Genotypic variation in biological nitrogen fixation by common bean

Field experiments were performed in Austria, Brazil, Chile, Colombia, Guatemala, Mexico and Peru as part of an FAO/IAEA Co-ordinated Research Programme to investigate the nitrogen fixing potential of cultivars and breeding lines of common bean (Phaseolus vulgaris L.). Each experiment included approximately 20 bean genotypes which were compared using the ¹⁵N isotope dilution method. Great differences in nitrogen fixation were observed between and within experiments, with average values of 35% N derived from atmosphere (% Ndfa) and highest values of 70% Ndfa being observed. These values which were larger than had been reported previously for common bean, were observed only when environmental factors were favorable. Therefore, common bean lines are available, which can support high biological nitrogen fixation. These can be used either directly as cultivars for production or in breeding programmes to enhance nitrogen fixation in other cultivars.     

Yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.) in Peru

Two field experiments were performed to evaluate the nitrogen fixation potential of twenty common bean cultivars and breeding lines during summer and winter seasons of 1986 and 1988, respectively. The ¹⁵N isotope dilution method was used to quantify N₂ fixation. The cultivars and breeding lines were variable in terms of their N₂ fixation. The cv. Caballero was very efficient, with more than 50% N derived from the atmosphere and 60–80 kg N ha^–1 fixed in both seasons. Other cultivars were less efficient, since the poorest ones derived less than 30% of their nitrogen from the atmosphere and fixed less than 20 kg N ha^–1. After additional testing the best cultivars may be used directly by the farmers for cultivation. The experiments have provided information about which genotypes may be used to breed for enhanced fixation in common bean.     

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

Common bean (Phaseolus vulgaris L.) is able to fix 20–60 kg N ha^–1 under tropical environments in Brazil, but these amounts are inadequate to meet the N requirement for economically attractive seed yields. When the plant is supplemented with N fertilizer, N₂ fixation by Rhizobium can be suppressed even at low rates of N. Using the ¹⁵N enriched method, two field experiments were conducted to compare the effect of foliar and soil applications of N-urea on N₂ fixation traits and seed yield. All treatments received a similar fertilization including 10 kg N ha^–1 at sowing. Increasing rates of N (10, 30 and 50 kg N ha^–1) were applied for both methods. Foliar application significantly enhanced nodulation, N₂ fixation (acetylene reduction activity) and yield at low N level (10 kg N ha^–1). Foliar nitrogen was less suppressive to nodulation, even at higher N levels, than soil N treatments. In the site where established Rhizobium was in low numbers, inoculation contributed substantially to increased N₂ fixation traits and yield. Both foliar and soil methods inhibited nodulation at high N rates and did not significantly increase bean yield, when comparing low (10 kg N ha^–1) and high (50 kg N ha^–1) rates applied after emergence. In both experiments, up to 30 kg N ha^–1 of biologically fixed N₂ were obtained when low rates of N were applied onto the leaves.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

Breding common bean for improved biological nitrogen fixation

Common bean (Phaseolus vulgaris L.), which is an important food crop in the Americas, Africa and Asia, usually is thought to fix only small amounts of atmospheric nitrogen. However, field data indicate considerable genetic variability for total N₂ fixation and traits associated with fixation. Studies have shown that selection to increase N₂ fixation will be successful if: (1) discriminating traits (selection criteria) are measured precisely, (2) variability in germplasm is heritable, (3) selected parents are also agronomically suitable, (4) units of selection facilitate quantification of selection criteria, and (5) a breeding procedure that allows maximum genetic gain for N₂ fixation and recombination with essential agronomic traits is chosen. Breeding lines capable of fixing enough atmospheric N₂ to support seed yields of 1000–2000 kg ha^–1 have been identified and new cultivars with high N₂ fixation potential are being released.     

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.     

Distribution of nitrogen in common bean (Phaseolus vulgaris L.) genotypes selected for differences in nitrogen fixation ability

The improvement of N₂ fixation in legumes may lead to increased yields and reduced fertilizer requirement. Levels of N₂ fixation were determined for three cultivars and nine progeny lines from two inbred backcross common bean (Phaseolus vulgaris L.) populations that were grown at Hancock, Wissconsin in 1984 and 1985 using ¹⁵N-depleted (NH₄)₂SO₄. The high N₂-fixing line Puebla 152 was the donor parent for both inbred backcross populations and the cultivars Porrillo Sintetico and Sanilac were the recurrent parents for populations 21 and 24, respectively. Total N yield, fixed N₂ and % N derived from the atmosphere were determined for whole plants and plant parts at the R3 (50% bloom) and R9 (maturity) growth stages. Significant year-by-line interactions were found for N₂ fixation traits among the population 21 lines and parents, but not for population 24 lines and their parents. Measures of N₂ fixation at R3 were inadequate to predict N₂ fixation at R9. Population 24 lines and parents differed for N₂ fixation ability at R9, and fixed N₂ was correlated with maturity. The recovery of an inbred backcross progeny line, 24-21, which matured earlier and fixed more N₂ than the recurrent parent Sanilac indicated that N₂ fixation was heritable and that favorable alleles, independent of maturity, were recovered from a late-maturing, high N₂-fixing donor parent by utilizing the inbred backcross breeding method. Since most fixed N₂ and non-fixed N (>80%) was found in the seeds at maturity, and most lines did not vary for the distribution of nitrogen throughout the plant, selection for improved remobilization of nitrogen to the seed to increase yield is impractical in this genetic material. The highest N₂-fixing lines tended to have high and similar % Ndfa in all plant parts.     

Methods for measuring biological nitrogen fixation in grain legumes

To assure proper management and fully realize the benefits of the legume-Rhizobium symbiosis it is necessary to be able to quantify the amount of nitrogen fixed. Having measured the effectiveness of atmospheric N₂ fixation the macro- or micro-symbionts as well as agronomic factors can be manipulated with the objective to maximize biological nitrogen fixation. A suitable method to quantify nitrogen fixation is therefore necessary in any programme aiming at increasing N₂ fixation, like the one being reported in this volume. There are several methods available to quantify nitrogen fixation and most of the commonly used ones are described in the present paper listing their advantages and disadvantages.     

Optimising biological N2 fixation by legumes in farming systems

Whether grown as pulses for grain, as green manure, as pastures or as the tree components of agro-forestry systems, the value of leguminous crops lies in their ability to fix atmospheric N₂, so reducing the use of expensive fertiliser-N and enhancing soil fertility. N₂ fixing legumes provide the basis for developing sustainable farming systems that incorporate integrated nutrient management. By exploiting the stable nitrogen isotope ¹⁵N, it has been possible to reliably measure rates of N₂ fixation in a wide range of agro-ecological field situations involving many leguminous species. The accumulated data demonstrate that there is a wealth of genetic diversity among legumes and their Rhizobium symbionts which can be used to enhance N₂ fixation. Practical agronomic and microbiological means to maximise N inputs by legumes have also been identified.     

Improvement of biological nitrogen fixation in Egyptian winter legumes through better management of Rhizobium

The diversity of rhizobia nodulating common bean ( Phaseolus vulgaris), berseem clover (Trifolium alexanderinum) and lentil (Lens culinaris) was assessed using several characterization techniques, including nitrogen fixation efficiency, intrinsic antibiotic-resistance patterns (IAR), plasmid profiles, serological markers and rep-PCR fingerprinting. Wide diversity among indigenous rhizobial populations of the isolates from lentil, bean and clover was found. Strikingly, a large percentage of the indigenous rhizobial population was extremely poor at fixing nitrogen. This emphasizes the need to increase the balance of highly efficient strains within the rhizobial population. Use of high-quality inocula strains that survive and compete with other less-desired and less-efficient N2-fixing rhizobia represents the best approach to increase biological nitrogen fixation of the target legume. In field-grown lentils, the inoculant strains were not able to outcompete the indigenous rhizobia and the native lentil rhizobia occupied 76–88% of the total nodules formed on inoculated plants. Nitrogen fixation by lentils, estimated using the ¹⁵N isotope dilution technique, ranged between 127 to 139 kg ha-1 in both inoculated and un-inoculated plants. With berseem clover, the inoculant strains were highly competitive against indigenous rhizobia and occupied 52–79% of all nodules. Inoculation with selected inocula improved N2 fixation by clover from 162 to 205 kg ha-1 in the three cuts as compared with 118 kg ha-1 in the un-inoculated treatment. The results also indicated the potential for improvement of N2 fixation by beans through the application of efficient N2-fixing rhizobia.     

Nitrogen fixation by white clover in pastures grazed by dairy cows: Temporal variation and effects of nitrogen fertilization

Effects of rate of nitrogen (N) fertilizer and stocking rate on production and N₂ fixation by white clover (Trifolium repens L.) grown with perennial ryegrass (Lolium perenne L.) were determined over 5 years in farmlets near Hamilton, New Zealand. Three farmlets carried 3.3 dairy cows ha^–1 and received urea at 0, 200 or 400 kg N ha^–1 yr^–1 in 8–10 split applications. A fourth farmlet received 400 kg N ha^–1 yr^–1 and had 4.4 cows ha^–1.There was large variation in annual clover production and total N₂ fixation, which in the 0 N treatment ranged from 9 to 20% clover content in pasture and from 79 to 212 kg N fixed ha^–1 yr^–1. Despite this variation, total pasture production in the 0 N treatment remained at 75–85% of that in the 400 N treatments in all years, due in part to the moderating effect of carry-over of fixed N between years.Fertilizer N application decreased the average proportion of clover N derived from N₂ fixation (P_N; estimated by ¹⁵N dilution) from 77% in the 0 N treatment to 43–48% in the 400 N treatments. The corresponding average total N₂ fixation decreased from 154 kg N ha^–1 yr^–1 to 39–53 kg N ha^–1 yr^–1. This includes N₂ fixation in clover tissue below grazing height estimated at 70% of N₂ fixation in above grazing height tissue, based on associated measurements, and confirmed by field N balance calculations. Effects of N fertilizer on clover growth and N₂ fixation were greatest in spring and summer. In autumn, the 200 N treatment grew more clover than the 0 N treatment and N₂ fixation was the same. This was attributed to more severe grazing during summer in the 0 N treatment, resulting in higher surface soil temperatures and a deleterious effect on clover stolons.In the 400 N treatments, a 33% increase in cow stocking rate tended to decrease P_N from 48 to 43% due to more N cycling in excreta, but resulted in up to 2-fold more clover dry matter and N₂ fixation because lower pasture mass reduced grass competition, particularly during spring.     

Soil moisture and potassium affect the performance of symbiotic nitrogen fixation in faba bean and common bean

Potassium (K) is reported to improve plant's resistance against environmental stress. A frequently experienced stress for plants in the tropics is water shortage. It is not known if sufficient K supply would help plants to partially overcome the effects of water stress, especially that of symbiotic nitrogen fixation which is often rather low in the tropics when compared to that of temperate regions. Thus, the impact of three levels of fertilizer potassium (0.1, 0.8 and 3.0 mM K) on symbiotic nitrogen fixation was evaluated with two legumes under high (field capacity to 25% depletion) and low (less than 50% of field capacity) water regimes. Plants were grown in single pots in silica sand under controlled conditions with 1.5 mM N (¹⁵N enriched NH₄NO₃). The species were faba bean (Vicia faba L.), a temperate, amide producing legume and common bean (Phaseolus vulgaris L.), a tropical, ureide producing species. In both species, 0.1 mM K was insufficient for nodulation at both moisture regimes, although plant growth was observed. The supply of 0.8 or 3.0 mM K allowed nodulation and subsequent nitrogen fixation which appeared to be adequate for respective plant growth. High potassium supply had a positive effect on nitrogen fixation, on shoot and root growth and on water potential in both water regimes. Where nodulation occurred, variations caused by either K or water supply had no consequences on the percentage of nitrogen derived from the symbiosis. The present data indicate that K can apparently alleviate water shortage to a certain extent. Moreover it is shown that the symbiotic system in both faba bean and common bean is less tolerant to limiting K supply than plants themselves. However, as long as nodulation occurs, N assimilation from the symbiotic source is not selectively affected by K as opposed to N assimilation from fertilizer.     

Measurements of N2 fixation by common bean in Central Brazil as affected by different reference crops

An experiment was conducted at EMBRAPA/CNPAF, Goiânia, Goias, Brazil, on a typic haplustox soil to evaluate growth and N₂ fixation-related parameters of Phaseolus vulgaris L. Bean lines, which had been selected for N₂ fixation at CNPAF, including production cultivars, germplasm bank entries, and parents and progenies of a cross made to improve this characteristic. Wheat (Triticum aestivum L.) and dwarf sorghum (Sorghum bicolor (L.) Moench) were evaluated as non-N₂-fixing reference crops for difference method (DM) and ¹⁵N isotope dilution technique (IDT) estimates of N₂ fixation. IDT estimates ranges from 4 to 18 kg N₂ fixed ha^-1. High variability associated with low levels of N₂ fixation precluded definitive identification of the best N₂ fixing bean lines. Due to differences in growth cycle and in patterns and amounts of soil N uptake during the season, neither of the reference crops tested appears to be an adequate control for either DM or IDT estimates of N₂ fixation. However, ranking of lines for effectiveness in N₂ fixation could be performed without the use of any reference crops.     

Potential for increasing biological nitrogen fixation in soybean

The importance of soybean as a source of oil and protein, and its ability to grow symbiotically on low-N soils, point to its continued status as the most valuable grain legume in the world. With limited new land on which to expand, and emphasis on sustainable systems, increases in soybean production will come mostly from increased yield per unit area. Improvements in biological nitrogen fixation can help achieve increased soybean production, and this chapter discusses research and production strategies for such improvement.The soybean-Bradyrhizobium symbiosis can fix about 300 kg N ha^-1 under good conditions. The factors which control the amount of N fixed include available soil N, genetic determinants of compatibility in both symbiotic partners and lack of other yield-limiting factors. Response to inoculation is controlled by the level of indigenous, competing bradyrhizobia, the N demand and yield potential of the host, and N availability in the soil.Research efforts to improve BNF are being applied to both microbe and soybean. While selection continues for effective, naturally occurring bradyrhizobia for inoculants and the use of improved inoculation techniques, genetic research on bradyrhizobia to improve effectiveness and competitiveness is advancing. Selection, mutagenesis and breeding of the host have focused on supernodulation, restricted nodulation of indigenous B. japonicum, and promiscuous nodulation with strains of bradyrhizobia from the cowpea cross-inoculation group. The research from the host side appears closer to being ready for practical use in the field.Existing knowledge and technology still has much to offer in improving biological nitrogen fixation in soybean. The use of high-quality inoculants, and education about their benefits and use can still make a significant contribution in many countries. The importance of using the best adapted soybean genotype with a fully compatible inoculant cannot be overlooked, and we need to address other crop management factors which influence yield potential and N demand, indirectly influencing nitrogen fixation. The implementation of proven approaches for improving nitrogen fixation in existing soybean production demands equal attention as received by research endeavours to make future improvements.     

Nitrogen fixation and plant growth of common bean (Phaseolus vulgaris L.) at different levels of phosphorus availability

The efficacy of the alumina system for differentiating between bean (Phaseolus vulgaris L.) genotypes for growth at different levels of phosphorus availability was determinated. In addition to response to P levels, comparisons were made between plants receiving N either from fertilizer or nitrogen fixation. When the cv. Carioca was provided with either 100 ppm of N or inoculated withRhizobium leguminosarum biovarphaseoli, differences in shoot dry weight and nodule number were related to P level. There was a greater proportion of green, ineffectivevs. red, active nodules at the low P concentration than at the higher P concentration. In a second experiment, two cvs., Puebla 152 and Carioca and the breeding line UW 24-21, either were inoculated with rhizobia or provided with 150ppm of N. Each genotype-nitrogen combination was grown at 8 levels of P. There was a positive effect of P level on shoot dry weight, nodule number and nodule mass. Root mass was affected less than nodule or shoot mass by the P level of the growth medium. Nodule mass, but not P concentration in the nodules, was affected by P level, whereas in the other plant tissues, P concentrations were lower at lower P levels in the media.     

Utilizing the potential for increased nitrogen fixation in common bean

Many variables affect the amount of N₂ fixation that occurs in field-grown common beans (Phaseolus vulgaris L.). When environmental conditions are optimized, genetically superior plants that are nodulated with efficient rhizobia are able to fix enough N₂ to support grain yields of up to 2000 kg ha^–1. All of the required components are available for common bean to fulfill its potential as a nitrogen-fixing food crop. However, these components must be assembled and presented to growers in a usable package that is economically attractive.     

The contribution of associative and symbiotic nitrogen fixation to the nitrogen nutrition of non-legumes

During the past 10 years estimates of N₂ fixation associated with sugar cane, forage grasses, cereals and actinorhizal plants grown in soil with and without addition of inoculum have been obtained using the ¹⁵N isotope dilution technique. These experiments are reviewed in this paper with the aim of determining the proportional and absolute contribution of N₂ fixation to the N nutrition of non-legumes, and its role as a source of N in agriculture. The review also identifies deficiencies in both the totality of data which are currently available and the experimental approaches used to quantify N₂ fixation associated with non-legumes.Field data indicate that associative N₂ fixation can potentially contribute agronomically-significant amounts of N (>30–40 kg N ha^-1 y^-1) to the N nutrition of plants of importance in tropical agriculture, including sugar cane (Saccharum sp.) and forage grasses (Panicum maximum, Brachiaria sp. and Leptochloa fusca) when grown in uninoculated, N-deficient soils. Marked variations in proportions of plant N derived from the atmosphere have been measured between species or cultivars within species.Limited pot-culture data indicate that rice can benefit naturally from associative N₂ fixation, and that inoculation responses due to N₂ fixation can occur. Wheat can also respond to inoculation but responses do not appear to be due to associative N₂ fixation. ¹⁵N dilution studies confirm that substantial amounts of N₂ can be fixed by actinorhizal plants.     

Nitrogen fixation and nodule occupancy by native strains of Rhizobium on different cultivars of common bean (Phaseolus vulgaris L.)

A field experiment under rainfed conditions was conducted in Durango, México, to assess N2-fixation of three cultivars of common bean (Phaseolus vulgaris L.) using ¹⁵N-methodology. In addition, diversity of rhizobial isolates obtained from nodules of the different plant genotypes was evaluated by intrinsic antibiotic resistance (IAR), PCR using enterobacterial repetitive intergenic consensus (ERIC) primers, PCR-RFLP analysis of the 16S rRNA gene and multilocus enzyme electrophoresis (MLEE). Selected isolates were used to determine acetylene reduction and competitive ability under greenhouse conditions. The three cultivars tested did not show high variation in N2-fixation, the %Ndfa values ranged from 19 to 26%. Variability in N2-fixation efficiency among various native rhizobial isolates was very high and our results indicate that differences in competitive abilitiy exist also. PCR-RFLP of the 16S rRNA gene and MLEE revealed that most of the isolates belong to the species Rhizobium etli. Intrinsic antibiotic resistance analysis and ERIC-PCR showed high diversity among isolates. In contrast, our results using MLEE show low genetic diversity (H = 0.105).     

A comparison between the acetylene reduction method,the isotope dilution method and the total nitrogen difference method for measuring nitrogen fixation in lucerne (Medicago sativa L.)

Summary In view of the increasing need for the exact estimation of the input of nitrogen in agroecosystems, an application of the acetylene-reduction technique was developed. The technique, consisting of a plastic bag incubation system, using propane as an internal standard of the apparent volume, made it possible to carry out repeated incubations on the same plant system. The evaluated technique included studies of the diffusion of ethylene and propane in a soil column, as well as studies of the optimal substrate concentration and grade of purity of the substrate. In addition, the conversion factor between amounts of reduced acetylene as compared wtih reduced dinitrogen was determined by¹⁵N₂ incubations to be 4.41. The developedin situ acetylene-reduction technique was compared with an isotope dilution method and a total nitrogen difference method. By comparing the derived total nitrogen fixation values from each with the value derived from the acetylene-reduction method; it was shown that the values differed significantly. The acetylene-reduction method gave the highest nitrogen fixation values, the isotope dilution the lowest values and the total nitrogen difference method was intermediate. No statistical significant difference existed between the two different reference crops used in the isotope dilution method.     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.