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)     

Strain of Rhizobium lupini determines natural abundance of 15N in root nodules of Lupinus spp

Discrepancies between French and Australian results in measurement of the natural enrichment of ¹⁵N in nodules of Lupinus spp were due to the strains of Rhizobium lupini used. The Australian inoculant strain, WU425, produced nodules enriched in ¹⁵N above values for the entire plants, on all lines of L. luteus and L. albus tested. The total nitrogen and the natural abundance of ¹⁵N in nodules produced by the French strain, LL13, were much lower. In tests in Australia and in France with L. luteus inoculated with 15 other strains of R. lupini from diverse origins, 9 produced nodules with elevated ¹⁵N abundance, but of these 8 were less than with strain WU425. Most strains producing ¹⁵N-enriched nodules were slower-growing on yeast extract-mannitol agar; most faster-growing strains produced little or no nodular ¹⁵N enrichment. For strains with ¹⁵N-enriched nodules, enrichment increased with increasing nodule N·plant⁻¹, and shoot nitrogen tended to be depleted in ¹⁵N. There was no relationship between symbiotic effectiveness of strains (mg N₂ fixed·plant⁻¹) and ¹⁵N enrichment of nodules.     

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.     

Symbiotic nitrogen contribution and biodiversity of root-nodule bacteria nodulating Psoralea species in the Cape Fynbos,South Africa

The genus Psoralea (tribe Psoraleae, family Leguminosae) is indigenous to the Cape fynbos of South Africa and little is known about its symbiosis and/or adaptation. The aim of this study was to assess root nodulation and N₂ fixation in eight of the 50 Psoralea species, as well as the biodiversity of their associated nodulating microsymbionts. The eight species studied (namely, Psoralea pinnata, Psoralea aphylla, Psoralea aculeata, Psoralea monophylla, Psoralea repens, Psoralea laxa, Psoralea asarina and Psoralea restioides) all had round-shaped, determinate type (desmodioid) nodules, and data from ¹⁵N natural abundance showed that they obtained 60–88% of their N nutrition from symbiotic fixation. These Psoralea species also transported their fixed-N as ureides (allantoin and allantoic acid) in the xylem stream, a symbiotic trait that links them very closely to the tribe Phaseoleae. Bacteria isolated from root nodules of the eight Psoralea species varied in phenotypic characteristics, nodulation promiscuity, and N₂-fixing efficacy. Furthermore, 16S rDNA gene sequence data showed that Psoralea species can form root nodules with different soil bacteria, including Rhizobium, Mesorhizobium and Burkholderia strains. This is not only evidence of nodulation promiscuity, but also an indication of the species’ adaptation to the nutrient-poor, low-N, sandy acidic soils of the Cape fynbos.     

Symbiotically-fixed and soil-derived nitrogen in legumes grown in pots in soils with different amounts of available nitrate

Symbiotically-fixed and soil-derived nitrogen have been measured in pot experiments for Medicago littoralis (medic), grown alone or with Lolium multiflorum (ryegrass) and for Pisum sativum (field pea). The four soils used contained organic matter labelled with ¹⁵N, and differed in their capacities to release available N. During a 4–12-week incubation each released inorganic N (NO^?₃) of approximately constant ¹⁵N atom% enrichment. In one soil, the mineralized N was supplemented by ¹⁵NO^?₃ of similar ¹⁵N atom% enrichment. Incubation of soils under intermittently moist and dry conditions increased N mineralization rates, but did not affect the ¹⁵N atom% enrichments of the released N.For all soils and treatments the amounts of soil-derived N taken up by plants equalled the amounts of available N in moist incubated, unplanted soils. The enrichment of ryegrass root N grown alone or with medic was slightly but consistently less than that of top N. Nitrogen of the legume nodules and pods (peas) was least enriched, followed by N of legume stems, leaves and roots; the ¹⁵N atom% enrichments of root N were 4–5 times those of nodule N.Peas generally outyielded and fixed more N than medic grown alone. Medic grown with ryegrass yielded least and fixed least N.For unamended soils, yields of legume dry matter and amounts of N fixed were greatest in Roseworthy or Avon sandy loam soils and least in Northfield clay loam. Addition of ¹⁵NO^?₃ to Avon soil decreased N fixed by peas and by medic grown alone or with ryegrass. For this soil, soil-derived N of plant tops exceeded fixed N of roots, even for unamended soil where fixation by legumes was relatively high. Thus, complete removal of plant tops would have produced a net loss of N from the soil, the net loss increasing with increasing amounts of ¹⁵NO^?₃ added.     

Natural abundance of 15N in nitrate,ureides, and amino acids from plant tissues

The natural ¹⁵N abundances (δ¹⁵N values) were measured for nitrate and free and bound amino acids from the leaves of field-grown spinach (Spinacia oleracea L.) and komatsuna (Brassica campestris L.), as well as ureides and free and bound amino acids in the leaves and roots of hydroponically grown soybean (Glycine max L.) totally depending on dinitrogen. Nitrate from the spinach and komatsuna leaves and ureides from leaves and roots of soybean showed higher δ¹⁵N values than the total tissue N and N in free or bound amino acid fractions. The δ¹⁵N values of individual free and bound amino acids, determined by GC/C/MS using their acetylpropyl derivatives, were similar in leaf tissues except for proline but varied in soybean root tissues. The order of ¹⁵N enrichment was similar in the four samples: aspartic acid > glutamic acid > threonine, proline, valine > glycine + alanine +serine, γ-amino butyric acid, and phenylalanine.     

Effect of successive cuttings on uptake and partitioning of 15N among plant parts of Leucaena leucocephala

Summary We studied the effect of three successive cuttings on N uptake and fixation and N distribution in Leucaena leucocephala. Two isolines, uninoculated or inoculated with three different Rhizobium strains, were grown for 36 weeks and cut every 12 weeks. The soil was labelled with 50 ppm KNO₃ enriched with 10 atom % ¹⁵N excess soon after the first cutting. Except for the atom % ¹⁵N excess in branches of K28 at the second cutting, both the L. leucocephala isolines showed similar patterns of total N, fixed N₂, and N from fertilizer distribution in different parts of the plant at each cutting. The Rhizobium strain did not influence the partitioning of ¹⁵N among the different plant parts. Significant differences in ¹⁵N enrichment occurred in different parts. Live nodules of both isolines showed the lowest atom % ¹⁵N excess values (0.087), followed by leaves (0.492), branches (0.552), stems (0.591), and roots (0.857). The roots contained about 60% of the total plant N and about 70% of the total N derived from fertilizer over the successive cuttings. The total N₂ fixed in the roots was about 60% of that fixed in the whole plant, while the shoots contained only 20% of the fixed N₂. We conclude that N reserves in roots and nodules constitute another N source that must be taken into account when estimating fixed N₂ or the N balance after pruning or cutting plants. ¹⁵N enrichment declined up to about fivefold in the reference and the N₂-fixing plants over 24 weeks following the ¹⁵N application. The proportion and the amounts of N derived from fertilizer decreased, while the amount derived from N₂ fixation increased with time although its proportion remained constant.     

N2 fixation and growth of legumes as affected by sulphur fertilization

The influence of three sulphur application rates in combination with two nitrogen application rates on N₂ fixation and growth of different legumes was investigated. N was applied as N-labelled ¹⁵NH₄ ¹⁵NO₃. The ¹⁵N isotope dilution technique was used to estimate N₂ fixation. At both N increments dry matter yield was highest with high S supply. Independently of the N supply, the high S application rate resulted in a significantly higher N accumulation, which was mainly caused by a higher N₂ fixation rate. With the grain legumes the weight of nodules was increased by the high S application rate. The higher number of nodules per pot with optimum S supply was the result of a better root growth. Rates of acetylene reduction correlated significantly with S supply.     

Effects of Multiple Reference Plants,Season, and Irrigation on Biological Nitrogen Fixation by Pasture Legumes using the Isotope Dilution Method

Abstract

The popular and widely used ¹⁵nitrogen (N)–isotope dilution method for estimating biological N fixation (BNF) of pasture and tree legumes relies largely on the ability to overcome the principal source of error due to the problem of selecting appropriate reference plants. A field experiment was conducted to evaluate the suitability of 12 non‐N₂‐fixing plants (i.e., nonlegumes) as reference plants for estimating the BNF of three pasture legumes (white clover, Trifolium repens L.; lucerne, Medicago sativa; and red clover, Trifolium pratense L.) in standard ryegrass–white clover (RWC) and multispecies pastures (MSP) under dry‐land and irrigation systems, over four seasons in Canterbury, New Zealand. The ¹⁵N‐isotope dilution method involving field ¹⁵N‐microplots was used to estimate BNF. Non‐N₂‐fixing plants were used either singly or in combination as reference plants to estimate the BNF of the three legumes. Results obtained showed that, on the whole, ¹⁵N‐enrichment values of legumes and nonlegumes varied significantly according to plant species, season, and irrigation. Grasses and herb species showed higher ¹⁵N‐enrichment than those of legumes. Highest ¹⁵N‐enrichment values of all plants occurred during late summer under dry‐land and irrigation conditions. Based on single or combined non‐N₂‐fixing plants as reference plants, the proportion of N derived from the atmosphere (% Ndfa) values were high (50 to 90%) and differed between most reference plants in the MSP pastures, especially chicory (Cichorium intybus), probably because it is different in phenology, rooting depth, and N‐uptake patterns compared to those of legumes. The percent Ndfa values of all plants studied also varied according to plant species, season, and irrigation in the MSP pastures. Estimated daily amounts of BNF varied according to pasture type, time of plant harvest, and irrigation, similar to those shown by percent Ndfa results as expected. Irrigation increased daily BNF more than 10‐fold, probably due to increased dry‐matter yield of pasture under irrigation compared to dry‐land conditions. Seasonal and irrigation effects were more important in affecting estimates of legume BNF than those due to the appropriate matching of N₂‐fixing and non‐N₂‐fixing reference plants.     

Elevated CO2 concentrations increase leaf nitrate reduction by strengthening sink activity in soybean plants

An experiment was conducted to examine the effect of CO₂ enrichment on the nitrate uptake, nitrate reduction activity, and translocation of assimilated-N from leaves at varying levels of nitrogen nutrition in soybean using ¹⁵N tracer technique. CO₂ enrichment significantly increased the plant biomass, apparent leaf photosynthesis, sugar and starch contents of leaves, and reduced-N contents of the plant organs only when the plants were grown at high levels of nitrogen. A high supply of nitrogen enhanced plant growth and increased the reduced-N content of the plant organs, but its effect on the carbohydrate contents and photosynthetic rate were not significant. However, the combination of high CO₂ and high nitrogen levels led to an additive effect on all these parameters. The nitrate reductase activity increased temporarily for a short period of time by CO₂ enrichment and high nitrogen levels. ¹⁵N tracer studies indicated that the increase in the amount of reduced-N by CO₂ enrichment was derived from nitrate-N and not from fixed-N of the plant. To examine the translocation of reduced-N from the leaf in more detail, another experiment was conducted by feeding the plants with ¹⁵NO₃-N through a terminal leaflet of an upper trifoliated leaf under depodding and/or CO₂ enrichment conditions. The export rate of ¹⁵N from the terminal leaflet to other plant parts decreased by depodding, but it increased by CO₂ enrichment. CO₂ enrichment increased the percentage of plant ¹⁵N in the stem and / or pods. Depodding increased the percentage of plant ¹⁵N in the leaf and stem. The results suggested that the increase in the leaf nitrate reduction activity by CO₂ enrichment was due to the increase of the translocation of reduced-N from leaves through the strengthening of the sink activity of pods and / or stem for reduced-N.     

Relationship between K15NO3 application period and 15N enrichment of apricot blossoms and developing fruit.

The relationship was assessed between the period of K¹⁵ NO₃ application and the level of ¹⁵N in the blossoms of ‘Royal’ apricot (Prunus armen iaca L.). Late summer applications of K¹⁵NO, resulted in a 34 fold greater ¹⁵N enrichment of apricot blossoms the following year than K¹⁵NO₃ applied during the dormant period. In contrast, ¹⁵N enrichment was 60% higher in vegetative shoots 30 days after anthesis when K¹⁵NO₃ was applied during the dormant period as occurred following summer applications. Thus, fertilizer nitrogen applied in summer may support the early development of fruit to a greater extent than nitrogen applied during the dormant period. When K¹⁵NO₃ was applied in Jan, ¹⁵N accumulation in reproductive organs occurred after anthesis and corresponded with the period of shoot elongation and leaf expansion. It appears that fertilizer N must be absorbed prior to leaf fall to reach reproductive organs during anthesis.     

Does labelling frequency affect N rhizodeposition assessment using the cotton-wick method?

The aim of the present study was to test and improve the reliability of the ¹⁵N cotton-wick method for measuring soil N derived from plant rhizodeposition, a critical value for assessing belowground nitrogen input in field-grown legumes. The effects of the concentration of the ¹⁵N labelling solution and the feeding frequency on assessment of nitrogen rhizodeposition were studied in two greenhouse experiments using the field pea (Pisum sativum L.). Neither the method nor the feeding frequency altered plant biomass and N partitioning, and the method appeared well adapted for assessing the belowground contribution of field-grown legumes to the soil N pool. However, nitrogen rhizodeposition assessment was strongly influenced by the feeding frequency and the concentration of labelling solution. At pod-filling and maturity, despite similar root ¹⁵N enrichment, the fraction of plants' belowground nitrogen allocated to rhizodeposition in both Frisson pea and the non-nodulating isoline P2 was 20 to more than 50% higher when plants were labelled continuously than when they were labelled using fortnightly pulses. Our results suggest that when ¹⁵N root enrichment was high, nitrogen rhizodeposition was overestimated only for plants that were ¹⁵N-fed by fortnightly pulses, and not in plants ¹⁵N-fed continuously. This phenomenon was especially observed for plants that rely on symbiotic N₂ fixation for N acquisition, and it may be linked to the concentration of the labelling solution. In conclusion, the assessment of nitrogen rhizodeposition was more reliable when plants were labelled continuously with a dilute solution of ¹⁵N urea.     

15N isotope dilution methodology for evaluating the dynamics of biologically fixed N in legume-non-legume associations

We examined the theoretical basis for estimating the transfer of N₂ fixed by legumes to companion cereals or grasses in intercropping or pasture systems using ¹⁵N isotope dilution methodology. A method was developed to calculate the symbiotic dependence of the legume in a mixed stand based on ¹⁵N enrichment of the associated non-legume and the estimate of fixed N transfer. Published field data were used to illustrate the application of the method. Complementary treatments for verifying N transfer and options for increasing the accuracy of estimates of N transfer are discussed.     

Ontogenic variation in nitrogen fixation and accumulation of nitrogen in mungbean,blackgram, cowpea,and groundnut

Ontogenic variations in N₂ fixation and accumulation of N by the mungbean (Vigna radiata L. Wilczek), blackgram (Vigna mungo L. Hepper), cowpea (Vigna unguiculata L. Walp.), and groundnut (Arachis hypogaea L.) were studied by a ¹⁵N-dilution technique. Pots filled with 7 kg of red yellow podzolic soil were used. Samples were taken 20, 40, 60, and 80 days after emergence which approximately corresponded to preflowering, flowering, early/mid-pod filling and late pod filling stages, respectively. During early growth (up to 40 days after emergence), the carryover of seed N accounted for a considerable fraction of the total plant N in the legumes, the highest being in the groundnut. With a correction for carryover, the groundnut derived over 45% of its N content from the atmosphere 20 days after emergence whereas the corresponding figures were 33% for the blackgram and about 28% for the cowpea and mungbean. Between flowering and early pod fill, there was a rapid increase in N₂ fixation in all legumes except in groundnut which showed highest fixation from 60 to 80 days after emergence. In the mungbean, N₂ fixation and uptake of soil N were insignificant 60 days after emergence while in other legumes these processes continued beyond this time. All legumes derived about 90% of their N from atmosphere by 80 days after emergence. However, due to considerable interspecific differences in total N yield the final amount of N₂ fixed showed an appreciable variation among legumes. It was highest in the groundnut (443 mg N plant^-1) followed by the cowpea (385), blackgram (273), and mungbean (145), respectively. The groundnut maintained nodules until the late pod filling stage while in other legumes, nodules senesced progressively following the mid-pod filling stage. During pod filling there was a net mobilization of N from vegetative tissues to developing pods in the mungbean, which amounted to about 20% of N in seeds. This mobilization was not evident in other legumes.     

Nodulation and estimation of symbiotic nitrogen fixation by herbaceous and shrub legumes in Guinea savanna in Nigeria

Twelve herbaceous and shrub legume species were grown in pot and field experiments in five sites representing three agroecological zones in moist savanna in Nigeria. The objectives were to: (1) assess natural nodulation of the legumes and characterize their indigenous rhizobia, (2) determine their need for rhizobia inoculation and (3) estimate the amount of N₂ fixed by each of these legumes. At 4 weeks after planting (WAP), Crotolaria verrucosa was not nodulated at any of the sites while Centrosema pascuorum had the highest number of nodules in all sites. At 8 WAP, all legumes were nodulated, with Mucuna pruriens having the least number of nodules and Stylosanthes hamata the highest. The number of nodules, however, was inversely correlated to the mass of nodules. Significant differences in nodulation of the legume species grown in the field also occurred between and within sites. Mucuna pruriens and Lablab purpureus produced more shoot and nodule biomass than the other legumes in all sites. Growth of most of these legumes responded to fertilizer application, except for C. verrucosa and Aeschynomene histrix. Except for C. verrucosa, average proportion of N₂ fixed was about 80% and this was reduced by about 20% with N fertilizer application. The majority of rhizobia isolates (60%) were slow growing, belonging to the Bradyrhizobia spp. group. Selected rhizobia isolates evaluated on Cajanus cajan, C. pascuorum, M. pruriens and Psophocarpus palustris varied from ineffective to highly effective in Leonard jar conditions. However, only growth of M. pruriens responded to inoculation in potted soils, whereas it was lower than that obtained with N fertilizer application. This indicated the need to screen more rhizobia in order to improve N₂ fixation and growth of legume species such as M. pruriens when it is introduced in soils deficient in N.     

Distillation solution with and without indicator for nitrogen‐15 measurement by optical emission spectrometry

Abstract

Optical emission spectroscopy provides a rapid and precise method for determining ¹⁵N/¹⁴N ratios of ¹⁵N‐enriched plant and/or soil samples. The objective of this study was to determine the effect of an indicator added in the distillation solution on the success rate of tube lighting in the optical emission spectrometer over a large range of N concentration in ¹⁵N enriched plant samples. One‐hundred‐eighty plant samples with large ranges of N concentration (4 to 30 g.kg^‐1 dry weight) and ¹⁵N atom enrichment (0.368 to 1.635%) were analyzed. Our data suggest that there was no difference in the success rate of tube lighting in emission spectroscopy and in ¹⁵N/¹⁴N ratio measured between samples prepared with and without addition of indicator in the distillation solution.     

Mineralization of nitrite fixed by soil organic matter

The biological availability of [¹⁵N]labelled nitrite fixed by the organic matter of two acid soils was determined by laboratory incubation and pot experiments. Fixed-N was resistant to mineralization, but was more readily available than the indigenous organic-N. Approximately 20% of the fixed-N was recovered as inorganic-N after 70 days of incubation, or was recovered in the roots and three cuttings of rye-grass grown for 197 days, whereas less than 10% of the indigenous organic-N was recovered. With increasing time, the fixed-N becomes more resistant to mineralization, becoming similar to the indigenous organic-N.     

Appraisal of 15N enrichment and 15N natural abundance methods for estimating N2 fixation by understorey Acacia leiocalyx and A. disparimma in a native forest of subtropical Australia

Purpose  

It is anticipated that global climate change will increase the frequency of wildfires in native forests of eastern Australia. Understorey legumes such as Acacia species play an important role in maintaining ecosystem nitrogen (N) balance through biological N fixation (BNF). This is particularly important in Australian native forests with soils of low nutrient status and frequent disturbance of the nutrient cycles by fires. This study aimed to examine ¹⁵N enrichment and ¹⁵N natural abundance techniques in terms of their utilisation for evaluation of N₂ fixation of understorey acacias and determine the relationship between species ecophysiological traits and N₂ fixation.     

Can differences in 15N natural abundance be used to quantify the transfer of nitrogen from legumes to neighbouring non-legume plant species?

Natural variations in the stable isotope ¹⁵N are often exploited in studies of N cycling in ecosystems. Lower ¹⁵N natural abundance in non-legume plants growing in association with legumes, compared with the non-legume grown alone in pure stands have been observed in cropping, forage, and agroforestry systems. Such observations have frequently been attributed to the transfer of biologically-fixed nitrogen (N) from the legume to the companion non-legume, and various methodologies have been employed to calculate the extent of the N transfer. While some of these ¹⁵N natural abundance-based estimates of N transfer were within the range previously reported using equivalent ¹⁵N-enriched techniques (<20% of non-legume plant N and <10 kg N ha⁻¹ derived from fixed N contributed by neighbouring legumes), many of the values obtained using natural abundance were much higher (30%–83% of the non-legume N derived from fixed N representing up to 30–40 kg N ha⁻¹) than generally measured by ¹⁵N-enriched methods; with even greater estimates being determined where data were available to allow N transfer to be re-calculated on the basis of total legume N rather than fixed N (42% to >100%, and up to 110 kg N ha⁻¹ per year). This review raises concerns about the assumptions behind the natural abundance approach, and provides some alternative interpretations for the observed differences in natural ¹⁵N abundance between plants grown in the presence and absence of legumes. It was concluded that simple comparative measures of non-legume δ¹⁵N alone cannot provide a quantitative estimate of N transfer between plant species if the dominant source and the isotopic identity of the transferred N cannot be validated, and if the extent of any isotopic fractionation associated with relevant N transformations occurring during transfer cannot be defined. To date this information is not forthcoming. There is a need to greatly improve our understanding of the transfer processes before the real value of the δ¹⁵N technology can be realized. In the first instance this will primarily be achieved by carefully executed experiments under controlled conditions, and in the field, employing both ¹⁵N natural abundance and enrichment approaches so estimates of transfer can be compared, and the data interrogated using modelling approaches to explore isotopic fractionation.     

Host-rhizobia interaction for effective inoculation: evaluation of the potential use of the ureide assay to monitor the symbiotic performance of tepary bean (Phaseolus acutifolius A. Gray)

Domesticated and wild-type tepary beans (Phaseolus acutifolius A. Gray) were grown with or without inoculation with rhizobia in pots under bacteriologically controlled conditions in a temperature-controlled glasshouse. Seeds were inoculated with a mixture of seven strains isolated from nodules collected from domesticated field-grown tepary bean in Arizona, USA, or with a commercial inoculant strain for Phaseolus vulgaris (CC511). Different degrees of plant reliance upon N₂ fixation for growth were generated by supplying the inoculated plants throughout growth with nutrients containing a range of concentrations of ¹⁵N-labeled NO₃ (0, 1, 2, 5 or 10 mM). An uninoculated treatment that received 10 mM ¹⁵N-labeled NO₃ was included to provide data for plants solely dependent upon NO₃ for growth. Six weeks after sowing, shoots were harvested for dry matter determination and subsequent ¹⁵N analysis, root-bleeding xylem sap was collected, and nodulation assessed. With regard to shoot biomass production, domesticated lines were more responsive to inoculation, but less responsive to applied N than wild types. All inoculated plants were nodulated, but the field isolates from tepary bean were more effective in N₂ fixation than strain CC511. It was concluded that tepary bean requires a specific inoculant to benefit from fixation of atmospheric N₂. Xylem sap samples were analysed for ureides (allantoin and allantoic acid), amino acid content (α-amino-N), and NO₃ concentration. The amount of ureide-N present in xylem sap was expressed as a percentage of total solute N, described as the relative abundance of ureide-N (RUN), for each N treatment and was compared to the proportion of plant N derived from N₂ fixation (%Ndfa) calculated using a ¹⁵N dilution technique. The RUN values ranged from 8% for saps collected from uninoculated plants provided with 10 mM NO₃ in the nutrient solution (%Ndfa=0) to 86-91% for nodulated plants grown in the absence of externally supplied NO₃ (%Ndfa=100). These data indicated that ureides were the principal product of N₂ fixation exported from the nodules to the shoot in xylem sap. Since RUN values were closely related to %Ndfa, it was proposed that N-solute analysis of xylem sap could provide a valuable analytical tool to monitor the symbiotic performance of tepary bean.