Citrate exudation from white lupin induced by phosphorus deficiency differs from that induced by aluminum

Both phosphorus (P) deficiency and aluminum (Al) toxicity induce root exudation of carboxylates, but the relationship between these two effects is not fully understood. Here, carboxylate exudation induced by Al in Lupinus albus (white lupin) was characterized and compared with that induced by P deficiency. Aluminum treatments were applied to whole root systems or selected root zones of plants with limited (1 microM) or sufficient (50 microM) P supply. Aluminum stimulated citrate efflux after 1-2 h; this response was not mimicked by a similar trivalent cation, La(3+). P deficiency triggered citrate release from mature cluster roots, whereas Al stimulated citrate exudation from the 5- to 10-mm subapical root zones of lateral roots and from mature and senescent cluster roots. Al-induced citrate exudation was inhibited by P limitation at the seedling stage, but was stimulated at later growth stages. Citrate exudation was sensitive to anion-channel blockers. Al treatments did not affect primary root elongation, but inhibited the elongation of lateral roots. The data demonstrate differential patterns of citrate exudation in L. albus, depending on root zone, developmental stage, P nutritional status and Al stress. These findings are discussed in terms of possible functions and underlying mechanisms.     

子叶磷在白羽扇豆缺磷适应性反应中的作用

实验用液体培养的方法,对比分析了在不同供磷条件下,白羽扇豆子叶中的磷对植物生长发育的影响,以及排根和根尖中有机酸积累和分泌的作用,结果表明,子叶中的磷能使白羽扇豆在完全缺磷２３d的环境中,不仅没有使干物质的积累减少,反而使干物质的积累略有增加,相反,如果没有子叶磷的供给,则使白羽扇豆在缺磷环境中产生强烈的抗胁迫反应,表现在干物质的积累明显下降,根系能产生大量的排根,排根能积累和分泌大量的柠檬酸,而根尖能积累和分泌萍果酸,在整个缺磷反应过程中,根尖中苹果酸的分泌要早于排根可柠檬酸的积累和分泌。     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.     

Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin

Release of large amounts of citric acid from specialized root clusters (proteoid roots) of phosphorus (P)-deficient white lupin (Lupinus albus L.) is an efficient strategy for chemical mobilization of sparingly available P sources in the rhizosphere. The present study demonstrates that increased accumulation and exudation of citric acid and a concomitant release of protons were predominantly restricted to mature root clusters in the later stages of P deficiency. Inhibition of citrate exudation by exogenous application of anion-channel blockers such as ethacrynic- and anthracene-9-carboxylic acids may indicate involvement of an anion channel. Phosphorus-deficiency-induced accumulation and subsequent exudation of citric acid seem to be a consequence of both increased biosynthesis and reduced metabolization of citric acid in the proteoid root tissue, indicated by increased in-vitro activity and enzyme protein levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31), and reduced activity of aconitase (EC 4.2.1.3) and root respiration. Similar to citric acid, acid phosphatase, which is secreted by roots and involved in the mobilization of the organic soil P fraction, was released predominantly from proteoid roots of P-deficient plants. Also ³³Pi uptake per unit root fresh-weight was increased by approximately 50% in juvenile and mature proteoid root clusters compared to apical segments of non-proteoid roots. Kinetic studies revealed a K _m of 30.7 μM for Pi uptake of non-proteoid root apices in P-sufficient plants, versus K _m values of 8.5–8.6 μM for non-proteoid and juvenile proteoid roots under P-deficient conditions, suggesting the induction of a high-affinity Pi-uptake system. Obviously, P-deficiency-induced adaptations of white lupin, involved in P acquisition and mobilization of sparingly available P sources, are predominantly confined to proteoid roots, and moreover to distinct stages during proteoid root development. Received: 10 September 1998 / Accepted: 22 December 1998     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

Effect of phosphorus supply on the formation and function of proteoid roots of white lupin (Lupinus albus L.)

We investigated (1) the effect of constant and altered inorganic phosphate (P_i) supply (1–100 mmol m^–3) on proteoid root production by white lupin ( Lupinus albus L.); and (2) the variation in citrate efflux, enzyme activity and phosphate uptake along the proteoid root axis in solution culture. Proteoid root formation was greatest at P_i solution concentrations of 1–10 mmol m^–3 and was suppressed at 25 mmol m^–3 P_i and higher. Except at 1 mmol m^–3 P_i, the formation of proteoid roots did not affect plant dry matter yields or shoot to root dry matter ratios, indicating that proteoid roots can form under conditions of adequate P supply and not at the expense of dry matter production. Plants with over 50% of the root system as proteoid roots had tissue P concentrations considered adequate for maximum growth, providing additional evidence that proteoid roots can form on P-sufficient plants. There was an inverse relationship between the P_i concentration in the youngest mature leaf and proteoid root formation. Citrate efflux and the activities of enzymes associated with citric acid synthesis (phosphoenolpyruvate carboxylase and malate dehydrogenase) varied along the proteoid root axis, being greatest in young proteoid rootlets of the 1–3 cm region from the root tip. Citrate release from the 0–1 and 5–9 cm regions of the proteoid root was only 7% (per unit root length) of that from the 1–3 cm segment. Electrical potential and ³²P_i uptake measurements showed that P_i uptake was more uniform along the proteoid root than citrate efflux.     

The influence of phosphorus deficiency on growth and nitrogen fixation of white clover plants

The effects of P deficiency on growth, N(2)-fixation and photosynthesis in white clover (Trifolium repens L.) plants were investigated using three contrasting relative addition rates of P, or following abrupt withdrawal of the P supply. Responses to a constant below-optimum P supply rate consisted of a decline in N(2)-fixation per unit root weight and a small reduction in the efficiency with which electrons were allocated to the reduction of N(2) in nodules. Abrupt removal of P arrested nodule growth and caused a substantial decline in nitrogenase activity per unit root weight, but not per unit nodule mass. Similarly, the rate of photosynthesis per unit leaf area was unaffected by abrupt P removal, whereas CO(2) acquisition for the plant as a whole decreased due to a decline in total leaf area, leaf area per unit leaf weight and utilization of incoming radiation. These changes followed the decline in tissue P concentrations. The ratio between CO(2)-fixation and N(2)-fixation was maintained under short-term P deprivation but increased under long-term low P supply, indicating a regulatory inhibition of nodule activity following morphological and growth adjustments. It is concluded that N(2)-fixation did not limit the growth of clover plants experiencing P deficiency. A low P status induced changes in the relative growth of roots, nodules and shoots rather than changes in N and/or C uptake rates per unit mass or area of these organs.     

Evidence of phloem boron transport in response to interrupted boron supply in white lupin (Lupinus albus L. cv. Kiev Mutant) at the reproductive stage

The present study investigates whether previously acquired boron(B) in mature leaves in white lupin can be retranslocated intothe rapidly growing young reproductive organs, in response toshort-term (3 d) interrupted B supply. In a preliminary experimentwith white lupin in soil culture, B concentrations in phloemexudates remained at 300–500 µM, which were substantiallyhigher than those in the xylem sap (10–30 µM). Thehigh ratios of B concentrations in phloem exudates to thosein the xylem sap were close to values published for potassiumin lupin plants. To differentiate ‘old’ B in theshoot from ‘new’ B in the root, an experiment wascarried out in which the plants were first supplied with 20µM ¹¹B (99.34% by weight) in nutrient solution for 48d after germination (DAG) until early flowering and then transferredinto either 0.2 µM or 20 µM ¹⁰B (99.47% by weight)for 3 d. Regardless of the ¹⁰B treatments, significant levelsof ¹¹B were found in the phloem exudates (200–300 µMin 20 µM ¹⁰B and 430 µM in 0.2 µM ¹⁰B treatment)and xylem sap over the three days even without ¹¹B supply tothe root. In response to the 0.2 µM ¹⁰B treatment, thetranslocation of previously acquired ¹¹B in the young (the uppermostthree leaves), matured, and old leaves was enhanced, coincidingwith the rise of ¹¹B in the xylem sap (to >15 µM) andphloem exudates (430 µM). The evidence supports the hypothesisthat previously acquired B in the shoot was recirculated tothe root via the phloem, transferred into the xylem in the root,and transported in the xylem to the shoot. In addition, somepreviously acquired ¹¹B in the leaves may have been translocatedinto the rapidly growing inflorescence. Phloem B transport resultedin the continued net increment of ¹¹B in the flowers over 3d without ¹¹B supply. However, it is still uncertain whetherthe amount of B available for recirculation is adequate to supportreproductive growth until seed maturation. Key words: ¹⁰B, ¹¹B, B recirculation, Lupinus albus L., phloem exudate, xylem sap Received 9 October 2007; Revised 28 November 2007 Accepted 30 November 2007     

Determination of lead in white lupin by anodic stripping voltammetry

An anodic stripping voltammetry method for the simultaneous determination of trace heavy metals in nutrient solutions, soils and plants has been developed at a hydrodynamic electrochemical sensor. Several parameters were optimized in order to enhance sensitivity. Calibration curves in different media are presented. The study of the uptake of lead by white lupin (Lupinus albus L.) was carried out. Toxicity symptoms were observed and compared with levels of lead measured in roots and leaves.     

Acclimation of white lupin to phosphorus deficiency involves enhanced expression of genes related to organic acid metabolism

White lupin (Lupinus albus L.) acclimates to phosphorus deficiency (–P) by the development of short, densely clustered lateral roots called proteoid (or cluster) roots. These specialized plant organs display increased exudation of citric and malic acid. The enhanced exudation of organic acids from P stressed white lupin roots is accompanied by increased in vitro phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activity. Here we report the cloning of full-length white lupin PEPC and MDH cDNAs. RNA blot analysis indicates enhanced expression of these genes in –P proteoid roots, placing higher gene expression at the site of organic acid exudation. Correspondingly, macroarray analysis of about 1250 ESTs (expressed sequence tags) revealed induced expression of genes involved in organic acid metabolism in –P proteoid roots. In situ hybridization revealed that PEPC and MDH were both expressed in the cortex of emerging and mature proteoid rootlets. A C₃ PEPC protein was partially purified from proteoid roots of P deficient white lupin. Native and subunit Mr were determined to be 440 kD and 110 kD, respectively. Citrate and malate were effective inhibitors of in vitro PEPC activity at pH 7. Addition of ATP partially relieved inhibition of PEPC by malate but had little effect on citrate inhibition. Taken together, the results presented here suggest that acclimation of white lupin to low P involves modified expression of plant genes involved in carbon metabolism.     

Nitrogen accumulation in sole and mixed stands of sweet-blue lupin (Lupinus angustifolius L.), ryegrass and oats

Nitrogen fixation was measured in monocropped sweet-blue lupin (Lupinus angustifolius), lupin intercropped with two ryegrass (Lolium multiflorum) cultivars or with oats (Avena sativa) on an Andosol soil, using the ¹⁵N isotope dilution method. At 117 days after planting and at a mean temperature below 10°C, monocropped lupin derived an average of 92% or 195 kg N ha⁻¹ of its N from N₂ fixation. Intercropping lupin with cereals increased (p<0.05) the percentage of N derived from atmospheric N₂ (% Ndfa) to a mean of 96%. Compared to the monocropped, total N fixed per hectare in intercropped lupin declined approximately 50%, in line with the decrease in seeding rate and dry matter yield. With these high values of N₂ fixation, selection of the reference crop was not a problem; all the cereals, intercropped or grown singly produced similar estimates of N₂ fixed in lupin. It was deduced from the ¹⁵N data that significant N transfer occurred from lupin to intercropped Italian ryegrass but not to intercropped Westerwoldian ryegrass or to oats. Doubling the ¹⁵N fertilizer rate from 30 to 60 kg N ha⁻¹ decreased % Ndfa to 86% (p<0.05), but total N fixed was unaltered. These results indicate that lupin has a high potential for N₂ fixation at low temperatures, and can maintain higher rates of N₂ fixation in soils of high N than many other forage and pasture legumes.     

Nitrogen fixation by white clover in pastures grazed by dairy cows: Temporal variation and effects of nitrogen fertilization

Effects of rate of nitrogen (N) fertilizer and stocking rate on production and N₂ fixation by white clover (Trifolium repens L.) grown with perennial ryegrass (Lolium perenne L.) were determined over 5 years in farmlets near Hamilton, New Zealand. Three farmlets carried 3.3 dairy cows ha^–1 and received urea at 0, 200 or 400 kg N ha^–1 yr^–1 in 8–10 split applications. A fourth farmlet received 400 kg N ha^–1 yr^–1 and had 4.4 cows ha^–1.There was large variation in annual clover production and total N₂ fixation, which in the 0 N treatment ranged from 9 to 20% clover content in pasture and from 79 to 212 kg N fixed ha^–1 yr^–1. Despite this variation, total pasture production in the 0 N treatment remained at 75–85% of that in the 400 N treatments in all years, due in part to the moderating effect of carry-over of fixed N between years.Fertilizer N application decreased the average proportion of clover N derived from N₂ fixation (P_N; estimated by ¹⁵N dilution) from 77% in the 0 N treatment to 43–48% in the 400 N treatments. The corresponding average total N₂ fixation decreased from 154 kg N ha^–1 yr^–1 to 39–53 kg N ha^–1 yr^–1. This includes N₂ fixation in clover tissue below grazing height estimated at 70% of N₂ fixation in above grazing height tissue, based on associated measurements, and confirmed by field N balance calculations. Effects of N fertilizer on clover growth and N₂ fixation were greatest in spring and summer. In autumn, the 200 N treatment grew more clover than the 0 N treatment and N₂ fixation was the same. This was attributed to more severe grazing during summer in the 0 N treatment, resulting in higher surface soil temperatures and a deleterious effect on clover stolons.In the 400 N treatments, a 33% increase in cow stocking rate tended to decrease P_N from 48 to 43% due to more N cycling in excreta, but resulted in up to 2-fold more clover dry matter and N₂ fixation because lower pasture mass reduced grass competition, particularly during spring.     

Excess cation uptake, and extrusion of protons and organic acid anions by Lupinus albus under phosphorus deficiency

In symbiotically-grown legumes, rhizosphere acidification may be caused by a high cation/anion uptake ratio and the excretion of organic acids, the relative importance of the two processes depending on the phosphorus nutritional status of the plants. The present study examined the effect of P deficiency on extrusions of H⁺ and organic acid anions (OA⁻) in relation to uptake of excess cations in N₂-fixing white lupin (cv. Kiev Mutant). Plants were grown for 49 days in nutrient solutions treated with 1, 5 or 25 mmol P m⁻³ Na₂HPO₄ in a phytotron room. The increased formation of cluster roots occurred prior to a decrease in plant growth in response to P deficiency. The number of cluster roots was negatively correlated with tissue P concentrations below 2.0 g kg⁻¹ in shoots and 3 g kg⁻¹ in roots. Cluster roots generally had higher concentrations of Mg, Ca, N, Cu, Fe, and Mn but lower concentrations of K than non-cluster roots. Extrusion of protons and OA⁻ (90% citrate and 10% malate) from roots was highly dependent on P supply. The amounts of H⁺ extruded per unit root biomass decreased with time during the experiment. On the equimolar basis, H⁺ extrusion by P-deficient plants (grown at 1 and 5 mmol P m⁻³) were, on average, 2–3-fold greater than OA⁻ exudation. The excess cation content in plants was generally the highest at 1 mmol P m⁻³ and decreased with increasing P supply. The ratio of H⁺ release to excess cation uptake increased with decreasing P supply. The results suggest that increased exudation of OA⁻ due to P deficiency is associated with H⁺ extrusion but contributes only a part of total acidification.     

Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

Nitrogen fixation by white clover when competing with grasses at moderately low temperatures

It was the aim of this study to determine the way in which low temperature modifies the effect of a competing grass on nitrogen fixation of a forage legume. White clover (Trifolium repens L.) was grown in monoculture or in different planting ratios with timothy (Phleum pratense L.) or perennial ryegress (Lolium perenne L.) in growth chambers at either 7.5/5°C (LoT) or 15/10°C (HiT) average day/night temperatures, and with 2.5 or 7.5 mM ¹⁵N-labelled nitrate in the nutrient solution.Competition with grass led to a marked increase in the proportion of clover nitrogen derived from symbiosis (% N_sym). This increase was slower at LoT where % N_sym was reduced considerably; it was closely related to the reduction in the amount of available nitrate as a result of its being utilized by the grass.Nitrogen concentration in white clover herbage and dry matter yield per clover plant were reduced, for the most part, when a competing grass was present. The amount of nitrogen fixed per plant of white clover decreased markedly with temperature. Low temperature consequently accentuated competition for nitrate. The capacity of white clover to compete successfully was limited by its slower growth and nitrogen accumulation.     

Na2CO3胁迫下星星草幼苗活性氧及保护酶活性的变化

对低浓度Na₂CO₃胁迫下星星草幼苗相对电导率、O^-₂产生速率、H₂O₂含量以及保护酶CAT、SOD和POD活性的研究结果表明,低盐胁迫1 d后,星星草幼苗细胞膜的通透性、O^-₂产生速率、H₂O₂含量及保护酶活性都随着盐胁迫的加剧而升高,其具体的变化规律与盐胁迫强度和幼苗细胞膜的受损伤程度密切相关,但相关关系的性质上具有差异。     

Nitrogen fixation in Pseudomonas stutzeri

A recently developed oxygen gradient system and a complex medium were used to isolate a microaerobically N₂-fixing heterotrophic bacterium from the rhizosphere of a high fixing Sorghum nutans cultivar. The isolate was identified as nif(+) phenotype of Pseudomonas stutzeri on the basis of cultural, physiological and biochemical characteristics, including DNA/DNA hybridization. N₂ fixation was demonstrated by assimilation of ¹⁵N₂ into cellular protein; the physiology of nitrogen fixation was studied. The isolate contains one 30 MD plasmid and can be cured with associated loss of N₂ fixation capability.Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday     

Nitrogen fixation and nitrogen balance studies in Rhizobium-VA mycorrhiza inoculated legume-grass association by15N isotope dilution technique under a field condition

A mixed pasture comprising of buffel grass and a legume siratro was studied under field condition for a two-year period to know the fodder yield increase, nitrogen fixation and nitrogen balance with and without the inoculation of VA mycorrhiza to grass and Rhizobium to legume component.¹⁵N dilution technique was followed using labelled ammonium sulphate. The data showed that during the first year of the above study combined inoculation of VA mycorrhiza and Rhizobium to grass and legume respectively significantly increased the total dry matter (DM) (23,900 kg ha^–1 yr^–1) and total N content (308 kg ha^–1 yr^–1) of the mixed pasture over the uninoculated mixture. However, the above increase due to combined inoculation was maximum during second year with respect to DM yield (28,200 kg ha^–1 yr^–1), but the total N harvested through grass-legume mixture was comparatively lower than the first year (297 kg ha^–1 yr^–1). The amount of biologically fixed N was highest in the first year (79 kg ha^–1 yr^–1) and showed a very drastic reduction at the end of second year (39 kg ha^–1 yr^–1). A positive nitrogen balance was observed in the grass-legume mixture irrespective of inoculation of VA mycorrhiza and/or Rhizobium.     

15N2 measurement of nitrogen fixation by legumes and actinorhizals: theory and practice

A gas-tight chamber has been constructed to calibrate the ¹⁵N isotope dilution method against direct ¹⁵N₂ measurements. The theoretical basis for such estimates is given, and the practical problems associated with the experiments are discussed.     

Evidence against associative N2 fixation as a significant N source in long-term wheat plots

In monocropped cereal systems, annual N inputs from non-fertilizer sources may be more than 30 kg ha^-1. We examined the possibility that these inputs are due to biological N₂ fixation (BNF) associated with roots or decomposing residues. Wheat was grown under greenhouse conditions in pots (34 cm long by 10 cm diameter) containing soil from a plot cropped to spring wheat since 1911 without fertilization. The roots and soil were sealed from the atmosphere and exposed to a¹⁵N₂-enriched atmosphere for three to four weeks during vegetative, reproductive or post-reproductive stages. This technique permitted detection of as little as 1 μg fixed N plant^-1 in plant material and 40 μg fixed N plant^-1 in soil. No fixation of¹⁵N₂ occurred during either of the first two labelling periods. In the final labelling period, straw returned to the soil was significantly enriched in¹⁵N, especially in a pot with a higher soil moisture content. Total BNF in this pot was 13 μg N plant^-1, or about 30 g N ha^-1. In a separate experiment with soil from the same plot, we detected BNF only when soil was amended with glucose at a high soil moisture content. Measured associative BNF was insufficient to account for observed N gains under field conditions. Lethbridge Research Centre contribution no. 3879488. Lethbridge Research Centre contribution no. 3879488.