Showing papers on "Nitrogen fixation published in 1979"

Nitrogen fixation in coral reef sponges with symbiotic cyanobacteria

Abstract: NITROGEN FIXATION by endosymbiotic cyanobacteria (blue-green algae) results in an important input of nitrogen into terrestrial ecosystems1; however, there is as yet little information on its significance in the marine environment2,3. Symbiosis of cyanobacteria with marine animals, although rare, is known to occur in an echiuroid worm4 and in sponges from coral reefs5 and the Mediterranean6. In the present study of sponge–cyanobacterial symbioses, several sponges from a coral reef in the Red Sea were tested, using the acetylene reduction technique7, immediately after their collection on reef-based platforms for their ability to fix nitrogen. Nitrogenase activity was detected in two sponges with cyanobacteria but not in a third with no cyanobacteria. This is the first demonstration of nitrogenase activity in an animal with symbiotic cyanobacteria. In two previous reports of nitrogen fixation in marine animals, the activity was attributed to bacteria in the gut8,9. Although these preliminary experiments are not sufficient to assess the significance of cyanobacterial nitrogenase activity in sponges, it is possible that any additional fixed nitrogen would be beneficial to sponges in tropical waters which are low in available nitrogen10 and in particulate nutrients11,12.

Hydrogenase in Rhizobium japonicum Increases Nitrogen Fixation by Nodulated Soybeans

Abstract: Some Rhizobium strains synthesize a unidirectional hydrogenase system in legume nodule bacteroids; this system participates in the recycling of hydrogen that otherwise would be lost as a by-product of the nitrogen fixation process. Soybeans inoculated with Rhizobium japonicum strains that synthesized the hydrogenase system fixed significantly more nitrogen and produced greater yields than plants inoculated with strains lacking hydrogen-uptake capacity. Rhizobium strains used as inocula for legumes should have the capability to synthesize the hydrogenase system as one of their desirable characteristics.

Tolerance of Rhizobia to Acidity, Aluminum, and Phosphate1

Abstract: Low levels of phosphorus and high levels of aluminum are important soil acidity factors for the growth of higher plants; however, very little is known about their effects on the soil rhizobia. The present study was conducted to determine the relative effects of acidity, P, and Al on rhizobia. Tolerance of low pH (4.5), low P (510 μM), and high AI (50 μM) was assessed for 10 strains of cowpea rhizobia by detailed growth studies in defined liquid media. Tolerances to these factors were determined for 65 strains of cowpea rhizobia and Rhizobium japonicum by a rapid method based on attainment of turbidity from a small inoculum. Strains varied in response. Low P (as compared with 1,000μM) limited total attainable population density to 5 X 10 cells/ml, and slowed the growth of some strains. Acidity generally increased lag time or slowed growth of most strains, and stopped growth of about 50% of them. Tolerance of acidity did not necessarily entail tolerance of Al. Aluminum (50 μM) increased the lag time or slowed growth of almost all strains tolerant of low pH. It virtually stopped growth of 40% of the strains. With our system the rhizobia had to make 1,000-fold growth in the stress media before they could significantly raise pH and precipitate Al. A valid rapid screening can be based on ability to attain visible turbidity in culture under acid or Al stress, so long as initial density is small («10 cells/ml). The cowpea rhizobia tended to have more tolerance to Al than R. japonicum and overall Al was a more severe stress than low pH or low P. Additional Index Words: acidity, phosphorus, aluminum, rhizobia, cowpea miscellany, Rhizobium japonicum. Munns, D. N., and H. H. Keyser. 1979. Tolerance of rhizobia to acidity, aluminum, and phosphate. Soil Sci. Soc. Am. J. 43: 519-523. EFFECTS of ACIDITY on species of Rhizobium are well documented. Pure culture studies (Graham and Parker, 1964) have shown the critical low pH range for growth is from about 4.0 to 6.0, with the slower growing R. japonicum, R. lupini, and cowpea miscellany being in general more acid tolerant than the others, and R. meliloti being the most acid sensitive. These 1 Contribution of the Department of land, Air and Water Res., University of Calif. Davis. Partly supported by grants from Agency for International Development (NiFrAL Project) and NSF/Rann. Received 21 July 1978. Approved 6 Feb. 1979. 2 Postgraduate Research Scientist and Professor of Soil Science, respectively. Senior author is currently with Cell Culture & Nitrogen Fixation Lab., USDA-SEA-AR, BARC-W, Beltsville, Md 20705. 51 9 observations on relative tolerance of the species agree with studies in acid soils (Damirgi et al., 1967; Jensen 1969). Within each species, important strain-to-strain variation has been demonstrated (Graham and Parker, 1964; Munns, 1965a). Besides low pH per se, acid mineral soils have low levels of phosphorus and high levels of aluminum (Kamprath, 1973; Pearson, 1975). There has been little research on the effects of these two soil factors on rhizobia. An early study of effects of P on rhizobia showed positive growth response to P additions in soil (Truesdell, 1917). Kamata (1962) related the ability to nodulate P-deficient soybeans with the rhizobial strains relative response to P in culture media. Werner and Berghauser (1976) demonstrated that three strains of Rhizobium were better than two other common bacteria at taking up P from very low concentrations in solution. Recent studies (Rerkasem, 1977; de Carvalho, 1978) have shown that some rhizobia can indeed survive high Al concentrations at low pH in both solution media and soil. However, there is still insufficient evidence to establish Al-tolerance in rhizobia as distinct from tolerance of low pH. There are no data concerning effects of Al on rhizobial growth rate. The objectives of this investigation were (i) to determine the effects of low P and high Al on the survival and growth rate of some rhizobia at low pH, (ii) to examine the relationship between acid-tolerance and Al-tolerance, and (iii) to rate the probable importance of the three stresses (acidity, P, and Al) according to their inhibitory effects on rhizobial growth. MATERIALS AND METHODS

Estimate of symbiotically fixed nitrogen in field grown soybeans using variations in15N Natural abundance

Abstract: The use of variations in natural abundance of15N between nitrogen fixing and non nitrogen fixing soybeans was investigated for quantitative estimate of symbiotic nitrogen fixation

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover II. COMPARISONS OF THE COST OF NITROGEN FIXATION AND THE UTILIZATION OF COMBINED NITROGEN

Abstract: Plants of soyabean, cowpea, and white clover were grown singly in pots in Saxcil growth cabinets at 23/18 °C, 30/24 °C, and 20/15 °C, respectively, until seed maturation or for 85 d (white clover). Two populations were produced within each species: one population effectively nodulated and wholly dependent for nitrogen on fixation in the root nodules, and a second population completely lacking nodules but receiving abundant nitrate nitrogen. In each species, the two populations were compared in terms of rate of gross photosynthesis, rate of shoot respiration, and rate of root respiration. Source of nitrogen had little or no effect on rate of photosynthesis or shoot respiration. In contrast, the rate of respiration of the nodulated roots of plants fixing their own nitrogen was greater, sometimes two-fold greater, than that of equivalent plants lacking nodules and utilizing nitrate nitrogen. This superiority in terms of rate of root respiration was generally confined to the period of intense nitrogen fixation. An analysis of the magnitude of this respiratory burden in terms of daily photosynthesis indicates that, in all three legumes, plants fixing their own nitrogen respire 11-13% more of their fixed carbon each day than equivalent plants lacking nodules and utilizing nitrate nitrogen. INTRODUCTION Biochemical studies of rhizobial metabolism indicate a relatively large energy requirement in the form of ATP and reductant for the synthesis in the nodule of ammonia from the dinitrogen molecule (Bulen and LeComte, 1966; Winter and Burris, 1968; Hardy and Havelka, 1975), but it seems generally to be thought that these energy costs, plus the ancillary costs of synthesizing and maintaining nodule tissue, do not perceptively decrease plant growth rate in comparison with plants utilizing a source of combined nitrogen such as nitrate, where the energy cost of reducing nitrate to ammonia within the plant is also thought to be substantial (Bergersen, 1971; Gibson, 1966, 1976; Minchin and Pate, 1973; Pate, 1976). However, an investigation of the influence of source of nitrogen on the carbon economy of Fiskeby soyabean (Ryle, Powell, and Gordon, 1978) indicated that 1 The Grassland Research Institute is financed through the Agricultural Research Council. This content downloaded from 157.55.39.235 on Fri, 07 Oct 2016 05:57:12 UTC All use subject to http://about.jstor.org/terms 146 Ryle, Powell, and Gordon—Cost of Nitrogen Fixation plants fixing atmospheric nitrogen respired 10-15% more of their fixed carbon than equivalent plants lacking nodules and utilizing nitrate nitrogen. In view of the important implications of this result, similar analyses were carried out on two other legumes, cowpea and white clover, to establish the generality of this response to the two sources of nitrogen. This paper reports the performance of the legumes in terms of three important physiological attributes: (i) photosynthesis of the whole plant, (ii) respiration of the shoot, and (iii) respiration of the root. The legumes were grown either wholly dependent on nitrogen fixation in their own nodules, or lacking nodules but receiving abundant nitrate nitrogen. Although the data from the soyabean experiment have been published in detail elsewhere (Ryle et ai., 1978), relevant measurements are given in the appropriate sections to facilitate comparisons with the cowpea and white clover data. MATERIALS AND METHODS The methods and conditions used to grow soyabean (Glycine max (L.). Merr. var. Fiskeby V), cowpea (Vigna unguiculata (L.) Walp var. K2809), and white clover (Trifolium repens L. var. Blanca) and the techniques used to measure their rates of photosynthesis and respiration have been described in detail elsewhere (Ryle, Powell, and Gordon, 1978; 1979). Briefly, plants were grown singly in 9-5 cm pots of Perlite in Saxcil growth cabinets at 23/18 °C (soyabean), 30/24 °C (cowpea), and 20/15 °C (white clover). Seeds were sterilized with u.v. radiation and, where appropriate, inoculated with an effective Rhizobium. The objective was to produce two populations of plants within each species: one population effectively nodulated and receiving a complete nutrient solution lacking only nitrogen, and a second population completely lacking nodules and receiving a very similar nutrient solution containing abundant nitrate nitrogen. The two populations from each species were grown in separate cabinets to minimize bacterial contamination. Nodulated plants were generally provided with 20 parts 10~6 of nitrate nitrogen until the nodules developed leghaemoglobin. Details of the growth conditions are summarized in Table 1. Measurements of photosynthesis and respiration were made at intervals of a few days until seed maturation in soyabean and cowpea or until day 85 in white clover; white clover remained vegetative in the 12 h photoperiod used here. An open, infrared gas analyser system was used to measure the flux of C02 into or out of the plant fractions under study (Ryle et al., 1978). After measurements of photosynthesis and respiration, the plants were harvested and their leaf areas determined with a Paton Industries electronic planimeter. Subsequently the plant fractions were dried at 100 °C and weighed, and their nitrogen contents determined (Ryle et ai, 1978). Table 1. Experimental material and conditions Species Soyabean Cowpea White clover Glycine max (L.) Vigna unguiculata (L.) Trifolium repens L. Merr. var. Fiskeby V Walp. var. K2809 var. Blanca Rhizobium CB 1809 CB 756 Rothamsted No. 5 Photoperiod 12 h 12 h 12 h Light 810 ± 10 //Einsteins 810 ± 10//Einsteins m~2 s~' m-2 s_1 (fluorescent) (fluorescent + incandescent) Day/night temperature 23/18 °C 30/24 °C 20/15 °C Growth medium Single plants in 9-5 cm pots of Perlite Nutrient solution Macroelements (parts 10~6) in demineralized water Ca K N S P Mg Na Fe 'Nitrate plants' 167 146 221 36 36 27 118 12 'Nodulated plants' 167 146 0 192 36 27 4 12 plus microelements Mn, Cu, B, Zn, Mo, and Co; pH adjusted 5-8-6-2; 150-400 ml d_1/plant according to plant size This content downloaded from 157.55.39.235 on Fri, 07 Oct 2016 05:57:12 UTC All use subject to http://about.jstor.org/terms Ryle, Powell, and Gordon—Cost of Nitrogen Fixation 147 RESULTS Plant growth The patterns of growth and the weights achieved by the three legumes given the two sources of nitrogen are shown in Fig. 1. In plants fixing their own nitrogen, the onset and, in the two annual legumes, the cessation of nitrogen fixation clearly set the limits for dry matter accumulation. However, the most important feature of the

Nitrogen Isotope Distribution as a Presumptive Indicator of Nitrogen Fixation

Abstract: The generally lower 15N/14N ratio of nitrogen-fixing organisms provides an opportunity to screen mixed populations for individuals fixing nitrogen. Samplings of associated California native species, along with the soil on which they were found, show differences in 15N/14N ratios suggesting a capability among some for the fixation of nitrogen. Not all suspected fixers were depleted in 15N, and two saprophytes had significant 15N enrichment. The method shows some promise, but further understanding of fractionation processes is needed before the method can be generally applied.

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover I. NITROGEN FIXATION AND THE RESPIRATION OF THE NODULATED ROOT

Abstract: Soyabean, cowpea, and white clover, inoculated with effective rhizobia, were grown singly with a standard mineral nutrition and light regime in controlled environments until seed maturation (in soyabean and cowpea) or late vegetative growth (white clover). Day/night temperature regimes were 23/18, 30/24, and 20/15 °C in soyabean, cowpea, and white clover, respectively. The respiratory losses of C02 from the nodulated root systems were studied in relation to the concurrent rate of fixation of atmospheric nitrogen. Despite differences in development, the effectiveness of the symbioses, and the temperature of growth, all three legumes exhibited similar respiratory losses from nodulated roots per unit of nitrogen fixed. During intense nitrogen fixation, the average respiratory losses for the three legumes varied between 6-3 and 6-8 mg C rng"1 N; within each species, the losses varied more widely at different stages of development. These respiratory burdens reflect the total cost to the plant of the nodule/nitrogen fixation syndrome including the subtending roots. The results are discussed in relation to the respiratory effluxes from nodules and roots, and to biochemical investigations of the costs of nitrogen fixation. INTRODUCTION Studies of the biochemistry of biological nitrogen fixation have repeatedly emphasized the energy-intensive nature of the reduction of the dinitrogen molecule to ammonia (Bulen and LeComte, 1966; Winter and Burris, 1968; Bergersen, 1971; Hardy and Havelka, 1975). Furthermore, there is increasing evidence that a proportion of the reducing power generated at the nitrogenase of the legume nodule may be utilized in the formation of hydrogen from protons (Dixon, 1968, 1972; Schubert and Evans, 1976; Schubert, Engelke, Russell, and Evans, 1977) and, moreover, that not many agricultural legume symbioses possess the appropriate biochemistry to recapture some or all of this energy—if indeed it can be recaptured (Evans, Ruiz-Argiieso, and Russell, 1978). Such studies indicate that the losses of fixed carbon from the nodulated roots of legumes are likely to be substantial, and that the respiratory costs of fixing nitrogen may vary with the specific biochemistry of the bacteroid. 1 The Grassland Research Institute is financed through the Agricultural Research Council. This content downloaded from 157.55.39.143 on Wed, 15 Jun 2016 04:50:37 UTC All use subject to http://about.jstor.org/terms 136 Ryle, Powell, and Gordon—Nitrogen Fixation and Respiration This paper reports experiments designed to measure the respiratory losses of fixed carbon from the nodulated roots of three legumes, soyabean, cowpea, and white clover, during their normal cycle of growth. The three legumes were deliberately chosen to provide contrasts in habit, growth cycle, geographical origin, and symbiotic effectiveness. All three were grown in the same light and nutritional conditions but the temperatures were selected to reflect their geographical origin. A detailed account of the carbon economy of the soyabean plants has been published elsewhere (Ryle, Powell, and Gordon, 1978). The respiratory losses of carbon from the nodulated root systems of the three legumes are considered in relation to their concurrent rates of fixation of atmospheric nitrogen. MATERIALS AND METHODS Plants of Fiskeby V soyabean (Glycine max (L.) Merr.), cowpea ( Vigna unguiculata (L.) Walp.) var. K28O9, and white clover (Trifolium repens L.) var. Blanca were grown singly in 9-5 cm diameter pots of Perlite. Seeds of soyabean and cowpea were sterilized with u.v. radiation and inoculated with Nitrogerm Rhizobium CB 1809 and CB 756. respectively, suspended in a sucrose milk-peat mixture. After sterilization with u.v. radiation, seeds of white clover were sown in shallow dishes of Perlite, inoculated at germination with Rothamsted Rhizobium 5 suspended in distilled water, and transplanted to pots at the time of emergence of the first leaf. They were subsequently reinoculated with the same Rhizobium about 7 d after transplantation. All plants were grown in Saxcil growth cabinets which provided a photoperiod of 12 h of light from daylight fluorescent tubes (soyabean only) or from daylight fluorescent tubes plus incandescent lamps (810 ± 10 /?Einsteins m~2 s_1: Lamda PAR meter). An automatic C02 injection system prevented the C02 levels from falling below 320 parts 10~6. Day/night temperature regimes were 23/18 °C (soyabean), 30/24 °C (cowpea), and 20/15 °C (white clover). All plants were provided daily with a nutrient solution containing essential macroand micronutrients with the exception of nitrogen (Ryle et al., 1978), which was normally completely absent. It proved impossible to grow cowpea and white clover from germination without any combined nitrogen, and so some nitrate nitrogen (20 parts 10~6) was added to these plants until the nodules developed leghaemoglobin, after which nitrate was completely withdrawn. As the plants increased in size the amount of nutrient solution provided was progressively increased to a maximum of 400 ml d_l per plant. Measurements of respiration were made by enclosing groups of plants (2-4, according to size) in Perspex chambers of up to 50 1 capacity, through which outside air was passed. Samples of air were drawn off before and after passing through the chamber and their C02 contents compared in an infrared gas analyser. The chamber was enclosed in a Saxcil cabinet adjusted to provide the appropriate temperature. Initially whole plants were held in the dark for 30-40 min at a temperature midway between that of day and night (e.g. in soyabean at 20-5 °C) for the effect of temperature to become stabilized. Subsequently, the plants were removed from the chamber, decapitated at Perlite level, returned to the chamber, and a measurement taken of the respiration of the nodulated root. The porous nature of the Perlite rooting medium ensured rapid equilibration of gas exchange. At intervals the validity of the decapitated root measurements was tested separately by comparison with root respiration measurements made on intact plants. Plants were removed from their pots and their roots gently shaken free of Perlite, and then sealed into 1 1 bottles of dilute nutrient solution with a 5 cm air gap immediately beneath the base of the shoots. Approximately 400 ml min-1 of outside air was bubbled through the solution continuously. After allowing 1-2 h for equilibration, respiration of the enclosed root system was determined by comparing the C02 content of the air with an infrared gas analyser before and after its passage through the solution. On some occasions, a second measurement of respiration was made after some of the more accessible nodules had been removed from the nodulated root system. The rate of respiration of the nodules removed from the nodulated root system could then be calculated by difference. Furthermore, after detaching the remaining nodules and determining their weight, the rate of respiration of root could also be calculated. These tests with intact plants indicated that the respiration of nodulated roots was generally unaffected for 30-60 min following decapitation, after which it usually declined. This content downloaded from 157.55.39.143 on Wed, 15 Jun 2016 04:50:37 UTC All use subject to http://about.jstor.org/terms Ryle, Powell, and Gordon—Nitrogen Fixation and Respiration 137 Cowpea was particularly sensitive in this respect and the respiration of its root system often began to decrease only 15-20 min after decapitation. Measurements of nodulated root respiration were made between the second and tenth hour of the photoperiod and, whenever possible, were successively rotated from early to late in the photoperiod at consecutive observations to eliminate potential bias. For the same reason, temperatures midway between those of day and night were chosen for the measurements so as to provide a mean rate of respiration as far as possible representative of a whole diurnal period. Ancillary measurements of root performance with decapitated plants and with sealed root systems both indicated that diurnal fluctuations in the rate of respiration (Fig. 1) and of acetylene reduction (Fig. 5) largely reflected the cycling of temperature in the rooting medium.

The Rhizobium-Legume Symbiosis

Abstract: Nitrogen is an essential nutrient. As N2 gas it is a major constituent of the atmosphere, but N2 is chemically inert and therefore unavailable as a source of nitrogen for use by most living organisms. However, some bacteria have the ability to reduce N2 and thereby “fix” atmospheric nitrogen using the enzyme nitrogenase. Many leguminous plants have capitalised on this special bacterial asset by going into partnership with nitrogen-fixing bacteria called rhizobia. In return for supplying nutrients to the bacteria, the plants receive a supply of reduced nitrogen. In essence, the legumes create a highly specialised environment within which the bacteria fix nitrogen. These specialised plant structures are called nodules; usually they are found on roots, but they also occur on the stems of some legumes.

Nitrogen-Fixing (Acetylene Redution) Activity and Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland Rice

Abstract: Nitrogen-fixing activity associated with different wetland rice varieties was measured at various growth stages by an in situ acetylene reduction method after the activities of blue-green algae (cyanobacteria) in the flood water and on the lower portion of the rice stem were eliminated. Nitrogen-fixing activities associated with rice varieties differed with plant growth stages. The activities increased with plant age, and the maximum was about at heading stage. The nitrogen fixed during the whole cropping period was estimated at 5.9 kg of N per ha for variety IR26 (7 days) and 4.8 kg of N per ha for variety IR36 (95 days). The population of aerobic heterotrophic N(2)-fixing bacteria associated with rice roots and stems was determined by the most-probable-number method, using semisolid glucose-yeast extract and semisolid malate-yeast extract media. The addition of yeast extract to the glucose medium increased the number and activity of aerobic heterotrophic N(2)-fixing bacteria. The glucose-yeast extract medium gave higher counts of aerobic N(2)-fixing bacteria associated with rice roots than did the malate-yeast extract medium, on which Spirillum-like bacteria were usually observed. The lower portion of the rice stem was also inhabited by N(2)-fixing bacteria and was an active site of N(2) fixation.

Nitrogen fixation by rhizosphere and free-living bacteria in salt marsh sediments1

Abstract: The rates of nitrogen fixation by rhizosphere and free-living bacteria are highest near the surface of a variety of salt marsh sediments and in the warm part of the year. The highest rates were found in vegetated habitats, reaching up to about 500 ng N.cm/sup -2/.h/sup -1/. Bacterial N/sub 2/ fixation for the entire marsh is more than 10 times larger than algal fixation and less than a third of the N required to support growth of the vegetation.

Interaction between a vesicular-arbuscular mycorrhiza and rhizobium and their effects on soybean in the field

Abstract: Summary Interaction between Glomus fasciculatus and Rhizobium japonicum and their effects on soybean in the field was studied in a phosphorus deficient sandy loam soil with pH 5·6. The number, dry weight and nitrogen content of the root nodules in plants inoculated with Glomus plus Rhizobium were significantly more compared to plants inoculated with only Rhizobium. Rhizobium inoculation did not have any significant effect on sporulation of G. fasciculatus in the rhizosphere. Although soybean plants inoculated with G. fasciculatus recorded increased phosphorus content, dry weight and grain yield than uninoculated plants the differences were not statistically significant. In Rhizobium only inoculation markedly increased the nitrogen content of the plant and grain yield. Dual inoculation with both the symbionts increased significantly the dry weight of the shoot and its nitrogen content over single inoculation with either Glomus or Rhizobium. These results suggest that vesicular–arbuscular (VA) mycorrhiza can greatly assist nodulation and nitrogen fixation in field growth soybean inoculated with rhizobia.

Low levels of fixed nitrogen required for isolation of free-living N 2 -fixing organisms from rice roots

Abstract: ISOLATION and counting of N2-fixing bacteria from the natural environment have until now been conducted on selective N-free media. It is likely that some N2-fixing bacteria are overlooked by using this procedure. We now report that most of the bacteria present in rice roots are N2-fixers, but that they require a supply of mineral or organic N for growth. In the presence of organic nitrogen, nitrogenase activity is detected in these organisms. The requirement for organic nitrogen for the detection of nitrogenase is also true for N2-fixing cultures of free-living rhizobia1–3.

Isolation by sucrose-density fractionation and cultivation in vitro of actinomycetes from nitrogen-fixing root nodules

Abstract: The fixation of atmospheric nitrogen by actinomycete-nodulated, woody dicotyledonous plants represents a substantial contribution to the global nitrogen cycle1, contributing especially to forested areas, wetlands, fields and disturbed sites of temperate and tropical regions2. The study of these nitrogen-fixing symbioses has been hampered by difficulties in isolating and culturing the microsymbionts in vitro. Recently, root nodule actinomycetes have been isolated by microdissection techniques and cultured in vitro3,4. We report here the isolation and culture for the first time of the actinomycetes associated with root nodules of Elaeagnus umbellata (Elaeagnaceae) and Alnus viridis ssp. crispa (Betulaceae) (shown in Fig. 1a and b, respectively), using a technique radically different from those used previously. For separating actinomycetes from root nodules, we have used the simple fractionation technique of sucrose-density sedimentation.

Economy of Photosynthate Use in Nitrogen-fixing Legume

Abstract: The economy of C use by root nodules was examined in two symbioses, Vigna unguiculata (L.) Walp. (cv. Caloona):Rhizobium CB756 and Lupinus albus L. (cv. Ultra):Rhizobium WU425 over a 2-week period in early vegetative growth. Plants were grown in minus N water culture with cuvettes attached to the nodulated zone of their primary roots for collection of evolved CO2 and H2. Increments in total plant N and in C and N of nodules, and C:N weight ratios of xylem and phloem exudates were studied by periodic sampling from the plant populations. Itemized budgets were constructed for the partitioning and utilization of C in the two species. For each milligram N fixed and assimilated by the cowpea association, 1.54 ? 0.26 (standard error) milligrams C as CO2 and negligible H2 were evolved and 3.11 milligrams of translocated C utilized by the nodules. Comparable values for nodules of the lupin association were 3.64 ? 0.28 milligrams C as CO2, 0.22 ? 0.05 milligrams H2, and 6.58 milligrams C. More efficient use of C by cowpea nodules was due to a lesser requirement of C for synthesis of exported N compounds, a smaller allocation of C to nodule dry matter, and a lower evolution of CO2. The activity of phosphoenolpyruvate carboxylase in nodule extracts and the rate of 14C02 fixation by detached nodules were greater for the cowpea symbiosis (0.56 ? 0.06 and 0.22 milligrams C as CO2 fixed per gram fresh weight per hour, respectively) than for the lupin 0.06 ? 0.02 and 0.01 milligrams C as CO2 fixed per gram fresh weight per hour. The significance of the data was discussed in relation to current information on theoretical costs of nitrogenase functioning and associated nodule processes. Several investigations have considered the energy relationships of N fixation by nodulated legumes. Some of these have concerned the thermodynamics of the reactions (3), the energetics of nitrogenase functioning (5, 6), and the effects of H2 evolution by nitrogenase on the efficiency of N fixation in specific symbioses (7, 20-23). Other investigations have assessed nodule functioning in terms of carbohydrate intake from the parent plant (9, 12, 15), CO2 loss by the below-ground organs of nodulated plants (9, 11, 14) or by a detailed C economy of nodules (1). This paper describes an experimentally based approach to determine the cost of symbiosis involving simultaneous measurements of N2 fixation, and CO2 and H2 evolution by attached nodules of intact legumes. These data are used to construct itemized budgets for the utilization of photosynthetically fixed C in two contrasting legume:

A Nodule-Specific Plant Protein (Nodulin-35) from Soybean

Abstract: Nodulin-35, a 35,000-molecular-weight protein, is present in soybean root nodules developed by different strains of Rhizobium japonicum, irrespective of their effectiveness in fixing atmospheric nitrogen. This protein is not detected in uninfected plants and bacteroids or in free-living Rhizobium and appears to be synthesized by the plant during the formation of root nodules.

Isolation of cyanobacteria from the aquatic fern, Azolla

Abstract: A procedure has been developed to isolate cyanobacteria from the aquatic fern Azolla. The method is based upon recovery of cyanobacterial “bundles” from digests of plants and use of this material as a massive inoculum for nitrogen-free media, followed by prolonged incubation in light. The procedure appears to select for those cells capable of growth in vitro. Isolated cyanobacteria were found to resemble Anabaena sp. morphologically but were capable of heterotrophic growth and had high nitrogenase activity when grown on fructose in the dark.

Nitrogen fixation (acetylene reduction) associated with communities of heterocystous and non-heterocystous blue-green algae in mangrove forests of Sinai

Abstract: High rates of nitrogen fixation (acetylene reduction) are associated with communities of heterocystous and non-heterocystous blue-green algae, which are widespread and abundant in the coastal mangrove forests of the Sinai Peninsula. Heterocystous forms, particularly representatives of the Rivulariaceae, grow in aerobic environments, where nitrogenase activity may be limited by the availability of nutrients such as Fe and PO4−P. Desiccated communities of Scytonema sp. reduce acetylene within ten minutes of wetting by tidal sea water. Communities dominated by the non-heterocystous Hydrocoleus sp., Hyella balani, Lyngbya aestuarii, Phormidium sp. and Schizothrix sp., occur in close contact with anaerobic sediments and reduce acetylene in the dark as well as in the light. Nitrogen fixation in all these communities is light dependant and may be supplemented by an alternative source of reductant in the dark. The indications are that nitrogen fixation by these communities of blue-green algae, makes a significant contribution to the overall nitrogen input of the mangrove ecosystem.

Assimilation and transport of nitrogenous compounds originated from 15N2 fixation and 15NO2 absorption

Abstract: Nodulated leguminous plants utilize both combined nitrogen absorbed by roots and gaseous nitrogen fixed by root nodules. In order to elucidate the physiological role of N2 fixation compared with NO2 absorption, the assimilation and transport of nitrogenous compounds from these nitrogen sources were studied. One part of soybean plants was administered with labeled nitrogen gas and unlabeled nitrate, and another part with unlabeled nitrogen gas and labeled nitrate. After feeding of labeled compounds to roots, ammonia, nitrate, amino acids, amides and allantoin in nodules, roots and stems were separated, and 15N content of them were determined optically using JASCO NIA-l 15N-analyzer. In nodules supplied with 15N2, high 15N contents were found in glutamate, alanine, serine, γ-amino-butyrate, but in roots supplied with 15NO2, asparagine showed the highest 15N content after 8 hr 15N feeding. In stems, aIIantoin showed the highest 15N content in the case of 15N2 treatment, and the ratio (15N from 15N2/...

Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation

Abstract: Mr\h.~~\~.fr. J. R.. and P. P. WONC. 1979. Nitrate reductase activities of rhizobia and the correlation between nitrate reduction and nitrogen fixation. Can. J. Microbial. 25: 1169- 1174. All species of Rllizobirrrtr except R. Irrpiiri had nitrate reductnse activity. Only R. Irrpiili was incapable of growth with nitrate as the sole source of nitrogen. However. the conditions necessary for the induction of nitr-ate reductase varied among species of Rhizohirrrti. R/lizohirrttr .jtrporrir~rrtlr and some Rhizol>irrrtr species of the cowpea strains expressed nitrate reductase activities both in the )root nodules of appropriate leguminous hosts and when grown in the presence of nitrate. Rl~Cohirrtt~ lrjfi~lii. R. ph(/.~~O/i, and R. Ir~grrt~~i~ro.sttr~~t~r did not express nitr'ate reductase activities in the root nodules, hut they did express them whcn grown in the PI-esencr of nitrate. In bacteroids of R. ,jc~pot~ic.t~rtl and some strains of cowpea R/rizo/~ilrrt~, high N, fixation activities were accompanied by high nitrate reductase activities. In bacteroids of R. ~rififolii. R. Ir~grrtt~itro,strrrrt)~. and R. phc~.cc,oli, high N, fixation activities were not ~~ccompanied by high nitrnte reductase activities.

Selecting Rhizobium for acid, infertile soils of the tropics

Abstract: AN increase in beef cattle production on the 350 million hectares of acid, infertile savannahs of tropical South America would have a major impact on world food production. Production in these regions is limited by the inadequate nutritional value of the native vegetation, and legume-based pastures are now being introduced1 to raise the protein content of available forage. However, to obtain full benefit from biological nitrogen fixation through nodulated legumes it is necessary that the introduced legume form an effective symbiosis with Rhizobium. The low pH, limited availability of phosphorus, and very high levels of aluminium and manganese in these soils, are likely to affect nodulation and nitrogen fixation. It is therefore essential that strains of Rhizobium adapted to acid conditions are used. Alkali production by rhizobia from tropical legumes and the consequent rise in pH of test media has made the development of a simple test to select such strains difficult2. We report here that interference from alkali production can be eliminated by minor changes in the screening medium. This finding challenges the view that alkali production is a distinguishing characteristic with ecological significance3, and is also a starting point for detailed study of the effects of acidity and related factors on growth of Rhizobium.

Nitrogen fixation by legumes in the tropics

Abstract: Cultivated plants continuously require nitrogen. This nitrogen is ordinarily supplied by mineralization of native soil nitrogen, but in sometimes supplemented by fertilizer nitrogen in the case of non-legumes or by these two sources plus dinitrogen fixation in the case of legumes...

Plasmids, Biological Properties and Efficacy of Nitrogen Fixation in Rhizobium japonicum Strains Indigenous to Alkaline Soils

Abstract: Summary: Plasmids were isolated from strains of Rhizobium japonicum, predominantly serogroup 135, obtained from soybean nodules collected at 15 sites in Nebraska, U.S.A. In addition to their serotype, these strains were indistinguishable from R. japonicum strain 3I1b135 in growth rate, sensitivity to phage Rhj781, antibiotic sensitivities, general colony characteristics and rates of nitrogen fixation per plant. All strains occupied soil habitats with similar characteristics, including a high pH (7.2 to 8.3), relatively high conductivity (0.04 to 0.32 mS), relatively high sodium saturation (0.32 to 12.7%), low iron content (3.2 to 14.8 p.p.m.) and low manganese content (5.1 to 18.7 p.p.m.). However, agarose gel electrophoresis analysis of plasmids enabled subdivision of these extra-slow-growing strains into four groups on the basis of differences in plasmid number and size. These strains carried combinations of two or more of four plasmids, ranging in mass from 49 to 118 megadaltons and comprising approximately 20% of the total DNA per cell. Biological and symbiotic data, along with plasmid analysis, were useful in identifying a wild-type strain (RJ23A) that shows potential as a soybean inoculant in alkaline soils.

Stem Nodules in Aeschynomene indica and Their Capacity of Nitrogen Fixation

Abstract: There has been no report on stem nodules with nitrogen fixing activity. Aeschynomene indica produce nodules on the aerial parts, and the stem nodules proved to be capable of considerable nitrogen fixation [C2H4 produced c. 4 μmol (g fresh weight)-1 h-1]. The stem nodules embed reddish tissues, and a rod-shaped bacterium was isolated from the tissues. The bacterium was ascertained to form root and stem nodules on seedlings of A. indica.

Temperature control of nitrogen fixation in Klebsiella pneumoniae.

Abstract: At growth temperatures above 37 degrees C, Klebsiella pneumoniae does not grow in a medium containing N2 or NO3- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N2 at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30 degrees C were shifted to 39 degrees C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39 degrees C. Thus, temperature seems to represent a third control system, besides NH4+ and O2, governing the expression of nif genes of K. pneumoniae.

The Respiratory Costs of Nitrogen Fixation in Soyabean, Cowpea, and White Clover

Abstract: Plants of soyabean, cowpea, and white clover were grown singly in pots in Saxcil growth cabinets at 23/18 °C, 30/24 °C, and 20/15 °C, respectively, until seed maturation or for 85 d (white clover). Two populations were produced within each species: one population effectively nodulated and wholly dependent for nitrogen on fixation in the root nodules, and a second population completely lacking nodules but receiving abundant nitrate nitrogen. In each species, the two populations were compared in terms of rate of gross photosynthesis, rate of shoot respiration, and rate of root respiration. Source of nitrogen had little or no effect on rate of photosynthesis or shoot respiration. In contrast, the rate of respiration of the nodulated roots of plants fixing their own nitrogen was greater, sometimes two-fold greater, than that of equivalent plants lacking nodules and utilizing nitrate nitrogen. This superiority in terms of rate of root respiration was generally confined to the period of intense nitrogen fixation. An analysis of the magnitude of this respiratory burden in terms of daily photosynthesis indicates that, in all three legumes, plants fixing their own nitrogen respire 11-13% more of their fixed carbon each day than equivalent plants lacking nodules and utilizing