Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

Biological nitrogen fixation associated with sugar cane

A recent¹⁵N dilution/N balance study confirmed that certain sugar cane varieties are capable of obtaining large contributions of nitrogen from plant-associated N₂ fixation. It was estimated that up to 60 to 80% of plant N could be derived from this source, and under good conditions of water and mineral nutrient supply, it may be possible to dispense with N fertilization of these varieties altogether. The recently discovered bacterium,Acetobacter diazotrophicus, apparently responsible for this N₂ fixation associated with the plants, has unique physiological properties for a diazotroph, such as tolerance to low pH, and high sugar and salt concentrations, lack of nitrate reductase, and nitrogenase activity which tolerates short-term exposure to ammonium. Furthermore, it also behaves as an endophyte, in that it is unable to infect sugar cane plants unless through damaged tissue or by means of VA mycorrhizae and is propagated via the planting material (stem pieces).     

Natural15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes

The¹⁵N abundance of plants usually closely reflects the¹⁵N abundance of their major immediate N source(s); plant-available soil N in the case of non-N₂-fixing plants and atmospheric N₂ in the case of N₂ fixing plants. The¹⁵N abundance values of these sources are usually sufficiently different from each other that a significant and systematic difference in the¹⁵N abundance between the two kinds of plants can be detected. This difference provides the basis for the natural¹⁵N abundance method of estimating the relative contribution of atmospheric N₂ to N₂-fixing plants growing in natural and agricultural settings. The natural¹⁵N abundance method has certain advantages over more conventional methods, particularly in natural ecosystems, since disturbance of the system is not required and the measurements may be made on samples dried in the field. This method has been tested mainly with legumes in agricultural settings. The tests have demonstrated the validity of this method of arriving at semi-quantitative estimates of biological N₂-fixation in these settings. More limited tests and applications have been made for legumes in natural ecosystems. An understanding of the limits and utility of this method in these systems is beginning to emerge. Examples of systematic measurements of differences in¹⁵N abundance between non-legume N₂-fixing systems and neighbouring non-fixing systems are more unusual. In principle, application of the method to estimate N₂-fixation by nodulated non-legumes, using the natural¹⁵N abundance method, is as feasible as estimating N₂-fixation by legumes. Most of the studies involving N₂-fixing non-legumes are with this type of system (e.g., Ceanothus, Chamabatia, Eleagnus, Alnus, Myrica, and so forth). Resuls of these studies are described. Applicability for associative N₂-fixation is an empirical question, the answer to which probably depends upon the degree to which fixed N goes predominantly to the plant rather than to the soil N pool. The natural¹⁵N abundance method is probably not well suited to assessing the contribution of N₂-fixation by free-living microorganisms in their natural habitat, particularly soil microorganisms.This work was supported in part by subcontracts under grants from the US National Science Foundation (DEB79-21971 and BSR821618)     

Bi-directional transfer of nitrogen between alfalfa and bromegrass: Short and long term evidence

Transfer of N from legumes to associated non-legumes has been demonstrated under a wide range of conditions. Because legumes are able to derive their N requirements from N₂ fixation, legumes can serve, through the transfer of N, as a source of N for accompanying non-legumes. Studies, therefore, are often limited to the transfer of N from the legume to the non-legume. However, legumes preferentially rely on available soil N as their source of N. To determine whether N can be transferred from a non-legume to a legume, two greenhouse experiments were conducted. In the short-term N-transfer experiment, a portion of the foliage of meadow bromegrass (Bromus riparius Rhem.) or alfalfa (Medicago sativa L.) was immersed in a highly labelled ¹⁵N-solution and following a 64 h incubation, the roots and leaves of the associated alfalfa and bromegrass were analyzed for ¹⁵N. In the long-term N transfer experiment, alfalfa and bromegrass were grown in an ¹⁵N-labelled nutrient solution and transplanted in pots with unlabelled bromegrass and alfalfa plants. Plants were harvested at 50 and 79 d after transplanting and analyzed for ¹⁵N content. Whether alfalfa or bromegrass were the donor plants in the short-term experiment, roots and leaves of all neighbouring alfalfa and bromegrass plants were enriched with ¹⁵N. Similarly, when alfalfa or bromegrass was labelled in the long-term experiment, the roots and shoots of neighbouring alfalfa and bromegrass plants became enriched with ¹⁵N. These two studies conclusively show that within a short period of time, N is transferred from both the N₂-fixing legume to the associated non-legume and also from the non-legume to the N₂-fixing legume. The occurrence of a bi-directional N transfer between N₂-fixing and non-N₂-fixing plants should be taken into consideration when the intensity of N cycling and the directional flow of N in pastures and natural ecosystems are investigated.     

15N-Determined effect of inoculation with N2 fixing bacteria on nitrogen assimilation in Western Canadian wheats

Summary A two-year field study was undertaken using¹⁵N isotope techniques to differentiate between stimulation of N uptake and N₂ fixation in Western Canadian cultivars of spring wheat (Triticum aestivum L. emend Thell) and durum (T. turgidum L. emend Bowden) in response to inoculation with N₂-fixing bacteria. Bacterial inoculation either had no effect or lowered the % N derived from the fertilizer and the fertilizer use efficiency. Despite the depression of fertilizer uptake, inoculants did not alter the relative uptake from soil and fertilizer-N pools indicating that bacterial inoculation did not alter rooting patterns. Nitrogen-15 isotope dilution indicated that N₂ fixation did occur. In 1984, % plant N derived from the atmosphere (% Ndfa) due to inoculation with Bacillus C-11-25 averaged 23.9% while that withAzospirillum brasilense ATCC 29729 (Cd) averaged 15.5%. In 1985, higher soil N levels reduced these values by approximately one-half. Cultivar x inoculant interactions, while significant, were not consistent across years. However, these interactions did not affect cultivars ‘Cadet’ and ‘Rescue’. In agreement with previous results, ‘Cadet’ performed well with all inoculants in both years while ‘Rescue’ performed poorly. Among 1984 treatments, the N increament in inoculated plants was positively correlated with % Ndfa but no such correlation existed in 1985. N₂ fixation averaged over all cultivars and strains was 17.9 and 6.7 kg N fixed ha⁻¹ in 1984 and 1985, respectively. Highest rates of N₂ fixation were estimated at 52.4 kg N ha⁻¹ for ‘Cadet’ in 1984 and 31.3 kg N ha⁻¹ for ‘Owens’ in 1985, both inoculated with Bacillus C-11-25, an isolate from southern Alberta soils. Inoculation with either ofAzospirillum brasilense strain Cd (ATCC29729) or 245 did not result in as consistent or as high N₂ fixation, suggesting that these wheats had not evolved genetic compatability with this exogenous microorganism. These agronomically significant amounts of N₂ fixation occurred under optimally controlled experimental conditions in the field. It is yet to be determined if N₂ fixation would occur in response to bacterial inoculation under dryland conditions commonly occurring in Western Canada. Contribution from Agriculture Canada Research Station, Lethbridge, Alberta, Canada.     

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.     

Nitrogen fixation associated with non-legumes in agriculture

Summary This review examines the nitrogen cycle in upland agricultural situations where nonlegume N₂-fixation is likely to be important for crop growth. Evidence for associative fixation is adduced from accumulation of N in the top 15 cm soil under grasses, from N balances for crop production obtained from both pot and field experiments, in tropical and temperate environments, measurements of nitrogen (C₂H₂ reduction) activity, uptake of¹⁵N₂ by plants and¹⁵N isotope dilution. Factors influencing the activity such as the provision of carbon substrate by the plant and the efficiency of its utilisation by the bacteria, plant cultivar, soil moisture and N levels, and inoculation with N₂-fixing bacteria are discussed. Crop responses to inoculation withAzospirillum are detailed. The breakdown of crop residues, particularly straw, can support large levels of N₂-fixation. Cyanobacteria as crusts on the soil surface also fix nitrogen actively in many environments. Fixation by the nodulated, non-legume treesCasuarina andParasponia has beneficial effects in some cropping systems in Asia. I conclude that nonlegume N₂-fixation makes a significant contribution to the production of some major cereal crops in both temperate and tropical environments.     

Alder and lupine enhance nitrogen cycling in a degraded forest soil in Northern Sweden

Positive effects of legumes and actinorhizal plants on N-poor soils have been observed in many studies but few have been done at high latitudes, which was the location of our study. We measured N₂ fixation and several indices of soil N at a site near the Arctic Circle in northern Sweden. More than 20 years ago lupine (Lupinus nootkatensis Donn) and gray alder (Alnus incana L. Moench) were planted on this degraded forest site. We measured total soil N, net N mineralization and nitrification with a buried bag technique, and fluxes of NH⁺ ₄ and NO^– ₃ as collected on ion exchange membranes. We also estimated N₂ fixation activity of the N₂-fixing plants by the natural abundance of ¹⁵N of leaves with Betula pendula Roth. as reference species. Foliar nitrogen in the N₂-fixing plants was almost totally derived from N₂ fixation. Plots containing N₂-fixing species generally had significantly higher soil N and N availability than a control plot without N₂-fixing plants. Taken together, all measurements indicated that N₂-fixing plants can be used to effectively improve soil fertility at high latitudes in northern Sweden.     

The role of root-associatedKlebsiella pneumoniae in the nitrogen nutrition ofPoa pratensis andTriticum aestivum as estimated by the method of15N isotope dilution

Summary The technique of¹⁵N isotope dilution was used to verify that nitrogen was fixed and transferred to the plant byKlebsiella pneumoniae strain Pp in association withPoa pratensis orTriticum aestivum. Surface sterilized, sprouting seeds were inoculated withK. pneumoniae and grown in sand in modified Leonard jars. Potassium nitrate enriched with¹⁵N was used to provide N concentrations ranging from 10–40 mg Nl^–1 nutrient solution. After 10–18 weeks the shoots and roots were analyzed separately for dry matter, N content, total N, and atom %¹⁵N excess. The acetylene reduction technique was used to test for the presence of N₂-fixing organisms on the roots. The data from¹⁵N isotope dilution demonstrated that up to 33.8% of N in the shoots ofP. pratensis and 15.9% in those ofT. aestivum were derived from associative N₂ fixation byK. pneumoniae. In most experiments the dry matter yield, N content, and total N yield of the shoots ofP. pratensis were increased byK. pneumoniae inoculation, whereas inoculation had no significant effect on the dry matter yield, N content or total N of the shoots ofT. aestivum.     

Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research

Over the last few years research in the area of biological nitrogen fixation (BNF) associated with cereals and grasses has become divided into two areas. On the one hand there have been a large number of reports of responses of field-grown plants to inoculation with N₂-fixing bacteria, principallyAzospirillum spp. On the other hand there have been several reports of significant contributions of associated BNF to the nutrition of several crops, including wetland rice, sugar cane and some forage grasses. However, where BNF contributions have definitely been established no certain information is available as to the diazotrophic organisms responsible. Furthermore, certain recent reports indicate that, at least in some cases, responses of plants to inoculation withAzospirillum spp. have been shown not to be due to BNF contributions. In this paper we review some recent progress in this field, particularly at our institute in Rio de Janeiro, concerning specificity of selected Azospirillum strains in the infection of cereal roots and the promotion of responses in the host plants. The possible mechanisms of plant response are discussed including the possibility that plant growth substances or bacterial nitrate reductase are involved. The application of¹⁵N and N balance techniques to the quantification of plant associated BNF are considered and the possible strategies that may be adopted to further the understanding of true N₂-fixing plant/diazotroph associations. The recent discovery of many more plant-associated N₂-fixing bacteria suggests that further research in this area may eventually lead to the development of such associations with applications for agricultural productivity.     

Nitrogen accumulation in sole and mixed stands of sweet-blue lupin (Lupinus angustifolius L.), ryegrass and oats

Nitrogen fixation was measured in monocropped sweet-blue lupin (Lupinus angustifolius), lupin intercropped with two ryegrass (Lolium multiflorum) cultivars or with oats (Avena sativa) on an Andosol soil, using the ¹⁵N isotope dilution method. At 117 days after planting and at a mean temperature below 10°C, monocropped lupin derived an average of 92% or 195 kg N ha⁻¹ of its N from N₂ fixation. Intercropping lupin with cereals increased (p<0.05) the percentage of N derived from atmospheric N₂ (% Ndfa) to a mean of 96%. Compared to the monocropped, total N fixed per hectare in intercropped lupin declined approximately 50%, in line with the decrease in seeding rate and dry matter yield. With these high values of N₂ fixation, selection of the reference crop was not a problem; all the cereals, intercropped or grown singly produced similar estimates of N₂ fixed in lupin. It was deduced from the ¹⁵N data that significant N transfer occurred from lupin to intercropped Italian ryegrass but not to intercropped Westerwoldian ryegrass or to oats. Doubling the ¹⁵N fertilizer rate from 30 to 60 kg N ha⁻¹ decreased % Ndfa to 86% (p<0.05), but total N fixed was unaltered. These results indicate that lupin has a high potential for N₂ fixation at low temperatures, and can maintain higher rates of N₂ fixation in soils of high N than many other forage and pasture legumes.     

Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

Growth and N2 fixation in mixed cropping of Medicago arborea and Atriplex halimus grown on a salt-affected soil using a 15N tracer technique

Abstract

The objective of this study was to evaluate dry matter (DM), nitrogen yield, N₂ fixation (Ndfa) and soil N uptake (Ndfs) in the shrubby medic (Medicago arborea) and saltbush (Atriplex halimus) grown in pots either solely or in a mixture on a salt-affected soil, using ¹⁵N dilution method. The combined DM of both species was considerably higher than that of solely grown shrubs. The inclusion of saltbush in the mixed cropping system decreased Ndfs by shrubby medic and enhanced % Ndfa without affecting amounts of N₂ fixed. It can be concluded that the use of mixed cropping system of shrubby medic and saltbush could be a promising bio-saline agricultural approach to utilize salt affected soils in terms of forage yield and N₂ fixation.     

Effect of plant genotype and nitrogen fertilizer on symbiotic nitrogen fixation by soybean cultivars

Summary Isotopic as well as non-isotopic methods were used to assess symbiotic nitrogen fixation within eight soybean [Glycine max (L.) Merr.] cultivars grown at 20 and 100 kg N/ha levels of nitrogen fertilizer under field conditions.The¹⁵N methodology revealed large differences between soybean cultivars in their abilities to support nitrogen fixation. In almost all cases, the application of 100 kg N/ha resulted in lower N₂ fixed in soybean than at 20 kg N/ha in the first year of the study. However, N₂ fixed in one cultivar, Dunadja, was not significantly affected by the higher rate of N fertilizer application. These results were confirmed by measurements of acetylene reduction activity, nodule dry weight and N₂ fixed as measured by the difference method. Further proof of differences in N₂ fixed within soybean cultivars and the ability of Dunadja to fix similar amounts of N₂ at 20 and 100 kg N/ha was obtained during a second year experiment. Dunadja yield was affected by N fertilizer and produced larger yield at 100 kg N/ha than at 20 kg N/ha. This type of cultivar could be particularly useful in situations where soil N levels are high or where there is need to apply high amounts of N fertilizer.The present study reveals the great variability between legume germplasms in the ability to fix N₂ at different inorganic N levels, and also the potential that exists in breeding for nitrogen fixation associative traits. The¹⁵N methodology offers a unique tool to evaluate germplasms directly in the field for their N₂ fixation abilities at different N fertilizer levels.     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

Estimation of biological nitrogen fixation associated withBrachiaria andPaspalum grasses using15N labelled organic matter and fertilizer

Summary Six pasture grasses,Paspalum notatum cv batatais,P. notatum cv pensacola,Brachiaria radicans, B. ruziziensis, B. decumbens andB. humidicola, were grown in concrete cylinders (60 cm diameter) in the field for 31 months. The soil was amended with either a single addition of¹⁵N labelled organic matter or frequent small (2 kg N. ha^–1) additions of¹⁵N enriched (NH₄)₂SO₄. In the labelled fertilizer treatment soil analysis revealed that there was a very drastic change in¹⁵N enrichment in plant-available nitrogen (NO ₃ ^– +NH ₄ ⁺ ) with depth. The different grass cultivars recovered different quantities of applied labelled N, and evidence was obtained to suggest that the roots exploited the soil to different depths thus obtaining different¹⁵N enrichments in soil derived N. This invalidated the application of the isotope dilution technique to estimate the contribution of nitrogen fixation to the grass cultivars in this treatment. In the labelled organic matter treatment the¹⁵N label in the plant-available N declined at a decreasing rate during the experiment until in the last 12 months the decrease was only from 0.274 to 0.222 atom % excess. There was little change in¹⁵N enrichment of available N with depth, hence it was concluded that although the grasses recovered different quantities of labelled N, they all obtained virtually the same¹⁵N enrichment in soil derived N. Data from the final harvests of this treatment indicated thatB. humidicola andB. decumbens obtained 30 and 40% respectively of their nitrogen from N₂ fixation amounting to an input of 30 and 45 kg N.ha^–1 year^–1 respectively.     

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.     

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.     

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

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