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

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

An inland and a coastal population of the Mediterranean xero-halophyte species Atriplex halimus L. differ in their ability to accumulate proline and glycinebetaine in response to salinity and water stress

Soil salinity and drought compromise water uptake and lead toosmotic adjustment in xero-halophyte plant species. These importantenvironmental constraints may also have specific effects onplant physiology. Stress-induced accumulation of osmocompatiblesolutes was analysed in two Tunisian populations of the Mediteraneanshrub Atriplex halimus L.—plants originating from a salt-affectedcoastal site (Monastir) or from a non-saline semi-arid area(Sbikha)—were exposed to nutrient solution containingeither low (40 mM) or high (160 mM) doses of NaCl or 15% polyethyleneglycol. The low NaCl dose stimulated plant growth in both populations.Plants from Monastir were more resistant to high salinity andexhibited a greater ability to produce glycinebetaine in responseto salt stress. Conversely, plants from Sbikha were more resistantto water stress and displayed a higher rate of proline accumulation.Proline accumulated as early as 24 h after stress impositionand such accumulation was reversible. By contrast, glycinebetaineconcentration culminated after 10 d of stress and did not decreaseafter the stress relief. The highest salt resistance of Monastirplants was not due to a lower rate of Na⁺ absorption; plantsfrom this population exhibited a higher stomatal conductanceand a prodigal water-use strategy leading to lower water-useefficiency than plants from Sbikha. Exogenous application ofproline (1 mM) improved the level of drought resistance in Monastirplants through a decrease in oxidative stress quantified bythe malondialdehyde concentration, while the exogenous applicationof glycinebetaine improved the salinity resistance of Sbikhaplants through a positive effect on photosystem II efficiency. Key words: Atriplex halimus, glycinebetaine, halophyte, NaCl, osmotic adjustment, proline, salinity, water stress     

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.     

The15N methodology in estimating N2 fixation by vetch and pea grown in pure stand or in mixtures with oat

Cereal-legume mixtures are frequently the best management decision for forage production instead of growing crops in pure stands. Nitrogen fertilization of cereal-legume mixtures is questionable since combined nitrogen could depress N₂ fixation by legumes. The objectives of this study were (1) to examine the effect of N fertilization on N₂ fixation by vetch and field peas in pure and in mixed stands with oats, and (2) to examine if there is any transfer of N from legumes to associated cereals. The field experiment was conducted for two growing seasons. The treatments were pure stands of vetch, pea and oats, and the mixtures of the two legumes with oats at the seeding ratios 90:10 and 75:25, fertilized with labelled¹⁵N at the rates of 15 and 90 kg N ha⁻¹. Nitrogen fertilization of 90 kg N ha⁻¹ suppressed N₂ fixation in both legumes grown in pure and in mixed stands. Crops grown in mixtures in many instances had lower atom %¹⁵N excess. Whether this was due to high N₂ fixation in the case of legume and transfer in the case of oat or the differences were due to practical problems of the¹⁵N technique is not clearly shown by the results, so based on the literature the aspect is discussed as well as the precautions which should be considered in using the¹⁵N technique in such studies.     

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.     

Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation

The ¹⁵N methods are potentially accurate for measuring N₂ fixation in plants. The only problem with those methods is, how to ensure that the ¹⁵N/¹⁴N ratio in the plant accurately reflects the integrated ¹⁵N/¹⁴N ratio (R) in soil which is variable in time and with soil depth. However, the consequences of using an inappropriate reference plant vary with the level of N₂ fixation and the conditions under which the study was made. For example, the errors introduced into the values of N₂ fixation are higher at low levels of fixation, and decrease with increasing rates of fixation. At very high N₂ fixation rates, the errors are often insignificant. Also, the magnitude of error is proportional to the rate of decline of the ¹⁵N/¹⁴N ratio with time. Since N₂ fixation in most plants would be expected to below 60%, the question of how to select a good reference plant is still pertinent. In this paper, we have discussed some of the criteria to adopt in selecting reference plants, e.g. how to ensure that the reference plant is not fixing N₂, is absorbing most of its N from the same zone as the fixing plant, and in the same pattern with time, etc. In addition, we have discussed ¹⁵N labelling materials and methods that are likely to minimize any errors even when the fixing and reference plants don't match well in certain important criteria. The use of slow release ¹⁵N fertilizer or ¹⁵N labelled plant materials results in slow changes in the ¹⁵N/¹⁴N ratio of soil, and is strongly recommended. Where ¹⁵N inorganic fertilizers are used, the application of the fertilizer in small splits at various intervals is recommended over a one-time application. The problem with the reference crop, which has sometimes discouraged potential users of the ¹⁵N methods, is surmountable, as discussed in this paper.     

Management of biological N2 fixation in alley cropping systems: Estimation and contribution to N balance

Alley cropping is being widely tested in the tropics for its potential to sustain adequate food production with low agricultural inputs, while conserving the resource base. Fast growth and N yield of most trees used as hedgerows in alley cropping is due greatly to their ability to fix N₂ symbiotically with Rhizobium. Measurements of biological N₂ fixation (BNF) in alley cropping systems show that some tree species such as Leucaena leucocephala, Gliricidia sepium and Acacia mangium can derive between 100 and 300 kg N ha^-1 yr^–1 from atmospheric N₂, while species such as Faidherbia albida and Acacia senegal might fix less than 20 kg N ha^-1 yr^-1. Other tree species such as Senna siamea and S. spectabilis are also used in alley cropping, although they do not nodulate and therefore do not fix N₂. The long-term evaluation of the potential or actual amounts of N₂ fixed in trees however, poses problems that are associated with their perennial nature and massive size, the great difficulty in obtaining representative samples and applying reliable methodologies for measuring N₂ fixed. Strategies for obtaining representative samples (as against the whole tree or destructive plant sampling), the application of ¹⁵N procedures and the selection criteria for appropriate reference plants have been discussed.Little is known about the effect of environmental factors and management practices such as tree cutting or pruning and residue management on BNF and eventually their N contribution in alley cropping. Data using the ¹⁵N labelling techniques have indicated that up to 50% or more of the tree's N may be below ground after pruning. In this case, quantification of N₂ fixed that disregards roots, nodules and crowns would result in serious errors and the amount of N₂ fixed may be largely underestimated. Large quantities of N are harvested with hedgerow prunings (>300 kg N ha^-1 yr^-1) but N contribution to crops is commonly in the range of 40–70 kg N ha^-1 season. This represents about 30% of N applied as prunings; however, N recoveries as low as 5–10% have been reported. The low N recovery in maize (Zea mays) is partly caused by lack of synchronization between the hedgerow trees N release and the associated food crop N demand. The N not taken up by the associated crop can be immobilized in soil organic matter or assimilated by the hedgerow trees and thus remain in the system. This N can also be lost from the system through denitrification, volatilization or is leached beyond the rooting zone. Below ground contribution (from root turnover and nodule decay) to an associated food crop in alley cropping is estimated at about 25–102 kg N ha^-1 season^-1. Timing and severity of pruning may allow for some management of underground transfer of fixed N₂ to associated crops. However many aspects of root dynamics in alley cropping systems are poorly understood. Current research projects based on ¹⁵N labelling techniques or ¹⁵N natural abundance measurements are outlined. These would lead to estimates of N₂ fixation and N saving resulting from the management of N₂ fixation in alley cropping systems.     

15N natural abundance in plants of the Amazon River floodplain and potential atmospheric N2 fixation

Summary The¹⁵N natural abundance values of various Amazon floodplain (várzea) plants was investigated. Samples of young leaf tissues were collected during three different periods of the river hydrography (low water, mid rising water and high water) and during one period in the Madeira River (high water). A large variation of¹⁵N abundance was observed, both among the different plant types and between the different flood stages. This variation probably, reflected, in part, the highly variable nature of the floodplain, sometimes dry and oxygenated and at other times inundated and anaerobic and, in part, changes in plant nitrogen metabolism. Comparison of the nitrogen isotopic composition of leguminous plants with that of non-leguminous plants showed that, on average, the¹⁵N abundance was lower in the legumes than non-legumes, suggesting active N-fixation. Also, the¹⁵N natural abundance in aquatic grasses of the generaPaspalum, was in general, lower than the¹⁵N abundance of aquatic grasses of the generaEchinochloa. As both of these grasses grow in the same general habitat, it appears thatPaspalum grasses may also be nitrogen fixers.     

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.     

Animal treading during wet soil conditions reduces N2 fixation in mixed clover-grass pasture

A field experiment was carried out over 12 months to determine the effect of animal treading on N₂ fixation in a mixed white clover-ryegrass pasture. The experimental site was defoliated by mowing for the duration of the study. A single treading event of moderate or severe pugging intensity was initiated in plots during wet spring conditions by using dairy cows at varying stocking rates (4.5 cows 100 m⁻² for 1.5 or 2.5 h, respectively). Inputs of dung and urine onto the plots was avoided by overnight housing of the cows and interception of excreta during the pugging event. Soil air-filled porosity decreased from 21% in the non-pugged control to 15–16% in pugged treatments by day 3. Bulk density of soil was not significantly affected by pugging. Soil inorganic N concentration increased in pugged treatments, and was 4-fold greater on day 28 in severely pugged plots compared to non-pugged plots. White clover plant density and plant size was markedly lower in pugged treatments (up to 85% and 72% reduction, respectively under severe pugging). White clover growth was most affected during the first 156 days after pugging (up to 90% decrease under severe pugging), leading to an annual clover dry matter production loss of 9% and 52%, respectively. The proportion of clover N derived from atmospheric N₂ (%Ndfa; estimated by ¹⁵N dilution) was initially reduced (to a lower limit of 43%) by severe pugging (days 28–71) before recovery to control levels (90%) by day 91. Annual N₂ fixation in clover herbage decreased significantly from 76 kg N ha⁻¹ yr⁻¹ in the non-pugged control, to 66 and 36 kg N ha⁻¹ yr⁻¹ under moderate and severe pugging, respectively. Most of this difference was evident within the first 156 days after pugging. Our data indicates that the major loss in fixed N₂ input under pugging was due to reduced clover growth and production resulting from pugging damage and loss of residual white clover biomass by hoof action.     

Amino acid content in xylem sap of regrowing alfalfa (Medicago sativa L.): Relations with N uptake,N2 fixation and N remobilization

During vegetative regrowth of Medicago sativa L., soil N, symbiotically fixed N₂ and N reserves meet the nitrogen requirements for shoot regrowth. Experiments with nodulated or non-nodulated plants were carried out to investigate the changes in N flows originating from the different N sources and in xylem transport of amino acids during regrowth. Exogenous N uptake, N₂ fixation and endogenous N remobilization were estimated by ¹⁵N labelling and amino acids in xylem sap were analysed. Removal of shoots resulted in great declines of exogenous N flows derived either from N₂ or from NH₄NO₃ during the first week of regrowth, thereafter recovery increased linearly. Mineral N uptake as well as N₂ fixation occurred mainly between the 10th and 18th day after removal of shoots while exogenous N assimilation in intact plants remained at a steady level. Nitrogen remobilization rates in defoliated plants increased by at least three to five-fold, especially during the first 10 days following shoot removal. Compared to control plants, contents of amino acids in xylem sap, during the first 10 days of regrowth, were reduced by about 72% and 82% in NH₄NO₃ grown and in N₂ fixing plants, respectively. Asparagine was the main amino acid transported in xylem sap of both treated plants. Its relative contents during this period significantly decreased from 75% to 59% and from 67% to 36% respectively in non-nodulated plants and in nodulated ones. This decline was accompanied by compensatory increase in the relative contents of aspartate and glutamine.     

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.     

The course of 15N-ammonium nitrate in a spring barley cropping system

¹⁵N-labelled ammonium nitrate was applied to spring barley growing on a Cambisol soil in western Switzerland. Immobilization, plant uptake and disappearance of inorganic nitrogen were followed at frequent intervals. Fertilizer nitrogen disappeared shortly after its application, mainly through immobilization by soil microorganisms and absorption by the crop. Some of the added nitrogen was probably denitrified as a result of humid conditions during the first days after fertilizer application. At the end of the growing season, 31% of the added nitrogen was recovered from the aerial barley plants, and 56% was immobilized by microorganisms. Most of the fertilizer nitrogen not used by the crop was immobilized in the upper 0–30 cm soil layer. This prevented downward movement of nitrate and limited nitrogen losses. Fertilizer efficiency was mainly determined by the competition between crop uptake and microbial immobilization. Careful consideration of the time of fertilization, taking into account plant growth and weather conditions, can result in an increase in fertilizer efficiency and minimal pollution.     

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.     

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.     

Protein degradation in different feedstuffs labelled with 15N by using the nylon bag technique

The nylon bag technique was used to determine the Nitrogen (N) and ¹⁵N degradation of ¹⁵N labelled feedstuffs in the rumen. The N and ¹⁵N degradation values were calculated according to ?rskov and McDonald (1979) and ranged from 46.8 to 92.0 and from 61.8 to 93.6%, respectively. The differences between N and ¹⁵N degradation values of high fibre content feedstuffs are the highest, thus the measuring errors were greatest here. But differences also existed in concentrates. This study indicated that especially barley had a higher proportion of microbial N in the bag residues after the washing than the other concentrates. Therefore it is necessary to correct the N degradation values not only in cases of high fibre content but also in cases of low nitrogen content of feedstuffs. The calculation of the N degradation values could be possible on the basis of crude fibre and crude protein contents of feedstuffs. But experiments with a much larger number of ¹⁵N labelled feedstuffs have to be realized to give an accurate prediction of N degradation.     

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.     

Design and development of a simple laboratory model to detect <Superscript>15</Superscript>N enrichment in cyanobacterial biomass and extra cellular ammonia using <Superscript>15</Superscript>N gas

A laboratory scale working model that could detect the ¹⁵N enrichment in cyanobacterial biomass and extracellular ammonia, using ¹⁵N gas under in vitro conditions was designed and fabricated. Using the model, ¹⁵N enrichment of 0.48% atom excess was detected in the cyanobacterial biomass on the 30 d after inoculation. The ¹⁵N enrichment increased linearly in the extracellular ammoniacal fraction from the 20 d onward. The model would prove to be a useful tool to quantify the extent of ¹⁵N enrichment under in vitro conditions using ¹⁵N gas.     

The use of mean pool abundances to interpret 15N tracer experiments

A pulse dilution ¹⁵N technique was used in the field to determine the effect of the ammonium to nitrate ratio in a fertilizer application on the uptake of ammonium and nitrate by ryegrass and on gross rates of mineralization and nitrification. Two experiments were performed, corresponding approximately to the first and second cuts of grass. Where no substantial recent immobilization of inorganic nitrogen had occurred, mineralization was insensitive to the form of nitrogen applied, ranging from 2.1–2.6 kg N ha^-1 d^-1. The immobilization of ammonium increased as the proportion of ammonium in the application increased. In the second experiment there was evidence that high rates of immobilization in the first experiment were associated with high rates of mineralization in the second. The implication was that some nitrogen immobilized in the first experiment was re-mineralized during the second. Whether this was nitrogen taken up, stored in roots and released following defoliation was not clear. Nitrification rates in this soil were low (0.1–0.63 kg N ha^-1 d^-1), and as a result, varying the ratio of ammonium to nitrate applied markedly altered the relative uptake of ammonium and nitrate. In the first experiment, where temperatures were low, preferential uptake of ammonium occurred, but where >90% of the uptake was as ammonium, a reduction in yield and nitrogen uptake was observed. In the second experiment, where temperatures and growth rates were higher, the proportion of ammonium to nitrate taken up had no effect on yield or nitrogen uptake.     

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.