Quantitative analysis of the initial transport of fixed nitrogen in nodulated soybean plants using 15N as a tracer

The quantitative analysis of the initial transport of fixed isotope 15-nitrogen (¹⁵N) in intact nodulated soybean plants (Glycine max [L.] Merr. cv. Williams) was investigated at the vegetative stage (36 days after planting, DAP) and pod-filling stage (91 DAP) by the ¹⁵N pulse-chase experiment. The nodulated roots were exposed to N₂ gas labeled with a stable isotope ¹⁵N for 1 h, followed by 0, 1, 3 and 7 h of exposure with normal air. Plant roots and shoots were separated into three sections (basal, middle and distal parts) with the same length of the main stem or primary root. Approximately 80 and 92% of fixed N was distributed in the basal part of the nodulated roots at the vegetative and pod-filling stages by the end of 1 h of ¹⁵N₂ exposure, respectively. In addition, about 90% of fixed ¹⁵N was retained in the nodules and 10% was exported to root and shoot after 1 h of ¹⁵N₂ exposure at 91 DAP. The percentage distribution of ¹⁵N in the nodules at the pod-filling stage decreased from 90% to 7% during the 7 h of the chase period, and increased in the roots (14%), stems (54%), leaves (12%), pods (10%) and seeds (4%). The ¹⁵N distribution was negligible in the distal root segment, suggesting that N fixation activity was negligible and recycling fixed N from the shoot to the roots was very low in the initially short time of the experiment.     

Ontogenic variation in nitrogen fixation and accumulation of nitrogen in mungbean,blackgram, cowpea,and groundnut

Ontogenic variations in N₂ fixation and accumulation of N by the mungbean (Vigna radiata L. Wilczek), blackgram (Vigna mungo L. Hepper), cowpea (Vigna unguiculata L. Walp.), and groundnut (Arachis hypogaea L.) were studied by a ¹⁵N-dilution technique. Pots filled with 7 kg of red yellow podzolic soil were used. Samples were taken 20, 40, 60, and 80 days after emergence which approximately corresponded to preflowering, flowering, early/mid-pod filling and late pod filling stages, respectively. During early growth (up to 40 days after emergence), the carryover of seed N accounted for a considerable fraction of the total plant N in the legumes, the highest being in the groundnut. With a correction for carryover, the groundnut derived over 45% of its N content from the atmosphere 20 days after emergence whereas the corresponding figures were 33% for the blackgram and about 28% for the cowpea and mungbean. Between flowering and early pod fill, there was a rapid increase in N₂ fixation in all legumes except in groundnut which showed highest fixation from 60 to 80 days after emergence. In the mungbean, N₂ fixation and uptake of soil N were insignificant 60 days after emergence while in other legumes these processes continued beyond this time. All legumes derived about 90% of their N from atmosphere by 80 days after emergence. However, due to considerable interspecific differences in total N yield the final amount of N₂ fixed showed an appreciable variation among legumes. It was highest in the groundnut (443 mg N plant^-1) followed by the cowpea (385), blackgram (273), and mungbean (145), respectively. The groundnut maintained nodules until the late pod filling stage while in other legumes, nodules senesced progressively following the mid-pod filling stage. During pod filling there was a net mobilization of N from vegetative tissues to developing pods in the mungbean, which amounted to about 20% of N in seeds. This mobilization was not evident in other legumes.     

Mobilization of labelled N in soybean leaves during early podfill

Soybeans accumulate N in vegetative tissues up to pod initiation after which total vegetative N may remain constant during early phases of pod development. Eventually much of the vegetative N is mobilized to the pods. The mobilization of N from vegetative tissue to pods during the first few days of pod development is poorly understood but is important to an overall understanding of soybean N nutrition. The vegetative tissues of field grown soybeans were labelled with ¹⁵N and sampled weekly during the reproductive phase of plant growth. Three foliar applications of (¹⁵NH₄)₂SO₄ were made prior to pod initiation at a combined rate of 3.3 kg N/ha. To immobilize soil N and to increase soybean dependance on N₂‐fixation, sawdust was applied at a rate of 52 t ha^‐1 . Irrigation was required almost weekly because of a shallow soil profile and below normal summer precipitation. Mobilization of vegetative N began immediately upon pod initiation and continued at a linear rate through pod development. It appeared that N₂‐fixation was able to provide approximately half of the N in pods during early podfill. Nitrogen content of vegetative tissue declined as soon as pods began developing.     

Time-course of nitrogen fixation in two bambara groundnut (Vigna subterranea L. Verdc.) cultivars

TheA-value method, involving the application of a higher¹⁵N rate to a reference non-N₂-fixing plant, was used to assess the magnitude of N₂ fixation in two bambara groundnut cultivars at four growth stages [vegetative, 0–47 days after planting (DAP); early pod-filling, 47–99 DAP; mid-pod-filling, 99–120 DAP; physiological maturity, 120–148 DAP). The cultivars were Ex-Ada, a bunchy type, and CS-88-11, a slightly spreading type. They were grown on a loamy sand. Uninoculated Ex-Ada and CS-88-11 were used as reference plants to measure the N₂ fixed in the inoculated bambara groundnuts. In this greenhouse study, soil was the major source of N in bambara groundnuts during vegetative growth, and during this period it accounted for over 80% of the N accumulaed in the plants. However, N₂ fixation became the major source of plant N during reproductive growth. There were significant differences between the two cultivars in the ability to fix N₂, and at physiological maturity, almost 75% of the N in CS-88-11 was derived from the atmosphere compared to 55% in Ex-Ada. Also, the total N fixed in CS-88-11 at physiological maturity was almost double that in Ex-Ada. Our data indicate that the higher N₂ fixation in CS-88-11 was due to two factors, a higher intensity of N₂ fixation and a longer active period of N₂ fixation. The results also suggest that bambara groundnut genotypes could be selected for higher N₂ fixation in farining systems.     

Effect of successive cuttings on uptake and partitioning of 15N among plant parts of Leucaena leucocephala

Summary We studied the effect of three successive cuttings on N uptake and fixation and N distribution in Leucaena leucocephala. Two isolines, uninoculated or inoculated with three different Rhizobium strains, were grown for 36 weeks and cut every 12 weeks. The soil was labelled with 50 ppm KNO₃ enriched with 10 atom % ¹⁵N excess soon after the first cutting. Except for the atom % ¹⁵N excess in branches of K28 at the second cutting, both the L. leucocephala isolines showed similar patterns of total N, fixed N₂, and N from fertilizer distribution in different parts of the plant at each cutting. The Rhizobium strain did not influence the partitioning of ¹⁵N among the different plant parts. Significant differences in ¹⁵N enrichment occurred in different parts. Live nodules of both isolines showed the lowest atom % ¹⁵N excess values (0.087), followed by leaves (0.492), branches (0.552), stems (0.591), and roots (0.857). The roots contained about 60% of the total plant N and about 70% of the total N derived from fertilizer over the successive cuttings. The total N₂ fixed in the roots was about 60% of that fixed in the whole plant, while the shoots contained only 20% of the fixed N₂. We conclude that N reserves in roots and nodules constitute another N source that must be taken into account when estimating fixed N₂ or the N balance after pruning or cutting plants. ¹⁵N enrichment declined up to about fivefold in the reference and the N₂-fixing plants over 24 weeks following the ¹⁵N application. The proportion and the amounts of N derived from fertilizer decreased, while the amount derived from N₂ fixation increased with time although its proportion remained constant.     

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.     

Evaluation of N2‐fixation measured by the 15N‐dilution and N‐difference methods in Nicaraguan and Ecuadorian Phaseolus vulgaris L. plants inoculated with Rhizobium leguminosarum biovar

Pot and field experiments were performed to assess N₂ fixation in Nicaraguan (R79 and R84) and Ecuadorian (Imba) common bean (Phaseolus vulgaris L.) cultivars, with the aim of improving their productivity by inoculating them with commercially produced Rhizobium phaseoli. With maize (Zea mays L.) as the non‐N₂‐fixing control, the percentage of N₂ fixed predicted by the ¹⁵N‐dilution method was significantly (P ≤ 0.05) higher than that predicted by the N‐difference method. However, the N₂ amounts predicted by the two methods were not significantly different. The correlation between the two methods was significant and positive (P ≤ 0.0001, n = 36). Compared with the native rhizobial strain, symbiotic associations of the bean cultivars with UMR1073, UMR1077 and UMR1899 rhizobial inoculants did not significantly (P ≤ 0.05) influence plant dry matter (DM) and N yields, the extent of N₂ fixation and uptake of soil and fertilizer N. Nevertheless, the UMR1077 and UMR1899 strains markedly increased the uptake of soil N by R84 plants, while decreasing N2 fixation. In contrast, the Imba‐UMR1899 association enhanced positive effects on all variables. About 60–70% of the total N taken up by the Imba plants was fixed N_2. The R79 and R84 plants fixed about 50% of their total N uptake. N₂ fixation rates were positively correlated with DM and total N yields, while being negatively correlated with soil N uptake (P ≤ 0.001, n = 36). Future research in Nicaragua should focus on selecting rhizobial strains suitable for indigenous common bean cultivars.     

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.     

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.     

Effect of nitrogen application on the A N value of soil

Pot experiments were conducted with two soils, from Rottenhaus and Seibersdorf in Austria, to ascertain whether the rate of fertilizer N application and the test crop would influence the amount of N available in the soil as assessed by the A-value method. ¹⁵N-labelled fertilizer was applied at rates of 10, 25, 40, 60, and 100 mg N kg^-1 soil, corresponding approximately to 20, 50, 80, 120 and 200 kg N ha^-1 respectively, and two crop species, barley (Hordeum vulgareL.) and non-nodulating soybean (Glycine max L.) were used to determine the soil A _N value under the various fertilizer regimes. The results showed that the Rottenhaus soil had a higher A _N value than the Seibersdorf soil, suggesting that the former was more fertile than the latter. The A _N values of both soils were significantly affected by the level of N application. When grown in the same soil, the two test crops showed significantly different fertilizer use efficiency and per cent N derived from fertilizer when the rate of N application exceeded 20 kg ha^-1. Thus, the A _N value as determined by the two test crops differed significantly for the same soil when the rate of N application was greater than 20 kg/ha. The difference was greater when the soil fertility level was high. The dependence of the A _N value on the level of N application and the species of crop seriously compromises the suitability of this method for determining plant-associated N₂ fixation. Hence, considerable caution is required when using this method to estimate plant-associated N₂ fixation.     

Field measurements of nitrogen fixation in leguminous trees used in agroforestry systems: Influence of 15N-labeling approaches and reference trees

Appropriate ¹⁵N-labeling methods are crucial for estimating N₂-fixation in trees used in agroforestry systems. A 4-year field experiment was conducted on an Alfisol in Southwestern Nigeria to compare the estimates of N₂ fixed in Leucaena leucocephala, using two non-N₂-fixing leguminous trees, Senna siamea and S. spectabilis, as reference plants and three different methods of introducing ¹⁵N into soil. The atom % ¹⁵N uptake pattern (as reflected in the leaves) was identical in both N₂- and non-N₂-fixing tree species irrespective of the ¹⁵N-application method. There was a significant decline in atom % ¹⁵N excess in the leaves of L. leucocephala (from 0.266 to 0.039), S. siamea (0.625 to 0.121), and S. spectabilis (from 0.683 to 0.118) from the first sampling 12 months after planting and the second sampling 18 months after sampling. From the second harvest in 1991 until the end of the experiment (fifth) harvest in 1993, however, the atom ¹⁵N % excess decline in leaves of the three species was less pronounced and depended on the method of ¹⁵N application. In those plants to which the tracer was applied once at planting, the ¹⁵N decline was steady between the second and the last prunings. In the split-application treatment, the atom ¹⁵N % excess increased slightly at the third pruning and decreased during the subsequent two prunings. The reference tree and the method of ¹⁵N application influenced the estimated proportion of N derived from atmospheric N₂ by L. leucocephala, calculated as 73 and 64%, corresponding to 119 and 98 kg N ha^-1 of N₂ fixed per 6 months, when S. spectabilis and S. siamea were used as reference trees, respectively. The approach by which ¹⁵N-labeled fertilizer was applied to the soil in three splits gave slightly higher estimates of N derived from the atmosphere but this was of little agronomic significance because total N₂ fixed was similar for all methods.     

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.     

Use of 15N isotope dilution for quantification of N2 fixation associated with roots of kallar grass (Leptochloa fusca (L.))

Summary Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using ¹⁵N isotopic dilution were carried out in nutrient solution and saline soil. In the nutrient solution, E. coli inoculated plants were taken as non-nitrogen-fixing control. It was observed that nearly 60%–80% of the plant N was derived from atmospheric fixation. Estimations based on the N difference method gave much lower values (18%–35%). In experiments with saline soil which was initially sterilized with chloroform fumigation, a mixed culture of N₂-fixing rhizospheric isolates from kallar grass roots was inoculated and planted to kallar grass. Uninoculated treatments were regarded as controls. The soil was previously labelled with ¹⁵N by adding cellulose and (¹⁵NH₄)₂SO₄. The results of these studies showed fixation values of 6%–32% when estimated by ¹⁵N dilution, whereas by the N difference method 54% of the plant N was estimated to be derived from fixation. This discrepancy is due to the increase in root proliferation due to inoculation, which results in greater uptake of soil N. The distribution of ¹⁵N in different fractions of the soil-N indicted isotopic dilution due to bacterial fixation of atmospheric N₂.     

Variation in nodulation and N2 fixation by the Gliricidia sepium/Rhizobium spp. symbiosis in a calcareous soil

Summary Variation in nodulation and N₂ fixation by the Gliricidia sepium/Rhizobium spp. symbiosis was studied in two greenhouse experiments. The first included 25 provenances of G. sepium inoculated with a mixture of three strains of Rhizobium spp. N₂ fixation was measured using the ¹⁵N isotope dilution method 12 weeks after planting. On average, G. sepium derived 45% of its total N from atmospheric N₂. Significant differences in fixation were observed between provenances. The percentage of N derived from atmospheric N₂ ranged from 26 to 68% (equivalent to 18–62 mg N plant^-1) and was correlated with total N in the plant (r=0.70; P=0.05). The second experiment included six strains of Rhizobium spp. and two methods of inoculation and the plants were harvested 14,35 and 53 weeks after planting. In the first harvest significant differences were found between the number of nodules and the percentage and amount of N₂ fixed. There was also a significant correlation between the number of nodules and the amount of N₂ fixed (r=0.92; P=0.05). In the final harvest no correlation was observed, although there were significant differences between the number of nodules and the percentage of N derived from the atmosphere. The amount of N₂ fixed increased with time (from an average of 27% at the first harvest to 58% at the final harvest) and was influenced by the Rhizobium spp. strain and the method of inoculation. It ranged from 36% for Rhizobium sp. strain SP 14 to 71% for Rhizobium SP 44 at the last harvest. Values for the percentage of atmosphere derived N₂ obtained by soil inoculation were slightly higher than those obtained by seed inoculation.     

Evaluation of nitrogen fixation by different strains of theAzolla-Anabaena symbiosis in the presence of a high level of ammonium

Summary The proportion of N derived from N₂ fixation for 99 strains ofAzolla spp. (comprising all known species) in the presence of ammonium (40 mg/1) was assessed using a¹⁵N-dilution technique. The percentage of N derived from air varied from 29.5% to 79.9%. Although the N concentration ofAzolla spp. was not correlated with fertilizer N, it correlated fairly well with N₂ fixation. Regression analysis suggests that the N yield ofAzolla spp. is more dependent on N₂ fixation than on ammonium assimilation. The high correlation between N yield and isotopically determined, fixed N₂ indicates that the N yield could be used as a parameter in the selection ofAzolla spp. strains that are capable of maintaining high N₂ fixation in the presence of a high level of ammonium.     

Yield,nodulation, and N2 fixation by cowpea cultivars at different phosphorus levels

A greenhouse experiment was conducted to investigate the effect of a P application (0 vs. 50 mg P kg^-1) on yield, nodulation, and N₂ fixation by three cowpea cultivars (Soronko, Amantin, and IT81D-1137) using the ¹⁵N isotope-dilution method. When P was not applied the inoculated cowpea genotypes showed significant differences (Soronko>Amantin> IT81D-1137) in N accumulation, in contrast to the uninoculated cowpea cultivars, which accumulated similar amounts of N. The differences in shoot N in inoculated plants were thus caused by differences in N₂ fixation. The average values of N fixed (for both P levels) were 74% in Soronko, 59% in Amantin, and 42% in IT81D-1137, corresponding to 80, 51, and 24 mg N plant^-1, respectively. Inoculation increased the total shoot-N accumulation in cv. Soronko by 270% without P and by 204% with P, cv. Amantin by 152 and 104%, and cv. IT81D-1137 by 74 and 58%, respectively. With P, the % N derived from atmosphere (%Ndfa) was 42% for IT81D-1137, 62% for Amantin, and 76% for Soronko. The high value for Soronko indicates that in a soil of medium fertility, certain cowpea cultivars are capable of satisfying their total N requirement through N₂ fixation. The P effect on N₂ fixation was mainly in the total amount of N fixed rather than on the percentage derived from the atmosphere.     

Quantification and potential availability of non-symbiotically fixed 15N in soil

Summary Non-symbiotic N₂ fixation was studied under laboratory conditions in two soils from Pakistan (Hafizabad silt loam and Khurrarianwala silt loam) and one from Illinois, USA (Drummer silty clay loam) incubated in a ¹⁵N-enriched atmosphere. N₂ fixation was greatest with the Drummer soil (18–122 g g^–1 soil, depending upon the soil treatment) and lowest with the Khurrarianwala soil (4–81 g g^–1 soil). Fixation was increased by the addition of glucose, a close correlation being observed between the amount of glucose added and the amount of N₂ fixed in the three soils (r = 0.96). Efficiency of N₂ fixation varied with soil type and treatment and was greatest in the presence of added inorganic P. Application of Mo apparently had a negative effect on the amount and efficiency of N₂ fixation in all the soils. The percentage of non-symbiotically fixed ¹⁵N in potentially mineralizable form (NH ₄ ⁺ -N released in soil after a 15-day incubation period under anaerobic conditions) was low (2%–18%, depending upon the soil treatment), although most of the fixed N (up to 90%) was recovered as forms hydrolysable with 6N HCl. Recovery in hydrolysable forms was much greater for the fixed N than for the native soil N, indicating that the former was more available for uptake by plants.     

Nodulation and N2 fixation of faba bean (Vicia faba L.) after soil amendment with crop residues

The effect of prior soil amendment with different N sources at 50 mg N (kg soil)^—1 on nodulation and N₂ fixation of faba bean (Vicia faba L. cv. Troy) using wheat (Triticum aestivum L. cv. Star) as reference crop was assessed in a pot experiment. Four treatments viz legume manure (LEGM) as clover shoots, cereal manure (CEREM) as barley straw, N fertilizer (FERT‐N) as Ca(NO₃)₂, and no‐manure control (NOMAN) were investigated consecutively at 45, 70, and 90 days after sowing (DAS). Faba bean nodulated profusely, with an increase on average from 629 nodules per pot at 45 DAS to nearly 2.3‐ and 3.3‐fold at 70 and 90 DAS, respectively. Low nodule numbers and nodule dry matter occurred under FERT‐N and CEREM, whereas high values were found for NOMAN and LEGM. Soil amendment affected percent N₂ fixation in relation to N source and plant age. Highest percent N₂ fixation (≥ 90 %) was found under the lowest N‐supplying amendments, no‐manure, and cereal manure, respectively. FERT‐N depressed N₂ fixation particularly at 45 DAS when N₂ fixation was reduced to as low as 23 %. The rise in N₂ fixation thereafter suggests that faba bean adjusted after depletion of mineral N in the soil. N₂ fixation was also decreased after cereal straw application, even though N concentration in faba bean plants was high. The results indicate that plant residues, both with high and low N concentration, applied to soil to raise its fertility may interfere with N₂ fixation of faba bean.     

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.     

Comparison of 15N natural abundance and difference methods for estimating N2 fixation of soybeans grown in a tropical soil

Abstract

A study was carried out to compare the difference or N-yield method with the ¹⁵N natural abundance method for the estimation of the fractional contribution of biological N₂ fixation in the different plant parts of nodulating and non-nodulating isolines of soybeans. The results indicated that the δ¹⁵N values of most plant parts of soybeans were significantly lower (p<0.05) in the nodulating than in the non-nodulating isoline. However, in the case of the root+nodule component, the δ¹⁵N value was higher in the nodulating than in the non-nodulating isoline possibly due to isotopic discrimination of ¹⁵N over ¹⁴N which may have occurred in the nodules. Inoculation of soybeans with the Bradyrhizobium japonicum strain CB 1809 increased significantly (p<0.05) the δ¹⁵N value of the root+nodule component implying that the effectiveness of the soybean-rhizobium symbiosis had increased by inoculation.

Percentage of plant N derived from atmospheric N₂ fixation (%Ndfa) estimated by the ¹⁵N natural abundance method was highly correlated (r=0.762, p<0.01) with that by the difference or N-yield method and the differences between the two methods were not statistically significant. The agreement between the two methods was closer at maturity than at the early reproductive stage.

The %Ndfa obtained by the difference method ranged from 48.4 to 92.6% whereas the %Ndfa obtained by the ¹⁵N natural abundance method ranged from 43.2 to 92.4% in the different plant parts. Based on the ¹⁵N natural abundance method, approximately 15% of the N in pod, shoot, grain, and shell was derived from the soil but in the case of stover, this fraction was about 55%.