P. M. Chalk

Quantifying below-ground nitrogen of legumes. 2. A comparison of 15N and non isotopic methods

Accurate information on below-ground nitrogen (N) of legumes is necessary for quantifying legume effects on soil N pools and on the N economies of crops following legumes in rotation systems. We report a series of glasshouse pot experiments to determine below-ground N (BGN) of the four legumes, fababean (Vicia faba), chickpea (Cicer arietinum), mungbean (Vigna radiata) and pigeonpea (Cajanus cajan) using both 15N shoot-labelling and 15N-labelled soil isotope-dilution methods, a mass N balance approach and the physical recovery of nodulated roots. Data from the 15N shoot-labelling experiment were manipulated in different ways in an attempt to counter errors associated with uneven 15N enrichment of roots and nodules. Values for BGN as percent of total plant N based on the physical recovery of nodulated roots ranged from 4 to 15%. With 15N shoot-labelling, a total of 8.11 mg 15N was supplied to each pot (six plants) as 0.5% 15N urea using either leaf-flap (fababean, mungbean and pigeonpea), petiole (chickpea) or leaf-tip (wheat) feeding. Calculations based on measurement of 15N enrichments of harvested plant parts and root-zone soil suggested that BGN represented 39% of total plant N for fababean, 53% for chickpea, 20% for mungbean and 47% for pigeonpea. The value for wheat was 60%. Adjustment for uneven nodulation patterns on the roots and nodule 15N depletion, resulting in different 15N enrichments between nodulated and unnodulated roots, reduced the fababean value to 37% and chickpea to 42%. Values using the other methods were generally in the same range, viz. 15–57% (simple 15N balance), 11–52% (soil 15N dilution) and 30–52% (mass N balance). We conclude that physical recovery of roots was the most inaccurate method for estimating BGN. Average values for BGN as percent of total plant N using all isotopic and mass N balance methods were 30% for fababean, 48% for chickpea, 28% for mungbean, and 43% for pigeonpea.15N shoot-labelling may be the best method for quantifying BGN of field-grown plants. The methodology is simple, apparently accurate provided care is taken in obtaining representative nodulated root samples and, unlike the soil 15N dilution method, does not require pre-treatment of the soil with 15N enriched material.

Nitrogen mineralisation, immobilisation and loss, and their role in determining differences in net nitrogen production during waterlogged and aerobic incubation of soils

Abstract Twenty air-dried soil samples were incubated for 14 days at 30°C to determine net N production ( np ) under aerobic and waterlogged conditions. The results showed that np was not always higher under waterlogged than under aerobic conditions, which differed from some previous reports. Five analytical equations were compared and used to estimate gross rates of N mineralisation, immobilisation and loss. It was elucidated that the equations based on changes in the AT and AL pools gave estimates of gross mineralization and consumption rates, with the values obtained with the equation of Shen et al. (Shen, S.M., Pruden, G., Jenkinson, D.S., 1984. Mineralization and immobilization of nitrogen in fumigated soil and the measurement of microbial biomass nitrogen. Soil Biology & Biochemistry 16, 437–444) ≥the equation of Kirkham and Bartholomew (Kirkham, D., Bartholomew, W.V., 1954. Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33–34) >the equation of Tiedje et al. (Tiedje, J.M., Sorensen, J., Chang, Y.-Y.L., 1981. Assimilatory and dissimilatory nitrate reduction: Perspectives and methodology for simultaneous measurement of several nitrogen cycle processes. Ecological Bulletin 33, 331–342) . The equations based on AT, AL and OL pools estimated gross immobilisation rates, of which the equation of Shen et al. (1984) gave higher values than the equation of Guiraud et al. (Guiraud, G., Marol, C., Thibaud, M.C., 1989. Mineralization of nitrogen in the presence of a nitrification inhibitor. Soil Biology & Biochemistry 21, 29–34) . The difference between gross consumption and immobilisation rates represented the rate of N loss from the exchangeable NH 4 + , NO 3 − and organic N pools. Gross N mineralization was not always higher under aerobic than under waterlogged conditions during the 14 days incubation of air-dried soils. Immobilisation was greater under aerobic conditions than under waterlogged conditions. Significant amounts of N were lost from some soils during the 2 weeks of incubation. Soils that lost N during aerobic incubation also lost substantial amounts of NH 4 + –N under waterlogged conditions. However, soils that lost NH 4 + –N during waterlogged incubation did not necessarily lose N when incubated aerobically. Mechanisms causing the difference in net N production between waterlogged and aerobic conditions are soil-dependent. Although the rate of gross mineralisation predominantly determines the amount of mineral N that may accumulate in soils, immobilisation and loss have the potential to significantly affect the quantity of mineral N accumulation.

Use of nitrification inhibitors to increase fertilizer nitrogen recovery and lint yield in irrigated cotton

This paper describes field experiments designed to evaluate the effectiveness of several nitrification inhibitors to prevent loss of fertilizer nitrogen (N) applied to cotton. The usefulness of nitrapyrin, acetylene (provided by wax-coated calcium carbide), phenylacetylene and 2-ethynylpyridine to prevent denitrification was evaluated by determining the recovery of N applied as15N labelled urea to a heavy clay soil in 1 m × 0.5 m microplots in north western N.S.W., Australia. In a second experiment, the effect of wax-coated calcium carbide on lint yield of cotton supplied with five N levels was determined on 12.5 m × 8 m plots at the same site.The15N balance study showed that in the absence of nitrification inhibitors only 57% of the applied N was recovered in the plants and soil at crop maturity. The recovery was increased (p < 0.05) to 70% by addition of phenylacetylene, to 74% by nitrapyrin, to 78% by coated calcium carbide and to 92% by 2-ethynylpyridine.In the larger scale field experiment, addition of the wax-coated calcium carbide significantly slowed the rate of NH4+ oxidation in the grey clay for approximately 8 weeks. Lint yield was increased (p < 0.05) by the addition of the inhibitor at all except the highest level of N addition. The inhibitor helped to conserve the indigenous N as well as the applied N.The research shows that the effectiveness of urea fertilizer for cotton grown on the heavy clay soils of N.S.W. can be markedly improved by using acetylenic compounds as nitrification inhibitors.

THE CONTRIBUTION OF ASSOCIATIVE AND SYMBIOTIC NITROGEN FIXATION TO THE NITROGEN NUTRITION OF NON-LEGUMES

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

Estimation of legume symbiotic dependence : an evaluation of techniques based on 15N dilution

Abstract An 15 N isotope dilution technique, first proposed more than 40 years ago, is now widely used to estimate of the proportion of legume N derived from biological N 2 fixation. The proposed attributes of the method are critically examined. The problems associated with the non-uniform distribution of applied isotope and the traditional use of a non-fixing reference plant are elaborated. The use of models which do not require a reference plant is a relatively recent development in the evolution of the methodology. The characteristics and efficacy of the modelling approach are reviewed.

Emission of nitrogen oxides (NO x ) from a flooded soil fertilized with urea: Relation to other nitrogen loss processes

Emissions of nitric oxide and other odd nitrogen oxides (NOx) from a flooded rice field were studied after urea had been broadcast into the floodwater.The NOx flux from the fertilized area was very low (0.2×10-9 g N m-2 s-1) for the first few days after application of urea and was high (0.95×10-9 g N m-2 s-1) in the subsequent period when significant nitrite and nitrate were present in the floodwater. At night, little if any NOx was exhaled but ambient NO2 was absorbed by the floodwater. An uptake velocity for NO2 of 3×10-4 m s-1 was measured during one night. Maximum NOx losses were observed near 1300 h when temperature and solar ultraviolet light were maximum.While the amounts of nitrogen oxides emitted are of little agronomic importance (∼2×10-3 per cent of the fertilizer nitrogen was lost as NOx during the 10-day study period), they may well be of significance as a source for some gas reactions in the atmosphere and for the global nitrogen cycle.Of the fertilizer nitrogen applied (as urea) approximately 30% was lost to the atmosphere by NH3 volatilization, 15% by denitrification, presumably as N2, and the remainder, less minor losses of NO and N2O, remained in the plant/soil/water system.

Characterization of the N benefit of a grain legume (Lupinus angustifolius L.) to a cereal (Hordeum vulgare L.) by an in situ 15N isotope dilution technique

SummaryA crop of barley was grown on plots which had previously supported pure stands of lupins, canola, ryegrass, and wheat. The plots were labelled with 15N-enriched fertilizers at the time of sowing of the antecedent crops. The crop of lupins, which derived 79% of its N from symbiotic N2 fixation at physiological maturity, conferred an N benefit to barley of 3.4 g N m-2 when compared to barley following wheat. Lupins used less fertilizer N and less unlabelled soil N compared to the other crops, but the ratios of these sources of N in the plant tops were similar. The apparent sparing of soil+fertilizer N under lupins compared with wheat was 13.6 g N m-2, which was much larger than the measured N benefit. Barley following lupins was less enriched in 15N compared to barley following wheat, and the measured isotope dilution was used to estimate the proportion of barley N derived from biologically fixed N in the lupin residues. This in turn enabled the N benefit to be partitioned between the uptake of spared N and the uptake of fixed N derived from the mineralization of legume residues. Spared N and fixed N contributed in approximately equal proportions to the N benefit measured in barley following lupins compared to barley following wheat.

Nitrogen fixation by nodulated soybean under tropical field conditions estimated by the 15N isotope dilution technique

Abstract The contribution of biological N 2 fixation to the N nutrition of nodulated soybean was estimated using the 15 N isotope dilution technique and a non-nodulating soybean isoline as a non-fixing control plant. The plants were grown in the field in concrete cylinders (60 cm dia) and harvested at seven stages of plant growth. Labelled N was added to the soil either as labelled organic matter before planting or in seven small additions (2kg N ha −1 ) of (NH 4 ) 2 SO 4 during the growing period. There was good agreement between isotope dilution estimates of nitrogen fixation for the two labelling methods. Acetylene reduction assays on intact root systems greatly underestimated N 2 fixing activity. The difference in total N between nodulated and non-nodulated plants generally gave higher estimates compared with the isotope technique. The data indicate that this was because nodulated plants recovered more N from the soil than the non-nodulated plants. After 92 days of growth, the soybean derived approximately 250kg N ha −1 from biological N 2 fixation.

The 15N-isotope dilution technique applied to the estimation of biological nitrogen fixation associated with Paspalum notatum cv. batatais in the field

Abstract The contribution of associated biological nitrogen fixation to the nitrogen nutrition of Paspaulum notatum cv. batatais was estimated using the 15 N-isotope dilution technique. The plants were grown in the field in concrete cylinders (60 cm dia) filled with soil, to which were added small quantities of 15 N-labelled fertilizer at frequent intervals over 12 months. The pensacola cultivar of P. notatum was used as a non-N 2 -fixing control plant. This was justified by the observation that nitrogenase (intact core C 2 H 2 reduction) activity associated with the pensacola cultivar was consistently much lower than that of the batatais cultivar. At the first harvest, no evidence for N 2 fixation associated with the batatais cultivar was obtained, probably because of slow establishment of the N 2 -fixing association. However, at the subsequent three harvests the batatais cultivar exhibited a lower 15 N-enrichment and yielded more N than the pensacola cultivar. These data together suggested that 8–25% of the N in the batatais cultivar originated from N 2 fixation. The grass Paspalum maritimum was also included in the experiment and exhibited low nitrogenase activity similar to that of the pensacola cultivar of P. notatum . However, the total N and 15 N data of these two grasses were not in good agreement indicating that it is important for the use of the isotope dilution technique that control plants are of very similar physiology and growth habit.

Integrated effects of abiotic stresses on inoculant performance, legume growth and symbiotic dependence estimated by 15N dilution

Temperature, water, salinity, sodicity, acidity and nutrient disorders are major abiotic stresses that can affect legume growth or the establishment and function of the legume-Rhizobium symbiosis. We have examined the literature where the application of the 15N isotope dilution methodology permits the effect of individual abiotic stresses to be independently and quantitatively separated into plant growth-mediated and BNF (biological N2 fixation)-mediated components. The response of the symbiosis to a particular stress depends on a host of factors, including legume genotype, cultivar, Rhizobium inoculant, climatic conditions, and the duration, timing and severity of the stress. Published data are analysed in terms of the above variables and their interactions. As a general rule, severe stress inhibits both legume dry matter (DM) and the proportional dependence of the legume on BNF as a source of N. The symbiosis is resilient to low to moderate stress, but there may still be a penalty on legume DM. Gaps in knowledge are identified, and general guidelines on the identification and amelioration of abiotic stresses are provided.