with a wide range of clover species, which made them valuable strains for inoculant manufacturers and they would be particularly useful for inoculation of seed mixtures in natural or cultivated pastures.

Cell wall formation pathways are differentially regulated in sugarcane contrasting genotypes associated with endophytic diazotrophic bacteria.

Differences in cell wall components between two BNF-contrasting sugarcane genotypes might result from genetic variations particular to the genotype and from the efficiency in diazotrophic bacteria association. Sugarcane is a plant of the grass family (Poaceae) that is highly cultivated in Brazil, as an important energy resource. Commercial sugarcane genotypes may be successfully associated with beneficial endophytic nitrogen-fixing bacteria, which can influence several plant metabolic pathways, such as cell division and growth, synthesis of hormones, and defense compounds. In this study, we investigated how diazotrophic bacteria associated with sugarcane plants could be involved in the regulation of cell wall formation pathways. A molecular and structural characterization of the cell wall was compared between two genotypes of sugarcane with contrasting rates of Biological Nitrogen Fixation (BNF): SP70-1143 (high BNF) and Chunee (low BNF). Differentially expressed transcripts were identified in transcriptomes generated from SP70-1143 and Chunee. Expression profiles of cellulose and lignin genes, which were more expressed in SP70-1134, and callose genes, which were more expressed in Chunee, were validated by RT-qPCR and microscopic analysis of cell wall components in tissue sections. A similar expression profile in both BNF-contrasting genotypes was observed in naturally colonized plants and in plants inoculated with G. diazotrophicus. Cell walls of the high BNF genotype have a greater cellulose content, which might contribute to increase biomass. In parallel, callose was concentrated in the vascular tissues of the low BNF genotype and could possibly represent a barrier for an efficient bacterial colonization and dissemination in sugarcane tissues. Our data show a correlation between the gene profiles identified in the BNF-contrasting genotypes and a successful association with endophytic diazotrophic bacteria.

Iberian Peninsula cowpea diversity: chloroplast, microsatellite and morpho-agronomic variability

Cowpea (Vigna unguiculata (L.) Walp.) is an important legume crop in Southern Europe and a valuable source of proteins, minerals, and vitamins. Moreover, cowpea has additional interest stemming from drought tolerance and high biological nitrogen fixation. In this research, the genetic diversity of cowpea landraces from Southern European countries was evaluated using molecular and morpho-agronomical approaches, with the objective of enhancing legumes diversity grown through sustainable cropping. A set of 10 chloroplast microsatellite primer pairs (cpSSRs) was used to evaluate genetic diversity and phylogenetic relationships among 113 Vigna accessions including Vigna unguiculata subsp. unguiculata, subsp. alba, subsp. pubescens, subsp. tenuis and var. spontanea and accessions from other Vigna species, as V. mungo, V. radiata and V. racemosa. This set of primers successfully established ten haplotypes, with the most frequent being shared by the V. unguiculata subsp. unguiculata cultigroups unguiculata and sesquipedalis and var. spontanea. Eight loci were polymorphic; nevertheless, a low level of polymorphism was verified within the cultivated cowpeas. Additionally, 10 agronomic traits were evaluated on cowpea landraces of the cultigroup unguiculata, mainly from Portugal. PCA clustered the landraces into three main groups, each one containing Portuguese landraces. Total seed weight per plant revealed the highest coefficient of variation, and 100 seed weight the highest heritability. The performed study shows the wide agro-morphological diversity still existing in cowpea in Iberian Peninsula and other Southern European countries, despite the low polymorphism detected in its chloroplast genome. The high variability detected in the collection of cowpea analysed and the sharing of haplotypes by cultivated and wild material is of great importance for breeding programs of this species.

Biological Nitrogen Fixation Response to Soil Fertility Is Species-Dependent in Annual Legumes

In low-input farming systems, the availability of nutrients can limit yields as well as biological nitrogen fixation (BNF). The adaptability of legume species to soil fertility conditions may be relevant for designing crop rotations. The aim of this study was to determine the effect of soil fertility on biological N fixation of two annual legumes (chickpea Cicer arietinum and bitter vetch Vicia ervilia) adapted to dry conditions growing in organically amended soils, and whether, this effect was species-dependent. Two legume species were sown in two fields with contrasting soil fertility, amended or non-amended with cow manure. We measured soil N and P pools and availability and estimated N derived from the atmosphere (%Ndfa) by 15N natural abundance. In low-fertility soils, both legume species showed high BNF (17–22 mg N2 fixed per kg of dry matter) that correlated with plant production. While chickpea BNF responded negatively to soil fertility and manure application, bitter vetch BNF remained virtually unchanged, irrespective of soil fertility, and did not correlate with soil N or P available forms. Bitter vetch productivity and BNF, both in low- and high-fertility scenarios, was not responsive to a single manure application event, while chickpea showed a strong response to added manure. Bitter vetch %Ndfa and BNF were always higher than chickpea and consequently this former species could be more appropriate for increasing soil N pools in low-input cropping systems.

Genetic Analysis and Mapping of QTLs for Soybean Biological Nitrogen Fixation Traits Under Varied Field Conditions.

Soybean is an important economic and green manure crop that is widely used in intercropping and rotation systems due to its high biological nitrogen fixation (BNF) capacity and the resulting reduction in N fertilization. However, the genetic mechanisms underlying soybean BNF are largely unknown. Here, two soybean parent genotypes contrasting in BNF traits and 168 F9:11 recombinant inbred lines (RILs) were evaluated under four conditions in the field. The parent FC1 always produced more big nodules, yet fewer nodules in total than the parent FC2 in the field. Furthermore, nodulation in FC1 was more responsive to environmental changes than that in FC2. Broad-sense heritability (h2b) for all BNF traits varied from 0.48 to 0.87, which suggests that variation in the observed BNF traits was primarily determined by genotype. Moreover, two new QTLs for BNF traits, qBNF-16 and qBNF-17, were identified in this study. The qBNF-16 locus was detected under all of the four tested conditions, where it explained 15.9–59.0% of phenotypic variation with LOD values of 6.31–32.5. Meanwhile qBNF-17 explained 12.6–18.6% of observed variation with LOD values of 4.93–7.51. Genotype group analysis indicated that the FC1 genotype of qBNF-16 primarily affected nodule size (NS), while the FC2 genotype of qBNF-16 promoted nodule number (NN). On the other hand, the FC1 genotype of qBNF-17 influenced NN and the FC2 genotype of qBNF-17 impacted NS. The results on the whole suggest that these two QTLs might be valuable markers for breeding elite soybean varieties with high BNF capacities.

Biological nitrogen fixation by irrigated dry bean (Phaseolus vulgaris L.) genotypes

Dry bean (Phaseolus vulgaris L.) is usually considered to be poor at biological nitrogen fixation (BNF), but large variations in this trait have been observed among bean genotypes. We evaluated 16 bean genotypes for N2 fixation ability in four N treatments: (i) uninoculated in low-N soil (30 kg N ha−1), (ii) inoculated with commercial Rhizobium leguminosarum bv. phaseoli inoculant Nitrastik-D® in low-N soil, (iii) inoculated with commercial R. leguminosarum bv. phaseoli inoculant Nodulator® in low-N soil, and (iv) uninoculated in high-N soil (100 kg N ha−1). There were differences between genotypes in all the plant parameters that were measured, but only nodulation was affected by N treatment. The 100 kg N ha−1 treatment suppressed nodulation. Seven genotypes nodulated well with either inoculant, two genotypes nodulated better with Nitrastik-D than with Nodulator, three nodulated better with Nodulator than with Nitrastik-D, and four nodulated poorly with either inoculant. Cultivars AC Redbond, Island, and Resolute, all currently commercially grown, did not fix much N2 at flowering (4–8 kg N ha−1) or maturity (19–34 kg N ha−1). By contrast, germplasm lines PI 136692 (red bean), GH-196 (pinto bean), and LEF2RB (carioca bean) had high BNF capability at flowering (10–11 kg N ha−1) and especially at maturity (60–72 kg N ha−1), in addition to high seed yield (2778–2897 kg ha−1), indicating their superior ability to support both of these economically important traits throughout plant growth. These three genotypes would be valuable to breeders for the genetic improvement of BNF in dry bean cultivars.

Screening Cowpea Genotypes for High Biological Nitrogen Fixation and Grain Yield under Drought Conditions

Core Ideas Cowpea nodule biomass was more sensitive to water stress compared to shoot biomass. Rainfed conditions resulted in 57% decrease in N2 fixation compared to well‐watered. Inter‐seasonal variability in rainfall accounted for 58% difference in cowpea grain yield. Future increase in drought and heat stress will challenge legume productivity in many regions. In Sub‐Saharan Africa, legumes are earmarked as an integral component in building resilience to climate change and food security in smallholder farming systems. Hence, the need for continuous screening of drought tolerant legume genotypes for sustained productivity. A 2‐yr field study was conducted to identify superior cowpea genotypes with high biological nitrogen fixation (BNF), grain yield, and drought tolerance. Four genotypes, selected from a pool of 91, were grown under four moisture levels ranging from well‐watered (WW) to severe water stress (SS). Grain yield, shoot biomass, nodule mass, and BNF were determined. There was significant effect of water stress on the measured parameters. Nodule mass and BNF were more sensitive to water stress compared to shoot biomass across genotypes. Water stress resulted in 80% reduction in nodule biomass between WW and SS. As a result, cowpea genotypes grown under SS conditions fixed 57% less N2 compared to those under WW. The study also revealed significant genotypic variation in shoot biomass, grain yield, and BNF. Genotype TVu4607 was low in grain yield but performed better for BNF (71 kg N ha−1 under WW and 30 kg N ha−1 under SS). Genotype IT00K‐1263, proved to be our superior genotype with a good balance of biomass, BNF and grain yield. Our findings suggest that future water stress may significantly affect cowpea productivity, mostly its capacity to fix nitrogen. Consequently, adaptation strategies to improve drought resistance in cowpea are needed, particularly improvement of BNF under water stress.

Do N‐fixing legumes promote neighbouring diversity in the tropics?

Abstract1. Although nitrogen‐fixing plants play a crucial role in maintaining high ecosystem productivity, their effects on forest diversity are greatly debated. Legumes can reduce local diversity because of the fertilization effect; however, they can also facilitate species coexistence through complementary resource utilization.2. In natural forests, nitrogen requirements and biological nitrogen fixation (BNF) vary widely among legumes. We used leaf nitrogen isotopic composition to differentiate the level of BNF activity among seven legumes across a steep gradient of soil available nitrogen within a 60‐ha stem‐mapping plot in a montane tropical rainforest in Hainan Island, China, and we evaluated their spatial distribution and neighbourhood diversity.3. Results show that the levels of BNF activity are tightly correlated with legume association to available soil nitrogen, where legumes associated with nitrogen‐rich habitats exhibit surprisingly greater BNF activity and a more diverse neighbourhood.4. Synthesis. These findings indicate that legumes satisfy their nitrogen‐demanding metabolism through a synergy of habitat preference and BNF activity. High BNF activity drives local diversity by promoting complementary resource utilization rather than intensifying above‐ground competition. This may be achieved by improving litter quality and stimulating mycorrhizal and microbial diversity. The abundance of nitrogen fixers in nitrogen‐rich habitats also explains how legumes contribute to maintaining high levels of soil nitrogen in tropical forests.

Comparative Analysis of the Combined Effects of Different Water and Phosphate Levels on Growth and Biological Nitrogen Fixation of Nine Cowpea Varieties.

Water deficit and phosphate (Pi) deficiency adversely affect growth and biological nitrogen fixation (BNF) of legume crops. In this study, we examined the impact of interaction between soil water conditions and available soil-Pi levels on growth, nodule development and BNF potential of nine cowpea varieties grown on dry savanna soils. In our experimental design, soils with different available soil-Pi levels, i.e., low, moderate, and high soil-Pi levels, collected from various farming fields were used to grow nine cowpea varieties under well-watered and water-deficit conditions. Significant and severe water deficit-damaging effects on BNF, nodulation, growth, levels of plant-nitrogen (N) and -phosphorus (P), as well as shoot relative water content and chlorophyll content of cowpea plants were observed. Under well-watered and high available soil-Pi conditions, cowpea varieties IT07K-304-9 and Dan'Ila exhibited significantly higher BNF potential and dry biomass, as well as plant-N and -P contents compared with other tested ones. Significant genotypic variations among the cowpeas were recorded under low available soil-Pi and water-deficit conditions in terms of the BNF potential. Principal component (PC) analysis revealed that varieties IT04K-339-1, IT07K-188-49, IT07K-304-9, and IT04K-405-5 were associated with PC1, which was better explained by performance for nodulation, plant biomass, plant-N, plant-P, and BNF potential under the combined stress of water deficit and Pi deficiency, thereby offering prospects for development of varieties with high growth and BNF traits that are adaptive to such stress conditions in the region. On another hand, variety Dan'Ila was significantly related to PC2 that was highly explained by the plant shoot/root ratio and chlorophyll content, suggesting the existence of physiological and morphological adjustments to cope with water deficit and Pi deficiency for this particular variety. Additionally, increases in soil-Pi availability led to significant reductions of water-deficit damage on dry biomass, plant-N and -P contents, and BNF potential of cowpea varieties. This finding suggests that integrated nutrient management strategies that allow farmers to access to Pi-based fertilizers may help reduce the damage of adverse water deficit and Pi deficiency caused to cowpea crop in the regions, where soils are predominantly Pi-deficient and drought-prone.

Draft Genome Sequence of Pseudomonas stutzeri Strain KMS 55, an Endophytic Diazotroph Isolated from Rice Roots.

ABSTRACTPseudomonas stutzeri strain KMS 55 (MTCC 12703) is an isolate from the root tissues of rice (Oryza sativa L.) that displays a high biological nitrogen fixation ability. Here, we report the complete genome sequence of this strain, which contains 4,637,820 bp, 4,289 protein-coding genes, 5,006 promoter sequences, 62 tRNAs, a single copy of 5S-16S-23S rRNA, and a genome average GC content of 51.18%. Analysis of the ~4.64-Mb genome sequence will give support to increased understanding of the genetic determinants of host range, endophytic colonization behavior, endophytic nitrogen fixation, and other plant-beneficial roles of Pseudomonas stutzeri.

Metabolism of nitrogen and carbon: Optimization of biological nitrogen fixation and cowpea development

Although the favorable role of plant growth-promoting bacteria (PGPB) in biological nitrogen fixation (BNF) is well established, their involvement in nodule metabolism is unknown. This study was performed to determine the relationship between C and N metabolism in cowpea nodules inoculated with only Bradyrhizobium sp. or with double and triple combinations of Bradyrhizobium sp., and two PGPB, Paenibacillus graminis and Paenibacillus durus during two critical phases of nodule development (flowering and beginning of senescence). The triple inoculation (Bradyrhizobium sp and two PGPB) induced higher N content in nodules, total-N accumulation per plant and shoot dry weight compared with other combinations at the beginning of senescence. This increased N performance was positively correlated with the nodule sucrose content but not with the content of total soluble carbohydrates, reduced sugars and starch. The higher BNF under triple inoculation conditions was not significantly associated with sucrose synthase activity but was slightly associated with soluble acid invertase activity in nodules at the beginning of senescence. These enzymes exhibited opposite responses between flowering and the beginning of senescence in all treatments. Glutamate synthase, glutamine synthetase and glutamate dehydrogenase were stimulated by double (Bradyrhizobium sp. + P. durus) and triple inoculation compared with other treatments. Our data revealed that inoculation with Bradyrhizobium sp. and PGPB is favorable for BNF activity in cowpeas. This positive interaction requires a complex balance involving enzymes and metabolites related to C and N metabolism.

Mapping of QTLs associated with biological nitrogen fixation traits in soybean

Biological nitrogen fixation (BNF) is a key process, but despite the economic and environmental importance, few studies about quantitative trait loci (QTL) controlling BNF traits are available, even in the economically important crop soybean Glycine max (L.) Merr. In this study, a population of 157 F2:7 RILs derived from crossing soybean cultivars Bossier (high BNF capacity) and Embrapa 20 (medium BNF capacity) was genotyped with 105 simple sequence repeat markers (SSRs). The genetic map obtained has 1231.2 cM and covers about 50% of the genome, with an average interval of 18.1 cM. Three traits, nodule number (NN), the ratio nodule dry weight (NDW)/NN and shoot dry weight (SDW) were used to evaluate BNF performance. A composite interval mapping for multiple traits method (mCIM) analysis mapped two QTLs for SDW (LGs E and L), three for NN (LGs B1, E and I), and one for NDW/NN (LG I); all QTLs were of small effect (R-values ranging from 1.7% to 10.0%) and explained 15.4%, 13.8% and 6.5% of total variation for these three traits, respectively.

Biological nitrogen fixation of some groundnuts as affected by genotype and applied phosphorus at Samaru, northern Guinea Savannah of Nigeria

Biological nitrogen fixation (BNF), brought about by both free living soil microorganisms and their symbiotic associations with higher plants, is the major source of N input into agricultural systems. Groundnuts (Arachis hypogaea L.), in symbiosis with rhizobia, in their root nodules, fix atmospheric nitrogen (N2). A field trial was carried out at Samaru, Nigeria, in 2008 with a view to evaluate BNF potentials of five groundnut genotypes (SAMNUT 10, 11, 21, 22 and 23). Each genotype received four rates of P (0, 20, 40 and 60 P2O5 ha-1) in an RCBD with three replications. N-Difference method was employed in assessing the BNF of the genotypes. Nodulation performance of the genotypes was observed. Results of the study showed that SAMNUT 11 and 22 significantly recorded thehighest nodulation, by number and weight. All the remaining genotypes statistically produced same nodule number. Moreover, the early maturing SAMNUT 21 fixed the highest (82 kg N ha-1) quantity of N2, while SAMNUT 11 (46 kg N ha-1) and 23 (31 kg N ha-1) were statistically similar and fixed the lowest N2. SAMNUT 11 and 22 were, therefore, found to significantly record the highest nodule number and weigh and SAMNUT 21 fixed the highest N2, and hence the highest BNF, indicating their genotypic desirability in terms of N2 addition to the soil environment, especially forsubsequent crops.Keywords: Arachis hypogaea, Biological Nitrogen Fixation, Genotype, Phosphorus

Yield and biological nitrogen fixation of cowpea varieties in the semi-arid region of Brazil

Cowpea is an important crop in small properties of the Brazilian semi-arid region, where it is cultivated without fertilizer application. In spite of the fundamental role played by biological Nitrogen fixation (BNF), little is known of the symbiosis between cowpea varieties and native rhizobia or recommended rhizobia strains. A field experiment was conducted aiming to estimate BNF and productivities of local varieties, in association with two previously described bradyrhizobial inoculant strains and native rhizobia (no inoculation). The plants received 20 kg ha−1 of enriched 15N fertilizer to allow the use of the isotopic dilution method. After harvest (80 days) straw and grain biomass was determined. The varieties differed in grain and straw productivity and in N and N derived from atmosphere (%Ndfa). Corujinha had the highest grain productivity (1147 kg ha−1), followed by Sempre Verde (920 kg ha−1), Azul (912 kg ha−1) and Cariri (889 kg ha−1). Costela de Vaca had the highest straw productivity (2258 kg ha−1), highest N content in the straw (28 g ha−1) and highest BNF (79 %Ndfa, corresponding to 45 kg ha−1 of N for total aboveground biomass and 39 kg ha−1 for the straw), but the lowest grain productivity (381 kg ha−1) and the lowest harvest index (0.14). The inoculations did not significantly alter productivities, N contents or %Ndfa but there was a tendency of lower grain productivities in the non-inoculated plants, which was reflected in lower total and biologically fixed N quantities, indicating that the native strains may be slightly less efficient.

Early responses of rice (Oryza sativa L.) seedlings to inoculation with beneficial diazotrophic bacteria are dependent on plant and bacterial genotypes

Rice cultivars, in combination with diazotrophic bacteria, can obtain variable contributions from Biological Nitrogen Fixation (BNF). Plant genetic controls and the genotype of the bacterial strains might regulate in particular ways the plant responses during these associations. Some of this regulation is likely to occur during the first stages of bacterial infection and plant colonization, and some of the early plant responses might involve ethylene signaling, as previously reported for sugarcane. The aim of this work was to investigate whether rice early responses to inoculation with beneficial diazotrophic bacteria and the expression of ethylene receptors (ERs) by the host are dependent on plant and bacterial genotypes. Bacterial colonization, lateral root development and expression of ERs were measured in seedlings of two rice cultivars, IR42 and IAC4440, which, respectively, are known to have higher and lower BNF capabilities. Inoculation experiments were performed with two strains of bacteria: Azospirillum brasilense sp245 and Burkholderia kururiensis M130. Cultivar IR42, which has a relatively high BNF capability, was more colonized by the bacteria early after inoculation in comparison with cv. IAC 4440, which has a lower BNF capability. However, although both cultivars showed a significant increase in lateral root numbers 10 days after inoculation with A. brasilense sp245, plants inoculated with B. kururiensis M130 did not. In addition, a differential ER expression pattern was observed between IR42 and IAC4440 in response to inoculation with the two strains. The expression of ERs was higher in the more BNF-effective cultivar IR42, than in the less BNF-effective cv. IAC4440, especially when cv. IR42 was inoculated with A. brasilense sp245. Moreover, plants associated with B. kururiensis M130 showed a different ER expression profile compared to those inoculated with A. brasilense sp245, with the latter exhibiting greater increases of ER mRNA levels three days after bacterial inoculation. We have shown that two rice genotypes contrasting in their BNF capability responded differentially to early inoculation with different strains of diazotrophic bacteria. The two rice genotypes were colonized with different bacterial numbers during their early association with two strains of diazotrophic bacteria, and the two strains had different effects on the promotion of lateral root development early after inoculation. They also triggered distinct ER expression patterns in the two rice cultivars. These data thus suggest that rice ethylene responses to beneficial diazotrophic bacteria are controlled by both plant and bacterial genotypes.