Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.     

Observation On The Germination Of The Seeds Of Crinum Macowani,Baker

Arid and semi-arid areas occupy an increasing fraction of the Earth's surface. Legume floras exist for most of these areas, but there is little information as to whether the plants nodulate and fix nitrogen (N) in their native habitats, although many have been used over millennia for food, forage and medicinal and other uses.

This review shows that, in those arid and semi-arid areas where data are available, the ability of legumes to nodulate is a significant attribute. It examines some host genera present and, where known, the bacteria that induce nodulation in them.

With some exceptions all legumes from well-studied arid areas have the potential to nodulate. Semi-arid areas vary between continents in terms of legume genera present, the probable extent of N fixation and in the endosymbionts (rhizobia) that induce nodulation in them.

With climate change and an increasing world population, there is an urgent need to develop the diverse range of nodulated legumes native to dry environments. With modern methods this goal is readily achievable.     

Role of legumes in providing N for sustainable tropical pasture systems

Forage legumes have long been lauded for their ability to fix atmospheric nitrogen and contribute to the sustainability of agricultural production systems. However despite the benefits they bring in terms of increased herbage and animal production they are not widely used in temperate or tropical regions. In this review the amounts of biological nitrogen fixation (BNF) needed to sustain the soil-plant-animal system are discussed and related to the amounts fixed in tropical pastures. The data suggest that tropical forage legumes have the capacity to meet the requirements to balance the N cycle of grazed pastures. The actual amounts required will depend on the rate of pasture utilization and the efficiency of recycling via litter, excreta and internal remobilization. The efficiency of nitrogen fixation (% of legume N derived from fixation) is usually high in tropical pastures (>80%) and is unlikely to be affected by inorganic soil N in the absence of N fertilizer. Thus an estimate of the amoutns of N fixed could be obtained from simple estimates of legume biomass provided tissue levels of other nutrients such as phosphorus and potassium are adequate. Key factors for the achievement of sustainable grass/legume pastures include the selection of appropriate germplasm adapted to the particular environment and the judicious use of fertilizers such as phosphorus, potassium, calcium, magnesium and sulphur on acid infertile soils typical of the sub-humid and humid tropics. The main constraints to the widespread adoption of forage legumes include a lack of legume persistence, the presence of anti-quality factors such as tannins, variable Bradyrhizobium requirements and lack of acceptability by farmers. Strategies for the alleviation of these constrainst are discussed. Forage legumes can be used to recuperate degraded soils via their ability to improve the physical, chemical and biological properties of soils and these benefits could be of particular use for small-scale resource-poor farmers. The incorporation of forage legumes into agropastoral systems is discussed as an environmentally and economically attractive means to encourage the widespread adoption of legumes in the humid tropics.     

A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L.f.) druce

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or RHIZOBIUM: The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.     

Ammonium fixation by some soils in the ussr and the availability of this ion to plants

Summary The ability of various soils to fix ammonium in unexchangeable form was studied. Soils in their natural state contain unexchangeably fixed NH₄. The amount of which considerably increases with systematic fertilization and in soils with high nutrient status, but the ability of these soils for extra fixation of ammonium decreases. Chernozem soils are able to fix ammonium in unexchangeable form in considerably greater quantities than soddy-podzolic soils. All soils show a little increase of ability to fix ammonium with profile depth. The heavier is the soil the greater is its ability for fixation of NH₄ in unexchangeable form.In the conditions of a greenhouse experiment unexchangeably fixed NH₄ was used by plants very poorly. Exchangeably absorbed ammonium is available to plants, although to a lesser extent than water soluble salts. Soils fix ammonium to a greater amount from NH₄OH than from ammonium nitrate, hence the latter is a better source of nitrogen for plants than ammonia liquor solution if applied to soils with a definite abolity to fix unexchangeable ammonium.     

Plant assimilation and nitrogen cycling

Nitrogen, an abundant and yet limiting nutrient for crop and food production, enters the plant as nitrate or ammonium, or as dinitrogen through biological fixation by procaryotes associated with the plant. Nitrogen incorporation into the soil-plant-animal system is ultimately restricted by rates of biological and industrial fixation. Biological fixation conserves fossil fuel, but fertilization is preferred in most present agriculture. Nitrogen-metabolism research has the practical objectives of allowing more efficient N-fertilizer utilization by plants, including those that fix N₂ but benefit from fertilizer_N supplements. Nitrogen accumulation by harvested crops results in changes in soil acidity, with the direction of change depending on the N-source. There is little information on long-term effects of crop N-nutrition on acidity, and acidity is a critical factor that affects agricultural productivity in many tropical soils. Thus, plant control of pH and the acid/base balance in the soil as a consequence of nitrogen uptake and assimilation are important areas of future research.     

Never too many? How legumes control nodule numbers

Restricted availability of nitrogen compounds in soils is often a major limiting factor for plant growth and productivity. Legumes circumvent this problem by establishing a symbiosis with soil-borne bacteria, called rhizobia that fix nitrogen for the plant. Nitrogen fixation and nutrient exchange take place in specialized root organs, the nodules, which are formed by a coordinated and controlled process that combines bacterial infection and organ formation. Because nodule formation and nitrogen fixation are energy-consuming processes, legumes develop the minimal number of nodules required to ensure optimal growth. To this end, several mechanisms have evolved that adapt nodule formation and nitrogen fixation to the plant's needs and environmental conditions, such as nitrate availability in the soil. In this review, we give an updated view on the mechanisms that control nodulation.     

Biology of theParasponia-Bradyrhizobium symbiosis

Parasponia remains the only non-legume known to nodulate withRhizobium/Bradyrhizobium. It is a pioneer plant that is capable of rapid growth and fixing large quantities of nitrogen. In addition to its high agronomic potential, the symbiosis offers the scientist the unique opportunity of studying differences at the molecular level of both partners, and to investigate any possible extension of the symbiosis to other non-legumes of importance. Haemoglobin has been found in the nodule tissue ofParasponia and other nodulated non-legumes and the gene for it has been found and expressed in non-nodulating plants such asTrema tomentosa andCeltis australis. Bradyrhizobium strains isolated from species ofParasponia growing in Papua New Guinea form a group that are more specific in their host requirements thanBradyrhizobium strains from tropical legumes from the same area. They do not effectively nodulate (except CP283) tropical legumes, andParasponia is not readily nodulated withRhizobium andBradyrhizobium strains from legumes. The effectiveness of the symbiosis is influenced by host species, theBradyrhizobium strain and the environment.Parasponia andersonii forms a more effective symbiosis than the other species tested. In competition studies with strains from legumes, isolates fromParasponia always dominate in nodules onParasponia.     

Legume-nodulating betaproteobacteria: diversity, host range, and future prospects

Rhizobia form specialized nodules on the roots of legumes (family Fabaceae) and fix nitrogen in exchange for carbon from the host plant. Although the majority of legumes form symbioses with members of genus Rhizobium and its relatives in class Alphaproteobacteria, some legumes, such as those in the large genus Mimosa, are nodulated predominantly by betaproteobacteria in the genera Burkholderia and Cupriavidus. The principal centers of diversity of these bacteria are in central Brazil and South Africa. Molecular phylogenetic studies have shown that betaproteobacteria have existed as legume symbionts for approximately 50 million years, and that, although they have a common origin, the symbiosis genes in both subclasses have evolved separately since then. Additionally, some species of genus Burkholderia, such as B. phymatum, are highly promiscuous, effectively nodulating several important legumes, including common bean (Phaseolus vulgaris). In contrast to genus Burkholderia, only one species of genus Cupriavidus (C. taiwanensis) has so far been shown to nodulate legumes. The recent availability of the genome sequences of C. taiwanensis, B. phymatum, and B. tuberum has paved the way for a more detailed analysis of the evolutionary and mechanistic differences between nodulating strains of alpha- and betaproteobacteria. Initial analyses of genome sequences have suggested that plant-associated Burkholderia spp. have lower G+C contents than Burkholderia spp. that are opportunistic human pathogens, thus supporting previous suggestions that the plant- and human-associated groups of Burkholderia actually belong in separate genera.     

Influence of ammonium and nitrate supply on growth,nitrate reductase activity and N-use efficiency in a natural hybrid pine and its parents

Aims Selection of tree species with a high capacity to assimilate N and efficiently utilize N resources would facilitate the success of initial tree seedling establishment in infertile soils. The preference for N forms was tested using three pine species (Pinus densata, Pinus tabuliformis and Pinus yunnanensis). Pinus densata is a natural diploid hybrid between P. tabuliformis and P. yunnanensis .Methods Seedlings of three pine species were supplied with nitrate-N, ammonium-N (at two different pH regimes) or combined ammonium and nitrate as a nitrogen source in perlite culture in a controlled environment.Important findings Seedlings of P. densata had higher total biomass and net photosynthesis when supplied with nitrate-N and ammonium nitrate than with ammonium-N. In parental species, total biomass and net photosynthesis for P. yunnanensis seedlings was higher in ammonium-N than in nitrate-N, whereas the other parental species P. tabuliformis had the highest total biomass among species for all treatments except ammonium with CaCO 3. Most morphological traits in P. densata seedlings were intermediate between its two parental species. However, N-use efficiency and photosynthetic N-use efficiency of P. densata significantly exceeded both parents when supplied with nitrate-N and ammonium nitrate. The results suggested that the diploid hybrid tree species P. densata has a preference for nitrate and is not well adapted to ammonium-N as a sole nitrogen source regardless of the growth medium pH. Based on changes in environmental conditions, such as predicted future temperature increases in high altitude areas associated with climate change, P. densata is likely to be increasingly competitive and have wide adaptation in high altitude regions.     

Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes

Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.     

Economic impact of future biological nitrogen fixation technologies on United States agriculture

The economic impact of some future biological nitrogen fixation (BNF) technologies are estimated using AGSIM, an economietric model of United States agriculture. Five separate scenarios were modeled: (1) legumes fix more nitrogen (N₂) with no yield increase, (2) legumes fix more N₂ with an increase in yields of 10%, (3) N fertilization requirements on all crops are reduced 50% with no yield changes, (4) total elimination of N fertilization, and (5) total elimination of N fertilization and non-legume yields decrease 10%. Results indicate that BNF technologies have a high value to society. Increasing the efficiency of legumes to fix N₂ may have an annual US benefit of $1,067 million while decreasing N fertilization by 1,547 thousand metric tons. Total elimination of N fertilization of the major crops has an annual US benefit of $4,484 million.     

Nitrogen Use Efficiency of Crop Plants: Physiological Constraints upon Nitrogen Absorption

Current global nitrogen fertilizer use has reached approximately one hundred billion kg per annum. In many agricultural systems, a very substantial portion of this applied nitrogen fertilizer is lost from soil to groundwaters, rivers and oceans. While soil physicochemical properties play a significant part in these losses, there are several characteristic features of plant nitrogen transporter function that facilitate N losses. Nitrate and ammonium efflux from roots result in a reduction of net nitrogen uptake. As external nitrate and ammonium concentrations, respectively, are increased, particularly into the range of concentrations that are typical of agricultural soils, elevated rates of nitrate and ammonium efflux result. The rapid down-regulation of high-affinity influx as plants become nitrogen replete further reduces the root's capacity to acquire external nitrogen; only nitrogen-starved roots absorb with both high capacity and high affinity. The results of studies using molecular biology methods demonstrate that genes encoding nitrate and ammonium transporters are rapidly down-regulated when nitrogen is resupplied to nitrogen-starved plants. Provision of ammonium to roots of plants actively absorbing nitrate imposes a block on nitrate uptake, the extent of which depends on the ammonium concentration, thus further reducing the efficient utilization of soil nitrate. During the daily variation of incoming light and during periods of low incident irradiation (i.e. heavy cloud cover) the expression levels of genes encoding nitrate and ammonium transporters, and rates of nitrate and ammonium uptake, are substantially reduced. Low temperatures reduce growth and nitrogen demand, and appear to discriminate against high-affinity nitrogen influx. In sum, these several factors conspire to limit rates of plant nitrogen uptake to values that are well below capacity. These characteristics of the plant's nitrogen uptake systems facilitate nitrogen losses from soils.     

Relationships Amongst the Fast-growing Rhizobia of Lablab purpureus, Leucaena leucocephala, Mimosa spp., Acacia farnesiana and Sesbania grandiflora and their Affinities with Other Rhizobial Groups

Colony characteristics, growth in litmus milk, precipitation in calcium glycerophosphate medium and utilization of carbon sources of the root-nodule bacteria isolated from the tropical legumes Leucaena, Mimosa, Acacia, Sesbania and Lablab were similar to fast-growing rhizobia of temperate legumes, particularly Rhizobium meliloti. In agglutination tests, isolates from each host shared antigens with one or more of five Rhizobium strains from Leucaena. Infective characteristics of the fast-growing rhizobia were studied in modified Leonard jars and in agar culture. Cross-infections by rhizobia between these plants were common and the association often effective. Lablab was effectively nodulated by its own fast-growing isolate but only formed root swellings, possibly ineffective pseudonodules, with the other isolates. Slow-growing rhizobia which were able to nodulate Macroptilium atropurpureus were unable to form nodules on these legumes except Lablab which was considered more akin to the cowpea group. All fast-growing isolates nodulated, often effectively, Vigna unguiculata and V. unguiculata ssp. sesquipedalis. The isolate from Lablab also effectively nodulated a number of other tropical legumes which have previously only been reported to nodulate with slow-growing nodule bacteria and it also produced ineffective nodulation on Medicago sativa. This is the first record of an effective fast-growing isolate from Lablab.     

Origins of cheating and loss of symbiosis in wild Bradyrhizobium

Rhizobial bacteria nodulate legume roots and fix nitrogen in exchange for photosynthates. These symbionts are infectiously acquired from the environment and in such cases selection models predict evolutionary spread of uncooperative mutants. Uncooperative rhizobia – including nonfixing and non‐nodulating strains – appear common in agriculture, yet their population biology and origins remain unknown in natural soils. Here, a phylogenetically broad sample of 62 wild‐collected rhizobial isolates was experimentally inoculated onto Lotus strigosus to assess their nodulation ability and effects on host growth. A cheater strain was discovered that proliferated in host tissue while offering no benefit; its fitness was superior to that of beneficial strains. Phylogenetic reconstruction of Bradyrhizobium rDNA and transmissible symbiosis‐island loci suggest that the cheater evolved via symbiotic gene transfer. Many strains were also identified that failed to nodulate L. strigosus, and it appears that nodulation ability on this host has been recurrently lost in the symbiont population. This is the first study to reveal the adaptive nature of rhizobial cheating and to trace the evolutionary origins of uncooperative rhizobial mutants.     

Symbiotic nitrogen fixation in legumes: Perspectives for saline agriculture

Saline agriculture provides a solution for at least two environmental and social problems. It allows us to return to agricultural production areas that have been lost as a consequence of salinization and it can save valuable fresh water by using brackish or salt water to irrigate arable lands. Sea water contains (micro) nutrients that can provide the additional benefit of a reduced need of fertilization in saline agriculture. However, nitrogen is only present in very low quantities in seawater. A salt tolerant nitrogen-fixing legume used as a vegetable crop, fodder or green manure could increase the availability of soil nitrogen as well as the sustainability of saline agriculture while minimizing the application of inorganic fertilizer. Besides the use of salt-tolerant legumes as green manure, such species could also be useful in salinized areas as fodder and/or human food.In this review, we assess the feasibility of the use of legumes in saline agriculture. Most legumes are sensitive to salinity, as is the process of nitrogen fixation by microorganisms in the nodules of the legumes. First, we identify different steps in nodulation and their respective sensitivity to salinity. We will then look at the sensitivity of the process of nitrogen fixation in various crop and non-crop legumes, differing in their tolerance to salinity. We will also look into the differential response of nitrogen fixation and biomass production to salinity. Finally, a list of salt tolerant legumes is presented (derived from the HALOPH database). We then evaluate the applicability and perspective of salt tolerant legumes in saline agriculture considering the diversity in growth forms, ecotypes and economic uses.     

Salinity and its effects on the functional biology of legumes

Salinity has plagued soil fertility and drastically affected growth and survival of glycophytes in irrigated regions of the world since the beginning of recorded history. It is particularly common in arid and semi-arid areas where evapotranspiration exceeds annual precipitation, and where irrigation is therefore necessary to meet crop water needs. Salt buildup in the soils and groundwater has threatened its productivity and sustainability. Plant responses to salt stress include an array of changes at the molecular, biochemical and physiological levels. Salt stress involves a water deficit induced by the salt concentration in the rhizosphere, resulting in disruption of homeostasis and ion distribution in the cell and denaturation of structural and functional proteins. As a consequence, salinity stress often activates cell signaling pathways including those that lead to synthesis of osmotically active metabolites, specific proteins, and certain free radical scavenging enzymes that control ion and water flux and support scavenging of oxygen radicals or chaperones. ROS detoxification forms an important defense against salt stress. Legumes are a key component of sustainable agriculture and can offer many economic and environmental benefits if grown more widely in crop rotations because of their ability to fix nitrogen in the root nodules in a symbiotic interaction with soil rhizobia. Due to their capacity to grow on nitrogen-poor soils, they can be efficiently used for improving saline soil fertility and help to reintroduce agriculture to these lands. However, in legumes, salt stress imposes a significant limitation of productivity related to the adverse effects on the growth of the host plant, the root-nodule bacteria, symbiotic development and finally the nitrogen fixation capacity. This paper reviews responses of legumes to salinity stress with emphasis on physiological and biochemical mechanisms of salt tolerance.     

Nitrate paradigm does not hold up for sugarcane

Modern agriculture is based on the notion that nitrate is the main source of nitrogen (N) for crops, but nitrate is also the most mobile form of N and easily lost from soil. Efficient acquisition of nitrate by crops is therefore a prerequisite for avoiding off-site N pollution. Sugarcane is considered the most suitable tropical crop for biofuel production, but surprisingly high N fertilizer applications in main producer countries raise doubt about the sustainability of production and are at odds with a carbon-based crop. Examining reasons for the inefficient use of N fertilizer, we hypothesized that sugarcane resembles other giant tropical grasses which inhibit the production of nitrate in soil and differ from related grain crops with a confirmed ability to use nitrate. The results of our study support the hypothesis that N-replete sugarcane and ancestral species in the Andropogoneae supertribe strongly prefer ammonium over nitrate. Sugarcane differs from grain crops, sorghum and maize, which acquired both N sources equally well, while giant grass, Erianthus, displayed an intermediate ability to use nitrate. We conclude that discrimination against nitrate and a low capacity to store nitrate in shoots prevents commercial sugarcane varieties from taking advantage of the high nitrate concentrations in fertilized soils in the first three months of the growing season, leaving nitrate vulnerable to loss. Our study addresses a major caveat of sugarcane production and affords a strong basis for improvement through breeding cultivars with enhanced capacity to use nitrate as well as through agronomic measures that reduce nitrification in soil.     

A New Species of Devosia That Forms a Unique Nitrogen-Fixing Root-Nodule Symbiosis with the Aquatic Legume Neptunia natans (L.f.) Druce

Rhizobia are the common bacterial symbionts that form nitrogen-fixing root nodules in legumes. However, recently other bacteria have been shown to nodulate and fix nitrogen symbiotically with these plants. Neptunia natans is an aquatic legume indigenous to tropical and subtropical regions and in African soils is nodulated by Allorhizobium undicola. This legume develops an unusual root-nodule symbiosis on floating stems in aquatic environments through a unique infection process. Here, we analyzed the low-molecular-weight RNA and 16S ribosomal DNA (rDNA) sequence of the same fast-growing isolates from India that were previously used to define the developmental morphology of the unique infection process in this symbiosis with N. natans and found that they are phylogenetically located in the genus Devosia, not Allorhizobium or Rhizobium. The 16S rDNA sequences of these two Neptunia-nodulating Devosia strains differ from the only species currently described in that genus, Devosia riboflavina. From the same isolated colonies, we also located their nodD and nifH genes involved in nodulation and nitrogen fixation on a plasmid of approximately 170 kb. Sequence analysis showed that their nodD and nifH genes are most closely related to nodD and nifH of Rhizobium tropici, suggesting that this newly described Neptunia-nodulating Devosia species may have acquired these symbiotic genes by horizontal transfer.     

Nodule growth and nitrogen fixation of <Emphasis Type="Italic">Calopogonium mucunoides</Emphasis> L. show low sensitivity to nitrate

It is well established that nitrate is a potent inhibitor of nodulation and nitrogen fixation in legumes. The objective of this study was to demonstrate the relative insensitivity of these processes to nitrate with Calopogonium mucunoides, a tropical South American perennial legume, native to the cerrado (savannah) region. It was found that nodule number was reduced by about half in the presence of high levels of nitrate (15 mM) but nodule growth (total nodule mass per plant) and nitrogen fixation (acetylene reduction activity and xylem sap ureide levels) were not affected. Other sources of N (ammonium and urea) were also without effect at these concentrations. At even higher concentrations (30 mM), nitrate did promote significant inhibition (ca. 50%) of acetylene reduction activity, but no significant reduction in xylem sap ureides was found. The extraordinary insensitivity of nodulation and N₂ fixation of C. mucunoides to nitrate suggests that this species should be useful in studies aimed at elucidating the mechanisms of nitrate inhibition of these processes.