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.     

Yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.) in Peru

Two field experiments were performed to evaluate the nitrogen fixation potential of twenty common bean cultivars and breeding lines during summer and winter seasons of 1986 and 1988, respectively. The ¹⁵N isotope dilution method was used to quantify N₂ fixation. The cultivars and breeding lines were variable in terms of their N₂ fixation. The cv. Caballero was very efficient, with more than 50% N derived from the atmosphere and 60–80 kg N ha^–1 fixed in both seasons. Other cultivars were less efficient, since the poorest ones derived less than 30% of their nitrogen from the atmosphere and fixed less than 20 kg N ha^–1. After additional testing the best cultivars may be used directly by the farmers for cultivation. The experiments have provided information about which genotypes may be used to breed for enhanced fixation in common bean.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

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.     

Measurements of nitrogen fixation in fababean at different N fertilizer rates using the 15N isotope dilution and ‘A-value’ methods

The ¹⁵N isotope dilution and A-value methods were used to measure biological nitrogen (N₂) fixation in field grown fababean (Vicia faba L.), over a 2-year period. Four N rates, 20, 100, 200 and 400 kg N ha^–1 were examined. The two isotope methods gave similar values of % N derived from the atmosphere (%Ndfa). With 20 kg N ha^–1, %Ndfa in fababean was about 85% in both years. Increasing the N rate to 100 kg N ha^–1 decreased N₂ fixation slightly to 75%. Further reductions in N₂ fixed to 60 and 43% occurred where 200 and 400 kg N ha^–1 were applied, respectively. Thus even higher rates of N than normally applied in farming practice could not completely suppress N₂ fixation in fababean.We also devised one equation for both the isotope dilution and A-value approaches, thereby (i) avoiding the need for different calculations for the ¹⁵N isotope methods, and (ii) showing once again that the isotope dilution and A-value methods are mathematically and conceptually identical.     

Seasonal N2 fixation by cool-season pulses based on several15N methods

Summary Accurate estimates of N₂ fixation by legumes are requisite to determine their net contribution of fixed N₂ to the soil N pool. However, estimates of N₂ fixation derived with the traditional¹⁵N methods of isotope dilution and A_N value are costly.Field experiments utilizing¹⁵N-enriched (NH₄)₂SO₄ were conducted to evaluate a modified difference method for determining N₂ fixation by fababean, lentil, Alaska pea, Austrian winter pea, blue lupin and chickpea, and to quantify their net contribution of fixed N₂ to the soil N pool. Spring wheat and non-nodulated chickpea, each fertilized with two N rates, were utilized as non-fixing controls.Estimates of N₂ fixation based on the two control crops were similar. Increasing the N rate to the controls reduced A_N values 32, 18 and 43% respectively in 1981, 1982 and 1983 resulting in greater N₂ fixation estimates. Mean seasonal N₂ fixation by fababean, lentil and Austrian winter pea was near 80 kg N ha^–1, pea and blue lupin near 60 kg N ha^–1, and chickpea less than 10 kg N ha^–1. The net effects of the legume crops on the soil N pool ranged from a 70 kg N ha^–1 input by lentil in 1982, to a removal of 48 kg N ha^–1 by chickpea in 1983.Estimates of N₂ fixation obtained by the proposed modified difference method approximate those derived by the isotope dilution technique, are determined with less cost, and are more reliable than the total plant N procedure.Scientific paper No. 6605. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164, U.S.A.     

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.     

Nitrogen fixation in breeding lines of Phaseolus vulgaris L.

Nitrogen fixation in nine common bean (Phaseolus vulgaris L.) lines was estimated using the ¹⁵N isotope dilution method at two locations in two seasons. In the first season at one location no N₂ fixation was detected while in the second season up to 51 kg N ha^–1 were estimated. There were significant differences between lines and correlations between trials were significant for the amounts of N₂ fixed, but not for total shoot nitrogen. The plants that fixed the most nitrogen nodulated rapidly after germination. Differences in maximum nodule mass, but not specific nodule activity, were detected also.     

N2 fixation in Thai soybeans: Effect of tillage and inoculation on15N-determined N2 fixation in recommended cultivars and advanced breeding lines

The practice of seeding soybeans following paddy rice in Thailand has encountered difficulties in seedling germination, nodulation and crop establishment. This research project evaluated the choice of a non-fixing control to quantify N₂ fixation by¹⁵N isotope dilution, and the effect of tillage regime, soybean cultivar, strain ofBradyrhizobium japonicum and P fertilization on yield and N₂ fixation after paddy rice in northern and central Thailand.Japanese non-nodulating lines Tol-0 and A62-2 were the most appropriatecontrol plants for¹⁵N isotope dilution for Thai soybeans in these soils which contained indigenous rhizobia. Cereals such as maize, sorghum and barley were also appropriate controls at some sites. The choice of the appropriate non-fixing control plant for the¹⁵N isotope dilution technique remains a dilemma and no alternative exists other than to use several possible controls with each experiment. Acetylene reduction assay (ARA) proved of little value for screening varieties on their N₂ fixing capacity.The recommended Thai soybean cultivars (SJ1, 2, 4, 5) and an advanced line 16–4 differed little in their ability to support N₂ fixation or yield, possibly due to similar breeding ancestry. The ten AVRDC (ASET) lines showed considerable genotypic control in their ability to utilize their three available N sources (soil, fertilizer, atmosphere) and to translate them into yields. None of these lines were consistently superior to Thai cultivars SJ4 or SJ5 although ASET lines 129, 209 and 217 showed considerable promise.Neither recommended Thai or ASET cultivars were affected by tillage regime. Zero tillage resulted in superior N₂ fixation and yield at two sites but conventional tillage was superior at another site. Soybean cultivars grown in Thailand were well adapted to zero tillage. Levels of N₂ fixation were similar to world figures, averaging more than 100 kg N ha^–1 and supplying over 50% of the plant's N yield. However, seed yields seldom exceeded 2 t ha^–1, well below yields for temperately-grown soybeans. It is not clear why Thai soybeans support N₂ fixation, but do not translate this into higher seed yields.     

Application of 15N and xylem ureide methods for assessing N2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N₂ fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N₂ fixation (%Pfix) and the amounts of N₂ fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N₂ fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the ¹⁵N natural abundance technique were compared with values obtained from ¹⁵N enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies.Inoculated seedlings of each of the three shrub legume species were planted in dense stands (1.5 m rows, 0.5 m between trees) in two randomised blocks. The northern block was used solely for natural abundance measurements, while ¹⁵N-enriched KNO₃ (10 atom % ¹⁵N excess) was applied four times over a 52 week period to plots in the southern block. The non-nodulating tree legume Senna spectabilis (formally Cassia spectabilis) was used as a non-N₂-fixing reference for the ¹⁵N-based procedures, with Guinea grass (Panicum maximum) included as an additional non-fixing check. Growth by the trees above 75 cm was first cut and removed after 22 weeks and regrowth was subsequently pruned periodically for another 95 weeks. Sampling for dry matter production, N yield and estimates of Pfix were restricted to the central four of the 32 plants which constituted each replicate plot. Information generated during the 117 week study indicated that estimates of Pfix by ¹⁵N natural abundance were closely similar to values derived with ¹⁵N-enrichment or sap ureides. The data indicated that Calliandra had a reduced reliance upon N₂ fixation relative to Gliricidia and Codariocalyx for the first 65 weeks after establishment. This appeared to be due to more prolifc root growth by Calliandra than either of the other N₂-fixing species and an ability to extract a greater proportion of its N requirements from soil mineral N. However, after week 65 and for the remainder of the experiment, estimates of Pfix for Calliandra were similar to the other shrub legumes. Over 117 weeks, prunings from Calliandra and Gliricidia had removed 52–58 t dry matter ha^-1, and between 1471 and 1678 kg N ha^-1, of which 1026–1063 kg N ha^-1 was estimated to have been derived from N₂ fixation. At the time of final harvest, 65–73% of the fixed N was present in shoot regrowth of the N₂ fixing shrubs, 9–18% in the roots, 15% in the trunk, and 2–6% in fallen leaves.     

The effect of a single application of cow urine on annual N2 fixation under varying simulated grazing intensity, as measured by four 15N isotope techniques

The effects of dairy cow urine and defoliation severity on biological nitrogen fixation and pasture production of a mixed ryegrass-white clover sward were investigated over 12 months using mowing for defoliation. A single application of urine (equivalent to 746 kg N ha^–1), was applied in late spring to plots immediately after light and moderately-severe defoliation (35 mm and 85 mm cutting heights, respectively) treatments were imposed. Estimates of percentage clover N derived from N₂ fixation (%Ndfa) were compared by labelling the soil with ¹⁵N either by applying a low rate of ¹⁵N-labelled ammonium sulphate, immobilising ¹⁵N in soil organic matter, adding ¹⁵N to applied urine, or by utilising the small differences in natural abundance of ¹⁵N in soil. Urine application increased annual grass production by 85%, but had little effect on annual clover production. However, urine caused a marked decline in %Ndfa (using an average of all ¹⁵N methods) from 84% to a low of 22% by 108 days, with recovery to control levels taking almost a year. As a result, total N fixed (in above ground clover herbage) was reduced from 232 to 145 kg N ha^–1 yr^–1. Moderately–severe defoliation had no immediate effect on N₂ fixation, but after 108 days the %Ndfa was consistently higher than light defoliation during summer and autumn, and increased by up to 18%, coinciding with an increase in growth of weeds and summer-grass species. Annual N₂ fixation was 218 kg N ha^–1 yr^–1 under moderately-severe defoliation compared to 160 kg N ha^–1 yr^–1 under light defoliation. Estimates of %Ndfa were generally similar when ¹⁵N-labelled or immobilised ¹⁵N were used to label soil regardless of urine and defoliation severity. The natural abundance technique gave highly variable estimates of %Ndfa (–56 to 24%) during the first 23 days after urine application but, thereafter, estimates of %Ndfa were similar to those using ¹⁵N-labelling methods. In contrast, in urine treated plots the use of ¹⁵N-labelled urine gave estimates of %Ndfa that were 20–30% below values calculated using conventional ¹⁵N-labelling during the first 161 days. These differences were probably due to differences in the rooting depth between ryegrass and white clover in conjunction with treatment differences in ¹⁵N distribution with depth. This study shows that urine has a prolonged effect on reducing N₂ fixation in pasture. In addition, defoliation severity is a potential pasture management tool for strategically enhancing N₂ fixation.     

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.     

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.     

Inhibition of N2 fixation by white clover (Trifolium repens L.) at low concentrations of NO inf3 sup− in flowing solution culture

The impact of sustained low external concentrations of NO ₃ ^– (0, 10, 100 and 1000 mmol m^–3) on plant growth and the relative acquisition of N through N₂ fixation and NO ₃ ^– uptake by established, nodulated white clover (Trifolium repens L. cv. Blanca) was studied over 28 days in flowing solution culture. Nitrogen fixation was measured by N difference and ¹⁵N dilution methods. Plants supplied with NO ₃ ^– achieved higher relative growth rates (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabmiEayaara% aaaa!3702!\[\bar x\]=0.091 d^–1) compared with control plants dependent on N₂ fixation (0.073 d^–1). Nitrate plants showed progressive increases in shoot: root d.w. ratios from 4 to 6.5–7.6 between days 0–28, compared with 5.1 on day 28 for control plants. Increases in both nodule d.w. and numbers per plant were inhibited after day seven at all concentrations of NO ₃ ^– . The severity of inhibition of N₂ fixation increased with increasing NO ₃ ^– concentration and with time. The total amounts of N₂ fixed per plant between days 0–7 after supplying 10, 100 and 1000 mmol m^–3 NO ₃ ^– , respectively, were 37–39, 28–30 and 0–13%, of the total N acquired. Between days 7–28 the proportional contributions of N₂ fixation to total N acquisition declined to 3, 0.5 and 0%, respectively, in these treatments. The corresponding mean specific rates of N₂ fixation between days 0–7 were, respectively, 5.4, 3.2, and 2.0 mmol N d^–1 g^–1 nodule d.w., compared with 7.9 mmol N d^–1 g^–1 nodule d.w. for zero NO ₃ ^– plants. There was no evidence of a transitory increase in N₂ fixation following the addition of NO ₃ ^– , even at the lowest supply concentration.     

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.     

Assessment of genetic variability for N2 fixation between and within provenances of Leucaena leucocephala and Acacia albida estimated by 15N labelling techniques

Nitrogen fixed in 13 provenances of Acacia albida and 11 isolines of Leucaena leucocephala inoculated with effective Rhizobium strains was measured by ¹⁵N techniques and the total N difference method. In the test soil, on the average, L. leucocephala derived about 65% of its total N from atmospheric N₂ fixation compared to about 20% by A. albida. Significant differences in the percentage of N derived from atmospheric N₂ (% Ndfa) occurred, between provenances or isolines within species. The % Ndfa ranged from 37 to 74% within L. leucocephala and from 6 to 37 within A. albida; (equivalent to 20–50 mg N plant^–1 and 4–37 mg N plant^–1 for the two species over three months, respectively) and was correlated with the nodule mass (r=0.91). The time course of N₂ fixation of three selected provenances (low, intermediate and good fixers) was followed at 12 weekly intervals over a 36 week period. The % Ndfa of all provenances and isolines increased with time; and except for one of the L. leucocephala provenances, % Ndfa was similar within species at the 36 weeks harvest. There was a significant correlation between % Ndfa and the amount of N₂ fixed (r=0.96). Significant interactions occurred between provenances and N treatments and often growth of uninoculated but N fertilized plants was less variable than for inoculated unfertilized plants.     

Influence of urea on biological N2 fixation and N transfer from Azolla intercropped with rice

The N₂ fixed by Azolla before and after urea application during the rice cycle, the mineralisation of Azolla-N as well as its availability to rice was studied in two greenhouse experiments conducted in 1996 and 1997 and in June 1998 in Goettingen (Germany). Dry matter production of the various rice parts of experiment 1 showed a clear positive synergism between treatment with Azolla and urea with a resulting apparent N recovery by rice increasing from 40% (without Azolla) to 57% in the presence of Azolla. Part of this increase may be due to N fixed biologically by Azolla and transferred to the rice. The second experiment shed some light on the role of BNF. Using an iterative method of estimation, the daily rate of N fixation was estimated at 0.6 – 0.7 kg N ha^–1. The rate was not so much affected by the age of the Azolla crop. At this rate, the BNF would amount to up to 100 kg N ha^–1 over a 130-day season. Assuming that BNF may be inhibited for a period of 5 – 10 days following urea application due to high levels of N in the floodwater, this might reduce the BNF by between 6 and 14 kg N ha over the season. Using the mean-pool-abundance concept, it was estimated that around 75 – 80% of the Azolla-N mineralized during the growth period was actually absorbed by the rice plants. Of the N taken up by rice around 28% was derived from the biologically fixed Azolla N, the remainder was urea N cycled through the Azolla. Azolla also seems to help sustain the soil N supply by returning N to the soil in quantities roughly equal to those extracted from the soil by the rice plant.     

Minimizing the effect of mineral nitrogen on biological nitrogen fixation in common bean by increasing nutrient levels

Although common bean (Phaseolus vulgaris L.) has good potential for N₂ fixation, some additional N provided through fertilizer usually is required for a maximum yield. In this study the suppressive effect of N on nodulation and N₂ fixation was evaluated in an unfertile soil under greenhouse conditions with different levels of soil fertility (low=no P, K and S additions; medium = 50, 63 and 10 mg kg^–1 soil and high = 200, 256 and 40 mg kg^–1 soil, respectively) and combined with 5, 15, 60 and 120 mg N kg^–1 soil of ¹⁵N-labelled urea. The overall average nodule number and weight increased under high fertility levels. At low N applications, nitrogen had a synergistic effect on N₂ fixation, by stimulating nodule formation, nitrogenase activity and plant growth. At high fertility and at the highest N rate (120 mg kg^–1 soil), the stimulatory effect of N fertilizer on N₂ fixation was still observed, increasing the amounts of N₂ fixed from 88 up to 375 mg N plant^–1. These results indicate that a suitable balance of soil nutrients is essential to obtain high N₂ fixation rates and yield in common beans.     

Seasonal accumulation and partitioning of nitrogen by lentil

Although wheat (Triticum aestivum L.) is the dominant crop of the semi-arid plains of Canada and the western United States, lentil (Lens culinaris Medik.) has become an important alternative crop. Sources and seasonal accumulation of N must be understood in order to identify parameters that can lead to increased N₂-fixing activity and yield. Inoculated lentil was grown in a sandy-loam soil at an irrigated site in Saskatchewan, Canada. Wheat was used as the reference crop to estimate N₂ fixation by the A-value approach. Lentil and wheat received 10 and 100 kg N ha⁻¹ of ammonium nitrate, respectively. Crops were harvested six times during the growing season and plant components analyzed. During the first 71 days after planting the wheat had a higher daily dry matter and N accumulation compared to lentil. However, during the latter part of the growing season, daily dry matter and N accumulation were greater for lentil. The maximum total N accumulation for lentil at maturity was 149 kg ha⁻¹. In contrast, wheat had a maximum N accumulation of 98 kg ha⁻¹ in the Feekes 11.1 stage, or 86 days after planting. The maximum daily rates of N accumulation were 3.82 kg N ha⁻¹ day⁻¹ for lentil and 2.21 kg N ha⁻¹ day⁻¹ for wheat. The percentage of N derived from N₂ fixation (% Ndfa) ranged from 0 at the first harvest to 92 % at final harvest. Generative plant components had higher values for % Ndfa than the vegetative components which indicates that N in the reproductive plant parts was derived largely from current N₂ fixation and lentil continued to fix N until the end of the pod fill stage. At final harvest, lentil had derived 129 kg N ha⁻¹ from N₂ fixation with maximum N₂-fixing activity (4.4 kg N ha⁻¹ day⁻¹) occurring during the early stages of pod fill. Higher maximum rates of N₂-fixing activity than net N accumulation (3.82 kg N ha⁻¹ day⁻¹) may have been caused by N losses like volatilization. In addition, lentil provided a net N contribution to the soil of 59 kg ha⁻¹ following the removal of the grain.     

15N-determined dinitrogen fixation potential of genetically diverse bean lines (Phaseolus vulgaris L.)

Improvement of dinitrogen fixation in beans (Phaseolus vulgaris L.) will depend on the selection of superior plant genotypes and the presence of efficient rhizobial strains. This study was conducted to evaluate diverse bean lines for N₂ fixation potential using the¹⁵N-depleted dilution technique under field conditions in Wisconsin, USA. Plants of 21 bean lines and three non-nodulating isolines of soybean received appliin Wisconsin, USA. Plants of 21 bean lines and three non-nodulating isolines of soybean received applications of¹⁵N-depleted ammonium sulphate. Shoots harvested at the V₆, R₃ and R₇ stages and dry seeds were analyzed for total N using the Kjeldahl procedure, and the ratio of¹⁵N to¹⁴N was determind on a MAT 250 mass spectrometer. Nodule occupancy of the applied strain ofR. leguminosarum biovarphaseoli, CIAT 899, was determined in five of the bean lines. Total shoot N content showed a pattern of accumulation similar to shoot dry weight and fixed N₂ in the shoot. Based on shoot total N, N₂ fixed in the shoot and shoot dry weight Riz 30 and Preto Cariri were identified as being as good fixers as Puebla 152 and Cargamanto appear to begin N₂ fixation early. Furthermore, some bean lines that fixed considerable N₂ did not translocate a large amount of N to the grains. Preto Cariri accumulated 21.2 kg N ha⁻¹ in the seeds compared to Puebla 152 which accumulated 43.8 kg N ha⁻¹ of the fixed N₂ into the grains. At the early sampling, Puebla 152 and 22–27 had a considerable higher percentage of their crown nodules formed by the inoculant strain CIAT 899, than did Rio Tibagi which has been considered a poor N₂ fixer.