Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Denitrifying ability of thirteen Rhizobium meliloti strains

The denitrifying ability of thirteen strains of Rhizobium meliloti was tested. Most of the strains were able to reduce nitrate to nitrous oxide or dinitrogen. However, they failed to use nitrate as electron acceptor for ATP generation or growth at low oxygen tensions. Under micro-aerobic conditions, free-living cells of R. meliloti 102-F-51 strain exhibited a constitutive nitrate reductase activity independent of the presence of nitrate. On the other hand, nitrite reductase activity was dependent not only on low levels of oxygen but also on the presence of a high nitrate concentration in the medium. Denitrification activity proceeded immediately once a threshold level of nitrite was accumulated in the medium or in cells incubated with 1mM nitrite. However, a lag period was required when cells were incubated with nitrate.     

Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants

The response ofAlnus glutinosa, Casuarina cunninghamiana, Elaeagnus angustifolia andMyrica cerifera to a range of substrate nitrogen levels in solution, in relation to plant growth, infection, nodulation and root fine structure was studied. Nine concentrations of potassium nitrate ranging from 0.05 to 3.0 mM, were tested on each of the species. Plants were inoculated withFrankia pure cultures after a two week exposure to one of the nine levels of added nitrate. After six more weeks with constant exposure to nitrate, plants were harvested and assayed. With the exception of Myrica, regression analyses of whole plant dry weights as a function of added nitrate were highly significant. There was a tendency for nodulated plants grown at intermediate levels of added nitrate to exhibit higher relative growth rates, probably due to the additive effect of substrate nitrogen and fixation of atmospheric nitrogen. The mean numbers of nodules per plant were, with the exception of Alnus, significantly higher at intermediate levels of added nitrate, as were mean nodule dry weights. A highly significant inverse relationship between nodule weight as a percentage of whole plant weight was found in Elaeagnus and Myrica. The observed response of Elaeagnus to added nitrate compared to other actinorhizal plants appears to demonstrate that root hair infected plants are much more sensitive to the inhibitory effects of added nitrate than plants infected by intercellular penetration. A sharp reduction in the presence of root hairs at high concentrations of nitrate was observed. This indicates that the inhibition of nodulation in some actinorhizal plant species results from nitrate induced root hair suppression.     

Development of the nitrogen fixing apparatus in the legumes,Centrosema pubescens Benth., and Vigna unguiculata L. Walp.

The sequence of events leading up to the establishment of symbiotic nitrogen-fixation were studied in two tropical legumes, Centrosema pubescens Benth, and Vigna unguiculata L. Walp. Parameters measured included fresh and dry weights, chlorophyll and leghaemoglobin contents, as well as the activities of NADH-nitrate reductase (EC 1.6.6.1), and nitrogenase (nitric-oxide reductase-EC 1.7.99.2) in plants that were inoculated with suitable rhizobia or which were watered with potassium nitrate. Dry weight and photosynthetic activity of both species followed the sigmoidal pattern which is characteristic of most plants. Growth was little different in either a qualitative or quantitative sense whether nitrogen was supplied as nitrate or through dinitrogen fixation. Although the biochemical sequence of events was dependent on the limiting sensitivities of the individual assays used, the data suggest that nitrate reductase is the first measurable enzymatic activity in the nodules (and roots), followed by acetylene reduction and leghaemoglobin in that order. It is possible therefore, that low levels of symbiotic nitrogen fixation occur in the nodules in the absence of leghaemoglobin. Nitrate reductase activity in C. pubescens nodules was negatively exponentially correlated with nitrogenase activity of the same nodules, suggesting a changing metabolism in old nodules. These data are discussed in terms of environmental and physical factors known to control nitrogen fixation.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Functional Activity of Exoglycans from Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Resistant Mutant M-71 during the Formation of Legume–Rhizobia Symbiosis against a High-Nitrogen Background

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71, capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO₃ ^–), was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of the same or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5- to 2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix⁺ phenotype of strain 250a, capable of forming symbiosis with pea plants only against a low-nitrogen background. In study of the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72–75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25–28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.     

The Effect of the Plant Growth Stimulant Bactozole on Rhizobium leguminosarum bv. viciae 250a and Its Nitrogen-Tolerant Mutant M-71 under Different Nitrogen Supply Conditions

The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied. At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold). At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect. At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration. The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8–2.5 times greater than it was at 6 mM nitrate. Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules. The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate. The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium are due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.     

Interaction of nitrate uptake and nitrogen fixation in faba beans

An experiment was carried out to determine the relationship between nitrate uptake and nitrogen fixation of faba beans. Therefore inoculated and uninoculated faba beans were grown in nutrient solution with different nitrate concentrations. Nitrate uptake was measured every two days during the growing period. At the end of the experiment the nitrate uptake kinetics were determined with a short time depletion technique and nitrogen fixation was measured with the acetylene reduction method. A limitation of nitrate uptake due to nitrogen fixation was relatively small. Nitrate concentrations of approximately 1 mol m^–3 and 5 mol m^–3 decreased nitrogen fixation to values of 16% and 1% of the control plants which received no nitrate nitrogen. A reduction of nitrogen fixation was mainly due to a decrease of specific nitrogen fixation per unit nodule weight and to a lesser extent due to a reduction of nodule growth. Only the maximum nitrate influx (I_max) seemed to be influenced by nitrogen fixation. Michaelis-Menten constants (K_m) and minimum NO _inf3 ^– -concentrations (C_min) were not significantly influenced by nitrogen fixation.     

Effect of nitrogen form at various levels of potassium,on the Meloidogyne-Fusarium wilt complex in muskmelon

Summary The influence of various potassium concentration and of nitrate or ammonium was evaluated on non inoculated muskmelon plants and on plants inoculated with theFusarium wilt pathogen (F), the root-knot nematode (Meloidogyne javanica) (Nem), or a combination of both pathogens (F+Nem). Increasing potassium concentrations raised top fresh weights (TFW) in all four groups. Nitrate fertilized plants weighed more than plants receiving ammonium, independent of the K⁺ level in the medium. In the Nem+F infected plants TFW values were one and half to two times greater in those receiving the nitrate than in those receiving the ammonium. Number of nematodes in the roots were not affected by nutritional difference. In the ammonium fertilized F and Nem+F plants 30% more wilting was found than after nitrate application. Irrespective of the form of nitrogen that was applied accumulation of N, P and K was found in the roots of the Nem+F and/or Nem plants, while in the shoots Ca, Mg, Na and P accumulated and K was depleted.Contribution from the Agricultural Research Organization, (ARO), No. 944-E, 1983 series.     

Ammonium sensing in nitrogen fixing bacteria: Functions of theglnB andglnD gene products

A plentiful supply of fixed nitrogen as ammonium (or other compounds such as nitrate or amino acids) inhibits nitrogen fixation in free-living bacteria by preventing nitrogenase synthesis and/or activity. Ammonium and nitrate have variable effects on the ability ofRhizobiaceae (Rhizobium, Bradyrhizobium andAzorhizobium) species to nodulate legume hosts and on nitrogen fixation capacity in bacteroid cells contained in nodules or in plant-free bacterial cultures. In addition to effects on nitrogen fixation, excess ammonium can inhibit activity or expression of other pathways for utilization of nitrogenous compounds such as nitrate (through nitrate and nitrite reductase), or glutamine synthetase (GS) for assimilation of ammonium. This paper describes the roles of two key genesglnB andglnD, whose gene products sense levels of fixed nitrogen and initiate a cascade of reactions in response to nitrogen status. While work onEscherichia coli and other enteric bacteria provides the model system,glnB and, to a lesser extent,glnD have been studied in several nitrogen fixing bacteria. Such reports will be reviewed here. Recent results on the identity and function of theglnB andglnD gene products inAzotobacter vinelandii (a free-living soil diazotroph) and inRhizobium leguminosarum biovarviciae, hereinafter designatedR.l. viciae will be presented. New data suggests thatAzotobacter vinelandii probably contains aglnB-like gene and this organism may have twoglnD-like genes (one of which was recently identified and namednfrX). In addition, evidence for uridylylation of theglnB gene product (the PII protein) ofR. l. viciae in response to fixed nitrogen deficiency is presented. Also, aglnB mutant ofR. l. viciae has been isolated; its characteristics with respect to expression of nitrogen regulated genes is described.     

Field measurement of symbiotic nitrogen fixation in an established lucerne ley using15N and an acetylene reduction method

Summary Lucerne is an important forage legume in the south and south-east of Sweden on well-drained soils. However, data is lacking on the apparent amount of nitrogen derived through N₂ fixation by field-grown lucerne. This report provides basic information on the subject. The experiment was performed in a lucerne ley grown 40 km north of Uppsala. The input of nitrogen through fixation to the above-ground plant material of an established lucerne (Medicago sativa L.) ley was estimate by¹⁵N methodology during two successive years. The amount of fixed N was 242 kg N ha^–1 in 1982 and 319 kg N ha^–1 in 1983. The proportion of N derived from the atmosphere (%Ndfa) was 70% and 80% for the two years respectively. The first harvest in both years contained a lower proportion fixed N. Both N₂ fixation and dry matter production were enhanced during the second year, particularly in the first harvest. The Ndfa was 61% in the first harvest in 1982, compared to 72% Ndfa during the same period in 1983. This demonstrates the strong influence of environment on both dry matter production and N₂ fixation capacity of the lucerne.In addition anin situ acetylene reduction assay was used in 1982 to measure the seasonal distribution of the N₂ fixation and in 1983 to study the effect of soil moisture on the N₂ fixation process. The seasonal pattern showed great dependence on physiological development and harvest pattern of the lucerne ley. The maximum rate of N₂ fixation occurred at the bud or early flower stage of growth and was followed by a rapid decline as flowering proceeded. After harvest the nitrogenase activity markedly decreased and remained low during at least two weeks until regrowth of new shoots began. Irrigation doubled the nitrogenase activity of the lucerne in late summer 1983, when soil moisture content in the top soil was near wilting point. No changes in nitrogenase activity did occur in response to watering earlier during the summer, when the soil matric potential was around –0.30 MPa.     

Contribution of dissolved dinitrogen in culture solutions to growth of Rhodopseudomonas capsulata with various sources of combined nitrogen

Fixation of dissolved dinitrogen in culture solutions by the photosynthetic bacterium Rhodopseudomonas capsulata, strain B10, reduced the lag phase associated with growth with glutamate. A comparable effect was not observed with ammonium chloride. This strain assimilated nitrate but nitrogen fixation was depressed during early growth on nitrate. It is shown that nitrite, the first product of nitrate assimilation, inhibits nitrogen fixation during the early stages of cell growth.     

Nitrogen fixation of field-inoculatedLeucaena leucocephala (Lam.) de Wit estimated by the15N and the difference methods

The amount of nitrogen fixed byLeucaena leucocephala (Lam.) de Wit was assessed on an Alfisol at the International Institute of Tropical Agriculture located in southwestern Nigeria. Estimated by the difference method, nitrogen fixation of leucaena inoculated with Rhizobium strain IRc 1045 was 133 kg ha^–1 in six months. Inoculation with Rhizobium strain IRc 1050 gave a lower nitrogen fixation of 76 kg ha^–1. Fertilization with 40 and 80 kg N ha^–1 inhibited nitrogen fixation by 43–76% and 49–71%, respectively. Estimates with the¹⁵N dilution method gave nitrogen fixation of 134 kg ha^–1 in six months when leucaena was inoculated with Rhizobium strain IRc 1045 and 98 kg ha^–1 for leucaena inoculated with Rhizobium strain IRc 1050. This nitrogen fixation represented 34–39% of the plant nitrogen. Inoculated leucaena derived 5–6% of its nitrogen from applied fertilizer and 56–54% from soil.     

Effect of low nitrate supply to nodulated lucerne on time course of activites of enzymes involved in inorganic nitrogen metabolism

Plants of lucerne ( Medicago sativa L. cv. Aragón) inoculated with several strains of Rhizobium meliloti were supplied with a low level of nitrate (5 m M ). After 1 week, normalised nodule mass, obtained by dividing nodule weight by shoot weight, was decreased by one-fourth. This result closely paralleled the bacteroid protein content of nodules, whereas the cytosolic content remained constant. Nitrate reductase activity (NRA, EC 1.7.99.4) of bacteroids increased rapidly after nitrate supply, with actual rates being highly dependent on the Rhizobium strain. The expression of cytosolic NR (EC 1.6.6.1) also varied depending on the bacterial strain but was largely insensitive to nitrate feeding. Nitrite reductase activity (NiRA, EC 1.7.2.2) of either bacteroid or plant origin was independent of the R. meliloti strain. Activation occurred after 3 and 7 days, respectively, of nitrate feeding. Significant amounts of nitrite were obtained throughout the experimental period from buffered extracts of both bacteroids and cytosol of nodules. However, when these nodules were ground in the presence of inhibitors of enzyme activity, nitrite was only found in nodules containing strain 102-F-51 after 1 week of treatment. These results agree with the recent hypothesis that nitrite plays a role in a secondary stage of nodule damage by nitrate. We propose that NiRA rather than NRA can be used as an internal probe of nitrate access to the infected region of nodules.     

Rhodopseudomonas sphaeroides forma sp. denitrificans,a denitrifying strain as a subspecies of Rhodopseudomonas sphaeroides

From polluted water of a lagoon pond a new type of denitrifying photosynthetic purple bacteria was isolated. With respect to morphology, fine structure, photopigments, requirement for growth factors, the range of utilization of organic substrates for phototrophic growth and DNA base ratio, the denitrifying strains show the closest resemblance to Rhodopseudomonas sphaeroides and were therefore described as a subspecies named R. sphaeroides forma sp. denitrificans. The new isolates grow well with nitrate anaerobically in the dark accompanying the evolution of nitrogen gas. They cannot assimilate nitrate as the nitrogen source for growth.     

Influence of combined nitrogen level andEucalyptus competition on dinitrogen fixation in nodulatedCasuarina

Summary Experiments were conducted to determine the effect on biological dinitrogen fixation byCasuarina of available nitrogen (N) in the substrate and competition by interplantedEucalyptus. In these experiments, combined N was applied to the plants after nodules were developed and functioning. Both environmental factors, nitrate and competition, were observed to influence biological dinitrogen fixation byCasuarina, but not yield (total dry weight). In one experiment, the proportion of nitrogen derived byCasuarina from atmospheric fixation (pNdfa) was observed to be inhibited by potassium nitrate in a linear fashion. However, substrate N did not significantly affect the weight of root nodules. Thus nodule dry weight was not highly correlated with the proportion of nitrogen fixed. In a second experiment, the presence of a non-fixing interplanted species,Eucalyptus, increased dinitrogen fixation inCasuarina.Casuarina interplanted withEucalyptus obtained a greater proportion of its nitrogen (94.75%) from fixation than didCasuarina grown alone (86.68%) suggesting that competition for substrate N influences the proportion of nitrogen fixed by this actinorhizal plant.Dedicated to the memory of Professor John G. Torrey     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Effect of nutrient medium pH on symbiotic nitrogen fixation byRhizobium leguminosarum andPisum sativum

Summary Experiments were performed to measure the pH-sensitive steps in nodulation and symbiotic fixation byPisum sativum and isolate RP-212-1 ofRhizobium leguminosarum. An aeroponic system with rigorous pH control was used to obtain numerous effective nodules. After exposure to various pH levels, the following responses were measured: (1) legume root growth and development, (2) survival and growth rate of a single effective bacterial isolate, (3) degree of nodulation, (4) rate of nitrogen fixation, (5) plant biomass, and (6) nitrogen content of plants. Both bacterial growth and root development were adequate at all pH levels from 4.4 to 6.6, but efficient nodulation and nitrogen fixation did not occur at pH 4.8 and below. The processes required for symbiosis were about 10 times as sensitive to acidity as either bacterial growth or root growth alone. Nodulation was the most acid-sensitive step.     

The potential for breeding white clover (Trifolium repens L.) with improved nodulation and nitrogen fixation when grown with combined nitrogen

Summary Sodium nitrate applications ranging from 0.36 to 22.84 mM N were shown to depress rates of nodule formation and reduce total nitrogen fixation (acetylene reduction) in white clover plants grown in aseptic test tube culture.Low nitrate levels gave an initial depression in symbiotic activity but the reduction was of short duration and these treatments were subsequently associated with enhanced rates of nodule formation and nitrogen fixation. As a result, phenotypic variation appeared to be strongly differentially affected by the amount of nitrate present. A subsequent experiment suggested that much of the variation was a consequence of early enhancement of plant growth rates by low levels of nitrate followed by rapid depletion thus giving a transitory inhibitory effect. This was confirmed in a third experiment in which the range of nitrate concentration was held constant. Differential effects on variability in nodule formation and nitrogen fixation were then greatly reduced but there was still a residual level of plant-to-plant variation. The results have clear implications for selecting genetic variants capable of fixing di-nitrogen in the presence of combined N. The provision of a single limiting dose of combined nitrogen to a population containing individuals with inherently different growth rates can bring about variations in the phenotypic expression of symbiotic characters. These variations are unlikely to be based on genetic factors which have a direct and stable effect on nodule development and nitrogenase activity. The implications of the results for plant breeding are discussed.     

Lack of effect of toxic aluminium concentrations on nitrogen fixation by nodulatedStylosanthes species

Summary Effects of three solution aluminium concentrations (0, 25, and 100M) on nitrogen fixation by well-nodulated plants ofStylosanthes hamata, Stylosanthes humilis andStylosanthes scabra are reported. Plants were inoculated with Rhizobium CB756 and grown for 21 days in an aluminium-free nutrient solution at pH 5.3 before imposition of the aluminium treatments.Nitrogen fixation was measured both by the increase in total nitrogen content of the plants and acetylene reduction in roots of plants harvested at 10 and 20 days after imposition of the aluminium treatments. Solution aluminium concentrations as high as 100M, had no detrimental effect on nitrogen fixation in any species.