N2 fixation in two Sesbania species and its transfer to rice (Oryza sativa L.) as revealed by 15N technology

Summary We used ¹⁵N technology to investigate N₂ fixation by Sesbania speciosa and Sesbania rostrata and its transfer to a lowland rice crop after incorporation of the Sesbania spp. into soil as green manure. During the first 50 days after establishment in November–December 1989, S. speciosa and S. rostrata produced 1126 and 923 kg dry matter ha^-1 respectively. They gathered 31 and 23 kg N ha^-1 respectively, of which 62%±5% and 55%±3% respectively, came from N₂ fixation. Both these species produced a greater biomass during September–October 1989, with S. rostrata producing more than S. speciosa. These results reflected differential responses by the plants to different day lengths at different times of the year. Furthermore, the dry matter yield and %N of ¹⁵N-labelled S. speciosa were smaller than those of the unlabelled plants, possibly due to inhibition of N₂ fixation in root nodules by the chemical N fertilizers added during labelling. These differences were not so pronounced in the stem-nodulated S. rostrata. The increased grain yield of rice fertilized with N in the form of chemical fertilizer or green manure was a result of an increased number of panicles per hill. The rice crop manured with S. speciosa produced a lower grain yield, with a lower grain weight than that manured with S. rostrata. This was due to a low uptake of soil N by rice manured with S. speciosa. Recovery of N from the green manure in rice straw with S. speciosa was significantly higher than from rice manured with S. rostrata, because of the higher applied N uptake by rice manured with the former.     

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.     

Symbiotic efficiency of Sesbania rostrata and S. cannabina as affected by agronomic practices

A field experiment was conducted to study the N₂ fixation efficiency of Sesbania rostrata and S. cannabina as affected by agronomic practices in semi-arid subtropical climate, Sowing seeds resulted in smaller numbers of nodules, lower dry weight, lower total biomass, less N uptake, and less N₂ fixation for S. rostrata than S. cannabina, while cut-stem planting improved the symbiotic efficiency. Flooding the soil increased the relative humidity of the crop micro-environment by 4–11% and induced early appearance of stem nodules in S. rostrata. Only 67 kg N ha^-1 was fixed by S. rostrata compared to 160 kg N ha^-1 by S. cannabina when normal agronomic practices (sowing and non-flooding) were followed. In contrast, planting stem cuttings and flooding resulted in greater biological N₂ fixation, 307 and 209 kg N ha^-1 by S. rostrata and S. cannabina, respectively. Therefore, S. rostrata can be successfully exploited as a green manure when stem cuttings are planted under flooded conditions.     

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.     

Sesbania rostrata as a green manure for lowland rice: Growth,N2 fixation,Azorhizobium sp. inoculation,and effects on succeeding crop yields and nitrogen balance

Summary Root and stem nodulation, nitrogen fixation (acetylene-reducing activity), growth and N accumulation bySesbania rostrata as affected by season and inoculation were studied in a pot experiment. The effects ofS. rostrata as a green manure on succeeding wet-season and dry-season rice yields and total N balance were also studied.S. rostrata grown during the wet season showed better growth, nodulation, and greater acetylene-reducing activity than that grown during the dry season. Inoculation withAzorhizobium caulinodans ORS 571 StrSpc® (resistant to streptomycin and spectinomycin) on the stem alone or on both root and stem significantly increased N₂ fixation by the plants. Soil and seed inoculation yielded active root nodules under flooded conditions. Plants that were not inoculated on the stem did not develop stem nodules. The nitrogenase activity of the root nodules was greater than that of the stem nodules in about 50-day-oldS. rostrata. S. rostrata incorporation, irrespective of inoculation, significantly increased the grain yield and N uptake of the succeeding wet season and dry season rice crops. The inoculated treatments produced a significantly greater N gain (873 mg N pot^–1) than the noinoculation (712 mg N pot^–1) treatment. About 80% of the N gained was transferred to the succeeding rice crops and about 20% remained in the soil. The soil N in the flooded fallow-rice treatment significantly declined (–140 mg N pot^–1) but significantly increased in bothS. rostrata-rice treatments (159 and 151 mg N pot^–1 in uninoculated and inoculated treatments respectively). The N-balance data gave extrapolated values of N₂ fixed per hectare at about 303 kg N ha^–1 per two crops forS. rostrata (uninoculated)-rice and 383 forS. rostrata (inoculated)-rice.     

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.     

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.     

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.     

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%.     

Landscape-scale estimates of dinitrogen fixation by Pisum sativum by nitrogen-15 natural abundance and enriched isotope dilution

Topography and slope position influence the soil and environmental factors that affect N₂ fixation by legumes. The present study was conducted to (1) estimate N₂ fixation by field peas in a gently rolling farm field using the natural ¹⁵N abundance and the ¹⁵N-enriched isotope dilution techniques and (2) identify soil and environmental factors that influence N₂ fixation at the landscape scale. Whereas soil available water capacity, available NH _inf4 ^sup+ , total crop yield, and percent N derived from N₂ fixation (% Ndfa) estimated using enriched N were significantly affected by landform patterns, soil NO _inf3 ^sup- levels, seed yield, and the % Ndfa estimated using natural abundance did not follow landform patterns. The % Ndfa using natural abundance was correlated with NH _inf4 ^sup+ but not with available soil water, pH, electrical conductivity, NO _inf3 ^sup- , or particle size. Estimates of the % Ndfa using enriched ¹⁵N ranged from 0 to 92.8%. The highest median value (68.6%) for % Ndfa using enriched N occurred on the divergent footslopes, with the lowest value (28.1%) on the convergent shoulders. Estimates of % Ndfa using natural abundance ranged from 13.2% to 96.9%. Smaller fluctuations during the growing season in the ¹⁵N of the available N pool may have resulted in less variability for % Ndfa using natural abundance compared to enriched ¹⁵N. Despite similar mean values for % Ndfa using natural abundance (44.5) and enriched ¹⁵N (49.6), no significant correlation between the two estimates was found. These results suggest that although topography may exert gross controls on N₂ fixation, large variations in N₂ fixation at the microsite level may preclude correlations between individual estimates and limit detection of landscape scale patterns of N₂ fixation.Contribution No. R754 of the Saskatchewan Center of Soil Research     

Influence of phosphorus or phosphorus-potassium fertilization on biomass and dinitrogen fixation of the stem-nodulating green-manure legume Sesbania rostrata in different marginally productive wetland rice soils

The performance of Sesbania rostrata varies widely from site to site. This makes it difficult to predict the N yield and biomass of this plant in marginally productive soils, and to arouse the interest of farmers in green manure technology. Three consecutive pot experiments were conducted in a greenhouse at the International Rice Research Institute (IRRI) to evaluate growth, nodulation, N₂ fixation (C₂H₂ reduction assay and ¹⁵N dilution method), and N yield of 6-week-old S. rostrata on 13 physicochemically different wetland rice soils of the Philippines and on three artificial substrates. The performance of S. rostrata on the unfertilized controls was compared with two fertilizer treatments containing either P (100 mg P kg^-1 dry soil) or P+K (100 mg P kg^-1 and 200 mg K kg^-1 dry soil). In the control soils and substrates, the N yield of S. rostrata varied between 20 and 470 mg N per pot, with the N rate from N₂ fixation ranging between 0 and 95%. In three of the nutritionally poor soils even Mn toxicity symptoms apparently occurred with S. rostrata. P application alleviated these symptoms and increased the overall N yield considerably, mainly through increased biological N₂ fixation. An additional increase in N yield was obtained by the PK treatment. Multiple regression analysis between soil characteristics and the N yield of S. rostrata showed that the original level of P (Olsen-extracted) and Mn in the soil accounted for 73% of the variance in biomass production by S. rostrata among the unfertilized soils and substrates.     

15N标记的土壤估测某些豆科植物N2固定的温室实验

Pot experiments were carried out to estimate N2 fixation by vetch,milk vetch,sickle alfalfa and broadbean in pure stand using a ^15N-labelled soil.Winter wheat was used as the non-fixing control.The 15N-labelled soil used was prepared by growing corn-wheat-corn successively on a nearly organic-matter-free Xiashu loess supplemented with adequate amounts of (15NH4)2SO4,P,K and micronutrients,then incorporating these 15N-labelled plant materials into the soil after each havest,and allowing the plant materials to be decomposed aerobically for 410d after incorporation of the plant material of the thire crop.The 15N enrichment of wheat plant-N varied slightly with organs,with a maximum difference of 9.8%,Based on 15N enrichment of soil N inferred from the mean value of the 15N enrichment in different organs of wheat 79%-91% of total N in the tops and 67%-74% of total N in the roots of legumes studied were derived from atmosphere .Estimate by isotope dilution method was in good agreement with that by the conventional difference method provided values obtained by the latter were corrected for seed N,and also with that from the measurement of N accumulated in the tops of the legumes.     

The fate of markerAzospirillum lipoferum inoculated into rice and its effect on growth,yield and N2 fixation of plants studied by acetylene reduction,15N2 feeding and15N dilution techniques

Summary A spontaneous mutant ofAzospirillum lipoferum, resistant to streptomycin and rifampicin, was inoculated into the soil immediately before and 10 days after transplanting of rice (Oryza sativa L.). Two rice varieties with high and low nitrogen-fixing supporting traits, Hua-chou-chi-mo-mor (Hua) and OS4, were used for the plant bacterial interaction study. The effect of inoculation on growth and grain and dry matter yields was evaluated in relation to nitrogen fixation, by in situ acetylene reduction assay,¹⁵N₂ feeding and¹⁵N dilution techniques. A survey of the population of marker bacteria at maximum tillering, booting and heading revealed poor effectivety. The population of nativeAzospirillum followed no definite pattern. Acetylene-reducing activity (ARA) did not differ due to inoculation at two early stages but decreased in the inoculated plants at heading. In contrast, inoculation increased tiller number, plant height of Hua and early reproductive growth of both varieties. Grain yield of both varieties significantly increased along with the dry matter. Total N also increased in inoculated plants, which was less compared with dry matter increase.¹⁵N₂ feeding of OS4 at heading showed more¹⁵N₂ incorporation in the control than in the inoculated plants. The ARA,¹⁵N and N balance studies did not provide clear evidence that the promotion of growth and nitrogen uptake was due to higher N₂ fixation.     

N2 fixation and growth of legumes as affected by sulphur fertilization

The influence of three sulphur application rates in combination with two nitrogen application rates on N₂ fixation and growth of different legumes was investigated. N was applied as N-labelled ¹⁵NH₄ ¹⁵NO₃. The ¹⁵N isotope dilution technique was used to estimate N₂ fixation. At both N increments dry matter yield was highest with high S supply. Independently of the N supply, the high S application rate resulted in a significantly higher N accumulation, which was mainly caused by a higher N₂ fixation rate. With the grain legumes the weight of nodules was increased by the high S application rate. The higher number of nodules per pot with optimum S supply was the result of a better root growth. Rates of acetylene reduction correlated significantly with S supply.     

A comparison of nitrogen fixation in genotypes of groundnut (Arachis hypogaea L.) using 15N-isotope dilution

Summary Nitrogen fixation in seven groundnut genotypes was measured by ¹⁵N-isotope dilution using a non-nodulating cultivar of groundnut as the nonfixing reference plant. Nitrogen fixation varied between 100 kg N ha^–1 in genotype J-11 and 153 kg N ha^–1 in Robut 33-1. The amount of plant-available soil N was small, so that 86%–92% of plant nitrogen was derived from N₂-fixation. Thus differences in N₂-fixation between genotypes closely reflected differences in their total N accumulation.ICRISAT Journal Article no. 600     

Nitrogen accumulation in three legumes and two cereals with emphasis on estimation of N2 fixation in the legumes by the natural 15N-abundance technique

Summary N accumulation and natural ¹⁵N abundance in three legumes (groundnuts, cowpeas, and soybeans) and in two cereals (sorghum and maize) were investigated over two seasons in Alfisols with and without N fertilization. Using the N uptake and natural ¹⁵N abundance of non-nodulating plants as the indication of N derived from soil and fertilizer, the per cent N derived from atmospheric N₂ was calculated for nodulated plants. In the first experiment, the groundnut genotype contained 85% atmosphere-derived N, but the percentage decreased with N application. Estimates of atmosphere-derived N by the N-difference and ¹⁵N-abundance techniques gave identical results. The percentages of atmosphere-derived N estimated by the two methods at different stages of groundnut growth were also similar. In the second experiment, atmosphere-derived N was estimated in plants grown with 0–200 kg ha^-1 applied N. The estimated atmosphere-derived N ranged from 42% to 61% for groundnuts from 33% to 77% for cowpeas, and from 24% to 48% for soybeans, depending on the amount of N applied. Inoculation with a Bradyrhizobium strain increased the percentage of atmospherederived N in soybean plants grown without any fertilizer N. The natural ¹⁵N abundance of sorghum and maize was very close to that of the non-nodulating groundnut, suggesting that these cereals can be used as reference plants in the estimation of atmosphere-derived N by the natural ¹⁵N-abundance method.ICRISAT Journal Article No. 876     

Biological nitrogen fixation in Azospirillum strain-maize genotype associations as evaluated by the 15N isotope dilution technique

Few studies of the inoculation of cereal crops with N₂-fixing bacteria have included more than one or two plant genotypes. In a recent study performed in Argentina using 12 different maize genotypes, it was found in 2 consecutive field experiments that several of them responded consistently, either negatively or positively, to inoculation with a mixture of strains of Azospirillum spp. The present study in post was performed to investigate the effect of inoculation of individual strains (and a mixture) of Azospirillum spp., and their nitrate reductase negative (NR-) mutants, on the growth of four of these maize genotypes. Two of these genotypes were grown in ¹⁵N-labelled soil with the aim of quantifying any contributions of biological N₂ fixation. Two genotypes (Morgan 318 and Dekalb 4D-70) produced similar increases in grain yield when they were inoculated with a mixture of Azospirillum spp. strains or fertilized with the equivalent of 100 kg N ha^-1. The other genotypes (Dekalb 2F-11 and CMS 22) showed little response to inoculation or N fertilization. The Morgan 318 and Dekalb 4D-70 genotypes showed a large increase in total N accumulation, suggesting that the response was due to increased N acquisition, but not due to bacterial nitrate reductase as the NR- mutants generally caused plant responses similar to those of the parent strains. Despite problems with the stabilization of the ¹⁵N enrichment in the soil, the ¹⁵N isotope dilution results indicated that there were very significant biological nitrogen fixation (BNF) contributions to the Dekalb 4D-70 and CMS 22 maize genotypes.Dedicated to Professor J.C.G. Ottow on the occasion of his 60th birthday     

Influence of mycorrhiza vs. soluble phosphate on growth,nodulation, and N2 fixation (15N) in alfalfa under different levels of water potential

Summary The legume Medicago sativa (+Rhizobium melilott) was grown under controlled conditions to study the interactions between soluble P in soil (four levels), or a mycorrhizal inoculum, and the degree of water potential (four levels) in relation to plant development and N₂ fixation. ¹⁵N-labelled ammonium sulphate was added to each pot for a qualitative estimate of N₂ fixation, in order to rank the effects of the different treatments.Dry-matter yield, nutrient content and nodulation increased with the amount of plant-available P in the soil, and decreased as the water stress increased, for each P-level. The mycorrhizal effect on dry matter, N yield, and on nodulation was little affected by the water potential. Since P uptake was affected by the water content in mycorrhizal plants, additional mechanisms, other than those mediated by P, must be involved in the mycorrhizal activity.There was a positive correlation between N yield and nodulation for the different P levels and the mycorrhizal treatment at all water levels. A high correlation between plant unlabelled N content and atom% ¹⁵N excess was also found for all levels of P. In mycorrhizal plants, however, the correlation between unlabelled N yield and ¹⁵N was lower. This suggests that mycorrhiza supply plants with other N sources in addition to those derived from the improvement on N₂ fixation.     

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.     

Evaluation of 15N-isotope dilution for measurement of nitrogen fixation in chickpea (Cicer arietinum L.)

Summary N accumulation, nodulation, and acetylene reduction activity were measured at frequent intervals during the growth of two chickpea genotypes, and N₂ fixation was estimated by an isotope-dilution method, using safflower as a non-N₂-fixing reference. Safflower was more efficient at N uptake than both the chickpea genotypes for at least the first 50 days and thus could not be used as an accurate reference control. We recommend that further work should employ non-nodulatiog genotypes of chickpea as reference plants and use slow-release forms of 15N fertilizer. Direct genotype comparison by isotope dilution estimated that genotype K 850 fixed 16–18 kg ha^–1 more N than G 130, and this difference was supported by the greater nodule mass and acetylene reduction activity in the K 850 cultivar. Inoculation with an ineffective chickpea Rhizobium sp. led to 69% nodulation on cultivar K 850 but only 33% on G 130. While nodule weight, N uptake, and acetylene reduction activity decreased with inoculation in K 850, the isotope dilutions were similar for both inoculation treatments. The lack of a significant effect on N₂ fixation was ascribed to the partial success of inoculant establishment.Published as Journal Article No. JA 692 of the International Crops Research Institute for the Semi Arid Tropics, Patancheru, A.P. 502324, India