Field measurement of symbiotic nitrogen fixation in an established lucerne ley using15N and an acetylene reduction method

Summary Lucerne is an important forage legume in the south and south-east of Sweden on well-drained soils. However, data is lacking on the apparent amount of nitrogen derived through N₂ fixation by field-grown lucerne. This report provides basic information on the subject. The experiment was performed in a lucerne ley grown 40 km north of Uppsala. The input of nitrogen through fixation to the above-ground plant material of an established lucerne (Medicago sativa L.) ley was estimate by¹⁵N methodology during two successive years. The amount of fixed N was 242 kg N ha^–1 in 1982 and 319 kg N ha^–1 in 1983. The proportion of N derived from the atmosphere (%Ndfa) was 70% and 80% for the two years respectively. The first harvest in both years contained a lower proportion fixed N. Both N₂ fixation and dry matter production were enhanced during the second year, particularly in the first harvest. The Ndfa was 61% in the first harvest in 1982, compared to 72% Ndfa during the same period in 1983. This demonstrates the strong influence of environment on both dry matter production and N₂ fixation capacity of the lucerne.In addition anin situ acetylene reduction assay was used in 1982 to measure the seasonal distribution of the N₂ fixation and in 1983 to study the effect of soil moisture on the N₂ fixation process. The seasonal pattern showed great dependence on physiological development and harvest pattern of the lucerne ley. The maximum rate of N₂ fixation occurred at the bud or early flower stage of growth and was followed by a rapid decline as flowering proceeded. After harvest the nitrogenase activity markedly decreased and remained low during at least two weeks until regrowth of new shoots began. Irrigation doubled the nitrogenase activity of the lucerne in late summer 1983, when soil moisture content in the top soil was near wilting point. No changes in nitrogenase activity did occur in response to watering earlier during the summer, when the soil matric potential was around –0.30 MPa.     

Remobilization of labelled nitrogen from vegetative tissues to pods of mungbean

Summary Remobilization of¹⁵N from vegetative tissue of mungbean (Vigna radiata (L.) Wilczek) into pods was measured during the reproductive phase of growth. Plant tissue was labelled with¹⁵N during vegetative development. Experiments were conducted in the field at two sites. At one site the soil provided cowpeas with most of their N but at the other site N fixation provided most of the N. Remobilized N from vegetative tissue to pods occurred soon after they began to develop. The quantity of the labelled N ultimately remobilized to the pods amounted to 50% for one cultivar (Tx33) at the high soil N site and 70% at the low N site. For the other cultivar (Tx13) the values were 25% and 30%, respectively. The two cultivars performed very differently with respect to partitioning of N into pods and the rate of N fixation. Even though more N was accumulated in the shoots of the high N fixing cultivar (Tx13) less total N was contained in the pods.     

Forests losing large quantities of nitrogen have elevated 15N:14N ratios

Summary Urea (U) and ammonium nitrate (AN) had been applied to a Scots pine (Pinus sylvestris L.) forest in northern Sweden for 18 consecutive years at four doses resulting in total N applications ranging from 0 to 1980 kg ha^–1. The ¹⁵N abundance ( ¹⁵N) of the grass Deschampsia flexuosa (L.) Trin. increased linearly (from –0.7 to 11.0) with application rate in the case of U. The response to AN was in the same direction but smaller. While others have shown that the initial response of nitrogen-limited systems to additions of N is a change of ¹⁵N abundance towards that of added N, this study shows that further and excessive additions leads to a retention of ¹⁵N. Monitoring ¹⁵N abundance over time in dose-response trials of this type thus opens new possibilities to estimate critical loads of N and the point of nitrogen saturation.     

Symbiotic N2 fixation by soybean in organic and conventional cropping systems estimated by 15N dilution and 15N natural abundance

Nitrogen (N) is often the most limiting nutrient in organic cropping systems. N₂ fixing crops present an important option to improve N supply and to maintain soil fertility. In a field experiment, we investigated whether the lower N fertilization level and higher soil microbial activity in organic than conventional systems affected symbiotic N₂ fixation by soybean (Glycine max, var. Maple Arrow) growing in 2004 in plots that were since 1978 under the following systems: bio-dynamic (DYN); bio-organic (ORG); conventional with organic and mineral fertilizers (CON); CON with exclusively mineral fertilizers (MIN); non-fertilized control (NON). We estimated the percentage of legume N derived from the atmosphere (%Ndfa) by the natural abundance (NA) method. For ORG and MIN we additionally applied the enriched ¹⁵N isotope dilution method (ID) based on residual mineral and organic ¹⁵N labeled fertilizers that were applied in 2003 in microplots installed in ORG and MIN plots. These different enrichment treatments resulted in equal %Ndfa values. The %Ndfa obtained by NA for ORG and MIN was confirmed by the ID method, with similar variation. However, as plant growth was restricted by the microplot frames the NA technique provided more accurate estimates of the quantities of symbiotically fixed N₂ (Nfix). At maturity of soybean the %Ndfa ranged from 24 to 54%. It decreased in the order ORG > CON > DYN > NON > MIN, with significantly lowest value for MIN. Corresponding Nfix in above ground plant material ranged from 15 to 26 g N m^-2, with a decreasing trend in the order DYN = ORG > CON > MIN > NON. For all treatments, the N withdrawal by harvested grains was greater than Nfix. This shows that at the low to medium %Ndfa, soybeans did not improve the N supply to any system but removed significant amounts of soil N. High-soil N mineralization and/or low-soil P availability may have limited symbiotic N₂ fixation.     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.     

Competition between siratro and kleingrass for15N labelled mineralized nitrogen

Summary Isotope dilution provides a method for measuring plant competition for mineral N and transfer of biologically fixed N from a legume to a grass. A plant growth medium was enriched with¹⁵N, and used to grow Siratro (Macropitilium atropurpureum D.C. Urb.) and Kleingrass 75 (Panicum coloratum L.) in 20 liter pots for 98 days in a glasshouse. The plants were grown in pure stand and in mixtures. When grown in 50∶50 mixture the grass obtained 59% of the labelled N and the legume obtained 41%. The grass produced nearly as much root mass as the legume even though biomass of the shoots were less than half that of the legume. Reducing the proportion of either plant species in the mixture reduced the proportion of the mineralized N absorbed by that species. The shoots of the grass were significantly more enriched (1.166 atom%¹⁵N excess) than the roots (1.036). The grass received 12% of its N as biologically fixed N from the legume.     

Measurement of biological dinitrogen fixation in grassland: Comparison of the enriched 15N dilution and the natural 15N abundance methods at different nitrogen application rates and defoliation frequencies

A plant mixture of white clover (Trifolium repens L.), red clover (Trifolium pratense L.), and ryegrass (Lolium perenne L.) was established in the spring of 1991 under a cover-crop of barley. Treatments were two levels of nitrogen (400 and 20 kg N ha^-1) and two cutting intensities (3 and 6 cuts per season). Fixation of atmospheric derived nitrogen was estimated by two ¹⁵N dilution methods, one based on application of ¹⁵N to the soil, the other utilising small differences in natural abundance of ¹⁵N.Both methods showed that application of 400 kg N ha^-1 significantly reduced dinitrogen fixation, while cutting frequency had no effect. Atmospheric derived nitrogen constituted between 50 and 64% of harvested clover nitrogen in the high-N treatment, while between 73% and 96% of the harvested clover nitrogen was derived from the atmosphere in the low-N treatment. The amounts of fixed dinitrogen varied between 31–72 kg N ha^-1 and 118–161 kg N ha^-1 in the high-N and low-N treatment, respectively. The highest values for biological dinitrogen fixation were estimated by the enriched ¹⁵N dilution method.Estimates of transfer of atmospheric derived nitrogen from clover to grass obtained by the natural ¹⁵N abundance method were consistently higher than those obtained by the enriched ¹⁵N dilution method. Neither mineral nitrogen application nor defoliation frequency affected transfer of atmospheric derived nitrogen from clover to grass.Isotopic fractionation of ¹⁴N and ¹⁵N (B value) was estimated by comparing results for nitrogen fixation obtained by the enriched ¹⁵N dilution and the natural ¹⁵N abundance method, respectively. B was on average +1.20, which was in agreement with a B value determined by growing white clover in a nitrogen free media.     

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

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

Effect of nitrogen fertilization and cropping system of the reference crop on estimation of N2 fixation by vetch using15N methodology

This study was conducted to examine the effects of varying N rates and cropping systems (mixedversus pure stand) on the suitability of oats (Avena sativa L.) for estimating N₂ fixed in sequentially harvested vetch (Vicia sativa L.) over two growing seasons (1984–85 and 1985–86). The N rates were, 20 and 100 kg N ha^–1 in 1984–85 and 15 and 60 kg N ha^–1 in 1985–86. In the 1984–85 season, vetch at maturity derived 76 and 63% N from fixation at the high and low N rates respectively. The corresponding values for the second season were 66 and 42%. Except in the 1985–86 season when some significantly higher values of % N₂ fixed were estimated by using the reference crop grown at the higher (A-value approach) than at the lower N rate (isotope-dilution approach), both approaches resulted in similar measurements of N₂ fixed. In the 1984–85 season, similar values of N₂ fixed were obtained using either the pure or mixed stand oats reference crops. Although in the 1985–86 season, the mixed reference crop occasionally estimated lower % N₂ fixed than pure oats, total N₂ fixed estimates were always similar (P<0.05). Thus, in general, N fertilization and cropping system of the reference crop did not significantly influence estimates of N₂ fixation.     

A field evaluation using the 15N isotope dilution method of lines of Phaseolus vulgaris L. bred for increased nitrogen fixation

N₂ fixation in lines of Phaseolus vulgaris was measured by ¹⁵N-isotope dilution to determine whether a programme of crossing and recurrent selection had resulted in enhanced nitrogen fixation. In field experiments on an isohyperthermic Aquic Hapludoll soil the amount of N₂ fixed by the different lines ranged from 18 to 36 kg ha^–1 (32 to 47% of plant N) in 56 days. The quantity of N₂ fixed and the proportion of plant N derived from fixation was not significantly greater in the lines selected for N₂ fixation (RIZ lines) than parental lines. Total shoot N ranged from 53 to 77 kg ha^–1 and partitioning of N to pods differed from 28 to 52% among the lines which all had similar growth habit and duration. Nodulation patterns were also distinct. Nodules formed early (10 to 15 plant^–1 at 13 days) in many lines, and smallest amounts of fixation were observed in those lines which nodulated slowly and did not form substantial nodule mass until after 40 days. The screening criteria used in the selection of the RIZ lines had been largely indirect with other factors such as disease resistance also being included. Progress for increasing N₂ fixation over good-fixing parental lines such as BAT76 was not significant and it is recommended that more attention be paid to early nodulation, to the use of soils with lower available N and to inter-crossing of lines having different good N₂ fixation traits in order to further enhance the potential for N₂ fixation in beans.     

The response of field grownPhaseolus vulgaris to Rhizobium inoculation and the quantification of N2 fixation using15N

Summary A field experiment was performed to assess the effects of Rhizobium inoculation and nitrogen fertilizer (100 kg N ha^–1) on four cultivars of Phaseolus beans; Carioca, Negro Argel, Venezuela 350 and Rio Tibagi. In the inoculated treatment 2.5 kg N ha^–1 of¹⁵N labelled fertilizer was added in order to apply the isotope dilution technique to quantify the contribution of N₂ fixation to the nutrition of these cultivars.Nodulation of all cultivars in the uninoculated treatments was poor, but the cultivars Carioca and Negro Argel were well nodulated when inoculated. Even when inoculated, nodulation of the cultivars Venezuela 350 and Rio Tibagi was poor and these cultivars showed little response to inoculation in terms of nitrogen accumulation or grain yield. The estimates of the contribution of N₂ fixation estimated using the isotope dilution technique, for the Carioca and Negro Argel cultivars, amounted to 31.7 and 18.4 kg N ha^–1 respectively. These two cultivars produced 991 and 883 kg ha^–1 of grain, respectively, when inoculated and 663 and 620 kg ha^–1 with the addition of 100 kg N ha^–1 of N fertilizer. The response to nitrogen was particularly poor due to high leaching losses in the very sandy soil at the experimental site.The Venezuela 350 and Rio Tibagi cultivars only responded to N fertilizer and not to inoculation with Rhizobium which stresses the great importance of selecting plant cultivars for nitrogen fixation in the field.     

Optimising biological N2 fixation by legumes in farming systems

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

Effects of plant breeding and selection on yields and nitrogen fixation in soybeans under two soil nitrogen regimes

Summary Soybeans (Glycine max (L.) Merr.) have a high N requirement which is fulfilled by soil N uptake and N₂-fixation. This study was concerned with the effects of past yield selection on N₂-fixation in soybeans.The soybean cultivars, Lincoln, Shelby, and Williams, which represent successive improvements in the Lincoln germplasm, and a non-nodulating control were planted in a soil containing¹⁵N labelled organic matter. Two replications occurred on soil previously cropped to alfalfa and two on soil previously cropped to soybeans. Plants were harvested at five growth stages and leaf area, plant weight, total N, and atom percent¹⁵N were determined. Mature grain was harvested and yield components were also determined, as well as the total N and¹⁵N content.Cultivar differences in total dry matter were only evident at physiological maturity, when Williams contained the greatest dry matter. Williams exhibited the longest period of seed formation and seed fill and also had the highest grain yield which resulted from a larger weight per seed.The N content of the cultivars did not vary until physiological maturity when Williams contained the highest percent N. The quantity of N fixed at physiological maturity was highest for Williams and lowest for Lincoln. Fixed N contained in the harvested grain was greater for Williams than for the other two cultivars. The fraction of the total plant N derived from fixation was not greatly affected by cultivar and all cultivars acquired an average of 50% of their total N through N₂-fixation.Previous cropping history greatly affected the quantity of N fixed and the fraction of the total plant N derived from fixation. Soybeans following soybeans were more dependent upon N₂-fixation than soybeans following alfalfa with the former deriving 65% of the total plant N from fixation and the latter only 32%. These soybean cultivars apparently utilized soil N first and then used N₂-fixation to satisfy their N requirement.The past selection for higher yield has resulted in soybean cultivars with improved capacities to fix atmospheric N₂ and an improved ability to take up available soil N.     

Variations and controls of nitrogen stable isotopes in particulate organic matter of lakes

Nitrogen stable isotope (δ¹⁵N) data of particulate organic matter (POM) from the literature were analyzed to provide an understanding of the variations and controls of δ¹⁵N_POM in lakes at the global scale. The δ¹⁵N_POM variability characterized by seasonal mean, minimum, maximum, and amplitude (defined as δ¹⁵N_POM maximum − δ¹⁵N_POM minimum) from 36 lakes with seasonal data did not change systematically with latitude, but was significantly lower in small lakes than in large lakes. The seasonal mean δ¹⁵N_POM increased from oligotrophic lakes to eutrophic lakes despite large variations that are attributed to the occurrences of nitrogen fixation across the trophic gradient and the differences in δ¹⁵N of dissolved inorganic nitrogen (DIN) in individual lakes. Seasonal mean δ¹⁵N_POM was significantly correlated with DIN concentration and δ¹⁵N_DIN in two subsets of lakes. Seasonal minimum δ¹⁵N_POM in individual lakes is influenced by nitrogen fixation and δ¹⁵N_DIN while seasonal maximum δ¹⁵N_POM is influenced by lake trophic state and δ¹⁵N_DIN. As a result of the dominance of non-living POM in the unproductive surface waters, seasonal δ¹⁵N_POM amplitude was small (mean = 4.2‰) in oligotrophic lakes of all latitudes. On the other hand, seasonal δ¹⁵N_POM amplitude in eutrophic lakes was large (mean = 10.3‰), and increased from low to high latitudes, suggesting that the seasonal variability of δ¹⁵N in the phytoplankton-dominated POM pool was elevated by the greater spans of solar radiation and thermal regimes at high latitudes. The δ¹⁵N_POM from 42 lakes with no seasonal data revealed no consistent patterns along latitude, lake area, and trophic gradients, and a greater than 2‰ depletion compared to the lakes with seasonal data. Along with the large seasonal variability of δ¹⁵N_POM within lakes, these results provide insightful information on sampling design for the studies of food web baseline in lakes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of isotope dilution to measure nitrogen fixation associated with the roots of sorghum and millet genotypes

Summary Genotypes of sorghum and millet have previously been found to have different amounts of root-associated acetylene reduction activity. Isotope dilution experiments using¹⁵N have been carried out to evaluate the amount of nitrogen fixed by bacteria which is incorporated into the different genotypes when grown in vermiculite in the glasshouse. Isotope dilution result indicated that the content of shoot nitrogen derived from biological nitrogen fixation varied by up to 27% between sorghum genotypes and 17% between millet genotypes. Considerable isotope dilution also resulted from uptake of non-exchangeable-N (as NH ₄ ⁺ ) in the vermiculite. It is possible that the genotypic differences in isotope dilution may reflect differences in the ability of plants to take up non-exchangeable-N, and that vermicultite is therefore an unsuitable growth medium for such studies.Published as Journal Article No. 493 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru P.O., Andhra Pradesh 502 324, India.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

Symbiotic N2 fixation in pea and field bean estimated by15N fertilizer dilution in field experiments with barley as a reference crop

Summary The total amount of nitrogen derived from symbiotic nitrogen fixation in two pea and one field bean cultivar, supplied with 50 kg N ha⁻¹ at sowing (‘starter’-N), was estimated to 165, 136, and 186 kg N ha⁻¹, respectively (three-year means). However, estimates varied considerably between the three years. At the full bloom/flat pod growth stage from 30 to 59 per cent of total N₂ fixation had taken place. The proportion of total N derived from N₂ fixation at maturity was higher in seeds than in vegetative plant parts and amounted to 59.5, 51.3 and 66.3 per cent of total above-ground plant N in the two pea cultivars and field bean, respectively (three-year means). The recovery of fertilizer N was 62.2, 70.2, 52.1, and 69.5 per cent in the two pea cultivars, field bean and barley, respectively. Growth analysis indicated that barley did not meet the claims for an ideal reference crop in the¹⁵N fertilizer dilution technique for estimating N₂ fixation in pea and field bean. ‘Starter’-N neither increased the seed yield nor the N content of the grain legumes.     

Variation in the natural abundance of 15N in the halophyte,Salicornia virginica,associated with groundwater subsidies of nitrogen in a southern California salt-marsh

To provide insight into the importance of the salt-marsh ecotone as a sink for inorganic nitrogen in perched groundwater, measurements were made of the natural abundance of ¹⁵N in dissolved NO₃-N and NH₄-N and in the salt-marsh halophyte, Salicornia virginica, along an environmental gradient from agricultural land into a salt-marsh. The increase in the natural abundance of ¹⁵N (expressed by convention as ¹⁵N) of NO₃-N, accompanied by the decrease in NO₃-N (and total dissolved inorganic N, DIN) concentration along the gradient, suggested that the salt-marsh ecotone is a site of transformation, most likely through denitrification, of inorganic nitrogen in groundwater. ¹⁵N enrichment in S. virginica (and the parasitic herb, Cuscuta salina), along the tidal marsh boundary, relative to high and middle marsh locations, indicated the retention of groundwater nitrogen as vegetative biomass. The correlation between ¹⁵N _Salicornia and ¹⁵N_NH4 suggested a preference for NH₄-N over NO₃-N during uptake by this plant. Groundwater inputs enhanced the standing crop, above-ground productivity, and nitrogen content of S. virginica but the ralative effects of pore water salinity and DIN concentration on these parameters were not determined. ¹⁵N enrichment of marsh plants by groundwater DIN inputs could prove useful in tracing the fate of these inputs in the marsh food web.     

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

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

Methods for measuring biological nitrogen fixation in grain legumes

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