Influence of dissimilarities in temporal and spatial N-uptake patterns on15N-based estimates of fixation and transfer of N in ryegrass-clover mixtures

The temporal N-uptake patterns of white clover (Trifolium repens L.) mixed with perennial ryegrass (Lolium perenne L.) and of red clover (Trifolium pratense L.) mixed with Italian ryegrass (Lolium multiflorum Lam.) were determined in successive harvests of herbage within the growth cycles of a ley established near Zürich (Switzerland). Rooting patterns were examined by injecting¹⁵N-fertilizer at soil depths ranging from 10 to 40 cm. The results were analyzed to determine the effect of variations in time and depth of N-uptake on the¹⁵N-based measurement of N from symbiosis (N_sym) and N from transfer (N_trans).Grasses in mixture appeared to have deeper rooting systems than grass monocultures, which led to an overestimation of N transfer from white clover to perennial ryegrass if¹⁵N was spread on the soil surface.White clover generally lagged behind grass in soil N- uptake. Soil N-uptake of red clover slowed down before that of the grass because % N_sym almost reached 100% during the second half of each growth cycle. However, the effect of these dissimilarities on the seasonal average of %N_sym did not exceed 2%.It is concluded that at the observed high levels of N₂ fixation, failure to account for the N-uptake patterns of the test and reference crops only slightly affected the estimates of % N_sym and % N_trans, and did not invalidate the observed differences between species.     

The response of white clover (Trifolium repens L.) seedlings to spring root temperatures: The relative roles of the plant and the Rhizobium bacteria

Three experiments are reported which examine the relative roles of host and Rhizobium genotypes as factors limiting clover (Trifolium repens L.) growth at low soil temperatures.In the first experiment un-nodulated clover and perennial ryegrass (Lolium perenne L.) were grown with non-limiting nitrate at root temperatures of 8, 10 and 12°C. The ryegrass had substantially better relative growth rates (RGR) than the clover with the biggest difference occurring at 8°C. Alterations in growth rate with temperature were more marked in clover than in ryegrass but the latter still produced several times more dry matter than clover at each temperature.In the subsequent experiments clover nodulated with different strains of rhizobia was grown with and without non-limiting additions of nitrate at root temperatures of 9, 12 and 15°C. Plants receiving nitrate generally produced more dry matter than those dependent upon Rhizobium for nitrogen but differences in yield between these treatments did not alter with temperature. This suggests that limitations imposed by nitrogen fixation are similar at both high and low temperatures. Indeed, there was some evidence that nitrogen limitations were rather more pronounced at the highest temperature. The first experiment clearly demonstrated that the clover genotype makes particularly poor use of nitrate at low root temperatures when compared to its common companion perennial ryegrass.It can be concluded that improvements in spring growth of clover will rest largely with alterations to the plant genotype and its ability to use combined nitrogen for growth at lower temperatures rather than with changes in rhizobia or any symbiotic characters.     

Interactions between nitrate uptake and N2 fixation in white clover

Summary White clover (Trifolium repens L.) plants grown in pots and supplied with the same concentration x days of¹⁵N labelled nitrate, but in contrasting patterns and doses had similar N concentrations but differed in the proportions devived from N₂ fixation and nitrate. N₂-fixation and nodule dry weight responded rapidly (2–3 days) to changes in nitrate availability. Plants exposed frequently to small doses of nitrate took up more nitrate (and hence relied less on N₂-fixation) and had greater dry weights and shoot: root ratios than those exposed to larger doses less often. In mixed ryegrass (Lolium perenne L.)/clover communities clover's ability to either successfully compete for nitrate or fix N₂ gave it consistently higher N concentrations than grass whether they were given high or low nitrate nutrient. This higher N concentration was accompanied by greater dry weights than grass in the low nitrate swards but not where high levels of nitrate were applied.     

Long- and short-term effects of crop residues on aluminum toxicity,phosphorus availability and growth of pearl millet in an acid sandy soil

Pasture swards containing perennial ryegrass (Lolium perenne L.) alone or with one of five different white clover (Trifolium repens L.) cultivars were examined for production and transfer of fixed nitrogen (N) to grass under dairy cow grazing. Grass-only swards produced 21% less than mixed clover-grass swards during the second year after sowing. Production from grass-only plots under a mowing and clipping removal regime was 44% less than from grass-only plots under grazing. Much of this difference could be attributed to N transfer. In swards without clover, the ryegrass component also decreased in favour of other grasses.The average amount of fixed N in herbage from all clover cultivars was 269 kg N ha^–1 yr^–1. Above-ground transfer of fixed N to grasses (via cow excreta) was estimated at 60 kg N ha^–1 yr^–1. Below-ground transfer of fixed N to grasses was estimated at 70 kg N ha^–1 yr^–1 by ¹⁵N dilution and was similar for all clover cultivars. Thus, about 50% of grass N was met by transfer of fixed N from white clover during the measurement year. Short-term measurements using a ¹⁵N foliar-labelling method indicated that below-ground N transfer was largest during dry summer conditions.     

Symbiotically fixed nitrogen from field- grown white and red clover mixed with ryegrasses at low levels of15N-fertilization

A field study was carried out near Zürich (Switzerland) to determine the yield of symbiotically fixed nitrogen (¹⁵N dilution) from white clover (Trifolium repens L.) grown with perennial ryegrass (Lolium perenne L) and from red clover (Trifolium pratense L.) grown with Italian ryegrass (Lolium multiflorum Lam.). A zero N fertilizer treatment was compared to a 30 kg N/ha per cut regime (90 to 150 kg ha⁻¹ annually). The annual yield of clover N derived from symbiosis averaged 131 kg ha⁻¹ (49 to 227 kg) without N fertilization and 83 kg ha⁻¹ (21 to 173 kg) with 30 kg of fertilizer N ha⁻¹ per cut in the seeding year. Values for the first production year were 308 kg ha⁻¹ (268 to 373 kg) without N fertilization and 232 kg ha⁻¹ (165 to 305 kg) with 30 kg fertilizer N ha⁻¹ per cut. The variation between years was associated mainly with the proportion of clover in the mixtures. Apparent clover-to-grass transfer of fixed N contributed up to 52 kg N ha⁻¹ per year (17 kg N ha⁻¹ on average) to the N yield of the mixtures. Percentage N derived from symbiosis averaged 75% for white and 86% for red clover. These percentages were affected only slightly by supplemental nitrogen, but declined markedly during late summer for white clover. It is concluded that the annual yield of symbiotically fixed N from clover/grass mixtures can be very high, provided that the proportion of clover in the mixtures exceeds 50% of total dry mass yield.     

Seasonal distribution of topsoil ammonium and nitrate under legume-grass and grass swards

Losses of soil N through leaching and N₂ fixation by legumes often are related to soil nitrate concentration. The seasonal distribution of soil ammonium and nitrate concentrations under ungrazed legume-grass and grass swards were evaluated on two experiments that were established in 1983 (Exp. 1) and in 1984 (Exp. 2). Treatments were white clover (Trifolium repens L.) (WC), red clover (Trifolium pratense L.) (RC), and birdsfoot trefoil (Lotus corniculatus L.) (BT), each grown with tall fescue (Festuca arundicacea Schreb.) (TF) at two legume proportions, and a pure stand of TF. The concentrations of both forms of N were measured in the top 20-cm layer during 2 years in Exp. 1 and for 1 year in Exp. 2. The concentrations of nitrate and ammonium were least in winter and spring, and greatest in summer. The concentration of nitrate for the mixtures decreased in the order WC-TF, RC-TF, and BT-TF in both summers of Exp. 1 but there were no mixture differences in Exp. 2. The concentration of soil ammonium was not affected by the treatments applied. We conclude that the concentration of soil nitrate usually was small for these swards but became greater and often dependent on species and legume proportion during summer. The concentration of soil ammonium also was greater in summer but was not affected by species or legume proportion. Journal of Paper no. J.-13359 of the Iowa Agric. and Home Econ. Exp Stn., Ames. Project 2281. Supported in part by the Facultad de Agronomía, Montevideo, Uruguay. Journal of Paper no. J.-13359 of the Iowa Agric. and Home Econ. Exp Stn., Ames. Project 2281. Supported in part by the Facultad de Agronomía, Montevideo, Uruguay.     

Nitrogen transfer from arrowleaf clover to ryegrass in field plantings

Arrowleaf clover (Trifolium vesiculosum Savi) and annual ryegrass Lolium multiflorum Lam.) commonly are overseeded in dormant bermudagrass (Cynodon dactylon L. Pers.) sod on coastal plain soils in the southeastern United States. Two field experiments were conducted in consecutive years at different sites to estimate the amount of N transferred from the clover to the annual grass. Nitrogen treatments included 50 kg N ha^-1 as ¹⁵N depleted ammonium nitrate applied in either February or April, and a check (no N applied). Three clippings were made during the cool-season from March to June. In both experiments, less than 5 kg N ha^-1 were transferred from the clover to the grass. Ryegrass yields of dry matter and total N were not increased by growing with clover. Clover growth was typical for the region; average dry matter yield in pure stand was 2,615 kg ha^-1 over the two-year period. Clover in mixed stand fixed between 20 and 60 kg N/ha. Less than 13% of N contained in ryegrass was transferred from arrowleaf clover to ryegrass at any clipping while clover was actively growing. The quantity of N transferred over the entire season was not statistically significant.     

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.     

Root exudates: a pathway for short-term N transfer from clover and ryegrass

The short-term transfer of nitrogen (N) from legumes to grasses was investigated in two laboratory studies. One study was done in pots where the roots of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were allowed to co-exist, and a second study was performed using a micro-lysimeter system designed to maintain nutrient flow from the clover to the grass, whilst removing direct contact between the root systems. The ¹⁵N-dilution technique was used to quantify the transfer of N between species. Levels of ammonia and amino acids were measured in root exudates. The amounts of N transferred were in the same order of magnitude in both the pot and micro-lysimeter experiments. In the micro-lysimeter experiment, 0.076 mg of N were transferred per plant from clover to ryegrass during the course of the experiment. Ammonium exudation was much higher than amino acid exudation. The most abundant amino acids in both clover and ryegrass root exudates were serine and glycine. However, there was no correlation between the free amino acid profile of root extracts and exudates for both plant species: Asparagine was the major amino acid in clover roots, while glutamine, glutamate and aspartate were the major amino acids in ryegrass roots. Comparison of exudates obtained from plants grown in non-sterile or axenic conditions provides evidence of plant origin of ammonium, serine and glycine.     

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.     

Dinitrogen fixation in white clover grown in pure stand and mixture with ryegrass estimated by the immobilized 15N isotope dilution method

Dinitrogen fixation in white clover (Trifolium repens L.) grown in pure stand and mixture with perennial ryegrass (Lolium perenne L.) was determined in the field using ¹⁵N isotope dilution and harvest of the shoots. The apparent transfer of clover N to perennial ryegrass was simultaneously assessed. The soil was labelled either by immobilizing ¹⁵N in organic matter prior to establishment of the sward or by using the conventional labelling procedure in which ¹⁵N fertilizer is added after sward establishment. Immobilization of ¹⁵N in the soil organic matter has not previously been used in studies of N₂ fixation in grass/clover pastures. However, this approach was a successful means of labelling, since the ¹⁵N enrichment only declined at a very slow rate during the experiment. After the second production year only 10–16% of the applied ¹⁵N was recovered in the harvested herbage. The two labelling methods gave, nonetheless, a similar estimate of the percentage of clover N derived from N₂ fixation. In pure stand clover, 75–94% of the N was derived from N₂ fixation and in the mixture 85–97%. The dry matter yield of the clover in mixture as percentage of total dry matter yield was relatively high and increased from 59% in the first to 65% in the second production year. The average daily N₂ fixation rate in the mixture-grown clover varied from less than 0.5 kg N ha⁻¹ day⁻¹ in autumn to more than 2.6 kg N ha⁻¹ day⁻¹ in June. For clover in pure stand the average N₂ fixation rate was greater and varied between 0.5 and 3.3 kg N ha⁻¹ day⁻¹, but with the same seasonal pattern as for clover in mixture. The amount of N fixed in the mixture was 23, 187 and 177 kg N ha⁻¹ in the seeding, first and second production year, respectively, whereas pure stand clover fixed 28, 262 and 211 kg N ha⁻¹ in the three years. The apparent transfer of clover N to grass was negligible in the seeding year, but clover N deposited in the rhizosphere or released by turnover of stolons, roots and nodules, contributed 19 and 28 kg N ha⁻¹ to the grass in the first and second production year, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

Quantification of N2-fixation and survival of inoculated diazotrophs associated with roots of Kallar grass

White clover plants were grown for 97 days under two temperature regimes (20/15°C and 8/5°C day/night temperatures) and were supplied with either small amounts (a total of 80 mg N pot^–1) of ammonium (NH ₄ ⁺ ) or nitrate (NO ₃ ^– ) nitrogen, or received no mineral N and relied on N₂ fixation. Greatest growth and total leaf area of clover plants occurred in N₂ fixing and NO ₃ ^– -fed plants grown at 20/15°C and poorest growth occurred in NH ₄ ⁺ -fed plants grown at 8/5°C. Nodule mass per plant was greater at 8/5°C due to increased nodule numbers rather than increased dry weight per nodule. This compensated to some extent for the reduced N₂-fixing activity per unit dry weight of nodule tissue found at the low growth temperature up to 116 d after sowing, but thereafter both activity per nodule dry weight and activity per plant were greater at the low temperature. Highest nitrate reductase activity (NRA) per g fresh weight and total activity per leaf, petiole or root occurred in NO ₃ ^– -fed plants at 8/5°C. Low growth temperature resulted in a greater partitioning of total plant NRA to the roots of NO ₃ ^– -fed plants. The results are considered in relation to the use of N fertiliser in the spring under field conditions.     

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.     

Effect of white clover cultivar on apparent transfer of nitrogen from clover to grass and estimation of relative turnover rates of nitrogen in roots

The apparent transfer of N from clover to associated grass was evaluated over a four year period both on the basis of harvested herbage and by taking account of changes in N in stubble and root (to 10 cm depth) in swards with perennial ryegrass and three different white clover cultivars differing in leaf size. The large leaved Aran transferred 15% of its nitrogen while Huia transferred 24% and the small leaved Kent Wild White transferred 34%. When changes in stubble and root N were taken into account the percentage of N transferred was calculated to be 5% less than in harvested herbage only, as the small leaved types had proportionately more N in the roots and stolons, but the large leaved type was probably more competitive towards the grass.Loss of N from clover roots from July to October was compared to that from grass roots in a grass/white clover sward continuously stocked with steers using a method which incorporated tissue turnover and ¹⁵N dilution techniques. Less than 1 mg N m^-2 d^-1 was lost from the grass roots. In contrast 8 mg m^-2 d^-1 were estimated to be lost from clover roots while 12 mg N m^-2 d^-1 were assimilated.It is concluded that clover cultivar and competitive ability on grass have to be taken into account together with the relationship between N turnover in roots and N available for grass growth when modelling N transfer in grass/clover associations.     

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.     

Effect of Nitrogen Supply on the Grass and Clover Components of Simulated Mixed Swards Grown under Favourable Environmental Conditions II. Nitrogen Fixation and Nitrate Uptake

Simulated mixed swards of Perennial Ryegrass (Lolium perenneL.) cv. S23 and White clover (Trifolium repens L.) cv. S100were grown from seed under a constant 20 °C day/15 °Cnight temperature regime and harvested at intervals over and88 d growht period. The swards received a nutrient solutiondaily, which was either High (220 mg l^–1) or Low (10 mgl^–1) in nitrate N. The nitrate was labelled with the ¹⁵Nisotope. An acetylene reduction assay was carried out on eachsward just prior to harvest. Rates of acetylene reduction agreed qualitatively with the ^l5Nanalyses but absolute values did not match (assuming a 4:1 C₂H₄:N₂ratio) and errors in the acetylene assay are discussed. In theLow-N swards clover relied almost entirely on symbioticallyfixed N₂, fixing more than ten times as much as the High-N cloverplants. In the Low-N treatment the grass was N-deficient despiteobtaining much more nitrate per unit root dry weight than clover.In the High-N swards, however, clover took up more nitrate perunit root weight than grass. The High-N clover plants also fixedsome N₂ and maintained a higher total-N content than grass throughoutthe period. There was no evidence of transfer of symbioticallyfixed N from the clover to the grass in either treatment. Trifolium repens, Lolium perenne, nitrate, nitrogen fixation, ¹⁵N, acetylene reduction     

Sitona weevils (Coleoptera: Curculionidae) as agents for rapid transfer of nitrogen from white clover (Trifolium repens L.) to perennial ryegrass (Lolium perenne L.)

The effects of root feeding by larvae of Sitona hispidulus (F.) (a common weevil pest of white clover) on the rate of transfer of nitrogen between plants of white clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) were investigated using a nutrient slant board technique. Clover plants, labelled with ¹⁵N were grown adjacent to ryegrass plants and were either infested with Sitona larvae or not infested. Ryegrass plants associated with the infested clover plants had a significantly higher dry matter yield and nitrogen content (75% and 74% respectively) than the uninvested plants, after 33 days exposure to insect herbivory. It was concluded that root feeding insects could play an important role in the cycling of nitrogen in grass/clover swards.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Effect of Temperature and Nitrogen Supply on the Growth of Perennial Ryegrass and White Clover. 2. A Comparison of Monocultures and Mixed Swards

White clover (Trifolium repens L.) and Perennial ryegrass (Loliumperenne L.) plants were grown, in Perlite, in simulated swardsas either monocultures or mixtures of equal plant numbers. Theywere supplied with a nutrient solution either high (220 µgg^–1) or low (40 µg g^–1) in ¹⁵N-labelled nitrateand grown to ceiling yield at either high (20°C day/15°Cnight) or low (10°C day/8°C night) temperature. Temperature had little effect on the maximum rates of grosscanopy photosynthesis which were similar in High-N grass andHigh-N and Low-N clover monocultures. However these maxima werereached more slowly in clover than grass, and more slowly atlow rather than high temperature. Nitrogen supply increasedphotosynthesis in grass but not in clover. Clover had higherN contents than grass in all four treatments, although in anygiven treatment its N content was lower, and contribution ofN₂-fixation relative to nitrate uptake higher, in mixture thanin monoculture. Conversely, grass had higher N contents in mixturethan monoculture, because more nitrate was available per plantand not because of transfer of biologically fixed N from clover. Under Low-N, clover outyielded grass in mixture, particularlyat high temperature. The grass plants in the Low-N mixtureshad higher N contents and higher SLA, LAR and shoot: root ratiosthan those in monoculture. It is proposed that competition forlight is the cause of the low relative yield and negative aggressivityof grass in these swards. Under High-N, grass outyielded cloverin monoculture and mixture, at both temperatures but particularlyat low temperature when grass had a high aggressivity. Nitrogenand yield component analyses shed no light on clover's apparentlylow competitive ability and evidence is drawn from the previouspaper to demonstrate that grass grew faster than clover onlyas spaced individuals during non-com petitive growth. The relativemerits of measures of competitive ability based on final harvestdata and physiological data taken over a growth period are discussed. Trifolium repens L., white clover, Lolium perenne, perennial ryegrass, competition, temperature, nitrogen     

Nitrogen fixation in perennial forage legumes in the field

Nitrogen acquisition is one of the most important factors for plant production, and N contribution from biological N₂ fixation can reduce the need for industrial N fertilizers. Perennial forages are widespread in temperate and boreal areas, where much of the agriculture is based on livestock production. Due to the symbiosis with N₂-fixing rhizobia, perennial forage legumes have great potential to increase sustainability in such grassland farming systems. The present work is a summary of a large number of studies investigating N₂ fixation in three perennial forage legumes primarily relating to ungrazed northern temperate/boreal areas. Reported rates of N₂ fixation in above-ground plant tissues were in the range of up to 373 kg N ha^–1 year^–1 in red clover (Trifolium pratense L.), 545 kg N ha^–1 year^–1 in white clover (T. repens L.) and 350 kg N ha^–1 year^–1 in alfalfa (Medicago sativa L.). When grown in mixtures with grasses, these species took a large fraction of their nitrogen from N₂ fixation (average around 80%), regardless of management, dry matter yield and location. There was a large variation in N₂ fixation data and part of this variation was ascribed to differences in plant production between years. Studies with experiments at more than one site showed that also geographic location was an important source of variation. On the other hand, when all data were plotted against latitude, there was no simple correlation. Climatic conditions seem therefore to give as high N₂ fixation per ha and year in northern areas (around 60°N) as in areas with a milder climate (around 40°N). Analyzing whole plants or just above-ground plant parts influenced the estimate of N₂ fixation, and most reported values were underestimated since roots were not included. Despite large differences in environmental conditions, such as N fertilization and geographic location, N₂ fixation (Nfix; kg N per ha and year) was significantly (P<0.001) correlated to legume dry matter yield (DM; kg per ha and year). Very rough, but nevertheless valuable estimations of Nfix in legume/grass mixtures (roots not considered) are given by Nfix = 0.026DM + 7 for T. pratense, Nfix = 0.031DM + 24 for T. repens, and Nfix = 0.021DM + 17 for M. sativa.