Influence of reference trees on N2-fixation estimates inLeucaena leucocephala andAcacia albida using15N-labelling techniques

Summary We examined the suitability of four reference crops, i.e., two non-fixing trees,Cassia siamea andEucalyptus grandis, and two uninoculated fixing trees,Leucaena leucocephala andAcacia albida, for measuring fixed N₂ fixed in inoculatedL. leucocephala andA. albida grown for 36 weeks in pots. The¹⁵N isotope-dilution (involving the addition of equal amounts of labelled N fertilizer to the non-fixing and the fixing plants) and theA-value (with different amounts of labelled N fertilizer added to the fixing and the non-fixing crops) methods were used. The isotope dilution approach gave several large negative estimates of fixed N₂ inA. albida. Positive and similar values of fixed N₂ were measured in all four reference crops using theA-value approach. ForL. leucocephala the isotope-dilution approach gave different estimates of fixed N₂, with the different reference crops; the uninoculated N₂-fixing crops indicated significantly less fixed N₂ than the non-fixing reference crops. Similar values for N₂ fixed inL. leucocephala were obtained using the two non-fixing trees, either by the isotope-dilution or theA-value method. On average,A. albida derived about twice as much N from fertilizer asL. leucocephala. In both species, the atom %¹⁵N excess declined by about 50% in successive harvests.     

Estimating N2 fixation by Sesbania rostrata and S. cannabina (syn. S. aculeata) in lowland rice soil by the 15N dilution method

Summary A field experiment in concrete-based plots was conducted to estimate the contribution of N derived from air (Ndfa) or biological N₂ fixation in Sesbania rostrata and S. cannabina (syn. S. aculeata), using various references, by the ¹⁵N dilution method. The two Sesbania species as N₂-fixing reference plants and four aquatic weed species as non-N₂-fixing references were grown for 65 days after sowing in two consecutive crops, in the dry and the wet seasons, under flooded conditions. Soil previously labeled with ¹⁵N at 0.26 atom % ¹⁵N excess in mineralizable N was further labeled by ammonium sulfate with 3 and 6 atom % ¹⁵N excess. The results showed that ¹⁵N enrichment of soil NH ₄ ⁺ -N dropped exponentially in the first crop to half the original level in 50 days while in the second crop, it declined gradually to half the level in 130 days. The decline in ¹⁵N enrichment, in both N₂-fixing and non-fixing species, was also steeper in the first crop than in the second crop. Variations in ¹⁵N enrichment among non-fixing species were smaller in the second crop. The ratio of the uptake of soil N to that of fertilizer N in N₂-fixing and non-fixing species was estimated by the technique of varying the ¹⁵N level. In the second crop, this ratio in non-fixing species was higher than that in N₂-fixing species. Comparable estimates of % Ndfa were obtained by using ¹⁵N enrichment of various non-fixing species. There was also good agreement between the estimates obtained by using ¹⁵N enrichment of non-fixing species and those by using soil NH ₄ ⁺ -N, particularly in the second crop. By 25 days after sowing, the first crop of both Sesbania spp. had obtained 50% of total N from the atmosphere and the second crop had obtained 75%. The contribution from air increased with the age of the plant and ranged from 70% to 95% in 45–55 days. S. rostrata fixed substantially higher amounts of N₂ due to its higher biomass production compared with S. cannabina. Mathematical considerations in applying the ¹⁵N dilution method are discussed with reference to these results.     

Nitrogen fixation by nodulated soybean under tropical field conditions estimated by the 15N isotope dilution technique

The contribution of biological N₂ fixation to the N nutrition of nodulated soybean was estimated using the ¹⁵N isotope dilution technique and a non-nodulating soybean isoline as a non-fixing control plant. The plants were grown in the field in concrete cylinders (60 cm dia) and harvested at seven stages of plant growth. Labelled N was added to the soil either as labelled organic matter before planting or in seven small additions (2kg N ha^?1) of (NH₄)₂SO₄ during the growing period.There was good agreement between isotope dilution estimates of nitrogen fixation for the two labelling methods. Acetylene reduction assays on intact root systems greatly underestimated N₂ fixing activity. The difference in total N between nodulated and non-nodulated plants generally gave higher estimates compared with the isotope technique. The data indicate that this was because nodulated plants recovered more N from the soil than the non-nodulated plants. After 92 days of growth, the soybean derived approximately 250kg N ha^?1 from biological N₂ fixation.     

Estimating N2-fixation in the field using 15N-labelled fertilizer: Some problems and solutions

The ¹⁵N-labelled fertilizer dilution technique provides a method of obtaining estimates of biological N₂-fixation in the field over the growing season. Field estimates of fixation obtained using peas, french beans, field beans and clover depended on the non-fixing control used. Differences in the N uptake patterns of the legume and control combinations, together with a decrease in the enrichment of plant available soil N with time, were major factors causing this dependency. A simple model of plant N accumulation at decreasing soil enrichment is presented, which explains these errors and allows a more rational choice of non-fixing control. The use of gypsum pelleted ¹⁵N fertilizer, or any other treatment which leads to a more stable soil enrichment, reduces errors caused by mismatched N uptake patterns in the two crops.     

Characterization of the N benefit of a grain legume (Lupinus angustifolius L.) to a cereal (Hordeum vulgare L.) by an in situ 15N isotope dilution technique

Summary A crop of barley was grown on plots which had previously supported pure stands of lupins, canola, ryegrass, and wheat. The plots were labelled with ¹⁵N-enriched fertilizers at the time of sowing of the antecedent crops. The crop of lupins, which derived 79% of its N from symbiotic N₂ fixation at physiological maturity, conferred an N benefit to barley of 3.4 g N m^-2 when compared to barley following wheat. Lupins used less fertilizer N and less unlabelled soil N compared to the other crops, but the ratios of these sources of N in the plant tops were similar. The apparent sparing of soil+fertilizer N under lupins compared with wheat was 13.6 g N m^-2, which was much larger than the measured N benefit. Barley following lupins was less enriched in ¹⁵N compared to barley following wheat, and the measured isotope dilution was used to estimate the proportion of barley N derived from biologically fixed N in the lupin residues. This in turn enabled the N benefit to be partitioned between the uptake of spared N and the uptake of fixed N derived from the mineralization of legume residues. Spared N and fixed N contributed in approximately equal proportions to the N benefit measured in barley following lupins compared to barley following wheat.     

Cultivar effects on nitrogen fixation in peas and lentils

Increasing nitrogen fixation in legume crops could increase cropping productivity and reduce nitrogen fertilizer use. Studies have found that crop genotype, rhizobial strain, and occasionally genotype-specific interactions affect N fixation, but this knowledge has not yet been used to evaluate or breed for greater N fixation in US crops. In this study five USDA varieties of lentils (Lens culinaris Medik.) and five varieties of peas (Pisum sativum L.) were tested with 13 to 15 commercially available strains of Rhizobium leguminoserum bv. viciae to identify the better N fixing rhizobial strains, crop varieties, and specific pairings. Peas and lentils inoculated with individual strains were grown in growth chambers for 6 week. Plants received (¹⁵NH₄)₂ SO₄ (5 at.%) starter fertilizer to measure N fixation by isotope dilution. Below- and above-ground biomass, numbers of nodules, and the proportion of plant N supplied by fixation (PNF) were determined. The percent of N fixed was significantly affected by crop variety and significantly correlated with number of nodules in both lentils and peas. This implies that one strategy for enhancing crop N fixation is developing varieties that have higher rhizobium infection rates. Total N fixation in lentils was significantly influenced by both crop variety and rhizobial strain. Eston variety lentil and Shawnee variety pea had the highest PNF of 80.8% and 91.3%, respectively. The different strains of R. leguminoserum affected PNF in lentils but not in peas. These findings suggest that N fixation improvement in lentils and peas may be addressed most effectively by breeding crops for greater N fixation hosting capacity.     

Effects of Multiple Reference Plants,Season, and Irrigation on Biological Nitrogen Fixation by Pasture Legumes using the Isotope Dilution Method

Abstract

The popular and widely used ¹⁵nitrogen (N)–isotope dilution method for estimating biological N fixation (BNF) of pasture and tree legumes relies largely on the ability to overcome the principal source of error due to the problem of selecting appropriate reference plants. A field experiment was conducted to evaluate the suitability of 12 non‐N₂‐fixing plants (i.e., nonlegumes) as reference plants for estimating the BNF of three pasture legumes (white clover, Trifolium repens L.; lucerne, Medicago sativa; and red clover, Trifolium pratense L.) in standard ryegrass–white clover (RWC) and multispecies pastures (MSP) under dry‐land and irrigation systems, over four seasons in Canterbury, New Zealand. The ¹⁵N‐isotope dilution method involving field ¹⁵N‐microplots was used to estimate BNF. Non‐N₂‐fixing plants were used either singly or in combination as reference plants to estimate the BNF of the three legumes. Results obtained showed that, on the whole, ¹⁵N‐enrichment values of legumes and nonlegumes varied significantly according to plant species, season, and irrigation. Grasses and herb species showed higher ¹⁵N‐enrichment than those of legumes. Highest ¹⁵N‐enrichment values of all plants occurred during late summer under dry‐land and irrigation conditions. Based on single or combined non‐N₂‐fixing plants as reference plants, the proportion of N derived from the atmosphere (% Ndfa) values were high (50 to 90%) and differed between most reference plants in the MSP pastures, especially chicory (Cichorium intybus), probably because it is different in phenology, rooting depth, and N‐uptake patterns compared to those of legumes. The percent Ndfa values of all plants studied also varied according to plant species, season, and irrigation in the MSP pastures. Estimated daily amounts of BNF varied according to pasture type, time of plant harvest, and irrigation, similar to those shown by percent Ndfa results as expected. Irrigation increased daily BNF more than 10‐fold, probably due to increased dry‐matter yield of pasture under irrigation compared to dry‐land conditions. Seasonal and irrigation effects were more important in affecting estimates of legume BNF than those due to the appropriate matching of N₂‐fixing and non‐N₂‐fixing reference plants.     

Effects of NPK fertilizer combinations on yield and nitrogen balance in sorghum or pigeonpea on a vertisol in the semi-arid tropics

A long-term experiment was carried out on a Vertisol from 1986 to 1992 to examine the combined effects of NPK fertilizers on yield using sorghum (Sorghum bicolor L. Moench cv. CSH 5) and short-duration pigeonpea (Cajanus cajan L. Millsp. cv. ICPL 87). The fertilizer treatments were as follows: 0 (no fertilization), N (150 kg N ha^-1 ), P (65.5 kg P₂O₅ ha^-1), K (124.5 kg K₂O ha^-1), and all possible combinations (NP, NK, PK, and NPK). In this study we continued this experiment during the period 1993 to 1994 and analyzed the crop yield response to fertilizers and the N balance. The amount of N derived from the atmosphere and fertilizer was estimated by the ¹⁵N natural abundance method and ^l5N isotope dilution method, respectively. A combined application of Nand P fertilizers gave the highest grain yield for the two crops under the 8th and 9th continuous croppings, unlike the application of K fertilizer. The values of total N for the two crops were significantly higher in the NP and NPK plots. These crops took up N mainly from soil. There was a significant positive relationship between the uptake of N_dff and N_dfs by each crop. Pigeonpea or sorghum took up more N from the soil in the N fertilizer plots than in the plots without N, suggesting that soil N fertility was enhanced and the amount of N supplied from soil increased in the plots with consecutive application of N fertilizer for 7 y. Even pigeonpea, which fixes atmospheric N inherently, needed N fertilizer to achieve high grain yield, suggesting that N fixation by the nodules was not always sufficient to meet the N requirements of the crop under these conditions. Although fertilizer N exerted a beneficial effect on plant growth and yield in the two crops, the values of fertilizer N recovery (FNR) by the two crops were considerably low. Therefore, it is suggested that the development of N fertilizer management which could maximize FNR of each crop should be promoted.     

Evaluation of N2 fixation by applying 15N labeled plant material and ammonium sulfate

Effect of different ¹⁵N labeled sources on the estimation of N₂ fixation was investigated. The combination of ¹⁵N labeled ammonium sulfate, ¹⁵N labeled plant material, and ¹⁵N labeled ammonium sulfate with unlabeled plant material, was examined in pot experiments. Two cultivars of soybean (Glycine max) and one of mungbean (Vigna radiata) were used. No significant difference was observed among the treatments for the estimation of N₂ fixation. This was due to the homogeneity and stability of the ¹⁵N abundance in soil which resulted in a similar N uptake from the soil by the N² fixing and reference crops. The plant yield, total N uptake and amount of N₂ fixed were higher in the Yellow Soil than in the Andosol. The amount of N₂ fixed was strongly influenced by the plant growth and consequently it affected the plant yield. The slow decomposition of plant material in the Andosol resulted in a low yield in both the N₂ fixing and reference crops. Thus, the artificial decrease of the available N content in soil, by application of plant material, did not stimulate N, fixation but suppressed plant growth and N₂ fixation.     

Isotope 15N enrichment of soil-ammonium N as a reference to estimate N2 fixation by stem and root-inoculated S. rostrata

Summary A pot experiment in the greenhouse was conducted to compare the contribution of N derived from the atmosphere or from biological N₂ fixation by Sesbania rostrata inoculated with Azorhizobium caulinodans, applied either to roots or to roots and stems (single or multiple stem inoculation). Two subsequent crops were grown for 50 days under flooded conditions. N derived from air was estimated by ¹⁵N dilution using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N and of Echinochloa crusgalli as the non-N₂-fixing reference datum and compared with estimates obtained by the N-difference method. The first crop was grown to stabilize the ¹⁵N into the soil organic N fraction. The ¹⁵N enrichment of soil NH _inf4 ^sup+ -N in the second crop declined slowly. The extractability ratio (¹⁵N enrichment of extractable soil N to ¹⁵N enrichment of total soil N) decreased from 4.8 to 4.1 50 days after planting. The enrichment of soil NH _inf4 ^sup+ -N was comparable to that of E. crus-galli, resulting in similar estimates of N derived from air when either soil NH _inf4 ^sup+ -N or enrichment of E. crus-galli was used as a non-fixing reference. The N-difference method did not always provide reliable estimates of N derived from air; percentages ranged from 75 to more than 80 by 50 days after planting in both crops and did not differ among treatments. The study demonstrates the potential of using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N as a non-N₂-fixing reference for reliable BNF estimates of crops in lowland puddled soil.     

A yield-independent, 15N-isotope dilution method to estimate legume symbiotic dependence without a non-N2-fixing reference plant

The proportional contribution of atmospheric N₂ to the N nutrition of lupin (P _atm) was estimated in a field experiment following addition of NH₄Cl of KNO₃ to unconfined microplots (1.5 m²) at 2.5 g N m^-2 (10 atom% ¹⁵N). The integrated ¹⁵N enrichment, or mean pool abundance, of nitrate extracted from 0- to 15-cm samples taken under the lupin crop on eight occasion between 28 and 190 days after sowing was used as the reference criterion to estimate P _atm by the ¹⁵N-isotope dilution technique. Estimates of P _atm were similar to those obtained using canola as a non-fixing reference plant, but were higher than estimates obtained using a yield-dependent model. Use of mean pool abundance obviates the need for a non-fixing reference plant, and the frequent sampling and isotope-ratio analysis of the legume biomass required with the yield-dependent model is unnecessary. However, further work is needed to validate a sampling strategy commensurate with the growth of the legume roots.     

Transfer of N fixed by a legume tree to the associated grass in a tropical silvopastoral system

Below-ground transfer of nitrogen (N) fixed by legume trees to associated non-N₂-fixing crops has received little attention in agroforestry, although the importance of below-ground interactions is shown in other ecosystems. We used ¹⁵N natural abundance to estimate N transfer from the legume tree Gliricidia sepium (Jacq.) Kunth ex Walp. to C₄ grass Dichanthium aristatum (Poir.) C.E. Hubb. in a silvopastoral system, where N was recycled exclusively by below-ground processes and N₂ fixation by G. sepium was the sole N input to the system. Finding a suitable reference plant, a grass without contact with tree roots or litter, was problematic because tree roots invaded adjacent grass monocrop plots and soil isotopic signature in soil below distant grass monocrops differed significantly from the agroforestry plots. Thus, we used grass cultivated under greenhouse conditions in pots filled with agroforestry soil as the reference. A model of soil ¹⁵N fractionation during N mineralization was developed for testing the reliability of that estimate. Experimental and theoretical results indicated that 9 months after greenhouse transplanting, the percentage of fixed N in the grass decreased from 35% to <1%, due to N export in cut grass and dilution of fixed N with N taken up from the soil. The effect of soil ¹⁵N fractionation on the estimate of the reference value was negligible. This indicates that potted grass is a suitable reference N transfer studies using ¹⁵N natural abundance. About one third of N in field-grown grass was of atmospheric origin in agroforestry plots and in adjacent D. aristatum grassland invaded by G. sepium roots. The concentration of fixed N was correlated with fine root density of G. sepium but not with soil isotopic signature. This suggests a direct N transfer from trees to grass, e.g. via root exudates or common mycorrhizal networks.     

Estimating symbiotic N2 fixation in Robinia pseudoacacia

Estimating symbiotic di‐nitrogen (N₂) fixation is challenging, especially when working with woody N₂ fixers in field trials. Fortunately, isotope methods based on ¹⁵N natural abundance or on ¹⁵N artificial enrichment (dilution method) make it possible to estimate the proportion of nitrogen derived from the atmosphere (Ndfa) in N₂‐fixing species. These methods have been extensively used in the field for herbaceous species, much less for tree species such as alder and acacia, and rarely for black locust (Robinia pseudoacacia). The objectives of this study were to characterize the fixation potential of black locust in a plantation by using the two ¹⁵N isotope methods in the field, and to document values of isotope fractionation occurring during N₂ fixation (the B value). B values were estimated both by growing trees on an N‐free medium in controlled conditions (B_lab) and by making Ndfa calculated with the natural abundance method converge with Ndfa calculated with the ¹⁵N dilution method in the field (B_field). The two methods gave consistent estimates of the B value. B values ranging between –1.4 and –3.2‰ were found, varying with the age of the plant material. Up to 76% of the N in the black locust trees came from the atmosphere, representing more than 45 kg N ha^?1 over five years and confirming that black locust may be well adapted to N‐poor soils.     

Estimating N2 fixation by field-grown lupins (Lupinus angustifolius L.) using soil and plant 15N enrichment

Summary Biological N₂ fixation was estimated in a field experiment following the addition of NH₄Cl or KNO₃ to unconfined microplots (1.5 m²) at 2.5 g N m^-2 (10 atom% ¹⁵N). A model of total N and ¹⁵N accumulation in lupins and decreasing ¹⁵N enrichment in the KCl-extractable soil-N pool (0–0.15 m depth) was used to estimate the proportion of N in lupins derived from biological N₂ fixation. Estimates of N₂ fixation derived from the model were compared with ¹⁵N isotope-dilution estimates obtained using canola, annual ryegrass, and wheat as nonfixing reference plants. Biomass, total N accumulation, or ¹⁵N enrichment in the lupin and reference crops did not differ whether NH _inf4 ^sup+ or NO _inf3 ^sup- was added as the labelled inorganic-N source. The decrease in soil ¹⁵N enrichment was described by first-order kinetics, whereas total N and ¹⁵N accumulation in the lupins were described by logistical equations. Using these equations, the uptake of soil N by lupins was estimated and was then used to calculate fixed N₂. Estimates of N₂ fixation derived from the model increased from 0 at 50 days after sowing to a maximum of 0.79 at 190 days after sowing. Those based on the ¹⁵N enrichment of the NO _inf3 ^sup- pool were 10% higher than those based on the mineral-N pool. ¹⁵N isotope-dilution estimates of N₂ fixation ranged from 0.37 to 0.55 at 68 days after sowing and from 0.71 to 0.77 at 190 days after sowing. Reference plant-derived values of N₂ fixation were all higher than modelled estimates during the early states of growth, but were similar to modelled estimates at physiological maturity. The use of the model to estimate N₂ derived from the atmosphere has the intrinsic advantage that the need for a non-fixing reference plant is avoided.     

The role of arbuscular mycorrhiza in legume symbiotic performance

Legumes may respond to non-rhizobial inoculants such as arbuscular mycorrhizal (AM) fungi either through an effect on plant growth or, in addition, through an effect on the function of the legume-Rhizobium symbiosis. We have examined the literature where the application of ¹⁵N isotope dilution methodology permits the effect of indigenous AM and AM inoculants to be quantitatively separated into plant-growth-mediated and biological N₂ fixation (BNF)-mediated components. These studies clearly demonstrate the beneficial effects that both indigenous and inoculated AM have on legume growth, N uptake and the proportional dependence of the legume on atmospheric N₂. While the published data allow an assessment of various biological, edaphic and environmental factors that affect the response of various legumes to AM inoculation, they also highlight the paucity of quantitative field data and the lack of understanding of the interaction of legume genotype with AM species with respect to legume symbiotic performance.     

Slow-release 15N fertilizer formulations to measure N2-fixation by isotope dilution

Pot experiments were carried out to examine the effects of slow-release fertilizer formulations on estimates of N₂-fixation determined by the isotope dilution method. Soybeans were used as the N₂-fixing plants, with non-nodulated soybeans and maize as the non-fixing controls. The ¹⁵N-fertilizer formulations used were (¹⁵NH₄)SO₄, K¹⁵NO₃, gypsum-pelleted K¹⁵NO₃, (¹⁵NH₄)₂SO₄ + glucose, ground plant material enriched with ¹⁵N or ¹⁵N-oxamide. The estimate of the amount of N₂ fixed by the nodulated soybean plants depended upon both the control plant and the fertilizer formulation used. Maize took up N later than non-nodulated soybean and estimates of soil N-pool (soil “A” value + fertilizer N added) calculated from the enrichment of this control were about twice as large as those calculated from the enrichment of non-nodulated soybean receiving the same fertilizer treatment. As a consequence, estimates of N₂-fixation relative to this control were lower than those relative to non-nodulating soybean (mean 140 mg N per pot compared with 292 mg N per pot). With unstablilized ¹⁵N salts errors were sufficient to produce negative estimates of fixation relative to maize. Even with a “well-matched” control (non-nodulated soybean) estimates of fixation varied with fertilizer formulation.     

Effect of phosphorus management in rice–mungbean rotations on sandy soils of Cambodia

Nitrogen (N) and phosphorus (P) deficiencies are key constraints in rainfed lowland rice (Oryza sativa L.) production systems of Cambodia. Only small amounts of mineral N and P or of organic amendment are annually applied to a single crop of rainfed lowland rice by smallholder farmers. The integration of leguminous crops in the pre‐rice cropping niche can contribute to diversify the production, supply of C and N, and contribute to soil fertility improvement for the subsequent crop of rice. However, the performance of leguminous crops is restricted even more than that of rice by low available soil P. An alternative strategy involves the application of mineral P that is destined to the rice crop already to the legume. This P supply is likely to stimulate legume growth and biological N₂ fixation, thus enhancing C and N inputs and recycling N and P upon legume residue incorporation. Rotation experiments were conducted in farmers' fields in 2013–2014 to assess the effects of P management on biomass accumulation and N₂ fixation (δ¹⁵N) by mungbean (Vigna radiata L.) and possible carry‐over effects on rice in two contrasting representative soils (highly infertile and moderately fertile sandy Fluvisol). In the traditional system (no legume), unamended lowland rice (no N, + 10 kg P ha^?1) yielded 2.8 and 4.0 t ha^?1, which increased to 3.5 and 4.7 t ha^?1 with the application of 25 kg ha^?1 of urea‐N in the infertile and the moderately fertile soil, respectively. The integration of mungbean as a green manure contributed up to 9 kg of biologically fixed N (17% Nfda), increasing rice yields only moderately to 3.5–4.6 t ha^?1. However, applying P to mungbean stimulated legume growth and enhanced the BNF contribution up to 21 kg N ha^?1 (36% Nfda). Rice yields resulting from legume residue incorporation (“green manure use”–all residues returned and “grain legume use”–only stover returned) increased to 4.2 and 4.9 t ha^?1 in the infertile and moderately fertile soil, respectively. The “forage legume use” (all above‐ground residues removed) provided no yield effect. In general, legume residue incorporation was more beneficial in the infertile than in the moderately fertile soil. We conclude that the inclusion of mungbean into the prevailing low‐input rainfed production systems of Cambodia can increase rice yield, provided that small amounts of P are applied to the legume. Differences in the attributes of the two major soil types in the region require a site‐specific targeting of the suggested legume and P management strategies, with largest benefits likely to accrue on infertile soils.     

Nitrogen use in maize-grain legume cropping systems in semi-arid Kenya

Locally suitable cultivars of maize, beans, and cowpeas were grown in field experiments for four seasons in semi-arid Kenya. For three seasons, the dry matter production and grain yield of maize and beans were not increased by N fertilizer additions up to 120 kg N ha^-1. Fertilizer recoveries measured by ¹⁵N isotope dilution techniques were low, less than 20%. Inoculated and uninoculated beans failed to fix N₂. By contrast the cowpea derived 50% of its N from fixation, equivalent to 197 kg N ha^-1. The N content of the grain generally exceeded 40 kg N ha^-1, and the N content of the seeds from the grain legumes were greater than those from the cereals. Large inputs of N fertilizer or N by fixation are required if maize-grain legume cropping system in semiarid Kenya are to be sustained in the long term.     

Determining Symbiotic Nitrogen Fixation in Dry Bean Cultivars Using Ureide Method and Isotope Dilution Techniques

Symbiotic nitrogen fixation (SNF) is an environmentally safe source of nitrogen (N) to the crop plants. In total, 12 dry bean (Phaseolus vulgaris L.) cultivars from pinto, navy, black, and kidney market classes were inoculated with rhizobia and grown in a greenhouse. SNF was estimated using isotope dilution technique and ‘ureide’ method. The amount of SNF ranged between 33 and 68 mg N plant^–1 when determined using ¹⁵N isotope dilution and followed the order: pinto > navy > black > kidney. Percent N derived from atmosphere (%Ndfa) significantly varied between 49% and 90% at V₃ and between 71% and 98% at R₂ stages. The outcomes of the experiment suggested that dry bean cultivars from different market classes have variable N₂ fixation ability, and fertilizer N required should be calculated according to their SNF potentials and N need of a specific market class or cultivar. Stable isotope dilution should be used as the standard procedure to estimate the SNF in dry bean.     

Interspecies competition and N transfer in a tropical grass-legume mixture

Competitiveness of Brachiaria decumbens cv. Basilisk and Stylosanthes guianensis cv. Minerão was investigated either without root restriction or by separating their root systems with a fine mesh or a solid barrier in the presence or absence of mycorrhiza (Glomus clarum). Nitrogen transfer between the legume and the grass was assessed with the ¹⁵N isotope dilution technique using a relatively stable ¹⁵N-enriched soil derived from a long-term labelling experiment. During establishment, legume development was severely restricted by competition from the grass in pots without a root barrier. However, as the system became N limited, the legume became dominant due to its access to atmospheric N₂ which contributed over 80% of the legume N requirements. S. guianensis was highly mycotrophic and inoculation with mycorrhiza favoured rapid establishment even in the treatments with no root barrier. Only in the presence of root barriers, either a mesh or a complete compartment separation, was the proportion of N derived from N₂ fixation positively affected by the presence of the fungus. No significant direct belowground N transfer from legume to grass was observed during the lifetime of the legume suggesting that the legume maintains a highly efficient recycling under N-limited conditions. However, after cutting the shoot at ground level, the grass assimilated significant amounts of N derived from decaying legume roots. We conclude that the main pathway of belowground N transfer from S. guianensis to associated B. decumbens occurred via decomposing roots rather than via root exudates or direct mycorrhizal hyphae transfer.