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.     

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.     

Soybean genotypic differences in sensitivity of symbiotic nitrogen fixation to soil dehydration

Nitrogen fixation activity by soybean (Glycine max (L.) Merr.) nodules has been shown to be especially sensitive to soil dehydration. Specifically, nitrogen fixation rates have been found to decrease in response to soil dehydration preceding alterations in plant gas exchange rates. The objective of this research was to investigate possible genetic variation in the sensitivity of soybean cultivars for nitrogen fixation rates in response to soil drying. Field tests showed substantial variation among cultivars with Jackson and CNS showing the least sensitivity in nitrogen accumulation to soil drying. Glasshouse experiments confirmed a large divergence among cultivars in the nitrogen fixation response to drought. Nitrogen fixation in Jackson was again found to be tolerant of soil drying, but the other five cultivars tested, including CNS, were found to be intolerant. Experiments with CNS which induced localized soil drying around the nodules did not result in decreases in nitrogen fixation rates, but rather nitrogen fixation responded to drying of the entire rooting volume. The osmotic potential of nodules was found to decrease markedly upon soil drying. However, the decrease in nodule osmotic potential occurred after significant decreases in nitrogen fixation rates had already been observed. Overall, the results of this study indicate that important genetic variations for sensitivity of nitrogen fixation to soil drying exist in soybean, and that the variation may be useful in physiology and breeding studies.     

Using an ecophysiological analysis to dissect genetic variability and to propose an ideotype for nitrogen nutrition in pea

BACKGROUNDS AND AIMS: Nitrogen nutrition of legumes, which relies both on atmospheric N2 and soil mineral N, remains a major limiting factor of growth. A decade ago, breeders tried to increase N uptake through hypernodulation. Despite their high nodule biomass, hypernodulating mutants were never shown to accumulate more nitrogen than wild types; they even generally displayed depressed shoot growth. The aim of this study was to dissect genetic variability associated with N nutrition in relation to C nutrition, using an ecophysiological framework and to propose an ideotype for N nutrition in pea. METHODS: Five pea genotypes (Pisum sativum) characterized by contrasting root and nodule biomasses were grown in the field. Variability among genotypes in dry matter and N accumulation was analysed, considering both the structures involved in N acquisition in terms of root and nodule biomass and their efficiency, in terms of N accumulated through mineral N absorption or symbiotic N2 fixation per amount of root or nodule biomass, respectively. KEY RESULTS: Nodule efficiency of hypernodulating mutants was negatively correlated to nodule biomass, presumably due to the high carbon costs induced by their excessive nodule formation. Root efficiency was only negatively correlated to root biomass before the beginning of the seed-filling stage, suggesting competition for carbon between root formation and functioning during the early stages of growth. This was no longer the case after the beginning of the seed-filling stage and nitrate absorption was then positively correlated to root biomass. CONCLUSIONS: Due to the high C costs induced by nodule formation and its detrimental effect on shoot and root growth, selecting traits for the improvement of N acquisition by legumes must be engineered (a) considering inter-relationships between C and N metabolisms and (b) in terms of temporal complementarities between N2 fixation and nitrate absorption rather than through direct increase of nodule and/or root biomass.     

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     

Effects of disease resistance genes on Rhizobium symbiosis in an annual legume

Summary The evolution of disease resistance in plants may be constrained if genes conferring resistance to pathogens interfere with plant responses toward other, nonpathogenic organisms. To test for such effects, we compared symbiotic nitrogen fixation in Amphicarpaea bracteata plants that differed at a major locus controlling resistance to the pathogen Synchytrium decipiens. Both resistant and susceptible plant genotypes nodulated successfully and grew significantly better in the presence of Rhizobium, although growth enhancement by Rhizobium was altered by different levels of nitrate fertilization. Plants homozygous for disease resistance achieved 2% higher growth than susceptible homozygotes across all treatments, but this difference was not significant. Resistant and susceptible plant genotypes did not differ in the mean number of nodules formed per plant or in nodule diameter. However, there was highly significant variation among replicate families within each disease resistance category for both nodulation characteristics. These results imply that genetic variation exists among A. bracteata plants both for diease resistance and for traits affecting symbiotic nitrogen fixation. However, there were no evident pleiotropic effects of disease resistance genes on the plant-Rhizobium symbiosis.     

Long-term effects of nitrate on lucerne (Medicago sativa L.) nitrogen fixation is not influenced by the denitrification status of the microsymbiont

Studies on the inhibitory effects of combined nitrogen on biological nitrogen fixation in legume crops have been usually carried out after short-term nitrate treatments at high concentrations. As these treatments are quite different from field conditions, a study was conducted to evaluate the effects of the continuous presence of nitrate (0, 1, 5 and 10 mM) throughout three months on lucerne (Medicago sativa L.). Plants were grown in a greenhouse with perlite as substrate and were inoculated with a denitrifying Sinorhizobium meliloti strain (102-F-51) and a non-denitrifying strain (102-F-65). During the first 60 days of growth, the highest nitrate treatment resulted in a complete inhibition of the main symbiotic parameters (nodule initiation and development and specific nitrogen fixation) in plants inoculated with either strain. However, after 3 months of growth in the presence of nitrate, this inhibition was partly abolished, with a high number of new functioning nodules being formed. Acetylene reduction activity (ARA) of these plants was 70% of the control plants. As this process was observed in plants nodulated with either strain, it is concluded that this was not related to the denitrifying ability of the strain, but is an intrinsic property of the lucerne nitrogen fixing system. As legume plants usually grow under natural field conditions in the continuous presence of nitrate, the ability to use simultaneously nitrate and atmospheric nitrogen could be of adaptive and agronomic importance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of ammonium and nitrate nitrogen on nitrogenase activity of pea plants as affected by light intensity and sugar addition

Summary Addition of ammonium chloride or potassium nitrate to nodulated pea plants resulted in a decrease in acetylene-reducing activity. Both nodule growth and specific activity of the nodules were diminished. Acetylene-reducing activity of isolated bacteroids, treated with EDTA-toluene and supplied with ATP and dithionite, had not decreased after a 3-day treatment of the plants with NH₄Cl or KNO₃. The effect of combined nitrogen could be counteracted by raising the light intensity or by the addition of sucrose to the growth medium. The latter treatment reduced the nitrogen uptake by the plants. It is concluded that combined nitrogen affects symbiotic nitrogen fixation via the carbohydrate supply to the bacteroids.     

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.     

Effects of drought stress on legume symbiotic nitrogen fixation: physiological mechanisms

Drought stress is one of the major factors affecting nitrogen fixation by legume-rhizobium symbiosis. Several mechanisms have been previously reported to be involved in the physiological response of symbiotic nitrogen fixation to drought stress, i.e. carbon shortage and nodule carbon metabolism, oxygen limitation, and feedback regulation by the accumulation of N fixation products. The carbon shortage hypothesis was previously investigated by studying the combined effects of CO2 enrichment and water deficits on nodulation and N2 fixation in soybean. Under drought, in a genotype with drought tolerant N2 fixation, approximately four times the amount of 14C was allocated to nodules compared to a drought sensitive genotype. It was found that an important effect of CO2 enrichment of soybean under drought was an enhancement of photo assimilation, an increased partitioning of carbon to nodules, whose main effect was to sustain nodule growth, which helped sustain N2 rates under soil water deficits. The interaction of nodule permeability to O2 and drought stress with N2 fixation was examined in soybean nodules and led to the overall conclusion that O2 limitation seems to be involved only in the initial stages of water deficit stresses in decreasing nodule activity. The involvement of ureides in the drought response of N2 fixation was initially suspected by an increased ureide concentration in shoots and nodules under drought leading to a negative feedback response between ureides and nodule activity. Direct evidence for inhibition of nitrogenase activity by its products, ureides and amides, supported this hypothesis. The overall conclusion was that all three physiological mechanisms are important in understanding the regulation of N2 fixation and its response of to soil drying.     

Large genotypic variation but small variation in N2 fixation among rhizobia nodulating red clover in soils of northern Scandinavia

AIMS: To analyse the symbiotic variations within indigenous populations of rhizobia nodulating red clover (Trifolium pratense L.) in soils of northern Norway and Sweden at different times of the growing season. METHODS AND RESULTS: A total of 431 nodule isolates sampled under field conditions in summer and autumn, were characterized genetically by targeting both chromosomal and symbiotic genes. The Enterobacterial Repetitive Intergenic Consensus polymerase chain reaction (PCR) fingerprinting of chromosomal DNA revealed considerable variation within the isolated populations that was more influenced by geographical origin than sampling time. Analysis of PCR amplified nodEF gene on the symbiotic plasmid by restriction fragment length polymorphism revealed a high proportion of nod types common to the two studied sites. The symbiotic efficiency of the isolates, representing both dominating and rare nodEF genotypes, showed high N(2) fixation rates in symbiosis with the host plant in a greenhouse experiment using the (15)N isotope dilution method. CONCLUSIONS: Effective N(2)-fixing strains of Rhizobium leguminosarum bv. trifolii nodulating red clover are common and genetically diverse in these northern Scandinavia soils. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the variability, stability and dynamics of resident populations of rhizobia nodulating red clover in Scandinavian soils which has practical implications for applying biological nitrogen fixation in subarctic plant production.     

Developing a breeding strategy to exploit quantitative variation in symbiotic nitrogen fixation

Summary This paper examines evidence which quantifies the relative importance of legume and Rhizobium genotypes as determinants of phenotypic variation in symbiotic nitrogen fixation. It demonstrates potentially large and unpredictable effects of the Rhizobium genotype. The likely importance of such effects on crop yield is considered. The information is then used to assess ways in which legume breeding programmes may be altered to encompass the effects of genetic variation in Rhizobium.     

The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis

A study was conducted to investigate the effects of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. The effects of P on growth and survival of Frankia in the rhizosphere was assessed by examing Frankia growth and survival in flasks of basal nutrient solution. There was no growth in the nutrient solution during the experimental period. However, the viability of Frankia in the nutrient solution without P supply was half that of the initial level, whereas, with P supply, there was only a minor decline during the first week. In a growth pouch experiment, supplying P increased plant and nodule growth, irrespective of P status of the inoculant Frankia culture. There were no effects of P status on any growth or nodulation parameters measured when the inoculants had been standardized on the basis of viability. In a split root experiment, Frankia inoculation and application of P together or separately did not cause any significant difference. This suggests that growth and nodulation respond only to total P supply. Increasing P from 0.1 to 10 M significantly increased plant growth but not N concentrations. Both nitrogen-fixation and nitrate supported growth were strongly increased as P increased from 0.1 to 1.0 M. This study indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes and P deficiency on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana operated indirectly via reducing host plant growth.     

Growth, nitrogen fixation and ammonium assimilation in common bean (Phaseolus vulgaris L) subjected to iron deficiency

A greenhouse experiment was carried out aiming to study the effect of iron deficiency on nitrogen fixation and ammonium assimilation in common bean nodules. Host-plant and nodule growth, symbiotic nitrogen fixation, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were analyzed in two common bean varieties subjected to iron deficiency. Results showed that host-plant and nodules growth, nitrogen fixation and GS activity decreased when under Fe-deficiency against an important increase of ammonium accumulation and GDH activity. Tolerant variety Flamingo is clearly less affected by iron deficiency than the sensitive one, Coco blanc. The allocation of iron to nodules and Fe use-efficiency for nodule growth and symbiotic nitrogen fixation were on the basis of the symbiotic performance of Flamingo under iron deprivation. Under Fe-deficiency, GDH take over GS the ammonium assimilation activity, particularly in the tolerant variety.     

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.     

马桑结瘤固氮与光合作用的关系

马桑(Coriaria sinica)植株的结瘤量、根瘤固氮活性和固氮能力均与植株叶面积和光合能力呈显著的直线相关关系,叶面积大、光合能力强的植株结瘤量大,根瘤固氮活性高,固氮能力强。马桑根瘤固氮活性呈白天升高夜间降低的昼夜变化特点,昼夜变幅为10～20μmol C2H2/g.h,光合作用是引起固氮活性昼夜变化的主要因素,同时受土壤温湿度的影响,遮阴或光照不足将引起马桑结瘤固氮能力的大幅度降低。     

An interdisciplinary research strategy to improve symbiotic nitrogen fixation and yield of common bean (Phaseolus vulgaris) in salinised areas of the Mediterranean basin

The main findings of a cooperative research group of agronomists, plant breeders, microbiologists, physiologists and molecularists to improve the symbiotic nitrogen fixation (SNF) and N2-dependent yield of common bean under moderate salinity in the Mediterranean basin are summarised. Agronomic surveys in reference production areas show large spatial and temporal variations in plant nodulation and growth, and in efficiency of utilisation of the rhizobial symbiosis. The latter was associated with a large rhizobial diversity, including new bean nodulating species. Macrosymbiont diversity in SNF and adaptation to NaCl was found. However, contrasts between plant genotypes could be altered by specific interactions with some native rhizobia. Therefore, variations in soil rhizobial population, in addition to agronomic practices and environmental constraints, may have contributed to erratic results observed in field inoculations. At the mechanistic level, nodule C and N metabolisms, and abcissic acid content, were related to SNF potential and tolerance to NaCl. Their relation with nodule conductance to O2 diffusion was addressed by in situ hybridisation of candidate carbonic anhydrase and aquaporin genes in nodule cortex. The limits and prospects of the cooperative strategy are discussed.     

Nodulation studies in legumes

Summary The influence of combined nitrogen (as ammonium nitrate) on the symbiotic performances of selected bacterial associations of four legumes was examined using sand culture.In barrel medic (Medicago tribuloides Desr.) and vetch (Vicia sativa L. andV. atropurpurea Desf.) bacterial partnerships of a host plant varied greatly in their nodulation responses to a range of amounts of nitrogen applied at sowing. Some bacterial strains exhibited varying degrees of stimulation of nodule number, growth and fixation by low or medium amounts of nitrogen. Higher levels of combined nitrogen depressed symbiosis. Other strain responses showed a severe restriction of symbiosis with any amount of added nitrogen.Seasonal influences conditioned symbiotic responses to combined nitrogen in an association of cowpea (Vigna sinensis End.) With a summer sowing small amounts of ammonium nitrate added at sowing benefited later symbiotic development. No such stimulation was evident in an autumn sowing and symbiotic injury from high levels of nitrogen was greater than in the summer sowing.The developing association of cowpea was found to be most sensitive to ammonium nitrate added just as the first leaves unfolded. Here damage was manifest in a permanent elevation of the top: root ratio with subnormal growth and functioning of nodules. Greatest benefit from added inorganic nitrogen followed applications made as the first nodules appeared on the primary root. In this case added combined nitrogen acted as an investment providing returns in additional fixation equivalent to 5–10 times the amount of nitrogen originally fed to the seedling and representing some 50 per cent greater total fixation than in minus-nitrogen controls.     

Manganese tolerance in subterranean clover (Trifolium subterraneum L.) genotypes grown with ammonium nitrate or symbiotic nitrogen

Summary A wide range of clover accessions were screened for reaction to manganese (Mn) in solution culture. Growth was supported with ammonium nitrate (NH₄NO₃) or symbiotic nitrogen to assess Mn effects on symbiosis and the suitability of NH₄NO₃ dependent growth for assessing Mn tolerance in clover. Reduction of dry matter at Mn 45 ppm varied 0–70%, at Mn 90 ppm, 38–92%, the extent depending on genotype. Tolerant clovers tended to restrict the movement of Mn from roots to shoots. Several previously untested lines were the most tolerant while some commercial lines possessed poor tolerance. Ranks of tolerance for the two nitrogen (N) sources at Mn 45 ppm were correlated suggesting no dominant, discriminatory effects of N source on Mn tolerance; but inclusion of symbiotic effectiveness in a multiple correlation improved the relation between relative tolerances of genotypes under different N sources. Mn affected some aspects of symbiosis. Total nodule nitrogenase activity mainly reflected effects of Mn on nodule number but nitrogenase activity per nodule also contributed. To establish relative tolerances of subterranean clover to Mn growth with NH₄NO₃ is suitable and useful when symbiotic effectiveness is unknown.     

Using a physiological framework for improving the detection of quantitative trait loci related to nitrogen nutrition in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Medicago truncatula is used as a model plant for exploring the genetic and molecular determinants of nitrogen (N) nutrition in legumes. In this study, our aim was to detect quantitative trait loci (QTL) controlling plant N nutrition using a simple framework of carbon/N plant functioning stemming from crop physiology. This framework was based on efficiency variables which delineated the plant’s efficiency to take up and process carbon and N resources. A recombinant inbred line population (LR4) was grown in a glasshouse experiment under two contrasting nitrate concentrations. At low nitrate, symbiotic N₂ fixation was the main N source for plant growth and a QTL with a large effect located on linkage group (LG) 8 affected all the traits. Significantly, efficiency variables were necessary both to precisely localize a second QTL on LG5 and to detect a third QTL involved in epistatic interactions on LG2. At high nitrate, nitrate assimilation was the main N source and a larger number of QTL with weaker effects were identified compared to low nitrate. Only two QTL were common to both nitrate treatments: a QTL of belowground biomass located at the bottom of LG3 and another one on LG6 related to three different variables (leaf area, specific N uptake and aboveground:belowground biomass ratio). Possible functions of several candidate genes underlying QTL of efficiency variables could be proposed. Altogether, our results provided new insights into the genetic control of N nutrition in M. truncatula. For instance, a novel result for M. truncatula was identification of two epistatic interactions in controlling plant N₂ fixation. As such this study showed the value of a simple conceptual framework based on efficiency variables for studying genetic determinants of complex traits and particularly epistatic interactions.