Nitrogen Fixation In Common Bean Research Articles

Influence of common bean genotypes and rhizobia interaction for nodulation and nitrogen fixation

Bacteria from the rhizobia group are able to associate symbiotically with bean crop, forming nodules in the root, in which the biological nitrogen-fixing process occurs. However, the efficiency of this process has been low and it can be attributed to genetic and environmental factors. Thus, the objective of this study was to evaluate the capacity of nodulation of local varieties and commercial common bean cultivars inoculated with a Rhizobium tropici strain used in commercial inoculants and rhizobia isolates from common bean root nodules. The experiment was carried out in a factorial scheme (2x4), in a randomized block design with four replicates. It was tested two local varieties and two commercial cultivars, inoculated with the reference Rhizobium tropici strain CIAT899 and the RBZ14 strain isolated from common bean nodules grown in soils of Southern Brazil, in adapted Leonard-type pots. The CIAT899 strain promoted either higher mass of viable nodules and higher nitrogen accumulation in the aerial part. The black group local variety showed better response than the cultivar TAA Dama for nodule viability, suggesting more efficiency for nodulation. The interaction between genotypes (local varieties and commercial cultivars) and bacteria showed the specificity of the complex symbiotic relationship of biological nitrogen fixation in common bean, requiring further studies of these interactions.

Implications of the Symbiotic Nitrogen Fixation in Common Bean under Seasonal Water Stress

The objective of this research was to study the performance of 10 common bean genotypes under water deficit stress and how it affects to their symbiotic relationship with 10 Rhizobium strains in both greenhouse and field conditions. PHA-0471, a small seeded genotype had the best yield under irrigation and under water stress. Other genotypes with tolerance to drought were the large-seeded PHA-0432 and PHA-0683. In the Rhizobium inoculation tests it was observed that the increase of dry nodular weight produced less seed yield in beans. PHA-0683 genotype presented a great uniformity on nodule size and an association with yield when it displays the big nodule phenotype. Further research about this would be interesting because this fact could be due to the existence of a plant blocking mechanism for inefficient strain nodules. The inoculated plants were productive in irrigated fields and in drought ones and their productivity was the same or even better than the N supplemented plant control. The genotype-strain relationship was very specific and the local strains achieved the greatest productivity with some genotypes in irrigated and drought conditions that make possible their use as inoculating strains, with relevance for the environmental impact of agriculture.

Evaluation of beneficial and inhibitory effects of nitrate on nodulation and nitrogen fixation in common bean (Phaseolus vulgaris)

AbstractThe effects of applied nitrate on symbiotic nitrogen fixation in legumes are complex. Both inhibition and promotion of nodulation by nitrate have been observed in a dose‐dependent manner. The objectives of this study were to determine the effects of nitrate at different concentrations on root nodulation in different genotypes in common bean (Phaseolus vulgaris). Six genotypes were inoculated with the same rhizobial strain and grown hydroponically in growth pouches in a growth chamber and exposed to six nitrate concentrations, including 0, 2.5, 5, 10, 15, and 20 mM for 4 weeks. The tested genotypes included three recombinant inbred lines (RILs, 25, 46, and 70) that differed in their responses to nitrogen (based on observations of one field growing season), their parents (Mist and Sanilac—registered varieties), which are different in N‐fixing abilities, and one nonnodulating mutant (R99). Our results showed that small amounts of nitrate (2.5 and 5 mM) promoted nodule formation and increased nodule biomass, compared with plants in the 0 nitrate control treatment. In contrast, nitrate concentrations over 10 mM inhibited nodulation, resulting in reductions in nodule number and nodule biomass. Nodulation was completely inhibited by 15‐mM nitrate in all the genotypes. Regression analyses indicated that 5‐mM nitrate is the optimum concentration for promoting nodulation as measured by the total number of nodules formed, the number of effective nodules formed, and the nodule biomass formed. In contrast, nitrogen fixation was inhibited by all levels of nitrate. No genotypic differences were observed in nodulation among the three RILs and their parental cultivars, but all were significantly different than R99, a nonnodulating mutant.

Identification of quantitative trait loci for symbiotic nitrogen fixation in common bean.

QTL were identified for symbiotic nitrogen fixation in common bean. These QTL were detected in both greenhouse and field studies, and many overlapped with previously reported QTL in diverse mapping populations. Common bean (Phaseolus vulgaris L.) productivity can be improved through the genetic enhancement of its symbiotic nitrogen fixation (SNF) capacity. This study was aimed at understanding the genetic architecture of SNF through QTL analysis of a recombinant inbred line (RIL) population contrasting for SNF potential. The mapping population consisted of 188 F4:5 RILs derived from a cross of Solwezi and AO-1012-29-3-3A that were evaluated for SNF in the greenhouse and field in Zambia. The population was genotyped with 5398 single-nucleotide polymorphism (SNP) markers. QTL for shoot biomass, nitrogen percentage in shoot biomass, nitrogen percentage in seed, total nitrogen derived from atmosphere (Ndfa) and percentage of nitrogen derived from the atmosphere (%Ndfa) were identified. Three QTL for %Ndfa were identified on chromosomes Pv01, Pv04 and Pv09. Five QTL for Ndfa were identified on Pv04, Pv06, Pv07, Pv09 and Pv11. The QTL Ndfa9.1SA identified in the current study overlapped with a previously reported QTL for SNF. A major QTL Ndfa7.1DB, SA (R2 = 14.9%) was consistently identified in two greenhouse studies and overlapped with previously reported QTL. The QTL Ndfa4.2SA identified from the greenhouse experiment is novel and overlapped with the QTL %NB4.3SA, %NS4.2SA and %Ndfa4.2SA from the field experiment. These QTL identified in both greenhouse and field experiments, which overlap with previously reported QTL, could potentially be deployed by marker-assisted breeding to accelerate development of bean cultivars with enhanced SNF.

Agronomic efficiency of Rhizobium strains from the Amazon region in common bean

ABSTRACT To maximize the contribution of biological nitrogen fixation in common bean, it is necessary to use bacterial strains that are more adapted, competitive, and efficient in the symbiotic process. In this regard, the aim of this study was to evaluate the agronomic efficiency (AE) of three bacterial strains isolated from acid soils with high Al content from the Amazon region in an Argissolo Vermelho Distrófico típico soil (Typic Rhodustults - USDA Classification) from the municipality of Formiga, MG, Brazil. We compared their AE to that of the reference strain CIAT 899T and of two controls without inoculation (one without and another with 80 kg ha-1 of N-urea). The results indicated that inoculation with the strains UFLA 02-100 and UFLA 02-127 provides grain yield equivalent to inoculation with the reference strain and to the control with mineral N. Thus, both have potential for recommendation as inoculants for common bean.

Response to selection for improved nitrogen fixation in common bean (Phaseolus vulgaris L.)

Breeding for high symbiotic nitrogen (N) fixation (SNF) in common bean (Phaseolus vulgaris L.) is expected to contribute to reduced application of chemical fertilizers in cropping systems involving common bean. The magnitude of variation and the genetic and phenotypic correlation among seed yield, SNF, estimated as the percentage of nitrogen derived from atmosphere, and related traits were studied in a population of 140 F4-derived F5 recombinant inbred lines, developed from a cross between low- and high-SNF bean genotypes ‘Sanilac’ and ‘Mist’, respectively. The experiment was conducted in a total of five location-years in Ontario, Canada, from 2011 to 2013. These location-years were grouped into stress- and optimum moisture test sites, based on the total precipitation during the growing season. In each test site two nitrogen supply management strategies, SNF-dependent and N fertilizer-dependent, were simulated separately in the field by inoculating the seed with a commercial Rhizobium leguminosarum bv. phaseoli and by application of N fertilizers at 100 kg ha−1, respectively. The genetic variation was significant for seed yield, SNF and related traits. The heritability of the traits ranged from 14 to 71% and 4 to 25% in optimum moisture and in stress environments, respectively. No significant correlation between SNF and seed yield indicated that selection for high SNF does not necessarily lead to greater seed yield and that selection for both traits should be performed simultaneously.

Natural rhizobial diversity helps to reveal genes and QTLs associated with biological nitrogen fixation in common bean.

Common bean is one of the most important crops for human feed, and the most important legume for direct consumption by millions of people, especially in developing countries. It is a promiscuous host legume in terms of nodulation, able to associate with a broad and diverse range of rhizobia, although the competitiveness for nodulation and the nitrogen fixation capacity of most of these strains is generally low. As a result, common bean is very inefficient for symbiotic nitrogen fixation, and nitrogen has to be supplied with chemical fertilizers. In the last years, symbiotic nitrogen fixation has received increasing attention as a sustainable alternative to nitrogen fertilizers, and also as a more economic and available one in poor countries. Therefore, optimization of nitrogen fixation of bean-rhizobia symbioses and selection of efficient rhizobial strains should be a priority, which begins with the study of the natural diversity of the symbioses and the rhizobial populations associated. Natural rhizobia biodiversity that nodulates common bean may be a source of adaptive alleles acting through phenotypic plasticity. Crosses between accessions differing for nitrogen fixation may combine alleles that never meet in nature. Another way to discover adaptive genes is to use association genetics to identify loci that common bean plants use for enhanced biological nitrogen fixation and, in consequence, for marker assisted selection for genetic improvement of symbiotic nitrogen fixation. In this review, rhizobial biodiversity resources will be discussed, together with what is known about the loci that underlie such genetic variation, and the potential candidate genes that may influence the symbiosis' fitness benefits, thus achieving an optimal nitrogen fixation capacity in order to help reduce reliance on nitrogen fertilizers in common bean.

Agronomic and Economic Efficiency of Common-Bean Inoculation with Rhizobia and Mineral Nitrogen Fertilization

Management of biological nitrogen fixation in common bean still requires improvement. The objective of this study was to verify the compatibility of nitrogen fertilization with biological N2 fixation to increase common bean yield and profitability. Four field experiments were performed in four municipalities of Minas Gerais, Brazil, during the 2009 and 2010 winter crop season. The 2009 crop experiments were set up under a no-till system and the 2010 crop was conducted using conventional tillage. A randomized block experimental design was used with four replications and seven treatments combining application rates of mineral N (at sowing and/or topdressing) and seed inoculation with Rhizobium tropici strain CIAT899. Inoculation with 20 kg ha-1 N-urea at sowing and seed inoculation does not interfere with nodule dry matter and promotes yield comparable to that observed with 80 kg ha-1 N-urea with economic profitability in both no-till and conventional tillage systems. These results show the possibility of economic savings with respect to N fertilizers, but also a significant ecological contribution by avoiding problems associated with misuse of these fertilizers, such as eutrophication of waterways and denitrification.

Responses of symbiotic nitrogen-fixing common bean to aluminum toxicity and delineation of nodule responsive microRNAs.

Aluminum (Al) toxicity is widespread in acidic soils where the common bean (Phaseolus vulgaris), the most important legume for human consumption, is produced and it is a limiting factor for crop production and symbiotic nitrogen fixation. We characterized the nodule responses of common bean plants inoculated with Rhizobioum tropici CIAT899 and the root responses of nitrate-fertilized plants exposed to excess Al in low pH, for long or short periods. A 43–50% reduction in nitrogenase activity indicates that Al toxicity (Alt) highly affected nitrogen fixation in common bean. Bean roots and nodules showed characteristic symptoms for Alt. In mature nodules Al accumulation and lipoperoxidation were observed in the infected zone, while callose deposition and cell death occurred mainly in the nodule cortex. Regulatory mechanisms of plant responses to metal toxicity involve microRNAs (miRNAs) along other regulators. Using a miRNA-macroarray hybridization approach we identified 28 (14 up-regulated) Alt nodule-responsive miRNAs. We validated (quantitative reverse transcriptase-PCR) the expression of eight nodule responsive miRNAs in roots and in nodules exposed to high Al for long or short periods. The inverse correlation between the target and miRNA expression ratio (stress:control) was observed in every case. Generally, miRNAs showed a higher earlier response in roots than in nodules. Some of the common bean Alt-responsive miRNAs identified has also been reported as differentially expressed in other plant species subjected to similar stress condition. miRNA/target nodes analyzed in this work are known to be involved in relevant signaling pathways, thus we propose that the participation of miR164/NAC1 (NAM/ATAF/CUC transcription factor) and miR393/TIR1 (TRANSPORT INHIBITOR RESPONSE 1-like protein) in auxin and of miR170/SCL (SCARECROW-like protein transcription factor) in gibberellin signaling is relevant for common bean response/adaptation to Al stress. Our data provide a foundation for evaluating the individual roles of miRNAs in the response of common bean nodules to Alt.

Genome-wide association analysis of symbiotic nitrogen fixation in common bean.

Significant SNPs and candidate genes for symbiotic nitrogen fixation (SNF) and related traits were identified on Pv03, Pv07 and Pv09 chromosomes of common bean. A genome-wide association study (GWAS) was conducted to explore the genetic basis of variation for symbiotic nitrogen fixation (SNF) and related traits in the Andean Diversity Panel (ADP) comprising 259 common bean (Phaseolus vulgaris) genotypes. The ADP was evaluated for SNF and related traits in both greenhouse and field experiments. After accounting for population structure and cryptic relatedness, significant SNPs were identified on chromosomes Pv03, Pv07 and Pv09 for nitrogen derived from atmosphere (Ndfa) in the shoot at flowering, and for Ndfa in seed. The SNPs for Ndfa in shoot and Ndfa in seed co-localized on Pv03 and Pv09. Two genes Phvul.007G050500 and Phvul.009G136200 that code for leucine-rich repeat receptor-like protein kinases (LRR-RLK) were identified as candidate genes for Ndfa. LRR-RLK genes play a key role in signal transduction required for nodule formation. Significant SNPs identified in this study could potentially be used in marker-assisted breeding to accelerate genetic improvement of common bean for SNF.

The micro-RNA72c-APETALA2-1 node as a key regulator of the common bean-Rhizobium etli nitrogen fixation symbiosis.

Micro-RNAs are recognized as important posttranscriptional regulators in plants. The relevance of micro-RNAs as regulators of the legume-rhizobia nitrogen-fixing symbiosis is emerging. The objective of this work was to functionally characterize the role of micro-RNA172 (miR172) and its conserved target APETALA2 (AP2) transcription factor in the common bean (Phaseolus vulgaris)-Rhizobium etli symbiosis. Our expression analysis revealed that mature miR172c increased upon rhizobial infection and continued increasing during nodule development, reaching its maximum in mature nodules and decaying in senescent nodules. The expression of AP2-1 target showed a negative correlation with miR172c expression. A drastic decrease in miR172c and high AP2-1 mRNA levels were observed in ineffective nodules. Phenotypic analysis of composite bean plants with transgenic roots overexpressing miR172c or a mutated AP2-1 insensitive to miR172c cleavage demonstrated the pivotal regulatory role of the miR172 node in the common bean-rhizobia symbiosis. Increased miR172 resulted in improved root growth, increased rhizobial infection, increased expression of early nodulation and autoregulation of nodulation genes, and improved nodulation and nitrogen fixation. In addition, these plants showed decreased sensitivity to nitrate inhibition of nodulation. Through transcriptome analysis, we identified 114 common bean genes that coexpressed with AP2-1 and proposed these as being targets for transcriptional activation by AP2-1. Several of these genes are related to nodule senescence, and we propose that they have to be silenced, through miR172c-induced AP2-1 cleavage, in active mature nodules. Our work sets the basis for exploring the miR172-mediated improvement of symbiotic nitrogen fixation in common bean, the most important grain legume for human consumption.

Partitioning the contributions of biochar properties to enhanced biological nitrogen fixation in common bean (Phaseolus vulgaris)

Studies document increases in biological nitrogen fixation (BNF) following applications of biochar. However, the underlying mechanisms for this response remain elusive. Greenhouse experiments were conducted to test the effects of biochar mineral nutrients, pH, and volatile matter (VM) on BNF in common beans (Phaseolus vulgaris L.). Biochars were produced from seven feedstocks pyrolyzed at either 350 or 550 °C. Biochars were treated with acid to reduce mineral nutrient contents, with acetone to remove acetone-soluble VM, with steam to reduce both the mineral and VM contents, or left untreated. The biochar additions at a rate of 15 t ha−1 resulted in an average 262 % increase in shoot biomass, 164 % increase in root biomass, 3575 % increase in nodule biomass, and a 2126 % increase in N derived from atmosphere (Ndfa) over the control. Simple mineral nutrients and soil acidity amelioration from the biochar were only to a minimal extent responsible for these increases (r 2 = 0.03; P = 0.0298, n = 201). Plant growth and Ndfa were significantly correlated with plant P uptake (r 2 = 0.22; P 0.05). Improved P nutrition resulted from 360 % greater mycorrhizal colonization with biochar additions. Removal of acetone-soluble VM increased plant growth and Ndfa, and VM extracted from the biochar produced at 350 °C reduced the growth of rhizobia in yeast extract mannitol agar (YMA) medium. In contrast, acetone-soluble VM extracted from seven biochars produced at 550 °C increased the growth of rhizobium in the YMA compared to an acetone-residue control, suggesting differential effects of VM forms on rhizobia.

Combined inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases phosphorus use efficiency for symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.)

This study compared the response of common bean (Phaseolus vulgaris L.) to arbuscular mycorrhizal fungi (AMF) and rhizobia strain inoculation. Two common bean genotypes i.e. CocoT and Flamingo varying in their effectiveness for nitrogen fixation were inoculated with Glomus intraradices and Rhizobium tropici CIAT899, and grown for 50days in soil–sand substrate in glasshouse conditions. Inoculation of common bean plants with the AM fungi resulted in a significant increase in nodulation compared to plants without inoculation. The combined inoculation of AM fungi and rhizobia significantly increased various plant growth parameters compared to simple inoculated plants. In addition, the combined inoculation of AM fungi and rhizobia resulted in significantly higher nitrogen and phosphorus accumulation in the shoots of common bean plants and improved phosphorus use efficiency compared with their controls, which were not dually inoculated. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve the efficiency in phosphorus use for symbiotic nitrogen fixation especially under phosphorus deficiency.

Effects of organic and inorganic phosphorus sources on nitrogen fixation by field grown common bean on an Alfisol and an Ultisol in Kakamega, Kenya

A field study was carried out in 2005 in Kakamega to quantify the effects of organic and inorganic sources of phosphorus on nitrogen fixation of common bean by the 15N Natural Abundance method. Field experiments were conducted on two different soil groups (Alfisol and Ultisol) in 20 farmers’ fields. Six treatments: control with no input, inorganic phosphorus, inorganic nitrogen, inorganic phosphorus plus nitrogen, seed priming with phosphorus and farmyard manure. These treatments were replicated three times in a randomized complete block design. Maize was used as a non-fixing reference plant for 15N analysis. The results showed that the total nitrogen accumulation by common bean ranged from 5 to 20 kg ha-1 in the Alfisol and from 8 to 26 kg ha-1 in the Ultisol and treatments in the Ultisol accumulated 20% more biomass comparedto treatments in the Alfisol. Further, the rate of nitrogen fixation was 50% to 54% Ndfa for the inorganic phosphorus sources and 40% to 51% Ndfa for the organic phosphorus source. Seed priming with phosphorus,inorganic nitrogen and the control obtained less than 40% Ndfa for nitrogen fixation. These results illustrate that phosphorus sources could differentially enhance nitrogen fixation by common bean in different soil groups in smallholder farming systems in Kakamega.

Contributions and limitations to symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.) in Romania

Common beans usually achieve grain yields less than the genotypic potential of the cultivar under Romanian field conditions. To understand better the contribution of nitrogen fixation to the yield formation, I made a long-term evaluation (1977–1994) of inoculation effects of 19 Rhizobium leguminosarum bv. phaseoli strains on common bean cultivated in several locations. Grain yields were significantly influenced by all selected strains, years and locations in all experimental cycles, and only partially by the interactions between strains and years or strains and locations. The average yield increases induced by these strains during the four cycles ranged from 6 to 20%. Four bacterial strains proved to be more stable in their field performances, taking into account the yield increases greater than 10% over controls observed in all individual trials. Mean yields and variation limits recorded during the long-term evaluation of strain efficacy in locations with different soil pH values showed similar patterns of yield increases from soils with acidic to neutral pH values. Linear regression between mean grain yields and average temperatures demonstrated the limiting effect of temperature on yield. The interaction between bacterial strain and nitrogen fertiliser rate demonstrated the ability of dinitrogen fixation to satisfy the crop requirements for this element. An evaluation of the amounts of nitrogen fixed in three common bean cultivars inoculated with two bacterial strains showed different N2-fixing capacities among plant genotypes.

Effects of common bean (Phaseolus vulgaris L.) cultivar and rhizobium strain on plant growth, seed yield and nitrogen content

The common bean is generally regarded as a relatively inefficient fixer of nitrogen. Information on the relative importance of host cultivar and rhizobium strain would be useful in guiding a breeding program to improve nitrogen fixation in common bean. Seventeen cultivars of common bean (15 of Meso-American and 2 of Andean origin, differing in nodulation potential, maturity, growth habit and market class) were investigated for symbiotic compatibility with 10 genetically diverse strains of bean rhizobia. In a greenhouse (27/22 °C), five sets of 170 pots were replicated over time: three sets were harvested after 30 d for determination of acetylene reduction activity, plant dry weights, and nitrogen content; two replications were grown to maturity for determination of seed dry weight and nitrogen content. There were highly significant effects of both strain and cultivar on most measured characteristics, but there was no interaction between cultivar and strain. Italian Barlotti produced the greatest plant weight after 30 d, but its final seed yield did not differ significantly from that of the highest yielding cv. BAT271. The strain TAL182 was associated with the greatest plant weight at 30 d, but the final seed yield associated with it was less than that of the most productive strain USDA9001. Strain USDA 2667 was characterized as type I by determination of its 16S rRNA gene nucleotide sequence. The nodules of plants in symbiosis with strains of type IIB had high levels (8.1%) of nitrogen and high levels (227 µmol g−1 h−1) of acetylene reduction activity (ARR). Nodules occupied by strains of type IIA and the type I USDA2667 had intermediate levels of nitrogen (6.9%) and ARR (166 µmol g−1 h−1), while nodules occupied by type 1 strains had low levels of nitrogen (5.2%) and ARR (144 µmol g−1 h−1). Screening of lines, parents and other breeding materials for improved N-fixation may be achieved with any effective strain since no host × strain interaction was detected. Key words: Common bean, Phaseolus vulgaris, nodulation, nitrogen fixation, rhizobium strains, cultivar × strain interaction

Effects of water stress on nitrogen fixation of common bean (Phaseolus vulgaris L.)

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Effect of low pH on nitrogen fixation of common bean grown at various calcium and nitrate levels

The influence of low pH (4.0–4.5) of the medium on nodulation, nodule biomass, nodule nitrogenase activity, and ultrastructure at various concentrations of calcium (Ca) (0.127 mM and 0.508 mM) and nitrate (NO3) (0.390 mM and 1.560 mM) at different stages of development (2nd, 3rd, 6th, and 7th compound leaf) of the common bean plants was studied. The experiment was conducted using Hellriegel's nutrient solution under greenhouse conditions. Bean root nodule nitrogen (N2) fixation was influenced favorably at a decrease of Ca (0.127 mM) and NO3 (0.390 mM) concentrations in the growth solution. In contrast, the biomass of the roots, stems, leaves, and pods decreased at low Ca and NO3 levels while the most sensitive were the leaves and pods of the plants. The number of nodules, their fresh and dry weight, nitrogenase activity, and ultrastructure was affected to a greater extent at a low pH of the medium when Ca and NO3 concentrations were low (0.127 mM and 0.390 mM, respectively). Under these conditions, the roots, stems, leaves, and pods were slightly affected. Nodule N2 fixation was more sensitive to low pH at the 2nd and 3rd leaf stage of plant development. The root and pod biomass had the same tendency of change as the nodules. On the contrary, at low pH and decreased Ca and NO3 levels (0.127 mM and 0.390 mM, respectively), the stem and leaf biomass increased significantly at the later stages of bean plant development (6th and 7th leaf). Our conclusion was that the stress effects of low pH of the medium had different affects at various Ca and NO3 concentrations in the nutrient solution and stages of common bean plant development.

Nitrogen fixation of common bean (Phaseolus vulgaris L.) as affected by mineral nitrogen supply at different growth stages

The effect of mineral nitrogen applications at different growth stages on N2 fixation, nodulation and shoot dry weight of two cultivars of common bean (Phaseolus vulgaris L.) was investigated in a greenhouse experiment. Mineral nitrogen was split into two unequal doses (5 and 25 mg N kg-1 soil). The main fertilizer dose was applied at sowing or at the vegetative stage, V6. N2 fixation was determined by the 15N dilution method combined with the single treatment method. By physiological maturity, shoot dry weights and grain yields did not differ between cultivars, but cv. Puebla fixed more N2 than cv. Negro Argel. The timing of the main nitrogen application (25 mg N kg-1 soil) did not affect final shoot dry weights and grain yields. At the V6 vegetative stage, 28 days after the application of 25 mg N kg-1 soil, the nodule numbers of cv. Puebla and cv. Negro Argel were 14% and 82%, respectively, of the nodule numbers of plants which received 5 mg N kg-1 soil at sowing. Also, nodule dry weight and acetylene reducing activity per plant at V6 were reduced following the application of 25 mg N kg-1 soil at sowing. However, when 25 mg N kg-1 soil were applied at sowing and 5 mg N kg-1 soil at the V6 stage, 55% and 45%, respectively, of the final (R8) nitrogen accumulation in the shoots of cv. Puebla and cv. Negro Argel was derived from N2 fixation. Less N was derived from the atmosphere (45% and 35% in the shoots of cv. Puebla and cv. Negro Argel at R8, respectively) when 5 mg N kg-1 soil was applied at sowing and 25 mg N kg-1 soil at V6. The delayed onset of N2 fixation of the Phaseolus vulgaris-Rhizobium symbiosis as a consequence of the main nitrogen application at sowing was less detrimental to the quantity of symbiotically fixed nitrogen of both cultivars than the effect of 25 mg N kg-1 soil during vegetative growth.

Response to inoculation and N fertilization for increased yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.)

Response to inoculation and N fertilization for increased yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.)