Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

Exploration of the nitrogen transport system of a nodulated legume using 15N

Summary Feeding experiments using ¹⁵N₂ or ¹⁵NO₃ are described investigating the transport of nitrogen in the field pea (Pisum arvense L.). Nitrogen assimilated by root or nodules moves preferentially upwards to the shoot through the xylem. Parts of the root below or distal to a region of assimilation can benefit from this nitrogen but do so to a much greater extent when the shoot is left attached than when it has been removed. A considerable proportion of the nitrogen received by a shoot from the root or nodules is apparently returned to the root in the translocation stream, this cycled nitrogen being especially important in the nutrition of outlying parts of nodulated roots growing in media lacking combined nitrogen.Nitrogen from nitrate fed to a mature leaf is exported in quantity to all parts of the plant except older regions of the shoot. Leaf and stem segments immediately above the fed leaf, and the root and its nodules receive large shares of this nitrogen, although the root's share declines noticeably as the plant ages.The root appears to be extremely inactive in transferring nitrogen from the downward translocation stream across to the stream of nitrogen leaving the root in the xylem. This may act as a major obstacle to the free circulation and mixing of nitrogen within the plant body.A scheme is proposed embracing the main quantitative features of the transport system for nitrogen in the species.     

Incorporation of 15N into allantoin in nodulated soybean plants supplied with 15N2

Incorporation of ¹⁵N into allantoin and allantoic acid in noduleswas higher than that in roots. This confirms that nodules produceallantoin. The ¹⁵N concentration in allantoin was slightly higherthan that in allantoic acid, suggesting that allantoin decomposedto allantoic acid. Allantoin and allantoic acid in nodules weretranslocated rapidly to roots. (Received August 25, 1976; )     

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.     

Nitrogen source influences natural abundance (15)N of Escherichia coli.

Escherichia coli cells were forced to mineralize or assimilate nitrogen in vitro by manipulating substrate carbon and nitrogen availability. When grown on an organic nitrogen source, E. coli cells released NH(4)(+) and were enriched in (15)N relative to the nitrogen source (1.6-3.1 per thousand). However, when cells were grown on an inorganic nitrogen source, the biomass was depleted (6.1-9.1 per thousand) relative to the source. By measuring (15)N enrichment of microorganisms relative to nitrogen pools, ecosystem ecologists may be able to determine if microorganisms are assimilating or mineralizing nitrogen.     

Natural 15N abundance in shrub and tree legumes,Casuarina, and non N2 fixing plants in Thailand

The leaves and nodules from the shrub and tree legumes, particularly, Aeschynomene spp., Sesbania spp., Mimosa spp. and Leucaena spp., and Casuarina spp. and the leaves from neighbouring non-fixing plants were analyzed for their natural abundances of ¹⁵N ( ¹⁵N).The ¹⁵N in the leaves of non-fixing plants was +5.9% on average, whereas those from shrub legumes and Casuarina spp. were lower and close to the values of atmospheric N₂, suggesting the large contribution of N₂ fixation as the N source in these plants. The ¹⁵N values of the leaves from tree legumes except for Leucaena spp. were between the shrub legumes and non-fixing plants, which suggests that the fractional contribution of fixed N₂ in tree legumes may be smaller than that in the shrub legumes. Casuarina spp. was highly dependent on N₂ fixation. The ¹⁵N values of the nodules from most of the shrub legumes investigated were higher than those of the leaves.     

Natural abundance of 15N and 13C in nodulated legumes and other plants in the cerrado and neighbouring regions of Brazil

Leaves from over 1000 Brazilian native plants growing in the cerrado and neighbouring regions were sampled for C and N content. Half of these were analysed for ¹⁵N and further samples for ¹³C and ash content. Nodulated legumes from all three sub-families were included, together with two types of reference plant, non-nodulated legumes and non-legumes. Particular emphasis was placed on the large caesalpinioid genus Chamaecrista which is here for the first time reported to fix nitrogen in its native habitats. Woody and herbaceous species of this and other nodulated genera, with the exception of the mimosoid tree Stryphnodendron, showed evidence of nitrogen fixation. Amounts fixed were site-specific as was the ¹⁵N signature of reference plants. There was no evidence that nodulated legumes had higher leaf N than non-nodulated legumes: both were higher than non-legumes. Several species of Chamaecrista from section absus and species of Stryphnodendron had carbon contents of 50–55%, higher than previously reported for leaves. This was coupled with low (1–3%) ash contents. The ¹³C values of plants with 49% C were significantly more negative than those with <49% C: most species in the former group were woody and most in the latter group herbaceous. Mimosa pudica was unusual in having a wide range of percent C, percent ash and ¹³C values; these parameters were significantly correlated. It is concluded that Brazilian native legumes can fix significant amounts of nitrogen in the nutrient-poor cerrado soils. Consideration of mineral and lipid nutrition will be necessary in order fully to understand relations between ¹³C, carbon content and other physiological parameters.     

The natural abundance of 15N in mat-forming lichens

Natural abundance of (15)N and [N] was studied in thalli of mat-forming lichens collected from tundra and heathland sites in the northern and southern hemispheres. The study includes samples of British Cladonia portentosa from sites in regions of high and low N-loading and in heathland growing both directly on peat and independently of the soil substratum, in a canopy of prostrate gorse ( Ulex minor). In the mat-forming lichens examined, a non-random pattern in [N] and delta(15)N was characterised by a minimum in delta(15)N, which occurred most frequently at 20-40 mm below the thallus apex. Nitrogen concentration increased above this point, towards the apex, though remained invariably low towards the thallus base. We discuss the significance of the pattern in [N] and delta(15)N for current theories describing the uptake and recycling of nitrogen by mat-forming lichens in oligotrophic habitats. Our data are incompatible with the suggested uptake of soil organic-N depleted in (15)N, though are consistent with possible internal recycling and the development of a structural necromass. The study emphasises the internal fractionation of nitrogen isotopes and provides a caveat against the assumption that values of delta(15)N provide an unequivocal indicator of source-sink relationships in nitrogen cycling.     

15N natural abundance studies in Australian commercial sugarcane

The measurement of natural ¹⁵N abundance is a well-established technique for the identification and quantification of biological N₂ fixation in plants. Associative N₂ fixing bacteria have been isolated from sugarcane and reported to contribute potentially significant amounts of N to plant growth and development. It has not been established whether Australian commercial sugarcane receives significant input from biological N₂ fixation, even though high populations of N₂ fixing bacteria have been isolated from Australian commercial sugarcane fields and plants. In this study, ¹⁵N measurements were used as a primary measure to identify whether Australian commercial sugarcane was obtaining significant inputs of N via biological N₂ fixation. Quantification of N input, via biological N₂ fixation, was not possible since suitable non-N₂ fixing reference plants were not present in commercial cane fields. The survey of Australian commercially grown sugarcane crops showed the majority had positive leaf ¹⁵N values (73% >3.00, 63% of which were >5.00), which was not indicative of biological N₂ fixation being the major source of N for these crops. However, a small number of sites had low or negative leaf ¹⁵N values. These crops had received high N fertiliser applications in the weeks prior to sampling. Two possible pathways that could result in low ¹⁵N values for sugarcane leaves (other than N₂ fixation) are proposed; high external N concentrations and foliar uptake of volatilised NH₃. The leaf ¹⁵N value of sugarcane grown in aerated solution culture was shown to decrease by approximately 5 with increasing external N concentration (0.5–8.0 mM), with both NO₃ ^– and NH₄ ⁺ nitrogen forms. Foliar uptake of atmospheric NH₃ has been shown to result in depleted leaf ¹⁵N values in many plant species. Acid traps collected atmospheric N with negative ¹⁵N value (–24.45±0.90) from above a field recently surface fertilised with urea. The ¹⁵N of leaves of sugarcane plants either growing directly in the soil or isolated from soil in pots dropped by 3.00 in the same field after the fertiliser application. Both the high concentration of external N in the root zone (following the application of N-fertilisers) and/or subsequent foliar uptake of volatilised NH₃ could have caused the depleted leaf ¹⁵N values measured in the sugarcane crops at these sites.     

N2 fixation in cowpea plants grown in farmers’ fields in the Upper West Region of Ghana, measured using15N natural abundance

Few studies have assessed the levels of symbiotic N nutrition in legumes grown by farmers in Africa. In this study, the shoots of cowpea plants were sampled from 63 farms in 12 villages within 5 districts of the Upper West Region of Ghana, and assessed for growth and symbiotic N nutrition. The data revealed considerable differences in cowpea plant density per m², plant growth,¹⁵N natural abundance (δ¹⁵N), %Ndfa, and N-fixed among different farms under one village, and between villages under the same district, and between districts in the Upper West Region. In farms where there were fewer cowpea plants per m², plant growth was better and dry matter yield per plant significantly greater, leading to strong variations in δ¹⁵N values. Except for four farms at Bamahu which had cowpea shoot Ndfa values of 12.1%, 30.0%, 36.5% and 46.6%, one farm at Babile with Ndfa value of 58.1%, and three farms at Silbelle with Ndfa values of 56.8%, 57.9% and 68.7%, the remaining 55 out of the 63 farms studied showed high shoot Ndfa values, ranging from 70.6% to 99.7%, which clearly indicates that cowpea cultivated by farmers in the Upper West Region of Ghana meet a large proportion of their N requirements from symbiotic fixation. At the district level, isotopic analysis showed that, on average, the¹⁵N natural abundance values (%0) of cowpea shoots were ?0.496±0.04 for Jirapa, ?0.083±0.06 for Nadowli, 0.368±0.08 for Lawra, J.333±0.29 for Wa and 0.365±0.09 for Sissala district. Estimates of the legume’s N derived from fixation were 66.3% for Wa district, 89.9% for Nadowli, 79.4% for Lawra, 78.9% for Sissala and 80.9% for Jirapa district. The amount of N-fixed ranged from 402.3 mg.plant^?1 for Nadowli, 176.5 mg.plant^?1 for Wa, 235.4 mg.plant^?1 for Sissala, 179.0 mg.plant^?1 for Lawra to 249.2 mg.plane^?1 for the Jirapa district. Expressed on per-hectare basis using cowpea density per m², the total amount of N-fixed was around 16.6 kg ha^?1 in the Nadowli district, 19.1 kg ha^?1 in Wa, 23.0 kg ha^?1 in Sissala, 2J.1 kg ha^?1 in Lawra and 17.6 kg ha^?1 in the Jirapa district. Averaged across all 5 districts, N-fixed by cowpea was about 19.5 kg ha^?1 in the Upper West Region of Ghana. These data suggest that, increasing N₂ fixation in fanners’ fields in Ghana would require optimization of cowpea plant density rather than biological manipulation of the symbiotic process (as %Ndfa values were generally very high).     

Natural abundance of 15N in actinorhizal plants and nodules

Substantial enrichment of some plant parts in ¹⁵N relative to the rest of the plant is unusual, but is found in the nitrogen-fixing nodules of many legumes. A range of actinorhizal plants was surveyed to determine whether the nodules of any of them are also substantially enriched in ¹⁵N. The nonlegume Parasponia, nodulated by a rhizobium, was also included. Four of the actinorhizal genera and Parasponia were grown in N-free culture, and three actinorhizal genera were collected from the field. Nodules of Parasponia, Casuarina and Alnus were¹⁵N enriched relative to other plant parts, but only Parasponia approached the degree of enrichment found in some legume nodules. The nodules of Datisca, Myrica, Elaeagnus, Shepherdia, and Coriaria were depleted in ¹⁵N. Thus many actinorhizal nodules are depleted in ¹⁵N compared to other plant parts and enrichment is modest when it does occur. Whole plant ¹⁵N content (¹⁵N) in four actinorhizal plants and Parasponia showed a relatively narrow range of –1.41 to –1.90. Hence regardless of the degree of nodule enrichment or depletion, whole plant ¹⁵N content appears to vary little in plants grown in N-free culture.     

Hepatoprotective effect of rooibos tea (Aspalathus linearis) on CCl4-induced liver damage in rats

Hepatoprotective properties of rooibos tea (Aspalathus linearis) were investigated in a rat model of liver injury induced by carbon tetrachloride (CCl(4)). Rooibos tea, like N-acetyl-L-cysteine which was used for the comparison, showed histological regression of steatosis and cirrhosis in the liver tissue with a significant inhibition of the increase of liver tissue concentrations of malondialdehyde, triacylglycerols and cholesterol. Simultaneously, rooibos tea significantly suppressed mainly the increase in plasma activities of aminotransferases (ALT, AST), alkaline phosphatase and billirubin concentrations, which are considered as markers of liver functional state. The antifibrotic effect in the experimental model of hepatic cirrhosis of rats suggests the use of rooibos tea as a plant hepatoprotector in the diet of patients with hepatopathies.     

Associative N2-fixation in plants growing in saline sodic soils and its relative quantification based on15N natural abundance

The interactive effect of low P supply (0, 10, 20 and 40 M) and plant age on nodule number, mass and functioning (ureide analysis technique), vegetative growth and pod production were investigated in glasshouse-grown nodulated cowpea (Vigna unguiculata L.cv. Kausband) in sand culture. Compared with 40 M P, P stress (0 M P) or very low (10 M P) supply markedly impaired nodulation, allantoin and amino-N concentrations and weight of N solutes in xylem exudates. Consequently, P stress reduced top growth and pod yields by 48 and 90%, respectively. N solutes in xylem exudates and total plant N assayed by Kjeldahl technique (as estimates of N₂ fixation) responded similarly to P supply. However, the relative ureide index [(ureide-N/ureide N+amino-N)×100] remained constant (99%), irrespective of P supply, indicating the plants' complete dependency on symbiosis for growth, without implying that growth was markedly increased by N₂ fixation. Although P concentrations in plant tops, roots and nodules increased with P supply, N concentrations in these plant tissues were unaffected by P supply. The concentrations of N and P in the nodules were 2–2 1/2 times higher than in plant tops. P application interacted strongly with plant age, with the largest P effect evidently achieved at the early podding stage. The significance and implications of these results are discussed.     

Measurement of nitrogen fixation by soybean in the field using the ureide and natural N abundance methods

Nitrogen fixation by field-grown soybean (Glycine max [L.] Merrill) was assessed by the natural ¹⁵N abundance and ureide methods. The field sites (five) and genotypes (six, plus two levels of inoculation on Bragg) were chosen to provide a range of proportions of plant N derived from nitrogen fixation (P). Genotypes K466, K468, nts1007, and nts1116 and Davis were included on the basis of their reported tolerance of the suppressive effects of nitrate on nodulation and nitrogen fixation. Bragg was included as a `nitrate-sensitive' genotype. Seeds of all genotypes were inoculated at sowing with Bradyrhizobium japonicum CB1809 (USDA136). Amounts of nitrate in the soil profile (0-1.2 meter depth) at sowing ranged from 70 (site 3) to 278 kilograms per hectare (site 5), resulting in large effects on plant nodulation, on the δ¹⁵N values of nodulated plants, on the relative abundance of ureide-N in vacuum-extracted sap (VES) and stem extracts, and finally on the estimates of P. There was no relationship between amount of soil nitrate at sowing and the δ¹⁵N of the plant-available soil N. Correlation matrices of the measured and calculated parameters indicated generally weak correlations between crop growth (dry matter and N) and the parameters of symbiotic activity (nodule weight, δ¹⁵N, relative ureide-N); correlations were strong and highly significant between nodulation and the measures of nitrogen fixation (δ¹⁵N, relative ureide-N; r = 0.79-0.92). Estimates of P ranged between 0 and 68% (δ¹⁵N) and between 6 and 56% (ureide) and were highly correlated (r = 0.97). Results indicated that the ureide method can be used with confidence to assess P by field-grown crops of soybean.     

Spatial variation of N2-fixation in field pea (Pisum sativum L.) at the field scale determined by the 15N natural abundance method

Grain legumes such as field pea are known to have high variability of yield and dinitrogen (N₂) fixation between seasons, but less is known about the yearly spatial variability within a field. The objective of this study was to improve the understanding of spatial field scale variability of field pea dry matter (DM) yield and nitrogen (N) acquisition from fixation and soil within a 10 ha farmer’s field. A 42 m systematic random grid providing 56 plant sampling locations across 10 ha supplemented by soil data provided from an existing database were used to determine whether the observed spatial variability could be explained by the variability in selected abiotic soil properties. All measured soil variables showed substantial variability across the field and the pea dry matter production ranged between 4.9 and 13.8 Mg ha^?1 at maturity. The percent of total N derived from the atmosphere (%Ndfa) at flowering, estimated using the ¹⁵N natural abundance method, ranged from 65% to 92% with quantitative N₂-fixation estimates from 93 kg to 202 kg N ha^?1. At maturity %Ndfa ranged from 26% to 81% with quantitative N₂-fixation estimates from 48 kg to 167 kg N ha^?1. Significant correlations were found between pea dry matter production and humus content, potassium content (collinear with humus) and total N in the 0–25 cm topsoil. No correlation was found between any individual soil property and %Ndfa or kg N fixed ha^?1. It was not possible to create a satisfactory global multi-regression model for the field dry matter production and N₂-fixation. A number of other models were tested, but the best was only able to explain less than 40% of the variance in %Ndfa using seven soil properties. Together with the use of interpolated soil data, high spatial variation of soil ¹⁵N natural abundance, a mean increase in pea ¹⁵N natural abundance of 1 δ unit between flowering and maturity and a reference crop decline of 1.3 δ¹⁵N unit over the same period increased noise of derived variables, making modeling of N₂-fixation difficult. Furthermore, complex interactions with other soil variables and biotic stresses not measured in this study may have contributed significantly to the variability of fixation and yield of pea within the field. Pea N₂-fixation obtained from two additional 10 ha farmer fields was in agreement with the other findings highlighting that N₂-fixation takes place under a range of physical and chemical soil properties and is controlled by local site specific conditions. In future studies addressing field scale variability we recommend that soil variables wherever possible should be measured in the same plots as the sampled crop. Sampling designs that optimize the use of a priori information about the field soil and landscape properties for positioning plots and that facilitate estimates of local variances should be considered.     

15N自然丰度法在陆地生态系统氮循环研究中的应用

随着氮沉降的不断增加以及人们对全球变化问题的日益关注, 稳定同位素技术在全球变化研究中得到广泛的应用。因为植物和土壤的氮同位素组成记录了氮循环影响因子的综合作用, 并且具有测量简单以及不受取样时间和空间限制的优点, 所以氮同位素自然丰度法被用于氮循环的研究中。该文从氮循环过程中植物和土壤的氮分馏入手, 总结国内外相关文献, 阐述了植物和土壤氮自然丰度在预测生态系统氮饱和和氮循环长期变化趋势中的应用; 总结了利用树轮δ ¹⁵N法研究氮循环过程中应该注意的事项以及目前尚未解决的问题。     

Estimates of seasonal nitrogen fixation of annual subterranean clover-based pastures using the15N natural abundance technique

Annual pasture legumes play a key role in ley farming systems of southern Australia, providing biologically fixed nitrogen (N) to drive the production of the pastures as well as subsequent crops grown in rotation. Seasonal inputs of biologically fixed N in shoot biomass of the subterranean clover (Trifolium subterraneum) component of grazed annual pastures were assessed using the¹⁵N natural abundance technique and appropriately timed sampling of herbage dry matter (DM) for N accumulation. At three study sites spanning a gradient across the Western Australian wheatbelt from 300 to 600 mm annual rainfall the performance of the clover and non-legume herbs and grasses was examined as paired comparisons involving two management treatments expected to give contrasting effects on pasture productivity, botanical composition and N₂ fixation. The proportion of clover N derived from atmospheric N₂ fixation (%Ndfa) ranged from 65 to 95% across sites, treatments and sampling times. Amounts of fixed N accumulated in clover shoot biomass ranged from 50 to 125 kg ha⁻¹, and paralleled trends in clover production. Substantial increases in pasture production in high yielding treatments generally occurred without decrease in %Ndfa, suggesting that N₂ fixation was essentially non-limiting to performance of the clover component. Seasonal profiles for accumulation of fixed N were skewed towards the late winter and spring period, particularly in low plant density pastures following a cereal crop. There were seasonal, site and treatment-specific effects on the proportion of clover and non-legume pasture components and consequently clover yield and N₂ fixation were variably affected by competition from non-legume species.     

Differences in contribution of biological nitrogen fixation to yield performance of common bean cultivars as assessed by the 15N natural abundance technique

Plant and Soil - Identification of variability in biological N2 fixation (BNF) contribution among common bean (Phaseolus vulgaris L.) cultivars under field conditions requires a reliable...     

Natural abundance of 15N in tropical plants with emphasis on tree legumes

Natural abundance of ¹⁵N ( ¹⁵N) of leaves harvested from tropical plants in Brazil and Thailand was analyzed. The ¹⁵N values of non-N₂-fixing trees in Brazil were +4.5±1.9, which is lower than those of soil nitrogen (+8.0±2.2). In contrast, mimosa and kudzu had very low ¹⁵N values (–1.4+0.5). The ¹⁵N values of Panicum maximum and leguminous trees, except Leucaena leucocephala, were similar to those of non-N₂-fixing trees, suggesting that the contribution of fixed N in these plants is negligible. The ¹⁵N values of non-N₂-fixing trees in Thailand were +4.9±2.0. Leucaena leucocephala, Sesbania grandiflora, Casuarina spp. and Cycas spp. had low ¹⁵N values, close to the value of atmospheric N₂ (0), pointing to a major contribution of N₂ fixation in these plants. Cassia spp. and Tamarindus indica had high ¹⁵N values, which confirms that these species are non-nodulating legumes. The ¹⁵N values of Acacia spp. and Gliricidia sepium and other potentially nodulating tree legumes were, on average, slightly lower than those of non-N₂-fixing trees, indicating a small contribution of N₂ fixation in these legumes.