A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

Acetylene reduction assay for nitrogen fixation under field conditions in remote areas

Summary A method for intensively sampling soil for nitrogen fixation potential using acetylene reduction assay is discussed. Acetylene was generated from calcium carbide. Soil cores were incubated in Mason jars with specially adapted lids. Air samples from the jars were stored and transported over dry KOH in 10 ml serum vials. The method overcomes many problems associated with other sampling procedures, and produces statistically reproducible data.Contribution #4 — Devon Island IBP Project and CCIBP contribution #173.Contribution #4 — Devon Island IBP Project and CCIBP contribution #173.     

Acetylene reduction (nitrogen fixation) associated with corn inoculated with Spirillum.

Sorghum and corn breeding lines were grown in soil in field and greenhouse experiments with and without an inoculum of N2-fixing in Spirillum strains from Brazil. Estimated rates of N2 fixation associated with field-grown corn and sorghum plants were less than 4 g of N2/ha per day. The mean estimated N2-fixation rates determined on segments of roots from corn inoculated with Spirillum and grown in the greenhouse at 24 to 27 degrees C were 15 g of N2/ha per day (16 inbreds), 25 g of N2/ha per day (six hybrids), and 165 g of N2/ha per day for one hybird which was heavily inoculated. The corresponding mean rates determined from measurements of in situ cultures of the same series of corn plants (i.e., 16 inbreds, six hybrids, and one heavily inoculated hybrid) were 0.4, 2.3, and 1.1 g of N2/ha per day, respectively. Lower rates of C2H2 reduction were associated with control corn cultures which had been treated with autoclaved Spirillum than with cultures inoculated with live Spirillum. No C2H2 reduction was detected in plant cultures treated with ammonium nitrate. Numbers of nitrogen-fixing bacteria on excised roots of corn plants increased an average of about 30-fold during an overnight preincubation period, and as a result acetylene reduction assays of root samples after preincubation failed to serve as a valid basis for estimating N2 fixation by corn in pot cultures. Plants grown without added nitrogen either with or without inoculum exhibited severe symptoms of nitrogen deficiency and in most cases produced significantly less dry weight than those supplied with fixed nitrogen. Although substantial rates of C2H2 reduction by excised corn roots were observed after preincubation under limited oxygen, the yield and nitrogen content of inoculated plants and the C2H2-reduction rates by inoculated pot cultures of corn, in situ, provided no evidence of appreciable N2 fixation.     

Nitrogenase from Rhodospirillum rubrum. Relation between 'switch-off' effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios.

Nitrogenase activity of 'membrane-free' extracts, produced from nitrogen-starved Rhodospirillum rubrum to which 4 mM NH4+ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this 'switch-off/switch-on' effect and the function of the membrane component is discussed. Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein rations. The ATP/2E- values are 4-5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.     

Dark anaerobic nitrogen fixation (acetylene reduction) in the cyanobacterium Oscillatoria sp.

The filamentous, non-heterocytous, nitrogen-fixing cyanobacterium Oscillatoria sp. strain 23 (Oldenburg) showed cycling of acetylene reduction in light-dark cycles. Under aerobic conditions nitrogenase activity is exclusively present during the dark period. However, if anaerobic conditions were applied during the dark period, two activity maxima were observed. A relatively small activity peak occurred during the first few hours of the dark period and a high peak as soon as the light was switched on. A low activity remained during the second half of the dark period. This pattern of acetylene reduction in Oscillatoria agrees well with the field data on nitrogen fixation [Stal, L.J. and Krumbein, W.E. (1984), Mar. Biol. 82, 217–224].     

Nitrogenase activity in cyanobacteria measured by the acetylene reduction assay: a comparison between batch incubation and on-line monitoring

A new on-line method for measuring acetylene reduction is described. It consists of a gas-flow cell connected to an electronic gas-mixing system and an automatic sample loop in the gas chromatograph. Alternatively, ethylene can be determined by using laser-based trace gas detection. The laser-based trace gas detection technique achieves a detection limit that is three orders of magnitude better than gas chromatography. We have applied the on-line method to the measurement of nitrogen fixation in a culture of the heterocystous cyanobacterium Nodularia spumigena and compared it with conventional batch-type incubations. Incubation of N. spumigena in the gas-flow cell resulted in very short response times with a steady-state flux of ethylene obtained within 2 min. Nitrogenase was shown to respond immediately to changes in light and oxygen. Monitoring of nitrogenase activity could be continued for several hours without having a negative impact on nitrogen fixation rates in N. spumigena . This was not the case in batch incubations, in which changes in nitrogenase activities were recorded during incubations, probably as a result of varying oxygen concentrations. It was therefore concluded that the on-line method is superior to batch incubations when rates of nitrogenase activity are to be measured. The method is suitable for natural samples (water or sediment).     

Aspects of nitrogen fixation in Lough Neagh. I. Acetylene reduction and the frequency of Aphanizomenon flos-aquae heterocysts

SUMMARY.

• 1 An investigation was made of the relationships between rates of nitrogen fixation (acetylene reduction), Aphanizomenon flosaquae heterocysts, NO₃-N concentration and other environmental factors which occurred in Lough Neagh between 1969 and 1972.
• 2 The maxima of cells differentiated as heterocysts (approximately 5% (1970), and approximately 7% (1971 and 1972) occurred when NO₃-N concentration was very low or undetectable.
• 3 Acetylene reduction was detectable between 16 May and 8 September 1972 but was not measured in the other years. Rates were greatest in the euphotic zone and it appeared that the nitrogenase activity was closely linked to a light dependent system.
• 4 Areal rates of acetylene reduction were highly correlated with Aphanizomenon heterocyst concentration (r=0.92). There was some indication that a release of phosphorus from the sediments might have stimulated Aphanizomenon nitrogen fixation on one occasion at the end of a calm period. Results show that the most likely conditions for the development of large crops of heterocystous species in Lough Neagh include a scarcity of NO₃-N and a plentiful supply of phosphorus.

     

High rates of nitrogen fixation (acetylene reduction) on coral skeletons following bleaching mortality

Nubbins of the coral Acropora aspera were artificially bleached and nitrogen fixation (acetylene reduction) rates were measured on the developing epilithic communities. Seasonal comparisons were made between corals that died in summer of heat stress and corals that died in winter from natural cold stress. Rates of acetylene reduction from artificially bleached corals peaked at 26.66 nmol cm⁻² h⁻¹ 2 weeks after summer mortality, while rates from natural winter mortality peaked at 18.07 nmol cm⁻² h⁻¹ 12 days after coral death. Comparative rates of acetylene reduction taken from live corals and coral rubble ranged between 0.56 and 1.16 nmol cm⁻² h⁻¹, and 0.15 and 12.77 nmol cm⁻² h⁻¹, respectively. N₂-fixation rates from dead corals were up to 30 times greater than those measured on live corals. The observed increase in N₂-fixation from dead corals may increase the availability of nitrogen for use in trophic processes within the reef for an extended period following the initial mortality event. If the spatial scale over which coral mortality has occurred in past thermal bleaching events is considered the ramifications of such an increase may be substantial.     

Nodulation and nitrogen fixation (Acetylene reduction) in desert ironwood (Olneya tesota)

Summary Acacia greggi, Cercidium floridium, and Olneya tesota seeds were inoculated with soil from beneath mature native desert trees and grown in the greenhouse on a nitrogen free media. Olneya tesota seedlings nodulated and reduced acetylene to ethylene. Nodulation or acetylene reduction was not observed in A. greggi or C. floridium. This is the first report of nodulation and nitrogen fixation in Olneya tesota.     

Absence of nitrogen fixation (acetylene reduction) by procaryotes in nectar of Banksias

Summary An earlier hypothesis that blue-green algae in the nectar ofBanksia telmatiaea contribute to the nitrogen economy of the host by fixing N₂ was tested. Field and laboratory experiments failed to demonstrate C₂H₄ production in C₂H₂-treated containers over extended periods. Soil N was not higher at the end of the flowering season and plants in which flower heads were removed prior to nectar production did not contain less N than the controls.     

Optimization of the 15N2 incorporation and acetylene reduction methods for free-living nitrogen fixation

Plant and Soil - To optimize assay conditions of two common methods for measuring potential free-living nitrogen-fixation (FLNF), acetylene reduction assay (ARA) and 15N2-incorporation (15N2), for...     

Physiological ecology of acetylene reduction (nitrogen fixation) in a Delaware salt marsh

The effects of several fixed nitrogen compounds on acetylene reduction activity (nitrogen fixation) of surface sediments from a Delaware salt marsh were studied. Ammonia addition caused little decrease in activity early in the summer but resulted in a considerable decrease (85–95%) in activity late in the summer and early in the fall. Nitrate caused a near complete suppression of activity at all times. Other compounds such as glutamate, urea, and yeast extract caused a slight increase in activity in tallSpartina sediments and caused more than a 2.5-fold increase in shortSpartina sediments. There was a lag period (1–2 days) before the commencement of in vitro acetylene reduction activity during the spring and early summer, but this lag period was not present in the late summer. The addition of chloramphenicol to samples from a shortSpartina zone caused decreases in activity similar to those obtained with ammonia, whereas chlorate amendments yielded results which, when compared on an electron basis, were comparable to those obtained with nitrate. These results indicated that the observed lag period may be the result of a physiological response to the in situ levels of ammonia and/or nitrate. It is suggested here that in situ nitrogenase activity may be controlled by two processes: (a) repression and derepression of nitrogenase synthesis mediated by the levels of ammonia, and (b) competition for reducing power (electrons) and energy (ATP) between the processes of nitrate reduction and nitrogen fixation.     

Nitrogenase phylogeny and the molybdenum dependence of nitrogen fixation in Methanococcus maripaludis.

We studied the effects of molybdenum, vanadium, and tungsten on the diazotrophic growth of Methanococcus maripaludis. Mo stimulated growth, with a maximal response at 4.0 microM, while V had no effect at any concentration tested. W specifically inhibited diazotrophic growth in the presence of Mo. Coupling the results of our analysis and other known metal requirements with phylogenies derived from nifD and nifK genes revealed distinct clusters for Mo-, V-, and Fe-dinitrogenases and suggested that most methanogens also have molybdenum-type nitrogenases.     

Conversion of acetylene reduction rates to nitrogen fixation rates in natural populations of blue-green algae

Twelve experiments are reported on naturally occurring groups of N₂-fixing blue-green algae in which reduction of acetylene and ¹⁵N₂ were determined simultaneously. Simple averaging of the $\text{C}{}_2\text{H}{}_2\text{N}{}_2$ reduction rates yielded a conversion factor of 4.4. Linear and log transform regression analyses yielded values of 4.8 and 4.2, respectively. Reports estimating nitrogen inputs based on acetylene reduction determinations are evaluated.     

Contribution from nitrogen fixation (acetylene reduction) to the nitrogen budget of Lake Tohopekaliga (Florida)

Nitrogen fixation, as assayed by the acetylene reduction technique, provided 44% of the input of nitrogen to a lake in central Florida (Lake Tohopekaliga) during 1984. Ninety-four percent of the lake total fixation was found in the water column and associated with Anabaena spp. The lake-wide average nitrogen fixation rate of 5.7 g N/m²-yr amounted to a mass loading of 497 metric tons of nitrogen for the year, and is one of the highest nitrogen fixation rates reported.     

Effect of ammonium nitrate on nodulation and nitrogen fixation (acetylene reduction) of the tropical legumeSesbania rostrata

Summary The effect of ammonium nitrate on nodulation and nitrogen fixation (acetylene reduction activity) was investigated usingSesbania rostrata, a tropical legume with the ability to nodulate both roots and stems. Plants were grown in plastic pots on silica sand and gravel, inoculated one month after sowing and then continuously irrigated with an automatic irrigation system. Four nitrogen treatments were applied: 0, 1.5, 3.0 and 6.0mm NH₄NO₃. Related symbiotic parameters were evaluated 20 days later. With 3.0mm NH₄NO₃, root nodulation was inhibited. At that concentration, stem nodulation was not affected, but related nitrogenase activity decreased 85% and was completely inhibited at 6.0mm NH₄NO₃. Increasing NH₄NO₃ concentration resulted in a diminution of stem nodule size. Stem and root acetylene reduction were strongly inhibited by high mineral nitrogen concentrations; however root nitrogen fixation was more affected than stem nitrogen fixation.

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Resumen Se estudió el efecto del nitrato amónico en la nodulación y la fijación de nitrógeno, medida mediante la reducción de acetileno, utilizandoSesbania rostrata, una leguminosa tropical que forma nódulos tanto en raíz como en tallo. Las plantas se cultivaron en arena de sílice y grava, en macetas de plástico, se inocularon un mes después de la siembra y a partir de este momento se regaron continua y automáticamente. Se aplicaron cuatro tratamientos de N; 0, 1.5, 3.0, y 6.0mm NH₄NO₃ Los parámetros relativos a la simbiosis se evaluaron 20 días después. La nodulación en raíz se inhibió con 3.0mm NH₄NO₃, sin embargo a esta concentración la nodulación en tallo no estaba inhibida aunque la actividad nitrogenasa disminuyó en un 85%, quedando totalmente inhibida a 6mm NH₄NO₃. El incremento en la concentración de nitrato amónico produjo una disminución del tamaño de los nódulos del tallo. Elevadas concentraciones de nitrógeno mineral inhibieron fuertemente la reducción de acetileno tanto a nivel de raíz como de tallo, aunque la fijación de nitrógeno resultó más afectada en la raíz.

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Résumé L'effet du nitrate d'ammonium sur la nodulation et la fixation d'azote (réduction de l'acétylène) a été étudié avec la légumineuse tropicaleSesbania rostrata, qui présente la particularité de noduler à la fois sur la tige et sur les racines. Les plantes ont été cultivées dans des pots en plastique sur un mélange constitué de sable siliceux et de gravier. Un mois après le semis, elles ont été inoculées et placées sous irrigation continue au moyen d'un système automatique permettant l'apport du milieu nutritif. Quatre concentrations en azote ont été utilisées: 0, 1,5, 3,0 et 6,0 mmol/l NH₄NO₃. Les paramètres symbiotiques ont été analysés 20 jours plus tard. La nodulation racinaire est totalement inhibée en présence de 3,0 mmol/l NH₄NO₃. Cette concentration en azote combiné n'a pas d'effet apparent sur la formation des nodules de tige, mais leur activité nitrogénase diminue de 85%; leur activité est totalement inhibée en présence de 6,0 mmol/l NH₄NO₃. L'augmentation de la concentration en nitrate d'ammonium, se traduit par une diminution de la taille des nodules de tige. Les fortes concentrations en azote combiné ont un effet inhibiteur très marqué sur les activités réductrices d'acétylène racinaire ou caulinaire deS. rostrata; toutefois cet effet est plus sensible au niveau racinaire qu'au niveau caulinaire.

     

Response to drought stress of nitrogen fixation (acetylene reduction) rates by field-grown soybeans

The effects of drought stress on soybean nodule conductance and the maximum rate of acetylene reduction were studied with in situ experiments performed during two seasons and under differing field conditions. In both years drought resulted in decreased nodule conductances which could be detected as early as three days after water was withheld. The maximum rate of acetylene reduction was also decreased by drought and was highly correlated with nodule conductance (r = 0.95). Since nodule conductance is equal to the nodule surface area times the permeability, the relationship of these variables to both whole-plant and unit-nodule nitrogenase activity was explored. Drought stress resulted in a decrease in nodule gas permeability followed by decreases in nodule surface area when drought was prolonged. Under all conditions studied acetylene reduction on a unit-nodule surface area basis was highly correlated with nodule gas permeability (r = 0.92). A short-term oxygen enrichment study demonstrated nodule gas permeability may limit oxygen flux into both drought-stressed and well-watered nodules of these field-grown soybeans.