α‐Aminoadipic Acid and α,ɛ‐Diaminopimelic Acid in Inoculated Pea Plants (Pisum sativum) and Root Nodule Bacteria (Rhizobium leguminosarum)

Summary: Pea plants that were nodulated by Rhizobium leguminosarum strains 248, 300 or 3622 and grown in the absence of combined nitrogen differed significantly in dry weight, leaf area, nitrogen content, nodule mass and nodule number. Transfer of plasmids from these field isolates to strain 16015, a non-nodulating derivative of strain 300, resulted in new strains capable of both nodulation and N2 fixation. In plants nodulated by these new strains the numbers of root nodules were significantly different, but there were no significant differences in N2 fixation, leaf area or dry weight. In several cases the introduction of additional plasmids into strain 300 or strain 16015 impaired symbiotic performance relative to strain 300 itself. Of all plant traits measured in symbiotic associations, leaf area was most highly correlated with the total Kjeldahl nitrogen content of plants after 25 d growth in the absence of combined nitrogen.

The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion-exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak. During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity. The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The Km values for malate (2.6 mM), NAD+ (27 microM), oxaloacetate (18 microM) and NADH (13 microM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 microM and 70 microM respectively). Binding of malate by the enzyme-NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex. From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacteroid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration. Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.

Lopetinsky, K. J., Lupwayi, N. Z., Olson, M. A., Akter, Z. and Clayton, G. W. 2014. Contrasting Rhizobium inoculation requirements of zero-tannin faba bean and narrow-leafed lupin in western Canada. Can. J. Plant Sci. 94: 1117-1123. Zero-tannin faba bean (Vicia faba minor) and narrow-leafed lupin (Lupinus angustifolius L.) have shown potential as new pulse crops in Alberta cropping systems, but their inoculation requirements to maximize biological N2 fixation (BNF) are unknown. We conducted a 6 site-year study to compare the effects of several commercial rhizobial inoculant products (eight for faba bean and three for lupin) in different formulations (granular, peat and liquid) on nodulation, N accumulation, grain yield and grain protein of the two crops. The liquid and peat formulations were applied to the seed, while the granular inoculant was applied to the soil. Inoculation had no significant effects on nodulation, grain yield and seed weight of faba bean in all site-years. Un-inoculated and inoculated plants nodulated equally well, suggesting the presence of adequate populations of effective indigenous Rhizobium leguminosarum bv. viciae for nodulation of untreated plants. The indingenous rhizobia could have originated from previous field pea (Pisum sativum L.) crops or leguminous native plants/weeds. By contrast, narrow-leafed lupin responded to inoculation in all site-years, and poor nodulation of un-inoculated plants indicated inadequate populations of indigenous R. lupini for nodulation in the soils. The seed-applied peat inoculant Nitragin Lupin and the soil-applied granular inoculant Soil Implant Lupin were equally effective in increasing nodulation relative to the un-inoculated control in 3 of 5 site-years (nodulation was not assessed in 1 site-year). However, relative to the un-inoculated control, Nitragin Lupin increased grain yields in 4 of 6 site-years compared with 1 of 5 for Soil Implant Lupin (and 2 of 6 for seed-applied TagTeam Lupin). These results show that faba bean probably does not require inoculation in these soils, although periodic checking is required to ensure that its high BNF potential is always realized, but narrow-leafed lupin needs to be inoculated with suitable inoculant products to increase BNF.

The experiment was carried out, under plastic house at the college of Agricultural Engineering Sciences, University of Sulaimani, Bakrajo, during 2018-2019 to determine influence of inoculation with arbuscular mycorrhizal fungi (AMF) and Rhizobium leguminosarum at different phosphors levels (0, 40, 80, 120 and 160 kgPha-1) on broad bean (Vicia faba L.) growth and nutrient uptake. The experiment was performed in a factorial experiment with completed randomized design (CRD) in a silty clay soil, with three replications for each treatment. After ten weeks of growth, the plants were harvested to determine plant growth (root colonization, shoot dry weight, root dry weight, nodule number, and shoot nutrient content N, P, K, Fe and Mo The results showed that inoculated broad bean plants with mycorrhiza or the bacterium Rhizobium leguminosarum increased plant growth and nutrient uptake compared with non-inoculated plant. Inoculated soil with AMF increased root colonization, shoot dry weight, root dry weight, and nodule number the highest value was (65%, 15.09gpot-1, 7.72gpot-1 and 128.67 nodule pot-1) respectively and the highest value for shoot N, P, K, Fe and Mo nutrients were (15.68gkg-1, 4.38gkg-1, 17.72 gkg-1, 184.00µgg-1 and 0.83 µgg-1) respectively recorded at highest P level (160 kgPha-1). But when the soil inoculated with the Rhizobium leguminosarum, the effect was increased plant growth, (root colonization ,shoot dry weight, root dry weight and nodule number) The highest value was(26.67% , 15, 60gpot-1, 8.03gpot-1 and 191.33nodule pot-1 ) recorded at highest P level, and inoculation with R. leguminosarum was increased significantly shoot N, P, K, Fe and Mo contents, the highest value were (20.35gkg-1, 3.72 gkg-1,16.78 gkg-1, 175.33 µgg-1 and 0.80 µgg-1 )respectively recorded at highest P level (160 kgPha-1).

The dependence of the nitrogen fixing system in the root nodules of pea plants (Pisum sativum) L. cv. Torsdag II) on light induced reactions was studied. The pots of the inoculated pea plants, after the nolules had fixed nitrogen for a fornight, were transferred to a dark room. The control plants were kept under normal lighting conditions.The decay of leghemoglobin was measured after photosynthesis had ceased. In the dark the red nodules turned green in three days, when about half of the haem had been broken down. The plants in normal lighting conditions had maintained the red nodules. The appearence of leghemoglobin and bacteroids was simultaneouos. In normal lighting conditions the number of bacteroids was about 1.6 × 108 per g fresh nodules. The appearance of leghemoglobin and bacteroids was simultaneous. In normal lighting conditons the number of bacteroids was bout 1.6 × 108 per g fresh nodules. At the same time as the nodules turned green in the dark most of the bacteroids disappeared and the number of rod‐shaped bacteria increased. After five days int the dark thenumber of bacteria of the green nodules was 2.2 × 108 per g fresh nodules. A large increase of of bacteria in the nodules is one of the results after the termination of effective symbiosis.Quantitative estimations were made with an automatic amino acid analysator of the amino acid composition in the root nodules of pea plants grown in the light and of pea plants grown in the dark. Altogether 27 amino acids and amides and 3 unknown ninhydrin positive compounds were found in the free amino acid fraction. In the red N‐fixing nodules asparagine, the amide of aspartic acid, was the most prominent (more than 50 per cent of the total amino acid fraction), indicating the energy charge of the nitrogen fixation. 5 days in the dark affected the proportions of the amino acids as follows. Asparagine, homoserine, γ‐aminobutyric acid and ethanolamine were decreased and the most of the others increased.In the hydrolysate of the non‐soluble protein fraction 25 amino acids could be detected. The proportions of the amino acids in the root nodules of light‐grown and dark‐grown pea plants were very similar. Hydroxyproline and α, γ‐diaminopimelic acid (DAP) were found in these fraction. Most of the DAP was contained in the peptide fraction. Also hydroxyproline was found to a small extent. It was assumed that the amino acids in this fraction were derived from the peptides of both plant cells and rhizobia.

Bacteria of the genus Rhizobium can form a symbiosis with plants of the family Leguminosae. Both bacteria and plant show considerable biochemical and morphological changes in order to develop and carry out the symbiosis. The Rhizobia induce special structures on the legumes, which are called root nodules. In these root nodules, the differentiated bacteria - so-called bacteroids - are localized. Within the root nodule the bacteroids are able to reduce atmospheric N 2 to NH 3 , which - after assimilation - is used by the plant. In turn, the plant supplies the bacteroids with carbon compounds from which the energy required for the N 2 -reduction is derived.The N 2 -reduction within the bacteroids is catalyzed by the enzyme nitrogenase. Nitrogenase requires for activity energy in the form of ATP and a low potential electron donor. An anaerobic environment at the site of nitrogen fixation is a requirement for nitrogenase because O 2 inhibits the activity of this enzyme. However O 2 is necessary for the respiration of the bacteroids. Without bacteroid respiration, no ATP is synthesized and no reducing equivalents are generated, which are both required for nitrogenase activity. This means that the O 2 supply to the bacteroids must be strictly regulated.As a side reaction during N-reduction, H +is reduced. Consequently, by reducing H +ATP and reducing equivalents are consumed. Under optimum condition, about 75 % of the electron flow through the nitrogenase reaction is utilized for the reduction of N2. The remainder is consumed in the reduction of H +. The apparent waste of energy through H +-reduction can be much greater than 25 %. The magnitude of loss is influenced by many factors.The aim of the experiments described in this thesis, is to identify the plant factors which determine the nitrogenase activity and the electron allocation to N 2 and H+ by nitrogenase. The experiments were performed with Rhizobium leguminosarum strain PRE and the host plants Pisum sativum cv. Rondo and Pisum sativum cv. Finale. Different physiological aspects underlying the functioning of the root nodule, were studied, namely:- the role of malate dehydrogenase in the supply of oxidizable substrates to the bacteroids- the role of glutamate oxaloacetate transaminase in the NH 3 assimilation and the exchange of metabolites between the symbionts in the root nodule- the influence of the external pH of bacteroids on bacteroid respiration and nitrogen fixation- the relationships between the bacteroid respiration, the intracellular ATP/ADP ratio and nitrogenase activity.In chapter 2, the presence of root nodule-stimulated forms of malate dehydrogenase is demonstrated. From a comparison of the kinetic properties of the predominant nodule-stimulated form and the main malate dehydrogenase form from uninfected root cells, it is concluded that the nodule-stimulated form is capable of catalyzing a high rate of malate formation from oxaloacetate. The second conclusion drawn from the kinetic data is that under physiological conditions the reduction of oxaloacetate to malate catalyzed by the nodule-stimulated form is inhibited at higher malate concentrations. Only the nodulestimulated form exhibits this kinetic property. This prevents the enzyme from catalyzing the reaction to equilibrium, which would lead to a very low oxaloacetate concentration in the cytoplasm of the root nodule cells. The malate concentration has to be controlled because malate is the main substrate of the bacteroids, it plays a central role in the metabolism of the mitochondria and malate -being a strong acid - affects the pH.In chapter 3, the action of malate/aspartate shuttle between the cytoplasm of the infected plant cell and the bacteroid has been demonstrated. The involvement of a nodule-stimulated glutamate oxaloacetate transaminase, present in the cytoplasm of root nodule cells, in the shuttle is suggested. The shuttle might have the following functions for nitrogen fixation.The shuttle can transport NADH from the cytoplasm of the nodule plant cells to the bacteroid, where NADH can be oxidized by the respiratory chain. The second function of the shuttle is the transamination of oxaloacetate to aspartate in the bacteroid. The aspartate formed in the bacteroid, is transported at high rates to the cytoplasm of the nodule. This is important because labelling studies of other investigators with 14C-labelled aspartate have demonstrated that aspartate is rapidly converted to malate in the cytoplasm of nodule plant cells. The aspartate formed in the bacteroid and transported to the plant cytoplasm by the shuttle, can replenish the loss in aspartate in the plant cytoplasm. The presence of a sufficient concentration of aspartate is necessary for the asparagine synthesis, a reaction of the NH 3 assimilation.In chapter 4, the effect of O 2 on nitrogenase activity and the electron allocation by nitrogenase in the root nodules and in the bacteroids, has been described. Oxygen limitation in bacteroids results in a decreased nitrogenase activity and a decreased electron allocation to N 2 by nitrogenase. In root nodules, the O 2 limitation causes also a decrease in nitrogenase activity, however the electron allocation remains constant. It is shown that the external pH of bacteroids determines the rate of respiration by the bacteroid and consequently the rate of nitrogenase activity, without affecting the electron allocation by nitrogenase. By comparing the electron allocation by nitrogenase in root nodules and that in isolated bacteroids, it is proposed that in the intact root nodule the nitrogenase activity is modulated by the pH.In chapter 5, the mechanism is studied by which the external pH of bacteroids changes the rate of respiration and the rate of nitrogenase activity at low O 2 concentrations. The relationships between the rate of respiration by the bacteroid, the nitrogenase activity and the intracellular ATP/ADP ratio are determined.The results demonstrate that a high rate of respiration of the bacteroids at low free O 2 concentrations is associated with an intracellular ATP/ADP ratio which is lower than ≈1.2 . A high rate of respiration is necessary to achieve maximum nitrogenase activity. When the intracellular ATP/ADP ratio increases above 1.2 , the respiration of the bacteroids decreases and the free O 2 concentration increases, which ultimately results in an inactivation of nitrogenase. From experiments with a H +-conducting ionophore, it is concluded that the lower rate of respiration at higher pH is caused by a higher intracellular ATP/ADP ratio. These observations demonstrate that the intracellular ATP/ADP ratio via the P i potential regulates the rate of respiration. This is similar with the classical mitochondrial respiratory control.In chapter 5 the ATP consumption by nitrogenase is compared with ATP synthesis by oxidative phosphorylation. The calculation shows that under conditions of nitrogen fixation the N 2 -reduction, is a major ATP-consuming process in the bacteroids. About 70 % of the ATP synthesized by oxidative phosphorylation is hydrolyzed by nitrogenase. Thus, nitrogenase by itself keeps the intracellular ATP/ADP ratio low and thereby stimulates the respiration.In chapter 6, the studied biochemical processes of the root nodule, are placed in a broader perspective. Four physiological processes in which malate is involved, are illuminated. A mechanism is postulated, which accounts for the balance between the supply of photosynthates and the supply of O 2 to the bacteroids. A change of the pH of the root nodule cells induced by changes of the malate concentration is the central theme of the proposal. The pH might influence the rate of respiration of the bacteroids and thus nitrogenase activity, but it also might regulate the O 2 influx into the central tissue of the root nodule. The pH changes are determined by the availability of sucrose for the root nodule cells. Finally the comparison between bacteroids and mitochondria is discussed.

A very significant increase in N2(C2H2) reduction by Visum sativum L. infected with Rhizobium leguminosarum occurred when plants were grown in the light with 6 hr of CO2 enrichment (0.00120 atm). Plants grown for 4 wk under 0.00120 atm CO2 showed significant increases over control plants at 0.00032 atm CO2 in plant dry weight, N content, root nodule mass, number of nodules, and mean nodule dry weight. Acetylene-reduction assays, however, revealed no reproducible increase in nitrogenase activity/mg nodule in plants subjected to long-term CO2 enrichment. Both control and CO2-enriched plants optimized the sink/source ratio between the mass of nodules and the extranodular plant mass. The optimum ratio for N2 reduction by 4-week-old peas was 0.05. Long-term CO2 enrichment did not promote root nodule formation to a greater degree than total plant development, and increases in N content were directly proportional to increases in nodule mass. Morphological data revealed significantly greater deposits of starch in root nodules of plants grown under CO2-enriched conditions. The results are interpreted as showing that short-term increases in CO2 levels promote N2 reduction by affecting root nodule functioning, whereas long-term CO2 enrichment promotes N2 reduction by increasing total plant and root nodule development.

Endophytes are a hyper diverse group of bacterial or fungal organisms residing within plant tissues without causing visible symptoms of disease. While their overall diversity and ecological functions are not well-understood, some endophytes can provide a range of important benefits to their host such as enhanced growth and increased tolerance to environmental stress. Nitrogen-fixing bacteria residing within nodules--specialized structures on the roots of legumes and actinorhizal plants--play an integral role in the plant microbiome by facilitating nitrogen availability in nutrient-poor environments. Remarkably, the patterns of diversity including both bacterial and fungal endophytes in root nodules are virtually known, except in a few agricultural crop legumes. This study represents the first comparison of the culturable microbial communities associated with the root nodules of red alder (Alnus rubra Bong.), an actinorhizal plant native to the Pacific Northwest of the United States, and Scotch broom (Cytisus scoparius L.), an invasive legume, which often co-occur in similar environments. Red alder and Scotch broom root nodules collected at a sympatric location near Sandy, OR, revealed high numbers of cultivable endophytes. Culturable bacterial endophytes were detected in 98% (n = 44) of red alder nodules and 93% (n = 42) of Scotch broom nodules. Out of the 306 bacterial sequences obtained from red alder and Scotch broom root nodules, a total of 88 operational taxonomic units (OTUs) were identified, with 58% (n = 51) unique to red alder, 25% (n = 22) unique to Scotch broom, and 17% (n = 15) shared between the two host species. Red alder hosted 30 bacterial genera, while Scotch broom hosted 20 genera, with red alder exhibiting significantly greater bacterial diversity than Scotch broom (PERMANOVA: p = 0.001, R² = 0.1158). Although red alder hosted a more diverse root nodule microbial community, both hosts displayed a high frequency of Bradyrhizobium and Mesorhizobium within their root nodules. While typically associated with legumes, these genera have previously been isolated from alder and shown to enhance its growth. However, this is the first time rhizobia have been reported as the most frequently observed bacterial taxa in red alder root nodules. Fungal endophytes were less prevalent, found in 53% (n = 24) of red alder nodules and 56% (n = 25) of Scotch broom nodules. Out of the 147 fungal sequences obtained from red alder and Scotch broom root nodules, a total of 51 operational taxonomic units (OTUs) were identified, with 55% (n =

This thesis deals with research on symbiotic nitrogen fixation of Pisumsativum and Rhizobium Leguminosarum. Nitrogen fixation takes place in the Rhizobium bacteroids which are located within root nodule cells. Two important proteins in nitrogen fixation are nitrogenase and leghemoglobin. Nitrogenase, the enzyme that reduces N 2 , is synthesized by Rhizobium. Leghemoglobin, which has a function in the O 2 supply of the bacteroids is synthesized by the plant. The molecular biology of symbiotic nitrogen fixation has hardly been the subject of investigation. The regulation of the synthesis in root nodules of e.g. nitrogenase and leghemoglobin is not at all completely clear.In this thesis some general aspects of nodule formation and the regulation of nitrogenase and leghemoglobin (Lb) synthesis have been studied.General aspectsThe transformation of Rhizobium bacteria into nitrogen fixing bacteroids was studied by investigating the DNA content of bacteroid cells by means of cytofluorometry (chapter I). Furthermore the RNA content and the quality of the RNA of R. leguminosarum (PRE) bacteroids were followed during pea development (chapter IV) and the protein synthesis in bacteroids and in the plant fraction of pea root nodules during nodule development was studied (chapter IV). The regulation of nitrogenase and leghemoglobin synthesis were studied in more detail.Regulation of nitrogenase and Lb synthesisNitrogenase and Lb synthesis were studied by 35SO42-labeling of intact pea plants. Intact pea plants are a rather complicated system to study Lb or nitrogenase synthesis, but in more simple systems like e.g. detached root nodules, nitrogenase and Lb synthesis are repressed. We did not succeed in developing a more simple system suitable for studying nitrogenase or Lb synthesis (chapter VII). The regulation of nitrogenase and Lb synthesis were studied by culturing pea plants under conditions that diminished in vivo nitrogenase activity and by determining nitrogenase and Lb synthesis during nodule formation and development. Growth conditions used to diminish in vivo nitrogenase activity were: 1. the addition of NH4+to the growth medium (chapter II) and 2. waterlogging (chapter III). The synthesis of the two nitrogenase components and Lb during nodule formation and development was studied in two different ways: 1. the synthesis of these three proteins was followed by 35SO42-labeling of pea plants of different ages, and analysis of bacteroid and plant proteins by polyacrylamide gel electrophoresis (chapter IV), 2. the sequence of appearance of the two nitrogenase components and Lb during pea nodule formation was determined with specific radioimmunoassays for these three proteins (chapter V).In principle, turnover of proteins can be an important factor in the regulation of protein composition in the bacteroid or plant fraction of root nodules, e.g. during nodule development or after changes in the environmental conditions. Therefore turnover rates of bacteroid and plant proteins of pea root nodules, especially nitrogenase and Lb, were determined (chapter VI).

Identification of rhizobial strains nodulating Egyptian grain legumes.

Photosynthesis, primary productivity, N content, and N(2) fixation were determined as a function of applied NH(4) (+) in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH(4) (+) was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH(4) (+), and total N content increased between 0 and 2 millimolar NH(4) (+) but did not change significantly with higher NH(4) (+) applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH(4) (+) concentration (16 millimolar). Symbiotic N(2) fixation by inoculated plants was maximal in peas grown with 2 millimolar NH(4) (+); and apparent relative efficiency of N(2) fixation, calculated from C(2)H(2) reduction and H(2) evolution, was maximal in the 2 to 4 millimolar NH(4) (+) concentration range. The capacity to fix N(2) through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH(4) (+) concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH(4) (+) concentration uninoculated plants had greater CER, N content, and dry weight.

Heavy metals accumulation has negative effects on plant growth and yields, especially in leguminous sensible species, as well as on soil processes, such as nutrients cycling, humification or degradation of various pollutants. Under the impact of heavy metals, inoculation with effective N2-fixing bacterial strains could positively influence plant development and soil processes. A greenhouse experiment has been carried out in order to assess changes in growth and nodulation of pea plants, cultivar CORINA inoculated with Rhizobium leguminosarum bv. leguminosarum strain Mz 805, under the influence of growing concentrations of cadmium in soil (1ppm, 3ppm and 30ppm), as compared with non-inoculated plants and non-polluted control. The paper presents the results concerning the effect of cadmium on plant growth, nodulation parameters and yields at main developing stages. These parameters significantly decreased when cadmium increased. The values of regression coefficients calculated were higher in non-inoculated variants than in inoculated ones. Chromatography revealed that in rhizosphere of pea plants inoculated with strain Mz 804, composition of humus fractions THAC, TFAC and FAC I was modified by the different nature of root exudates.

Although mineral N generally has a negative effect on legume-rhizobia symbioses, experiments in hydroponic culture in our laboratory (Waterer et al., 1992) have shown that low concentrations of NH+ 4 can stimulate nodulation in pea (Pisum sativum L.). The objectives of the current study were to determine the immediate and residual effects of NH+ 4 on nodulation and N2 fixation in pea in sand culture. Peas (cv. Express) were exposed to 0.0, 0.5, 1.0, and 2.0 mM of 15N-labelled (NH4)2SO4 for 28 days after inoculation (DAI). From 28 to 56 DAI the plants were grown on a NH+ 4-free nutrient solution. Plants were harvested at 7, 14, 21, 28 and 56 DAI and nitrogenase activity was measured by gas exchange at 28 and 56 DAI. Root, shoot, and nodule dry weight (DW) and total N content were obtained, in addition to nodule counts and 15N enrichment of plant composites. The 1.0 and 2.0 mM/NH+ 4 treatments consistently resulted in higher total plant DW accumulation than the control (0.0 mMNH+ 4). At 28 DAI, plants exposed to 1.0 and 2.0 mM/NH+ 4had 1.8 to 2.8 times more nodules plant−1, respectively, and plants exposed to 2.0 mM NH+ 4 had 1.7 fold higher specific nodulation [nodule number (g root DW)−1]. However, individual nodule DW was greater in control plants, such that there were no differences in nodule DW per plant among treatments. Ammonium treatment resulted in a more nitrogen derived from the atmosphere (NDFA) in peas early in the experiment, but by 28 DAI there were no treatment effects on NDFA. Whole plant and nodule specific nitrogenase activity [μmol H2 (g nodule DW)−1 h−1] was higher in control plants at 28 DAI.

Although mineral N generally has a negative effect on legume-rhizobia symbioses, experiments in hydroponic culture in our laboratory (Waterer et al., 1992) have shown that low concentrations of NH+4 can stimulate nodulation in pea (Pisum sativum L.). The objectives of the current study were to determine the immediate and residual effects of NH+4 on nodulation and N2 fixation in pea in sand culture. Peas (cv. Express) were exposed to 0.0, 0.5, 1.0, and 2.0 mM of 15N-labelled (NH4)2SO4 for 28 days after inoculation (DAI). From 28 to 56 DAI the plants were grown on a NH+4-free nutrient solution. Plants were harvested at 7, 14, 21, 28 and 56 DAI and nitrogenase activity was measured by gas exchange at 28 and 56 DAI. Root, shoot, and nodule dry weight (DW) and total N content were obtained, in addition to nodule counts and 15N enrichment of plant composites. The 1.0 and 2.0 mM NH+4 treatments consistently resulted in higher total plant DW accumulation than the control (0.0 mM NH+4). At 28 DAI, plants exposed to 1.0 and 2.0 mM NH+4 had 1.8 to 2.8 times more nodules plant-1, respectively, and plants exposed to 2.0 mM NH+4 had 1.7 fold higher specific nodulation (nodule number g-1 root DW). However, individual nodule DW was greater in control plants, such that there were no differences in nodule DW per plant among treatments. Ammonium treatment resulted in more nitrogen derived from the atmosphere (NDFA) in peas early in the experiment, but by 28 DAI there were no treatment effects on NDFA. Whole plant and nodule specific nitrogenase activity (µmol H2 g-1 nodule DW h-1) was higher in control plants at 28 DAI. However, by 56 DAI, after an additional 4 weeks of NH+4-free nutrition, no differences in nitrogenase activity nor whole plant or specific nodulation were detectable. This study indicates that nodulation in pea is stimulated in sand culture while exposed to NH+4. However, once NH+4 is removed, relative growth rate, nodulation and nitrogenase activity becomes similar to plants that were never exposed to NH+4.

SUMMARY Aeschynomene fluminensis Veil., originally obtained from flooded areas of the Pantanal Matogrossense region of Brazil, was grown under stem‐flooded or non‐flooded conditions for 70 d after inoculation with isolates of photosynthetic stem nodule rhizobia obtained from native A. fluminensis. Stem nodules formed only on submerged stems of flooded plants (mean of 25 per plant), and did not form on aerial parts, although they were capable of growing and fixing N2 after drainage of the stems. Root nodules formed on both non‐flooded and flooded plants but were usually decreased in number by flooding (from means of 124 to 51 per plant, respectively). Flooding (and stem‐nodulation) resulted in an increase in shoot (and a decrease in root) dry weight, regardless of rhizobial isolate.Stem nodules were attached by a wide collar of aerenchymatous tissue at the base of the nodule. There were large air spaces in the stem where nodules were subtended and these were continuous with nodule aerenchyma/outer cortex. In addition, aerenchyma and spongy tissue at the base of the nodule connected both flooded and non‐flooded root nodules to large intercellular spaces in the root cortex. The stem and root nodules were ovoid in shape, and essentially aeschynomenoid in type, i.e. the central infected tissue was without uninfected, interstitial cells. Root nodules had a similar structure to stem nodules (although stem nodules were generally larger), and flooded root nodules were approximately twice the size of non‐flooded nodules. The infected tissue of root and stem nodules consisted of spherical, bacteroid‐containing cells containing one or two rod‐shaped bacteroids per peribacteroid unit and prominent organelles. Infection threads were observed in root but not in stem nodules.The cortex of stem and root nodules had an apparent oxygen diffusion barrier, consisting of concentric layers of small cells with interlocking cell walls and few intercellular spaces. Cell layers external to these consisted of larger cells and intercellular spaces, with some spaces being occluded with an electron‐dense material that contained a glycoprotein recognized by the monoclonal antibodies MAC236 and MAC265. The amount of glycoprotein occlusions did not appear to differ between nodule types or treatments, although stem nodules contained intracellular glycoprotein vesicles adjacent to cell walls. The exterior of the nodules consisted of an epidermis of thin flattened cells with occasional lenticels. Amyloplasts were common in lower stem and hypocotyl nodules, but fewer in flooded or non‐flooded root nodules. Upper stem nodules (i.e. those within 6 cm of the water surface) differed from more profoundly submerged stem nodules by having chloroplasts throughout the cortex. Root nodules did not contain chloroplasts, and undifferentiated plastids were found mainly in lower stem nodules.