Field measurements of symbiotic nitrogen fixation in an established pasture using acetylene reduction and a 15N method

The C₂H₂ reduction (A.R.) assay was investigated for quantitative measurement of symbiotic N₂ fixation in established legume-based pastures under field conditions.It was found that the rate of C₂H₄ production was relatively constant for approx 6 h. For an accurate estimation of the N₂ fixing activity, many field samples are required to overcome the errors due to the inherent spatial distribution of white clover (Trifolium repens L.) within the pasture. Furthermore, it is necessary to physically integrate the day-to-day and diurnal variations in the N₂ fixing activity of the legume to obtain a reliable estimate of the rate of C₂H₄ production and hence symbiotic N₂ fixation.The A.R. assay and an ¹⁵N technique were compared for measuring symbiotic N₂ fixation in established pastures. A 3 h incubation in the A.R. assay gave the best estimate of symbiotic N₂ fixation relative to the ¹⁵N technique. The 1 h incubation over-estimated and the 6 h incubation under-estimated the rate of symbiotic N₂ fixation.     

Slow-release 15N fertilizer formulations to measure N2-fixation by isotope dilution

Pot experiments were carried out to examine the effects of slow-release fertilizer formulations on estimates of N₂-fixation determined by the isotope dilution method. Soybeans were used as the N₂-fixing plants, with non-nodulated soybeans and maize as the non-fixing controls. The ¹⁵N-fertilizer formulations used were (¹⁵NH₄)SO₄, K¹⁵NO₃, gypsum-pelleted K¹⁵NO₃, (¹⁵NH₄)₂SO₄ + glucose, ground plant material enriched with ¹⁵N or ¹⁵N-oxamide. The estimate of the amount of N₂ fixed by the nodulated soybean plants depended upon both the control plant and the fertilizer formulation used. Maize took up N later than non-nodulated soybean and estimates of soil N-pool (soil “A” value + fertilizer N added) calculated from the enrichment of this control were about twice as large as those calculated from the enrichment of non-nodulated soybean receiving the same fertilizer treatment. As a consequence, estimates of N₂-fixation relative to this control were lower than those relative to non-nodulating soybean (mean 140 mg N per pot compared with 292 mg N per pot). With unstablilized ¹⁵N salts errors were sufficient to produce negative estimates of fixation relative to maize. Even with a “well-matched” control (non-nodulated soybean) estimates of fixation varied with fertilizer formulation.     

Characterization and extract ability of immobilized 15N from the soil microbial biomass

The microbial biomass of a typical Illinois Mollisol (Flanagan silt loam) was labeled with ¹⁵N, and several extradants were tested to determine their effectiveness in separating the immobilized ¹⁵N from the native soil N. From 3 to 5% of the total N and from 7 to 11% of the tracer N were removed by the milder extradants (i.e. hot water, hot 10mM CaCl₂, hot 5mm NaHCO₃, and cold 10mm NaHCO₃). Acidified permanganate (0.1 m KMnO₄ in 2 m H₂SO₄) and anhydrous formic acid were the most intensive extradants tested; they removed from 10 to 13% of the total N and about 14% of the immobilized ¹⁵N. An inverse relationship was observed between the amount of N extracted and selectivity of the extradants for removing the microbial ¹⁵N, indicating that the milder procedures were more selective in extracting the immobilized ¹⁵N.Distinct differences in the chemical distribution of organic N were observed for the immobilized ¹⁵N and native soil N. Lower proportions of the immobilized ¹⁵N were accounted for as acid-insoluble N and ammonia N (NH₃-N); while higher proportions were found in the amino acid and hydrolyzable unknown (HUN) N fractions.     

Influence of arbuscular mycorrhizae and phosphorus fertilization on growth, nodulation and N2 fixation (15N) inMedicago sativa at four salinity levels

The rose of an isolate of the arbuscular mycorrhizal (AM) fungusGlomus mosseae in the protection ofMedicago sativa (+Rhizobium meliloti) against salt stress induced by the addition of increasing levels of soluble salts was studied. The interactions between soluble P in soil (four levels), mycorrhizal inoculum and degree of salinity in relation to plant growth, nutrition and infective parameters were evaluated. Salt stress was induced by sequential irrigation with saline water having four concentrations of three salts (NaCl, CaCl₂, and MgCl₂).¹⁵N-labelled ammonium sulphate was added to provide a quantitative estimate of N₂ fixation under moderate to high salinity levels. N and P concentration and nodule formation increased with the amount of plant-available P or mycorrhizal inoculum in the soil and generally declined as the salinity in the solution culture increased from a moderate to a high level. The mycorrhizal inoculation protected the plants from salt stress more efficiently than any amount of plant-available P in soil, particularly at the highest salinity level applied (43.5 dS m^–1). Mycorrhizal inoculation matched the effect on dry matter and nutrition of the addition in the soil of 150 mg P kg^–1. Nevertheless the highest saline solution assayed (43.5 dS m^–1) affected more severely plants supplemented with phosphorus than those with the addition of mycorrhizal inoculum. Such a saline-depressing effect was 1.5 (biomass), 1.4 (N) and 1.5 (P) times higher in plants supplied with soluble phosphate than with AM inoculum. Mechanisms beyond those mediated by P must be involved in the AM-protectioe effect against salinity. The¹⁵N methodology used allowed the determination of N₂ fixation as influenced by different P applications compared to mycorrhizal inoculation. A lack of correlation between nodule formation and function (N₂ fixation) was evidenced in mycorrhizal-inoculated plants. In spite of the reduced activity per nodule in mycorrhizal-inoculated In spite of the reduced activity per nodule in mycorrhizal-inoculated plants, the N contents determined indicated the highest acquisition of N occurred in plants with the symbiotic status. Moreover, N and P uptake increased while Ca and Mg decreased in AM-inoculated plants. Thus P/Ca ratios and cation/anion balance in general were altered in mycorrhizal treatments. This study therefore confirms previous findings that AM-colonized plants have optional and alternative mechanisms available to satisfy their nutritive requirements and to maintain their physiological status in stress situations and in disturbed ecosystems.     

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

The contribution of biological N₂ fixation to the N nutrition of nodulated soybean was estimated using the ¹⁵N 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 (NH₄)₂SO₄ 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 N₂ 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 N₂ fixation.     

Confirmation of nitrogen fixation in two tropical grasses by 15N2 incorporation

Intact soil cores containing plants of Paspalum notatum or Digitaria decumbens were selected with the acetylene reduction method, and then exposed to ¹⁵N₁₅ to confirm nitrogen fixation in tropical grass-bacteria associations. In a preliminary experiment with P. notatum¹⁵N₂ incorporation was slow but progressive during 24 h in roots but translocation to rhizomes and leaves ceased after 17h. With improved assay chambers, enrichments of 0.151 and 0.563 ¹⁵N atom % excess were obtained in roots of D. decumbens cv transvala and P. notatum systems respectively, after 3 days. Enrichments in rhizomes were similar to those of roots; however in the leaves only 8% of root enrichment was observed. The addition of sucrose to the soil doubled N₂-fixation in roots in both grass species studied, but did not result in increased incorporation into the leaves of P. notatum.     

Nitrogen fixation by intact grass-soil cores using 15N2 and acetylene reduction

To determine N₂ fixation by intact grass-soil cores, samples were collected from 25 sites in central Texas during the summer. Three cores (32 cm² each) were extracted immediately adjacent to one another from single grass clumps or sods. Two of these cores were incubated under 10% C₂H₂ in air and the third core was incubated for 12 h in an atmosphere with 10% ¹⁵N₂ enrichment. Following incubation with ¹⁵N₂ the same core was assayed for rate of C₂H₂ reduction (AR). Rates of AR were generally low and quite variable (0–7.6 μmol C₂H₄ core^?1 day^?1). ¹⁵N₂ was incorporated into root and shoot tissues within 12–24 h. Extrapolated values of N₂ fixation based on ¹⁵N₂ incorporation ranged from 0 to 20 kg N ha^?1100 day^?1. The ratio of C₂H₂ reduced (μ mol C₂H₄ core^?1 day^?1) to N₂ fixed (μ mol N₂ fixed core^?1 day^?1) was highly variable ranging from 0 to 12. This study confirmed that N₂ is fixed in the rhizosphere of grasses grown in Texas through the use of ¹⁵N₂ and demonstrated that incorporation of fixed N into shoots was relatively rapid.     

Immobilization of 15N in forest litter studied by 15N CPMAS NMR spectroscopy

Solutions labelled with ¹⁵N were applied as (¹⁵NH₄)₂SO₄ or K¹⁵NO₃ to isolated microplots in the floor of mountain beech forest (Nothofagus solandri var. cliffortioides) and incubated for 135 days under field conditions of moisture and temperature. Solid state ¹⁵N CPMAS NMR spectra of the forest litter layer showed that more than 80% of the total signal intensity was attributable to the secondary amide-peptide peak. The degree of ¹⁵N enrichment or form of N did not alter the relative intensity of signals attributable to ¹⁵N in peptides, nucleic acids and aliphatic amine groups (amino sugars and free NH₂ on amino acids). Combinations of ¹³C and ¹⁵N-NMR spectra, edited by a process that exploited differences in proton spin properties between distinct categories of organic matter, indicated incorporation of ¹⁵N in humified organic matter rather than partly degraded plant material. This application demonstrated that solid state ¹⁵N CPMAS NMR has potential for use in studies of N immobilization under field conditions and with materials containing little N and small ¹⁵N enrichment.     

Assessment of N2 fixation by Casuarina equisetifolia inoculated with Frankia ORS021001 using 15N methods

Casuarina equisetifolia seedlings, uninoculated or inoculated with Frankia strain ORS021001 were grown for 4.5 months in pouches, then transplanted into 1 m³ concrete containers forming 1 m² microplots. Trees were harvested 6.5 months later when they were 11 months old. N₂ fixation was measured using three methods of assessment: the direct isotopic method, the A-value method and the difference method. Estimations of N₂ fixation during the 6.5 months following transplantation were respectively 3.27, 2.31 and 3.07g N₂ per tree. From these values it was calculated that about 40–60 kg N₂ would be fixed per hectare in a year at normal densities of 10,000 trees ha^?1. The results of this experiment confirm that Frankia strain ORS021001 can be confidently recommended to inoculate casuarinas in the field. Means to improve nodulation and subsequently N₂ fixation by casuarinas are discussed.     

Assessment of the relative uptake of added and indigenous soil nitrogen by nodulated legumes and reference plants in the 15N dilution measurement of N2 fixation: Derivation of method

Current methods for measuring N₂ fixation by nodulated legumes involve the addition of small amounts of ¹⁵N-labelled plant-available N compounds to soil so that plant N derived from the soil may be identified. All such methods assume that the proportions of added N and indigenous soil N assimilated by N₂-fixing and non-fixing plants grown in the same soil are the same, irrespective of the amount of soil N assimilated. The development of a method for assessing these proportions is described.Nodulated legumes and reference plants are grown in soils receiving none or one rate of addition of labelled N compound containing several (two or more) concentrations of ¹⁵N. The proportions of added and indigenous N assimilated, are determined from the intercepts and slopes of regression lines relating isotopic composition of plant N to that of added N, together with some other readilly-obtainable plant N measurements.     

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

The contribution of associated biological nitrogen fixation to the nitrogen nutrition of Paspaulum notatum cv. batatais was estimated using the ¹⁵N-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 ¹⁵N-labelled fertilizer at frequent intervals over 12 months. The pensacola cultivar of P. notatum was used as a non-N₂-fixing control plant. This was justified by the observation that nitrogenase (intact core C₂H₂ reduction) activity associated with the pensacola cultivar was consistently much lower than that of the batatais cultivar.At the first harvest, no evidence for N₂ fixation associated with the batatais cultivar was obtained, probably because of slow establishment of the N₂-fixing association. However, at the subsequent three harvests the batatais cultivar exhibited a lower ¹⁵N-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 N₂ 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 ¹⁵N 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.     

Natural abundance of 15N in root nodules of soybean,lupin, subterranean clover and lucerne

The total nitrogen of root nodules of yellow lupins (Lupinus luteus) and soybeans (Glycine max), when grown with N₂ of air as the sole source of nitrogen, became progressively enriched with ¹⁵N relative to other parts of the plants. Nodules of subterranean clover (Trifolium subterraneum) and lucerne (Medicago sativa) were not enriched with ¹⁵N. Analysis of the distributions of ¹⁵N amongst nodule fractions showed highest specific enrichment in coarse plant cell fragments and bacteroids in soybean and in lupins the soluble protein was also highly enriched. In terms of the total μg of ¹⁵N excess, the bacteroids contained most in soybean nodules and the soluble protein contained most in lupin nodules.     

Use of 15N isotope dilution for quantification of N2 fixation associated with roots of kallar grass (Leptochloa fusca (L.))

Summary Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using ¹⁵N isotopic dilution were carried out in nutrient solution and saline soil. In the nutrient solution, E. coli inoculated plants were taken as non-nitrogen-fixing control. It was observed that nearly 60%–80% of the plant N was derived from atmospheric fixation. Estimations based on the N difference method gave much lower values (18%–35%). In experiments with saline soil which was initially sterilized with chloroform fumigation, a mixed culture of N₂-fixing rhizospheric isolates from kallar grass roots was inoculated and planted to kallar grass. Uninoculated treatments were regarded as controls. The soil was previously labelled with ¹⁵N by adding cellulose and (¹⁵NH₄)₂SO₄. The results of these studies showed fixation values of 6%–32% when estimated by ¹⁵N dilution, whereas by the N difference method 54% of the plant N was estimated to be derived from fixation. This discrepancy is due to the increase in root proliferation due to inoculation, which results in greater uptake of soil N. The distribution of ¹⁵N in different fractions of the soil-N indicted isotopic dilution due to bacterial fixation of atmospheric N₂.     

Root-associated nitrogen-fixing bacteria and their role in the nitrogen nutrition of wheat estimated by 15N isotope dilution

The ability of two cultivars of spring wheat, Cadet and Rescue, and their reciprocal chromosome substitution lines, C-R5D and R-C5D, to obtain significant quantities of N from atmospheric N₂ was investigated in glasshouse experiments using ¹²⁵N dilution. The wheat was inoculated with N₂-fixing bacteria, including Azotohacter, Azospirillum, Klebsiella and Bacillus spp, in pure and mixed culture, at N concentrations ranging from 1 to 56 mg N plant^?1 (14–168 μg N ml^?1), in sand culture and in three soils of differing N content. Root-ussociutcd N₂-fixalion was negligible unless carbohydrate was added to the rooting medium. Atmospheric N₂ was incorporated into wheat roots and translocated to the tops, when plants inoculated with Azotobacter beijerinckii or Azospirillum brasilense sp. 107 were amended with glucose and malate respectively, under monoxenic conditions in sand culture.     

Evaluation of N2 fixation by applying 15N labeled plant material and ammonium sulfate

Effect of different ¹⁵N labeled sources on the estimation of N₂ fixation was investigated. The combination of ¹⁵N labeled ammonium sulfate, ¹⁵N labeled plant material, and ¹⁵N labeled ammonium sulfate with unlabeled plant material, was examined in pot experiments. Two cultivars of soybean (Glycine max) and one of mungbean (Vigna radiata) were used. No significant difference was observed among the treatments for the estimation of N₂ fixation. This was due to the homogeneity and stability of the ¹⁵N abundance in soil which resulted in a similar N uptake from the soil by the N² fixing and reference crops. The plant yield, total N uptake and amount of N₂ fixed were higher in the Yellow Soil than in the Andosol. The amount of N₂ fixed was strongly influenced by the plant growth and consequently it affected the plant yield. The slow decomposition of plant material in the Andosol resulted in a low yield in both the N₂ fixing and reference crops. Thus, the artificial decrease of the available N content in soil, by application of plant material, did not stimulate N, fixation but suppressed plant growth and N₂ fixation.     

Estimating N2-fixation in the field using 15N-labelled fertilizer: Some problems and solutions

The ¹⁵N-labelled fertilizer dilution technique provides a method of obtaining estimates of biological N₂-fixation in the field over the growing season. Field estimates of fixation obtained using peas, french beans, field beans and clover depended on the non-fixing control used. Differences in the N uptake patterns of the legume and control combinations, together with a decrease in the enrichment of plant available soil N with time, were major factors causing this dependency. A simple model of plant N accumulation at decreasing soil enrichment is presented, which explains these errors and allows a more rational choice of non-fixing control. The use of gypsum pelleted ¹⁵N fertilizer, or any other treatment which leads to a more stable soil enrichment, reduces errors caused by mismatched N uptake patterns in the two crops.     

Quantification and potential availability of non-symbiotically fixed 15N in soil

Summary Non-symbiotic N₂ fixation was studied under laboratory conditions in two soils from Pakistan (Hafizabad silt loam and Khurrarianwala silt loam) and one from Illinois, USA (Drummer silty clay loam) incubated in a ¹⁵N-enriched atmosphere. N₂ fixation was greatest with the Drummer soil (18–122 g g^–1 soil, depending upon the soil treatment) and lowest with the Khurrarianwala soil (4–81 g g^–1 soil). Fixation was increased by the addition of glucose, a close correlation being observed between the amount of glucose added and the amount of N₂ fixed in the three soils (r = 0.96). Efficiency of N₂ fixation varied with soil type and treatment and was greatest in the presence of added inorganic P. Application of Mo apparently had a negative effect on the amount and efficiency of N₂ fixation in all the soils. The percentage of non-symbiotically fixed ¹⁵N in potentially mineralizable form (NH ₄ ⁺ -N released in soil after a 15-day incubation period under anaerobic conditions) was low (2%–18%, depending upon the soil treatment), although most of the fixed N (up to 90%) was recovered as forms hydrolysable with 6N HCl. Recovery in hydrolysable forms was much greater for the fixed N than for the native soil N, indicating that the former was more available for uptake by plants.     

Isotopologue ratios of N2O emitted from microcosms with NH4 fertilized arable soils under conditions favoring nitrification

Soils represent the major source of the atmospheric greenhouse gas nitrous oxide (N₂O) and there is a need to better constrain the total global flux and the relative contribution of the microbial source processes. The aim of our study was to determine variability and control of the isotopic fingerprint of N₂O fluxes following NH₄⁺-fertilization and dominated by nitrification. We conducted a microcosm study with three arable soils fertilized with 0–140 mg NH₄⁺–N kg⁻¹. Fractions of N₂O derived from nitrification and denitrification were determined in parallel experiments using the ¹⁵N tracer and acetylene inhibition techniques or by comparison with unfertilized treatments. Soils were incubated for 3–10 days at low moisture (30–55% water-filled pore space) in order to establish conditions favoring nitrification. Dual isotope and isotopomer ratios of emitted N₂O were determined by mass spectrometric analysis of δ¹⁸O, average δ¹⁵N (δ¹⁵N^bulk) and ¹⁵N site preference (SP = difference in δ¹⁵N between the central and peripheral N positions of the asymmetric N₂O molecule). N₂O originated mainly from nitrification (>80%) in all treatments and the proportion of NH₄⁺ nitrified that was lost as N₂O ranged between 0.07 and 0.45%. δ¹⁸O and SP of N₂O fluxes ranged from 15 to 28.4‰ and from 13.9 to 29.8‰, respectively. These ranges overlapped with isotopic signatures of N₂O from denitrification reported previously. There was a negative correlation between SP and δ¹⁸O which is opposite to reported trends in N₂O from denitrification. Variation of average ¹⁵N signatures of N₂O (δ¹⁵N^bulk) did not supply process information, apparently because a strong shift in precursor signatures masked process-specific effects on δ¹⁵N^bulk. Maximum SP of total N₂O fluxes and of nitrification fluxes was close to reported SP of N₂O from NH₄⁺ or NH₂OH conversion by autotrophic nitrifiers, suggesting that SP close to 30‰ is typical for autotrophic nitrification in soils following NH₄⁺-fertilization. The results suggest that the δ¹⁸O/SP fingerprint of N₂O might be used as a new indicator of the dominant source process of N₂O fluxes in soils.     

盆栽和田间条件下土壤15N标记肥料氮的转化

利用¹⁵N在盆栽条件下研究了铵的矿物固定作用对肥料氮在三种土壤中转化的影响.结果表明,红壤性水稻土不固定肥料铵,但在白土和夹沙土中,56-77%的肥料氮被土壤矿物所固定,这些“新固定”的固定态铵的有效性很高,其中90%以上在30-50天内即被水稻所吸收,或者为微生物所利用转变为生物固定态氮.生物固定态氮对当季作物的有效性远较“新固定”的固定态铵的低.田间微区试验的结果还表明,甚至第二、三季作物吸收的残留肥料氮中,20-86%的氮也系来自固定态铵.作者认为,对具有较强固铵能力的土壤来说,只有了解铵的矿物固定作用,才能正确了解肥料氮的其它转化过程.     

Influence of mycorrhiza vs. soluble phosphate on growth,nodulation, and N2 fixation (15N) in alfalfa under different levels of water potential

Summary The legume Medicago sativa (+Rhizobium melilott) was grown under controlled conditions to study the interactions between soluble P in soil (four levels), or a mycorrhizal inoculum, and the degree of water potential (four levels) in relation to plant development and N₂ fixation. ¹⁵N-labelled ammonium sulphate was added to each pot for a qualitative estimate of N₂ fixation, in order to rank the effects of the different treatments.Dry-matter yield, nutrient content and nodulation increased with the amount of plant-available P in the soil, and decreased as the water stress increased, for each P-level. The mycorrhizal effect on dry matter, N yield, and on nodulation was little affected by the water potential. Since P uptake was affected by the water content in mycorrhizal plants, additional mechanisms, other than those mediated by P, must be involved in the mycorrhizal activity.There was a positive correlation between N yield and nodulation for the different P levels and the mycorrhizal treatment at all water levels. A high correlation between plant unlabelled N content and atom% ¹⁵N excess was also found for all levels of P. In mycorrhizal plants, however, the correlation between unlabelled N yield and ¹⁵N was lower. This suggests that mycorrhiza supply plants with other N sources in addition to those derived from the improvement on N₂ fixation.