Enrichment of natural 15N abundance in frankia-infected nodules

Abstract

The enrichment of ¹⁵N in the nodules of some N₂-fixing leguminous plants is an interesting finding (Shearer et al. 1982). The extent of ¹⁵N enrichment differed depending on the plant species (Shearer et al. 1982; Yoneyama 1987) and bacterial strains (Steele et al. 1983), and in soybeans it was apparently related to the nitrogen fixation efficiency (Shearer et al. 1984)     

Landscape-scale estimates of dinitrogen fixation by Pisum sativum by nitrogen-15 natural abundance and enriched isotope dilution

Topography and slope position influence the soil and environmental factors that affect N₂ fixation by legumes. The present study was conducted to (1) estimate N₂ fixation by field peas in a gently rolling farm field using the natural ¹⁵N abundance and the ¹⁵N-enriched isotope dilution techniques and (2) identify soil and environmental factors that influence N₂ fixation at the landscape scale. Whereas soil available water capacity, available NH _inf4 ^sup+ , total crop yield, and percent N derived from N₂ fixation (% Ndfa) estimated using enriched N were significantly affected by landform patterns, soil NO _inf3 ^sup- levels, seed yield, and the % Ndfa estimated using natural abundance did not follow landform patterns. The % Ndfa using natural abundance was correlated with NH _inf4 ^sup+ but not with available soil water, pH, electrical conductivity, NO _inf3 ^sup- , or particle size. Estimates of the % Ndfa using enriched ¹⁵N ranged from 0 to 92.8%. The highest median value (68.6%) for % Ndfa using enriched N occurred on the divergent footslopes, with the lowest value (28.1%) on the convergent shoulders. Estimates of % Ndfa using natural abundance ranged from 13.2% to 96.9%. Smaller fluctuations during the growing season in the ¹⁵N of the available N pool may have resulted in less variability for % Ndfa using natural abundance compared to enriched ¹⁵N. Despite similar mean values for % Ndfa using natural abundance (44.5) and enriched ¹⁵N (49.6), no significant correlation between the two estimates was found. These results suggest that although topography may exert gross controls on N₂ fixation, large variations in N₂ fixation at the microsite level may preclude correlations between individual estimates and limit detection of landscape scale patterns of N₂ fixation.Contribution No. R754 of the Saskatchewan Center of Soil Research     

Nitrogen accumulation in three legumes and two cereals with emphasis on estimation of N2 fixation in the legumes by the natural 15N-abundance technique

Summary N accumulation and natural ¹⁵N abundance in three legumes (groundnuts, cowpeas, and soybeans) and in two cereals (sorghum and maize) were investigated over two seasons in Alfisols with and without N fertilization. Using the N uptake and natural ¹⁵N abundance of non-nodulating plants as the indication of N derived from soil and fertilizer, the per cent N derived from atmospheric N₂ was calculated for nodulated plants. In the first experiment, the groundnut genotype contained 85% atmosphere-derived N, but the percentage decreased with N application. Estimates of atmosphere-derived N by the N-difference and ¹⁵N-abundance techniques gave identical results. The percentages of atmosphere-derived N estimated by the two methods at different stages of groundnut growth were also similar. In the second experiment, atmosphere-derived N was estimated in plants grown with 0–200 kg ha^-1 applied N. The estimated atmosphere-derived N ranged from 42% to 61% for groundnuts from 33% to 77% for cowpeas, and from 24% to 48% for soybeans, depending on the amount of N applied. Inoculation with a Bradyrhizobium strain increased the percentage of atmospherederived N in soybean plants grown without any fertilizer N. The natural ¹⁵N abundance of sorghum and maize was very close to that of the non-nodulating groundnut, suggesting that these cereals can be used as reference plants in the estimation of atmosphere-derived N by the natural ¹⁵N-abundance method.ICRISAT Journal Article No. 876     

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 by field-grown lupins (Lupinus angustifolius L.) using soil and plant 15N enrichment

Summary Biological N₂ fixation was estimated in a field experiment following the addition of NH₄Cl or KNO₃ to unconfined microplots (1.5 m²) at 2.5 g N m^-2 (10 atom% ¹⁵N). A model of total N and ¹⁵N accumulation in lupins and decreasing ¹⁵N enrichment in the KCl-extractable soil-N pool (0–0.15 m depth) was used to estimate the proportion of N in lupins derived from biological N₂ fixation. Estimates of N₂ fixation derived from the model were compared with ¹⁵N isotope-dilution estimates obtained using canola, annual ryegrass, and wheat as nonfixing reference plants. Biomass, total N accumulation, or ¹⁵N enrichment in the lupin and reference crops did not differ whether NH _inf4 ^sup+ or NO _inf3 ^sup- was added as the labelled inorganic-N source. The decrease in soil ¹⁵N enrichment was described by first-order kinetics, whereas total N and ¹⁵N accumulation in the lupins were described by logistical equations. Using these equations, the uptake of soil N by lupins was estimated and was then used to calculate fixed N₂. Estimates of N₂ fixation derived from the model increased from 0 at 50 days after sowing to a maximum of 0.79 at 190 days after sowing. Those based on the ¹⁵N enrichment of the NO _inf3 ^sup- pool were 10% higher than those based on the mineral-N pool. ¹⁵N isotope-dilution estimates of N₂ fixation ranged from 0.37 to 0.55 at 68 days after sowing and from 0.71 to 0.77 at 190 days after sowing. Reference plant-derived values of N₂ fixation were all higher than modelled estimates during the early states of growth, but were similar to modelled estimates at physiological maturity. The use of the model to estimate N₂ derived from the atmosphere has the intrinsic advantage that the need for a non-fixing reference plant is avoided.     

Determination of 15N abundance of amino acids in soil hydrolysates

¹⁵N abundance of amino acids in soil hydrolysates was determined by emission spectroscopic method.     

Quantification of the contribution of biological nitrogen fixation to Cratylia mollis using the N natural abundance technique in the semi-arid Caatinga region of Brazil

This study was performed to investigate the capacity of the woody perennial Cratylia mollis, a legume endemic to the semi-arid region of the North-East of Brazil, to nodulate, and obtain N from BNF using the ¹⁵N natural abundance technique. To estimate the ¹⁵N abundance of the N-derived from soil, the leaves of several (4-6) non-legume and non-nodulating legume species growing in close proximity to the Cratylia were analysed for δ¹⁵N. The δ¹⁵N values of these reference plants were high (from +9 to +16‰) and showed relatively small differences between species at each site/sampling time. At the irrigated site at both samplings, and at the non-irrigated site sampled in the rainy season, the ¹⁵N abundance of the Cratylia mollis leaves was far lower (+1 to +5‰) strongly suggesting that the legume obtained large proportional contributions from BNF. As was to be expected, no nodules were found on the Cratylia plants at the non-irrigated site in the dry season, and the ¹⁵N abundance of the Cratylia mollis plants were very similar to that of the reference plants, consistent and nodulation and BNF being limited by water deficit at this time.     

Application of N2/Ar ratio method for N2 fixation studies in submerged soil

Abstract

Recently there has been developments in the measurement of N₂ fixation due mainly to the C₂H₂ reduction method (1). This method, however, has several disadvantages, especially for submerged soil, and the estimated amount of fixed N₂ on the basis of the C₂H₂ reduction activity is not very reliable. The tracer ¹⁵N₂ technique which gives a reliable estimation of the fixed N₂ is too expensive for common use. Development of an alternative method suitable for submerged soil would therefore be desirable. The present authors expected that the measurement of the ratio N₂/Ar in the soil solution might provide advantages for the estimation of the fixed N₂ in submerged soil.     

15N标记的土壤估测某些豆科植物N2固定的温室实验

Pot experiments were carried out to estimate N2 fixation by vetch,milk vetch,sickle alfalfa and broadbean in pure stand using a ^15N-labelled soil.Winter wheat was used as the non-fixing control.The 15N-labelled soil used was prepared by growing corn-wheat-corn successively on a nearly organic-matter-free Xiashu loess supplemented with adequate amounts of (15NH4)2SO4,P,K and micronutrients,then incorporating these 15N-labelled plant materials into the soil after each havest,and allowing the plant materials to be decomposed aerobically for 410d after incorporation of the plant material of the thire crop.The 15N enrichment of wheat plant-N varied slightly with organs,with a maximum difference of 9.8%,Based on 15N enrichment of soil N inferred from the mean value of the 15N enrichment in different organs of wheat 79%-91% of total N in the tops and 67%-74% of total N in the roots of legumes studied were derived from atmosphere .Estimate by isotope dilution method was in good agreement with that by the conventional difference method provided values obtained by the latter were corrected for seed N,and also with that from the measurement of N accumulated in the tops of the legumes.     

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.     

N2O产生法测定土壤无机态氮15N丰度

用化学方法分别将土壤中微量的铵、硝酸盐和亚硝酸盐转化为N2O气体,然后用带自动预浓缩装置的同位素比值质谱仪测定N2O中的15N丰度.N2O中的15N丰度测量值完全符合铵、硝酸盐和亚硝酸盐的15N参考值.方法快速、简单和准确,不受空气氮的污染.特别是方法的检测限很低,每批次样品中只需含5～ 20μg N.它将有助于土壤氮素的矿化作用、硝化作用和反硝化作用的研究.     

C and N natural abundance of the soil microbial biomass

Stable isotope analysis is a powerful tool in the study of soil organic matter formation. It is often observed that more decomposed soil organic matter is ¹³C, and especially ¹⁵N-enriched relative to fresh litter and recent organic matter. We investigated whether this shift in isotope composition relates to the isotope composition of the microbial biomass, an important source for soil organic matter. We developed a new approach to determine the natural abundance C and N isotope composition of the microbial biomass across a broad range of soil types, vegetation, and climates. We found consistently that the soil microbial biomass was ¹⁵N-enriched relative to the total (3.2 ‰) and extractable N pools (3.7 ‰), and ¹³C-enriched relative to the extractable C pool (2.5 ‰). The microbial biomass was also ¹³C-enriched relative to total C for soils that exhibited a C3-plant signature (1.6 ‰), but ¹³C-depleted for soils with a C4 signature (−1.1 ‰). The latter was probably associated with an increase of annual C3 forbs in C4 grasslands after an extreme drought. These findings are in agreement with the proposed contribution of microbial products to the stabilized soil organic matter and may help explain the shift in isotope composition during soil organic matter formation.     

Incorporation of 15N into various nitrogenous compounds in intact soybean nodules after exposure to 15N2 gas

The intact nodules attached to the upper part of soybean roots were exposed to ¹⁵N₂ and the incorporation of ¹⁵N into various soluble nitrogen constituents was investigated. Results indicated that ammonia, a primary product of N₂ fixation, was located in more than two compartments. Ammonia reduced from N₂ gas seemed to be incorporated firstly into glutamine especially amido-group nitrogen. Newly fixed nitrogen was secondly incorporated into glutamic acid and alanine in this sequence. These results suggested that fixed ammonia was assimilated by glutamine synthetase/glutamate synthase pathway. Turn-over rate of allantoin plus allantoic acid and serine was relatively high, although apparently these compounds were not primary products of newly fixed ammonia. ¹⁵N content of allantoin was always higher than that of allantoic acid. ¹⁵N incorporation to aspartic acid and asparagine was relatively slow, especially in early period. In bacteroid fraction there is much amount of ammonia comparing with other compounds, while allantoin and asparagine were presented exclusively in cytosol. ¹⁵N was incorporated into nitrate within a few minutes especially in bacteroids.     

Nitrogen fixation and soil N level during maturation affect the contents of storage compounds of soybean seeds

We compared the concentrations and contents of protein and oil in mature seeds from nodulated and non-nodulated soybean plants grown on soils with four different N levels during maturation. We observed a positive correlation between the contents of protein and oil in seeds from nodulated plants. Seeds from nodulated plants grown on urea-treated soil showed higher protein and lower oil contents than those from plants grown on soil treated with coated slow release N fertilizer (LP-100). Contents of these compounds in seeds from nodulated plants grown on LP-100 soil were almost the same as those from non-nodulated plants on the same soil. These observations indicated that N economy in roots during seed maturation affects the contents of storage compounds. We suggested that the control of the N₂ fixation activity of soybean plants and management of soil N level during seed maturation are important to determine the contents of protein and oil in seeds.     

Isotope 15N enrichment of soil-ammonium N as a reference to estimate N2 fixation by stem and root-inoculated S. rostrata

Summary A pot experiment in the greenhouse was conducted to compare the contribution of N derived from the atmosphere or from biological N₂ fixation by Sesbania rostrata inoculated with Azorhizobium caulinodans, applied either to roots or to roots and stems (single or multiple stem inoculation). Two subsequent crops were grown for 50 days under flooded conditions. N derived from air was estimated by ¹⁵N dilution using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N and of Echinochloa crusgalli as the non-N₂-fixing reference datum and compared with estimates obtained by the N-difference method. The first crop was grown to stabilize the ¹⁵N into the soil organic N fraction. The ¹⁵N enrichment of soil NH _inf4 ^sup+ -N in the second crop declined slowly. The extractability ratio (¹⁵N enrichment of extractable soil N to ¹⁵N enrichment of total soil N) decreased from 4.8 to 4.1 50 days after planting. The enrichment of soil NH _inf4 ^sup+ -N was comparable to that of E. crus-galli, resulting in similar estimates of N derived from air when either soil NH _inf4 ^sup+ -N or enrichment of E. crus-galli was used as a non-fixing reference. The N-difference method did not always provide reliable estimates of N derived from air; percentages ranged from 75 to more than 80 by 50 days after planting in both crops and did not differ among treatments. The study demonstrates the potential of using ¹⁵N enrichment of soil NH _inf4 ^sup+ -N as a non-N₂-fixing reference for reliable BNF estimates of crops in lowland puddled soil.     

Effect of soil physical properties on soybean nodulation and N2 fixation at the early growth stage in heavy soil field in Hachirougata Polder,Japan

We studied the effect of the soil physical properties on soybean nodulation and N₂ fixation in the heavy soil of an upland field (UF) and an upland field converted from a paddy field (UCPF) in the Hachirougata polder, Japan. Seeds of the soybean cultivar Ryuho were sown in each field with or without inoculation of Bradyrhizobium japonicum A1017. The soybean plants were sampled at 35 (V3) and 65 (Rl) d after sowing (DAS), and then nodulation and the percentage of N derived from N₂ fixation in the xylem sap were determined. The soil physical properties were different between UF and UCPF, especially the air permeability and soil water regime. Nodule growth was restricted in UCPF irrespective of rhizobial inoculation, though rhizobial infection was not inhibited by the unfavorable soil physical conditions. Soybean plant growth was closely related to the nodule mass and N₂ fixation activity, and the inoculation of a superior rhizobium strain was effective only at 35 DAS. These results indicate that soybean nodulation and N₂ fixation was considerably affected by the physical properties of heavy soil, and that it is important to maintain the N₂ fixation activity and inoculate the soybean plants with a superior rhizobium strain at a later growth stage in order to increase soybean production in heavy soil fields.     

Plant nitrate use in deciduous woodland: the relationship between leaf N, N natural abundance of forbs and soil N mineralisation

Our aim was to study whether the in situ natural abundance ¹⁵N (δ¹⁵N)-values and N concentration of understory plants were correlated with the form and amount of mineral N available in the soil. Also to determine whether such differences were related to earlier demonstrations of differences in biomass increase in the same species exposed to nutrient solutions with both and or to alone. Several studies show that the δ¹⁵N of in soil solution generally is isotopically lighter than the δ¹⁵N of due to fractionation during nitrification. Hence, it is reasonable to assume that plant species benefiting from in ecosystems without significant leaching or denitrification have lower δ¹⁵N-values in their tissues than species growing equally well, or better, on We studied the δ¹⁵N of six understory species in oak woodlands in southern Sweden at 12 sites which varied fivefold in potential net N mineralisation rate The species decreased in benefit from in the following order: Geum urbanum, Aegopodium podagraria, Milium effusum, Convallaria majalis, Deschampsia flexuosa and Poa nemoralis. Four or five species demonstrated a negative correlation between and leaf δ¹⁵N and a positive correlation between and leaf N concentration. In wide contrast, only D. flexuosa, which grows on soils with little nitrification, showed a positive correlation between and the leaf N concentration and δ¹⁵N-value. Furthermore, δ¹⁵N of plants from the field and previously obtained indices of hydroponic growth on relative to were closely correlated at the species level. We conclude that δ¹⁵N may serve as a comparative index of uptake of among understory species, preferably in combination with other indices of N availability. The use of δ¹⁵N needs careful consideration of known restrictions of method, soils and plants.     

Estimating N2 fixation by Sesbania rostrata and S. cannabina (syn. S. aculeata) in lowland rice soil by the 15N dilution method

Summary A field experiment in concrete-based plots was conducted to estimate the contribution of N derived from air (Ndfa) or biological N₂ fixation in Sesbania rostrata and S. cannabina (syn. S. aculeata), using various references, by the ¹⁵N dilution method. The two Sesbania species as N₂-fixing reference plants and four aquatic weed species as non-N₂-fixing references were grown for 65 days after sowing in two consecutive crops, in the dry and the wet seasons, under flooded conditions. Soil previously labeled with ¹⁵N at 0.26 atom % ¹⁵N excess in mineralizable N was further labeled by ammonium sulfate with 3 and 6 atom % ¹⁵N excess. The results showed that ¹⁵N enrichment of soil NH ₄ ⁺ -N dropped exponentially in the first crop to half the original level in 50 days while in the second crop, it declined gradually to half the level in 130 days. The decline in ¹⁵N enrichment, in both N₂-fixing and non-fixing species, was also steeper in the first crop than in the second crop. Variations in ¹⁵N enrichment among non-fixing species were smaller in the second crop. The ratio of the uptake of soil N to that of fertilizer N in N₂-fixing and non-fixing species was estimated by the technique of varying the ¹⁵N level. In the second crop, this ratio in non-fixing species was higher than that in N₂-fixing species. Comparable estimates of % Ndfa were obtained by using ¹⁵N enrichment of various non-fixing species. There was also good agreement between the estimates obtained by using ¹⁵N enrichment of non-fixing species and those by using soil NH ₄ ⁺ -N, particularly in the second crop. By 25 days after sowing, the first crop of both Sesbania spp. had obtained 50% of total N from the atmosphere and the second crop had obtained 75%. The contribution from air increased with the age of the plant and ranged from 70% to 95% in 45–55 days. S. rostrata fixed substantially higher amounts of N₂ due to its higher biomass production compared with S. cannabina. Mathematical considerations in applying the ¹⁵N dilution method are discussed with reference to these results.     

Natural 15N and 13C abundance in Andisols influenced by long-term fertilization management in Japan

Two field experiments were conducted on Andisols in Japan to evaluate the changes in the natural ¹⁵N and ¹³C abundance in the soil profile and to determine whether the values of δ¹⁵N could be used as an indicator of fertilizer sources or fertilizer fate. The 6-year experiment conducted at the National Agricultural Research Center (NARC) consisted of the following treatments: application of swine compost (COMPOST), slow-release nitrogen fertilizer (SRNF), readily available nitrogen fertilizer (RANF), and absence of fertilization (CONTROL). Experimental plots located at the Nippon Agricultural Research Institute (NARI) received cattle compost at different rates for 12 years; a forest soil at this site was sampled for comparison. Swine compost application led to a considerable change in the δ¹⁵N distribution pattern in the soil profile, with the highest δ¹⁵N values recorded in the top 20 cm layers of the COMPOST plot, decreasing in the sequence of CONTROL >- RANF > SRNF, mainly due to the relatively high δ¹⁵N value of swine compost and its subsequent decomposition. In contrast, SRNF application resulted in the lowest δ¹⁵N values in soil, indicating the presence of negligible nitrogen losses relative to input and low nitrogen cycling rates. Values of δ¹⁵N increased with compost application rates at NARI. In the leachate collected at 1-m depth, the δ¹⁵N values decreased in the sequence of COMPOST > RANF ≥ CONTROL > SRNF. The δ¹³C values in soil peaked in the 40–60 cm layers for all the fertilizers. The δ¹³C value was lowest in forest soil due to the presence of plant residues in soil organic matter. These results indicated that the δ¹⁵N values in the upper soil layers or leachate may enable to detect pollution sources of organic or inorganic nitrogen qualitatively in Andisols.     

Areal variation in the relationship between natural 15N abundances of rice (Oryza sativa L.) and soil

This study was performed to clarify whether areal variation exists in the relationship between natural ¹⁵N abundances (δ¹⁵N values) of rice (Oryza sativa L.) and soil without an applied nitrogen (N) source, and to explore possible reasons for any areal variation. We investigated the relationships between δ¹⁵N values of rice and those of unamended soil with no applied N source in two locations; Daisen and Ogata, in Akita Prefecture, Japan. The δ¹⁵N values of rice in Daisen were higher than those in Ogata from 2007 to 2009, irrespective of the cropping year. Results demonstrated areal variation in the relationship between δ¹⁵N values of rice and those of unamended soil. The variation might be attributed to variation in the δ¹⁵N of natural N input and to ammonia nitrification and subsequent denitrification. When the relationship between δ¹⁵N values of rice and those of unamended soil is used to discriminate between organic and conventional rice, the areal variation of the relationship in the target area should be taken into account, from the point of the δ¹⁵N value of natural N input and N transformation in the soil.