Natural 15N abundance of presumed N2-fixing and non-N2-fixing plants from selected ecosystems

Summary The ¹⁵N/¹⁴N ratios of plant and soil samples from Northern California ecosystems were determined by mass spectrometry. The ¹⁵N abundance of 176 plant foliar samples averaged 0.0008 atom % ¹⁵N excess relative to atmospheric N₂ and ranged from-0.0028 to 0.0064 atom % ¹⁵N excess relative to atmospheric N₂. Foliage from reported N₂-fixing species had significantly lower mean ¹⁵N abundance (relative to atmospheric N₂ and total soil N) and significantly higher N concentration (% N dry wt.) than did presumed non-N₂-fixing plants growing on the same sites. The mean difference between N₂-fixing species and other plants was 0.0007 atom % ¹⁵N. N₂-fixing species had lower ¹⁵N abundance than the other plants on most sites examined despite large differences between sites in vegetation, soil, and climate. The mean ¹⁵N abundance of N₂-fixing plants varied little between sites and was close to that of atmospheric N₂. The ¹⁵N abundance of presumed non-N₂-fixing species was highest at coastal sites and may reflect an input of marine spray N having relatively high ¹⁵N abundance. The ¹⁵N abundance of N₂-fixing species was not related to growth form but was for other plants. Annual herbaceous plants had highest ¹⁵N abundance followed in decreasing order by perennial herbs, shrubs, and trees. Several terrestrial ferns (Pteridaceae) had ¹⁵N abundances comparable to N₂-fixing legumes suggesting N₂-fixation by these ferns. On sites where the ¹⁵N abundance of soil N differs from that of the atmosphere, N₂-fixing plants can be identified by the natural ¹⁵N abundance of their foliage. This approach can be useful in detecting and perhaps measuring N₂-fixation on sites where direct recovery of nodules is not possible.     

Bi-directional transfer of nitrogen between alfalfa and bromegrass: Short and long term evidence

Transfer of N from legumes to associated non-legumes has been demonstrated under a wide range of conditions. Because legumes are able to derive their N requirements from N₂ fixation, legumes can serve, through the transfer of N, as a source of N for accompanying non-legumes. Studies, therefore, are often limited to the transfer of N from the legume to the non-legume. However, legumes preferentially rely on available soil N as their source of N. To determine whether N can be transferred from a non-legume to a legume, two greenhouse experiments were conducted. In the short-term N-transfer experiment, a portion of the foliage of meadow bromegrass (Bromus riparius Rhem.) or alfalfa (Medicago sativa L.) was immersed in a highly labelled ¹⁵N-solution and following a 64 h incubation, the roots and leaves of the associated alfalfa and bromegrass were analyzed for ¹⁵N. In the long-term N transfer experiment, alfalfa and bromegrass were grown in an ¹⁵N-labelled nutrient solution and transplanted in pots with unlabelled bromegrass and alfalfa plants. Plants were harvested at 50 and 79 d after transplanting and analyzed for ¹⁵N content. Whether alfalfa or bromegrass were the donor plants in the short-term experiment, roots and leaves of all neighbouring alfalfa and bromegrass plants were enriched with ¹⁵N. Similarly, when alfalfa or bromegrass was labelled in the long-term experiment, the roots and shoots of neighbouring alfalfa and bromegrass plants became enriched with ¹⁵N. These two studies conclusively show that within a short period of time, N is transferred from both the N₂-fixing legume to the associated non-legume and also from the non-legume to the N₂-fixing legume. The occurrence of a bi-directional N transfer between N₂-fixing and non-N₂-fixing plants should be taken into consideration when the intensity of N cycling and the directional flow of N in pastures and natural ecosystems are investigated.     

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.     

Short-range spatial variability of soil δ15N natural abundance – effects on symbiotic N2-fixation estimates in pea

The δ¹⁵N natural abundance (‰) of the total soil N pool varies at the landscape level, but knowledge on short-range variability and consequences for the reliability of isotopic methods are poorly understood. The short-range spatial variability of soil δ¹⁵N natural abundance as revealed by the ¹⁵N abundance in spring barley and N₂-fixing pea was measured within the 0.15–4 m scale at flowering and at maturity. The short-range spatial variability of soil δ¹⁵N natural abundance and symbiotic nitrogen fixation were high at both growth stages. Along a 4-m row, the δ¹⁵N natural abundance in barley reference plants varied up to 3.9‰, and sometimes this variability was observed even between plants grown only 30 cm apart. The δ¹⁵N natural abundance in pea varied up to 1.4‰ within the 4-m row. The estimated percentage of nitrogen derived from the atmosphere (%Ndfa) varied from 73–89% at flowering and from 57–95% at maturity. When increasing the sampling area from 0.01 m² (single plants) and up to 0.6 m² (14 plants) the %Ndfa coefficient of variation (CV) declined from 5 to 2% at flowering and from 12 to 2% at maturity. The implications of the short-range variability in δ¹⁵N natural-abundance are that estimates of symbiotic N₂-fixation can be obtained from the natural abundance method if at least half a square meter of crop and reference plants is sampled for the isotopic analysis. In fields with small amounts of representative reference crops (weeds) it might be necessary to sow in reference crop species to secure satisfying N₂-fixation estimates.     

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.     

Short-term Nitrogen Fixation by Legume Seedlings and Resprouts After Fire in Mediterranean Old-fields

Fires may greatly alter the N budget of a plant community. During fire nitrogen is lost to the atmosphere. Although high light availability after fire promotes N₂-fixation, the presumably high soil N availability could limit N₂-fixation activity. The latter limitation might be particularly acute in legume seedlings compared with resprouts, which have immediate access to belowground stored carbon. We wished to learn whether early post-fire conditions were conducive to N₂-fixation in leguminous seedlings and resprouts in two types of grassland and in a shrubland and whether seedlings and resprouts incurred different amounts of N₂-fixation after fire. We set 18 experimental fires in early autumn on 6 plots, subsequently labelling 6 subplots (2 × 2 m²) in each community with ¹⁵NH₄⁺-N (99 atom % excess). For 9 post-fire months we measured net N mineralisation in the top 5 cm of soil and we calculated the fraction of legume N derived from the atmosphere (%Ndfa) in seedlings and resprouts. We used two independent estimates of the amounts of N derived from non-atmospheric sources in potentially N₂-fixing plants: mean soil pool abundance and the ¹⁵N-enrichment of non-legumes. Despite substantial soil net N mineralisation in all burned community types (about 2.6 g Nm⁻² during the first nine months after fire), the %Ndfa of various legume species was 52–99%. Legumes from both grasslands showed slightly higher N₂-fixation values than shrubland legumes. As grassland legumes grew in more belowground dense communities than shrubland legumes, we suggest that higher competition for soil resources in well established grass-resprouting communities may enhance the rate of N₂-fixation after fire. In contrast to our hypothesis, legume seedlings and resprouts from the three plant communities studied, had similar %Ndfa and apparently acquired most of their N from the atmosphere rather than from the soil.     

Application of 15N and xylem ureide methods for assessing N2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N₂ fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N₂ fixation (%Pfix) and the amounts of N₂ fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N₂ fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the ¹⁵N natural abundance technique were compared with values obtained from ¹⁵N enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies.Inoculated seedlings of each of the three shrub legume species were planted in dense stands (1.5 m rows, 0.5 m between trees) in two randomised blocks. The northern block was used solely for natural abundance measurements, while ¹⁵N-enriched KNO₃ (10 atom % ¹⁵N excess) was applied four times over a 52 week period to plots in the southern block. The non-nodulating tree legume Senna spectabilis (formally Cassia spectabilis) was used as a non-N₂-fixing reference for the ¹⁵N-based procedures, with Guinea grass (Panicum maximum) included as an additional non-fixing check. Growth by the trees above 75 cm was first cut and removed after 22 weeks and regrowth was subsequently pruned periodically for another 95 weeks. Sampling for dry matter production, N yield and estimates of Pfix were restricted to the central four of the 32 plants which constituted each replicate plot. Information generated during the 117 week study indicated that estimates of Pfix by ¹⁵N natural abundance were closely similar to values derived with ¹⁵N-enrichment or sap ureides. The data indicated that Calliandra had a reduced reliance upon N₂ fixation relative to Gliricidia and Codariocalyx for the first 65 weeks after establishment. This appeared to be due to more prolifc root growth by Calliandra than either of the other N₂-fixing species and an ability to extract a greater proportion of its N requirements from soil mineral N. However, after week 65 and for the remainder of the experiment, estimates of Pfix for Calliandra were similar to the other shrub legumes. Over 117 weeks, prunings from Calliandra and Gliricidia had removed 52–58 t dry matter ha^-1, and between 1471 and 1678 kg N ha^-1, of which 1026–1063 kg N ha^-1 was estimated to have been derived from N₂ fixation. At the time of final harvest, 65–73% of the fixed N was present in shoot regrowth of the N₂ fixing shrubs, 9–18% in the roots, 15% in the trunk, and 2–6% in fallen leaves.     

Adaptation of methods for evaluating N2 fixation in food legumes and legume cover crops

Methods for partitioning the nitrogen assimilated by nodulated legumes, between nitrogen derived from soil sources and from N₂ fixation, are described as applied in peninsular Malaysia. The analysis of nitrogenous components translocated from the roots to the shoots of nodulated plants in the xylem sap is outlined, with some precautions to be observed for applications in the tropics. Some examples of the use of the technique in surverying apparent N₂ fixation by tropical legumes, in studying interrow cropping in plantation systems and in assessing effects of experimental treatments on N₂ fixation by food legumes, are described. Techniques for assesing N₂ fixation by means of¹⁵N abundance have been used to show that applications of nitrogenous fertilizers commonly used in Malaysia for soybeans depress N₂ fixation, that similar results are obtained with natural abundance and¹⁵N-enrichment methods and that, in at least two locations in Malaysia, differences between the natural abundance of¹⁵N in plant-available soil nitrogen and in atmospheric N₂ are great enough to permit application to measurement of N₂ fixation by leguminous crops.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

Alder and lupine enhance nitrogen cycling in a degraded forest soil in Northern Sweden

Positive effects of legumes and actinorhizal plants on N-poor soils have been observed in many studies but few have been done at high latitudes, which was the location of our study. We measured N₂ fixation and several indices of soil N at a site near the Arctic Circle in northern Sweden. More than 20 years ago lupine (Lupinus nootkatensis Donn) and gray alder (Alnus incana L. Moench) were planted on this degraded forest site. We measured total soil N, net N mineralization and nitrification with a buried bag technique, and fluxes of NH⁺ ₄ and NO^– ₃ as collected on ion exchange membranes. We also estimated N₂ fixation activity of the N₂-fixing plants by the natural abundance of ¹⁵N of leaves with Betula pendula Roth. as reference species. Foliar nitrogen in the N₂-fixing plants was almost totally derived from N₂ fixation. Plots containing N₂-fixing species generally had significantly higher soil N and N availability than a control plot without N₂-fixing plants. Taken together, all measurements indicated that N₂-fixing plants can be used to effectively improve soil fertility at high latitudes in northern Sweden.     

Nitrogen Transfer Between Plants: A 15N Natural Abundance Study with Crop and Weed Species

An increasing amount of evidence indicates that N can be transferred between plants. Nonetheless, a number of fundamental questions remain. A series of experiments was initiated in the field to examine N transfer between N₂-fixing soybean (Glycine max [L.] Merr.) varieties and a non-nodulating soybean, and between N₂-fixing peanut (Arachis hypogaea L.) or soybean and neighboring weed species. The experiments were conducted in soils with low N fertilities and used differences in N accumulation and/or ¹⁵N natural abundance to estimate N transfer. Mixtures of N₂-fixing and non-nod soybean indicated that substantial inter-plant N transfer occurred. Amounts were variable, ranging from negligible levels to 48% of the N found in the non-nod at maturity. Transfer did not appear to strongly penalize the N₂-fixing donor plants. But, in cases where high amounts of N were transferred, N content of donors was noticeably lowered. Differences were evident in the amount of N transferred from different N₂-fixing donor genotypes. Results of experiments with N₂-fixing crops and the weed species prickly sida (Sida spinosa L.) and sicklepod (Senna obtusifolia [L.] Irwin & Barneby) also indicated substantial N transfer occurred over a 60-day period, with amounts accounting for 30–80% of the N present in the weeds. Transfer of N, however, was generally very low in weed species that are known to be non-hosts for arbuscular mycorrhizae (yellow nutsedge, Cyperus esculentus L. and Palmer amaranth, Amaranthus palmeri [S.] Watson). The results are consistent with the view that N transfer occurs primarily through mycorrhizal hyphal networks, and they reveal that N transfer may be a contributing factor to weed problems in N₂-fixing crops in low N fertility conditions.     

Nitrogen Transfer Within and Between Plants Through Common Mycorrhizal Networks (CMNs)

Mycorrhizae play a critical role in nutrient capture from soils. Arbuscular mycorrhizae (AM) and ectomycorrhizae (EM) are the most important mycorrhizae in agricultural and natural ecosystems. AM and EM fungi use inorganic NH₄ ⁺ and NO₃ ^?, and most EM fungi are capable of using organic nitrogen. The heavier stable isotope ¹⁵N is discriminated against during biogeochemical and biochemical processes. Differences in ¹⁵N (atom%) or δ¹⁵N (‰) provide nitrogen movement information in an experimental system. A range of 20 to 50% of one-way N-transfer has been observed from legumes to nonlegumes. Mycorrhizal fungal mycelia can extend from one plant's roots to another plant's roots to form common mycorrhizal networks (CMNs). Individual species, genera, even families of plants can be interconnected by CMNs. They are capable of facilitating nutrient uptake and flux. Nutrients such as carbon, nitrogen and phosphorus and other elements may then move via either AM or EM networks from plant to plant. Both ¹⁵N labeling and ¹⁵N natural abundance techniques have been employed to trace N movement between plants interconnected by AM or EM networks. Fine mesh (25～45 μm) has been used to separate root systems and allow only hyphal penetration and linkages but no root contact between plants. In many studies, nitrogen from N₂-fixing mycorrhizal plants transferred to non-N₂–fixing mycorrhizal plants (one-way N-transfer). In a few studies, N is also transferred from non-N₂–fixing mycorrhizal plants to N₂-fixing mycorrhizal plants (two-way N-transfer). There is controversy about whether N-transfer is direct through CMNs, or indirect through the soil. The lack of convincing data underlines the need for creative, careful experimental manipulations. Nitrogen is crucial to productivity in most terrestrial ecosystems, and there are potential benefits of management in soil-plant systems to enhance N-transfer. Thus, two-way N-transfer warrants further investigation with many species and under field conditions.     

Inoculation of Barley with efficient mycorrhizal fungi stimulates seed yield

Summary This short communication proposes the use of soils whose organic N has been lightly enriched in¹⁵N to screen plants for N₂-fixing activity. The major advantage of this approach is that it provides a reliable, integrated estimate of N₂-fixation up to any point in the life cycle of the plant, while using very few plants. The feasibility of the method, from the points of view of cost and availability of suitable soils, is discussed.     

Evaluation of N2-fixation and nitrogen economy of a maize/cowpea intercrop system using15N dilution methods

Yields of above ground biomass and total N were determined in summer-grown maize and cowpea as sole crops or intercrops, with or without supplementary N fertilizer (25 kg N ha⁻¹, urea) at an irrigated site in Waroona, Western Australia over the period 1982–1985. Good agreement was obtained between estimates of N₂ fixation of sole or intercrop cowpea (1984/85 season) based on the¹⁵N natural abundance and¹⁵N fertilizer dilution techniques, both in the field and in a glasshouse pot study. Field-grown cowpea was estimated to have received 53–69% of its N supply from N₂-fixation, with N₂-fixation onlyslightly affected by intercropping or N fertilizer application. Proportional reliance on N₂-fixation of cowpea in glasshouse culture was lower (36–66%) than in the field study and more affected by applied N. Budgets for N were drawn up for the field intercrops, based on above-ground seed yields, return of crop residues, inputs of fixed N and fertilizer N. No account was taken of possible losses of N through volatilization, denitrification and leaching or gains of N in the soil from root biomass. N₂-fixation was estimated tobe 59 kg N ha⁻¹ in the plots receiving no fertilizer N, and 73 kg N ha⁻¹ in plots receiving 25 kg N ha⁻¹ as urea. Comparable fixation by sole cowpea was higher (87 and 82 kg N ha⁻¹ respectively) but this advantage was outweighed by greater land use efficiency by the intercrop than sole crops.     

Estimation of symbiotic N2 fixation in an Amazon floodplain forest

Sustainable management for existing Amazonian forests requires an extensive knowledge about the limits of ecosystem nutrient cycles. Therefore, symbiotic nitrogen (N₂) fixation of legumes was investigated in a periodically flooded forest of the central Amazon floodplain (Várzea) over two hydrological cycles (20 months) using the ¹⁵N natural abundance method. No seasonal variation in ¹⁵N abundance (δ ¹⁵N values) in trees which would suggest differences in N₂ fixation rates between the terrestrial and the aquatic phase was found. Estimations of the percentage of N derived from atmosphere (%Ndfa) for the nodulated legumes with Neptunia oleracea on the one side and Teramnus volubilis on the other resulted in mean %Ndfa values between 9 and 66%, respectively. More than half of the nodulated legume species had %Ndfa values above 45%. These relatively high N gains are important for the nodulated legumes during the whole hydrological cycle. With a %Ndfa of 4–5% for the entire Várzea forest, N₂ fixation is important for the ecosystem and therefore, has to be taken into consideration for new sustainable land-use strategies in this area.     

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.     

Does nitrogen transfer between plants confound 15N-based quantifications of N2 fixation?

Background and aims

Transfer of fixed N from legumes to non-legume reference plants may alter the ¹⁵N signature of the reference plant as compared to the soil N available to the legume. This study investigates how N transfer influences the result of ¹⁵N-based N₂ fixation measurements.

Methods

We labelled either legumes or non-legumes with ¹⁵N and performed detailed analyses of ¹⁵N enrichment in mixed plant communities in the field. The results were used in a conceptual model comparing how different N transfer scenarios influenced the ¹⁵N signatures of legumes and reference plants, and how the resulting N₂ fixation estimate was influenced by using reference plants in pure stand or in mixture with the legume.

Results

Based on isotopic signatures, N transfer was detected in all directions: from legume to legume, from legume to non-legume, from non-legume to legume, from non-legume to non-legume. In the scenario of multidirectional N transfer, N₂ fixation was overestimated by using a reference plant in pure stand.

Conclusions

Fixed N transferred to neighbouring reference plants modifies the ¹⁵N signature of the soil N available both to the reference plant and the N₂-fixing legume. This provides strong support for using reference plants growing in mixture with the legumes for reliable quantifications of N₂ fixation.     

Estimation of symbiotic dinitrogen fixation in alder forest by the method based on natural15N abundance

Annual N₂-fixation in virgin forest ecosystems has been measured using a¹⁵N natural abundance (¹⁵N) procedure. This method was compared to a¹⁵N labelled fertilizer isotopic dilution method. For young alders (5–6 years old), ¹⁵N of leaves gave results in good agreement with the isotopic dilution of fertilizer method. Since ¹⁵N variability was expected according to plant physiology, for alder trees, leaves were collected at various heights after the end of the growing season, and, to take account of isotopic variations coming from derived inputs, ¹⁵N of leaves of a large number of other plants in the same are were measured to give control values. Following this procedure, the ¹⁵N method gave reliable evaluation of the nitrogen supply, by through N₂-fixation, to alders, which were found to maintain high nitrogen fixing capacity in a sequence ranging from first stage of establishment of climactic formation. Moreover, the same method is reported to discriminate various origins ofAlnus glutinosa grown in natural conditions, possibly in relation to the genetic diversity of this species.     

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.     

Growth and nitrogen acquisition strategies of Acacia senegal seedlings under exponential phosphorus additions

There remains conflicting evidence on the relationship between P supply and biological N₂-fixation rates, particularly N₂-fixing plant adaptive strategies under P limitation. This is important, as edaphic conditions inherent to many economically and ecologically important semi-arid leguminous tree species, such as Acacia senegal, are P deficient. Our research objective was to verify N acquisition strategies under phosphorus limitations using isotopic techniques. Acacia senegal var. senegal was cultivated in sand culture with three levels of exponentially supplied phosphorus [low (200 μmol of P seedling⁻¹ over 12 weeks), mid (400 μmol) and high (600 μmol)] to achieve steady-state nutrition over the growth period. Uniform additions of N were also supplied. Plant growth and nutrition were evaluated. Seedlings exhibited significantly greater total biomass under high P supply compared to low P supply. Both P and N content significantly increased with increasing P supply. Similarly, N derived from solution increased with elevated P availability. However, both the number of nodules and the N derived from atmosphere, determined by the ¹⁵N natural abundance method, did not increase along the P gradient. Phosphorus stimulated growth and increased mineral N uptake from solution without affecting the amount of N derived from the atmosphere. We conclude that, under non-limiting N conditions, A. senegal N acquisition strategies change with P supply, with less reliance on N₂-fixation when the rhizosphere achieves a sufficient N uptake zone.