Effect of nitrogen fertilization and cropping system of the reference crop on estimation of N2 fixation by vetch using15N methodology

This study was conducted to examine the effects of varying N rates and cropping systems (mixedversus pure stand) on the suitability of oats (Avena sativa L.) for estimating N₂ fixed in sequentially harvested vetch (Vicia sativa L.) over two growing seasons (1984–85 and 1985–86). The N rates were, 20 and 100 kg N ha^–1 in 1984–85 and 15 and 60 kg N ha^–1 in 1985–86. In the 1984–85 season, vetch at maturity derived 76 and 63% N from fixation at the high and low N rates respectively. The corresponding values for the second season were 66 and 42%. Except in the 1985–86 season when some significantly higher values of % N₂ fixed were estimated by using the reference crop grown at the higher (A-value approach) than at the lower N rate (isotope-dilution approach), both approaches resulted in similar measurements of N₂ fixed. In the 1984–85 season, similar values of N₂ fixed were obtained using either the pure or mixed stand oats reference crops. Although in the 1985–86 season, the mixed reference crop occasionally estimated lower % N₂ fixed than pure oats, total N₂ fixed estimates were always similar (P<0.05). Thus, in general, N fertilization and cropping system of the reference crop did not significantly influence estimates of N₂ fixation.     

Successional changes in soil nitrogen availability,non-symbiotic nitrogen fixation and carbon/nitrogen ratios in southern Chilean forest ecosystems

Vast areas of southern Chile are now covered by second-growth forests because of fire and logging. To study successional patterns after moderate-intensity, anthropogenic fire disturbance, we assessed differences in soil properties and N fluxes across a chronosequence of seven successional stands (2–130 years old). We examined current predictions of successional theory concerning changes in the N cycle in forest ecosystems. Seasonal fluctuations of net N mineralization (N_min) in surface soil and N availability (N_a; N_a=NH ₄ ⁺ –N+NO ₃ ^– –N) in upper and deep soil horizons were positively correlated with monthly precipitation. In accordance with theoretical predictions, stand age was positively, but weakly related to both N_a (r ²=0.282, P<0.001) and total N (N_tot; r ²=0.192, P<0.01), and negatively related to soil C/N ratios (r ²=0.187, P<0.01) in surface soils. A weak linear increase in soil N_min (upper plus deep soil horizons) was found across the chronosequence (r ²=0.124, P<0.022). N_min occurred at modest rates in early successional stands, suggesting that soil disturbance did not impair microbial processes. The relationship between N fixation (N_fix) in the litter layer and stand age best fitted a quadratic model (r ²=0.228, P<0.01). In contrast to documented successional trends for most temperate, tropical and Mediterranean forests, non-symbiotic N_fix in the litter layer is a steady N input to unpolluted southern temperate forests during mid and late succession, which may compensate for hydrological losses of organic N from old-growth ecosystems.     

Response to inoculation and N fertilization for increased yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is able to fix 20–60 kg N ha^–1 under tropical environments in Brazil, but these amounts are inadequate to meet the N requirement for economically attractive seed yields. When the plant is supplemented with N fertilizer, N₂ fixation by Rhizobium can be suppressed even at low rates of N. Using the ¹⁵N enriched method, two field experiments were conducted to compare the effect of foliar and soil applications of N-urea on N₂ fixation traits and seed yield. All treatments received a similar fertilization including 10 kg N ha^–1 at sowing. Increasing rates of N (10, 30 and 50 kg N ha^–1) were applied for both methods. Foliar application significantly enhanced nodulation, N₂ fixation (acetylene reduction activity) and yield at low N level (10 kg N ha^–1). Foliar nitrogen was less suppressive to nodulation, even at higher N levels, than soil N treatments. In the site where established Rhizobium was in low numbers, inoculation contributed substantially to increased N₂ fixation traits and yield. Both foliar and soil methods inhibited nodulation at high N rates and did not significantly increase bean yield, when comparing low (10 kg N ha^–1) and high (50 kg N ha^–1) rates applied after emergence. In both experiments, up to 30 kg N ha^–1 of biologically fixed N₂ were obtained when low rates of N were applied onto the leaves.     

Nitrogen fixation by white clover in pastures grazed by dairy cows: Temporal variation and effects of nitrogen fertilization

Effects of rate of nitrogen (N) fertilizer and stocking rate on production and N₂ fixation by white clover (Trifolium repens L.) grown with perennial ryegrass (Lolium perenne L.) were determined over 5 years in farmlets near Hamilton, New Zealand. Three farmlets carried 3.3 dairy cows ha^–1 and received urea at 0, 200 or 400 kg N ha^–1 yr^–1 in 8–10 split applications. A fourth farmlet received 400 kg N ha^–1 yr^–1 and had 4.4 cows ha^–1.There was large variation in annual clover production and total N₂ fixation, which in the 0 N treatment ranged from 9 to 20% clover content in pasture and from 79 to 212 kg N fixed ha^–1 yr^–1. Despite this variation, total pasture production in the 0 N treatment remained at 75–85% of that in the 400 N treatments in all years, due in part to the moderating effect of carry-over of fixed N between years.Fertilizer N application decreased the average proportion of clover N derived from N₂ fixation (P_N; estimated by ¹⁵N dilution) from 77% in the 0 N treatment to 43–48% in the 400 N treatments. The corresponding average total N₂ fixation decreased from 154 kg N ha^–1 yr^–1 to 39–53 kg N ha^–1 yr^–1. This includes N₂ fixation in clover tissue below grazing height estimated at 70% of N₂ fixation in above grazing height tissue, based on associated measurements, and confirmed by field N balance calculations. Effects of N fertilizer on clover growth and N₂ fixation were greatest in spring and summer. In autumn, the 200 N treatment grew more clover than the 0 N treatment and N₂ fixation was the same. This was attributed to more severe grazing during summer in the 0 N treatment, resulting in higher surface soil temperatures and a deleterious effect on clover stolons.In the 400 N treatments, a 33% increase in cow stocking rate tended to decrease P_N from 48 to 43% due to more N cycling in excreta, but resulted in up to 2-fold more clover dry matter and N₂ fixation because lower pasture mass reduced grass competition, particularly during spring.     

Limitations to measuring riverine denitrification at the whole reach scale: effects of channel geometry,wind velocity,sampling interval,and temperature inputs of N<Subscript>2</Subscript>-enriched groundwater

A model-based approach was recently introduced for measuring riverine denitrification based on measured changes in dissolved N₂ concentration during riverine transport (Laursen & Seitzinger, 2002a). Inputs to the model, including water temperature, channel depth, wind velocity, and time-of-travel between sampling locations, vary greatly among natural systems. Simulations were run by varying the values of these inputs and determining rates of N₂ accumulation in river water and the detection limits for measuring denitrification using this method. Dinitrogen was found to accumulate most rapidly in streams that were shallow, particularly under conditions of low wind velocity. Dissolved N₂ concentrations, modeled in rivers with a diurnal temperature variation of 5 °C and under conditions of no denitrification or 1 mmol N m⁻² h⁻¹, showed that sensitivity of the method can vary as temperatures change. Under low wind conditions and in rivers <1m in depth, this method is capable of detecting denitrification rates as low as 30–100 μmol N m⁻² h⁻¹. This limit of detection should be adequate to measure in situ rates in many North American streams, particularly in agricultural watersheds. In deeper rivers N₂ accumulated more slowly and the method became less sensitive. The results of this study should guide decisions regarding the application of this method based on the specific characteristics of a study reach (channel geometry) and the physical conditions (i.e. wind velocity and water temperature) under which measurements are to be made. The input of N₂-enriched groundwater along a study reach can result in N₂ accumulation that could be misinterpreted as denitrification. Some knowledge of the inputs of groundwater along a reach should also guide decisions regarding the application of this method.     

Effects of elevated CO<Subscript>2</Subscript> and nitrogen on wheat growth and photosynthesis

The effects of nitrogen [75 and 150 kg (N) ha⁻¹] and elevated CO₂ on growth, photosynthetic rate, contents of soluble leaf proteins and activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and nitrate reductase (NR) were studied on wheat (Triticum aestivum L. cv. HD-2285) grown in open top chambers under either ambient (AC) or elevated (EC) CO₂ concentration (350 ± 50, 600 ± 50 μmol mol⁻¹) and analyzed at 40, 60 and 90 d after sowing. Plants grown under EC showed greater photosynthetic rate and were taller and attained greater leaf area along with higher total plant dry mass at all growth stages than those grown under AC. Total soluble and Rubisco protein contents decreased under EC but the activation of Rubisco was higher at EC with higher N supply. Nitrogen increased the NR activity whereas EC reduced it. Thus, EC causes increased growth and P_N ability per unit uptake of N in wheat plants, even if N is limiting.     

Gas Exchange and Epidermal Characteristics of Miscanthus Populations in Taiwan Varying with Habitats and Nitrogen Application

Seventeen clones of C₄ grass Miscanthus spp. collected from different climatic regions and elevations of Taiwan were transplanted in pots. 15–16 months after collection the plants received 0, 1, and 2 g of nitrogen fertiliser (N₀, N₁, and N₂, respectively) per pot. All the measurements were done 10–12 d after N application. The relationships between net photosynthetic rate (P _N) and photon flux density (PFD) showed a saturated curve, with PFD saturation at about 1 000 µmol m^–2 s^–1. The ranges of PFD saturated P _N (P _sat) for all the tested clones with N₀, N₁, and N₂ were 8–16, 11–18, and 12–21 µmol m^–2 s^–1, respectively. The clones from southern Taiwan, a tropical region, showed the highest P _sat, followed by the clones from northern Taiwan, a subtropical region, while those from mountainous area showed the lowest P _sat. The clones collected from southern Taiwan showed the highest frequency of stomata on the adaxial surface, and those collected from the high mountainous area showed the lowest frequency. Also the adaxial surface of leaves from the higher mountainous area had more wax deposited than the leaves from the lowland. Thus the low P _sat in mountain clones is limited by both stomatal and non-stomatal factors. Further, the lower leaf conductance and different epidermal characteristics of mountain clones might prevent excessive loss of heat through transpiration and provide production against ultraviolet-B radiation.     

Light-Limited Growth Rate Modulates Nitrate Inhibition of Dinitrogen Fixation in the Marine Unicellular Cyanobacterium Crocosphaera watsonii

Biological N₂ fixation is the dominant supply of new nitrogen (N) to the oceans, but is often inhibited in the presence of fixed N sources such as nitrate (NO₃ ⁻). Anthropogenic fixed N inputs to the ocean are increasing, but their effect on marine N₂ fixation is uncertain. Thus, global estimates of new oceanic N depend on a fundamental understanding of factors that modulate N source preferences by N₂-fixing cyanobacteria. We examined the unicellular diazotroph Crocosphaera watsonii (strain WH0003) to determine how the light-limited growth rate influences the inhibitory effects of fixed N on N₂ fixation. When growth (µ) was limited by low light (µ = 0.23 d⁻¹), short-term experiments indicated that 0.4 µM NH₄ ⁺ reduced N₂-fixation by ∼90% relative to controls without added NH₄ ⁺. In fast-growing, high-light-acclimated cultures (µ = 0.68 d⁻¹), 2.0 µM NH₄ ⁺ was needed to achieve the same effect. In long-term exposures to NO₃ ⁻, inhibition of N₂ fixation also varied with growth rate. In high-light-acclimated, fast-growing cultures, NO₃ ⁻ did not inhibit N₂-fixation rates in comparison with cultures growing on N₂ alone. Instead NO₃ ⁻ supported even faster growth, indicating that the cellular assimilation rate of N₂ alone (i.e. dinitrogen reduction) could not support the light-specific maximum growth rate of Crocosphaera. When growth was severely light-limited, NO₃ ⁻ did not support faster growth rates but instead inhibited N₂-fixation rates by 55% relative to controls. These data rest on the basic tenet that light energy is the driver of photoautotrophic growth while various nutrient substrates serve as supports. Our findings provide a novel conceptual framework to examine interactions between N source preferences and predict degrees of inhibition of N₂ fixation by fixed N sources based on the growth rate as controlled by light.     

Acid dissociation constants and pH values for standard “bes” and “tricine” buffer solutions in 30, 40, and 50 mass% dimethyl sulfoxide/water between 25 and −25 °C

Information is given concerning two standard buffer solutions suitable as pH references in 30, 40, and 50 mass% dimethyl sulfoxide (DMSO)/H₂O mixed solvents at subzero temperatures from −20 to 0 °C, with the intention of establishing a pH (designated pH^*) scale. The two buffers selected were the ampholytes N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid (“bes”) and N-tris(hydroxymethyl)methylglycine (“tricine”), and the reference standard consisted of equal molal quantities of the buffer and its respective sodium salt. The assignment of pH^* values was based on measurements of the emf of cells without liquid junction of the type: Pt;H₂(g,1 atm) ¦Bes, Na Besate, NaCl ¦ AgCl;Ag and Pt;H₂(g,1 atm) ¦Tricine, Na Tricinate, NaCl ¦AgCl;Ag and the pH^* was derived from a determination of K₂, the equilibrium constant for the dissociation process (Buffer)^±/ai (Buffer)⁻ + H⁺.     

Synthesis and GABAA receptor activity of 2,19-sulfamoyl analogues of allopregnanolone

The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the β-face of ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy. The methodology also allows the synthesis of 3α-substituted analogues such as the 3α-fluoro derivative. GABA_A receptor activity of the synthetic analogues was evaluated by assaying their effect on the binding of [³H]flunitrazepam and [³H]muscimol. The 3α-hydroxy-2,19-sulfamoyl analogue and its N-benzyl derivative were more active than allopregnanolone for stimulating binding of [³H]flunitrazepam. For the binding of [³H]muscimol, both synthetic analogues and allopregnanolone stimulated binding to a similar extent, with the N-benzyl derivative exhibiting a higher EC₅₀. The 3α-fluoro derivative was inactive in both assays.     

Measurements of nitrogen fixation in fababean at different N fertilizer rates using the 15N isotope dilution and ‘A-value’ methods

The ¹⁵N isotope dilution and A-value methods were used to measure biological nitrogen (N₂) fixation in field grown fababean (Vicia faba L.), over a 2-year period. Four N rates, 20, 100, 200 and 400 kg N ha^–1 were examined. The two isotope methods gave similar values of % N derived from the atmosphere (%Ndfa). With 20 kg N ha^–1, %Ndfa in fababean was about 85% in both years. Increasing the N rate to 100 kg N ha^–1 decreased N₂ fixation slightly to 75%. Further reductions in N₂ fixed to 60 and 43% occurred where 200 and 400 kg N ha^–1 were applied, respectively. Thus even higher rates of N than normally applied in farming practice could not completely suppress N₂ fixation in fababean.We also devised one equation for both the isotope dilution and A-value approaches, thereby (i) avoiding the need for different calculations for the ¹⁵N isotope methods, and (ii) showing once again that the isotope dilution and A-value methods are mathematically and conceptually identical.     

Symbiotic N2-fixation in alpine tundra: ecosystem input and variation in fixation rates among communities

Annual inputs of symbiotic N₂-fixation associated with 3 species of alpine Trifolium were estimated in four alpine communities differing in resource supplies. We hypothesized that fixation rates would vary according to the degree of N, P, and water limitation of production, with the higher rates of fixation in N limited communities (dry meadow, moist meadow) and lower rates in P and water limited communities (wet meadow, fellfield). To estimate N₂-fixation rates, natural abundance of N isotopes (¹⁵N) were measured in field collected Trifolium and reference plants and in Trifolium plants grown in N-free medium in a growth chamber. All three Trifolium species relied on a large proportion of atmospherically-fixed N₂ to meet their N requirements, ranging from 70 to 100%. There were no apparent differences in the proportion of plant N derived from fixation among the communities, but differences in the contribution of the Trifolium species to community cover resulted in a wide range of annual N inputs from fixation, from 127 mg m^–2 year^–1 in wet meadows to 810 mg m^–2 year^–1 in fellfields. Annual spatially integrated input of symbiotic N₂-fixation to Niwot Ridge, Colorado was estimated at 490 mg m^–2 year^–1 (5 kg ha^–1 year^–1), which is relatively high in the context of estimates of net N mineralization and N deposition.     

Effects of planted tree fallows on soil nitrogen dynamics,above-ground and root biomass,N2-fixation and subsequent maize crop productivity in Kenya

The effects of planted fallows of Sesbania sesban (L.) Merr. and Calliandra calothyrsus (Meissner) on soil inorganic nitrogen dynamics and two subsequent maize crops were evaluated under field conditions in the highlands of eastern Kenya. Continuous unfertilised maize, maize/bean rotation and natural regrowth of vegetation (weed fallow) were used as control treatments. The proportion of symbiotic N₂-fixation was estimated by measuring both leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment by the ¹⁵N dilution technique for Sesbania and Calliandra, using Eucalyptus saligna (Sm.) and Grevillea robusta (A. Cunn) as reference species. Above- and below-ground biomass and N contents were examined in Sesbania, Calliandra, Eucalyptus and Grevillea 22 months after planting. Both the content of inorganic N in the topsoil and the quantity of N mineralised during rainy seasons were higher after the Sesbania fallows than after the other treatments. Compared to the continuous unfertilised maize treatment, both residual crop yields were significantly higher when mineral N (one application of 60 kg N ha^–1) was added. Furthermore, the second crop following the Sesbania fallow was significantly higher than the continuous maize crop. The above-ground biomass of the trees at final harvest were 31.5, 24.5, 32.5 and 43.5 Mg ha^–1 for the Sesbania, Calliandra, Grevillea and Eucalyptus, respectively. For the total below-ground biomass the values for these same tree species were 11.1, 15.5, 17.7, and 19.1 Mg ha^–1, respectively, of which coarse roots (>2 mm), including tap roots, amounted to 70–90%. About 70–90% of the N in Sesbania, and 50–70% in Calliandra, was derived from N₂-fixation. Estimates based on leaf ¹⁵N enrichment and whole-plant ¹⁵N enrichment were strongly correlated. The N added by N₂-fixation amounted to 280–360 kg N ha^–1 for Sesbania and 120–170 kg N ha^–1 for Calliandra, resulting in a positive N balance after two maize cropping seasons of 170–250 kg N ha^–1 and 90–140 kg N ha^–1, for Sesbania and Calliandra, respectively. All the other treatments gave negative N balances after two cropping seasons. We conclude that Sesbania sesban is a tree species well suited for short duration fallows due to its fast growth, high nutrient content, high litter quality and its ability to fix large amounts of N₂ from the atmosphere.     

Nitrogen fixation in perennial forage legumes in the field

Nitrogen acquisition is one of the most important factors for plant production, and N contribution from biological N₂ fixation can reduce the need for industrial N fertilizers. Perennial forages are widespread in temperate and boreal areas, where much of the agriculture is based on livestock production. Due to the symbiosis with N₂-fixing rhizobia, perennial forage legumes have great potential to increase sustainability in such grassland farming systems. The present work is a summary of a large number of studies investigating N₂ fixation in three perennial forage legumes primarily relating to ungrazed northern temperate/boreal areas. Reported rates of N₂ fixation in above-ground plant tissues were in the range of up to 373 kg N ha^–1 year^–1 in red clover (Trifolium pratense L.), 545 kg N ha^–1 year^–1 in white clover (T. repens L.) and 350 kg N ha^–1 year^–1 in alfalfa (Medicago sativa L.). When grown in mixtures with grasses, these species took a large fraction of their nitrogen from N₂ fixation (average around 80%), regardless of management, dry matter yield and location. There was a large variation in N₂ fixation data and part of this variation was ascribed to differences in plant production between years. Studies with experiments at more than one site showed that also geographic location was an important source of variation. On the other hand, when all data were plotted against latitude, there was no simple correlation. Climatic conditions seem therefore to give as high N₂ fixation per ha and year in northern areas (around 60°N) as in areas with a milder climate (around 40°N). Analyzing whole plants or just above-ground plant parts influenced the estimate of N₂ fixation, and most reported values were underestimated since roots were not included. Despite large differences in environmental conditions, such as N fertilization and geographic location, N₂ fixation (Nfix; kg N per ha and year) was significantly (P<0.001) correlated to legume dry matter yield (DM; kg per ha and year). Very rough, but nevertheless valuable estimations of Nfix in legume/grass mixtures (roots not considered) are given by Nfix = 0.026DM + 7 for T. pratense, Nfix = 0.031DM + 24 for T. repens, and Nfix = 0.021DM + 17 for M. sativa.     

Seasonal Photochemical Transformations of Nitrogen Species in a Forest Stream and Lake

The photochemical release of inorganic nitrogen from dissolved organic matter is an important source of bio-available nitrogen (N) in N-limited aquatic ecosystems. We conducted photochemical experiments and used mathematical models based on pseudo-first-order reaction kinetics to quantify the photochemical transformations of individual N species and their seasonal effects on N cycling in a mountain forest stream and lake (Plešné Lake, Czech Republic). Results from laboratory experiments on photochemical changes in N speciation were compared to measured lake N budgets. Concentrations of organic nitrogen (N_org; 40–58 µmol L⁻¹) decreased from 3 to 26% during 48-hour laboratory irradiation (an equivalent of 4–5 days of natural solar insolation) due to photochemical mineralization to ammonium (NH₄ ⁺) and other N forms (N_x; possibly N oxides and N₂). In addition to N_org mineralization, N_x also originated from photochemical nitrate (NO₃ ⁻) reduction. Laboratory exposure of a first-order forest stream water samples showed a high amount of seasonality, with the maximum rates of N_org mineralization and NH₄ ⁺ production in winter and spring, and the maximum NO₃ ⁻ reduction occurring in summer. These photochemical changes could have an ecologically significant effect on NH₄ ⁺ concentrations in streams (doubling their terrestrial fluxes from soils) and on concentrations of dissolved N_org in the lake. In contrast, photochemical reactions reduced NO₃ ⁻ fluxes by a negligible (<1%) amount and had a negligible effect on the aquatic cycle of this N form.     

Modelling of seasonal variation in nitrogen retention and in-lake concentration: A four-year mass balance study in 16 shallow Danish lakes

The mass balance for total nitrogen (N) was studied over a four-year period in 16 shallow mainly eutrophic 1st order Danish lakes. Water was sampled in the main inlet of each lake 18–26 times annually, and from the outlets and the lake 19 times annually. Water was also sampled from minor inlets, although less frequently. N input and output were calculated using daily data on discharge (Q), the latter being obtained either from the Q/H relationship based on automatic recordings of water level (H) for the main in- and outlet, or by means of Q/Q relationships for the minor inlets. Annual mean N retention in the lakes ranged from 47 to 234 mg N m^–2 d^–1, and was particularly high in lakes with high N loading. Annual percentage retention (N _ret –y%) ranged from 11 to 72%. Non-linear regression analysis revealed that hydraulic retention time and mean depth accounted for 75% of the variation in annual mean N _ret –y% and, in combination with inlet N concentration, accounted for 84% of the variation in the in-lake N concentration. N _ret % varied according to season, being higher in the second and third quarter than in the first and fourth quarter (median 18–19%). A simple model was developed for predicting monthly nitrogen retention (N _ret –m) on the basis of external N loading, the lake water pool of nitrogen N _pool , hydraulic loading and lake water temperature. Calibration of only two parameters on data from the randomly selected 8 out of 16 lakes rendered the model capable of accurately simulating seasonal dynamics of the in-lake N concentration and N _ret –m in all 16 lakes. We conclude that with regard to shallow, eutrophic lakes with a relatively low hydraulic retention time, it is now possible to determine not only annual mean nitrogen retention, but also the seasonal variation in N _ret–m . Prediction of seasonal variation in N loading of downstream N-limited coastal areas is thereby rendered much more reliable.     

Interactive effects of exogenous combined nitrogen and phosphorus on growth and nitrogen fixation by azolla

Effects of N source and media-N and P levels were examined on growth, N uptake, and N₂ fixation ofAzolla pinnata withAnabaena azollae association (azolla) at two inoculum-P concentrations. Each expeiment was conducted for 7 days in a growth chamber using azolla at a predetermined inoculum-P concentration and the growth media containing a combination of four levels of P (0, 15, 75, and 200 M) and three levels (0, 1, and 5 mM) of either¹⁵N-enriched NH ₄ ⁺ as ammonium sulfate or¹⁵N-enriched NO ₃ ^– as potassium nitrate. Nitrogen uptake and N₂ fixation were measured by¹⁵N isotopic dilution method. Tissue P and N, N uptake, and N₂-fixation increased with increasing P concentration in the media regardless of the inoculum-P level of azolla. Increasing P concentration in the media increased growth of azolla at low inoculum P, but the effect on high inoculum-P azolla was either small or absen. High inoculum-P concentration resulted in increased growth, tissue-N and P concentrations, N uptake, and N₂ fixation by azolla. Ammonium in the growth media caused larger increase in tissue-N and greater repression of N₂ fixation than equimolar concentration of NO ₃ ^– . In the presence of NH ₄ ⁺ or NO ₃ ^– , in the growth media, N uptake by azolla exceeded the corresponding decrease in N₂ fixation, resulting in an overall increase in tissue-N concentration. Phosphorus in the media tended to negate the inhibitory effect of NH ₄ ⁺ or NO ₃ ^– on N₂ fixation. A multiple regression model showed that the effect of tissue-N on N₂ fixation was negative while that of tissue-P was positive. Therefore, a relative change in tissue-N and P appeared to regulate N₂ fixation. Tissue-N and P had similar effects on relative growth rate of azolla also. Inoculum-P level of azolla was important in determining the response to media-P.This research was supported by a grant from USAID under Indo-US Science and Technology Initiative.     

Minimizing the effect of mineral nitrogen on biological nitrogen fixation in common bean by increasing nutrient levels

Although common bean (Phaseolus vulgaris L.) has good potential for N₂ fixation, some additional N provided through fertilizer usually is required for a maximum yield. In this study the suppressive effect of N on nodulation and N₂ fixation was evaluated in an unfertile soil under greenhouse conditions with different levels of soil fertility (low=no P, K and S additions; medium = 50, 63 and 10 mg kg^–1 soil and high = 200, 256 and 40 mg kg^–1 soil, respectively) and combined with 5, 15, 60 and 120 mg N kg^–1 soil of ¹⁵N-labelled urea. The overall average nodule number and weight increased under high fertility levels. At low N applications, nitrogen had a synergistic effect on N₂ fixation, by stimulating nodule formation, nitrogenase activity and plant growth. At high fertility and at the highest N rate (120 mg kg^–1 soil), the stimulatory effect of N fertilizer on N₂ fixation was still observed, increasing the amounts of N₂ fixed from 88 up to 375 mg N plant^–1. These results indicate that a suitable balance of soil nutrients is essential to obtain high N₂ fixation rates and yield in common beans.     

Effect of N Source on the Steady State Growth and N Assimilation of P-limited Anabaena flos-aquae

Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH₃, NO₃⁻, or N₂. Expressed as a function of relative growth rate, steady state cellular P contents and PO₄ assimilation rates did not vary with N-source. However, N-source did alter the maximal PO₄-limited growth rate achieved by the cultures: the NO₃⁻ and N₂ cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH₃ grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO₃⁻-grown cells were the smallest (627 ± 34 micromoles C · 10⁻⁹ cells), while NH₃-grown cells were largest (900 ± 44 micromoles C · 10⁻⁹ cells) and N₂-fixing cells were intermediate (726 ± 48 micromoles C · 10⁻⁹ cells) in size. In the NO₃⁻-and N₂-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH₃-grown cells. Heterocysts were absent in NH₃-grown cultures but were present in both the N₂ and NO₃⁻ cultures. In the NO₃⁻-grown cultures C₂H₂ reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N₂-fixing cultures the acetylene:N₂ ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal.

Compared with NH₃, the assimilation of NO₃⁻ and N₂ resulted either in a decrease in cellular C (NO₃⁻ and N₂ cultures) or in a lower maximal growth rate (N₂ culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO₃⁻ or N₂ compared with NH₃.

     

Application of 15N and xylem ureide methods for assessing N2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N₂ fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N₂ fixation (%Pfix) and the amounts of N₂ fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N₂ fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the ¹⁵N natural abundance technique were compared with values obtained from ¹⁵N enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies.Inoculated seedlings of each of the three shrub legume species were planted in dense stands (1.5 m rows, 0.5 m between trees) in two randomised blocks. The northern block was used solely for natural abundance measurements, while ¹⁵N-enriched KNO₃ (10 atom % ¹⁵N excess) was applied four times over a 52 week period to plots in the southern block. The non-nodulating tree legume Senna spectabilis (formally Cassia spectabilis) was used as a non-N₂-fixing reference for the ¹⁵N-based procedures, with Guinea grass (Panicum maximum) included as an additional non-fixing check. Growth by the trees above 75 cm was first cut and removed after 22 weeks and regrowth was subsequently pruned periodically for another 95 weeks. Sampling for dry matter production, N yield and estimates of Pfix were restricted to the central four of the 32 plants which constituted each replicate plot. Information generated during the 117 week study indicated that estimates of Pfix by ¹⁵N natural abundance were closely similar to values derived with ¹⁵N-enrichment or sap ureides. The data indicated that Calliandra had a reduced reliance upon N₂ fixation relative to Gliricidia and Codariocalyx for the first 65 weeks after establishment. This appeared to be due to more prolifc root growth by Calliandra than either of the other N₂-fixing species and an ability to extract a greater proportion of its N requirements from soil mineral N. However, after week 65 and for the remainder of the experiment, estimates of Pfix for Calliandra were similar to the other shrub legumes. Over 117 weeks, prunings from Calliandra and Gliricidia had removed 52–58 t dry matter ha^-1, and between 1471 and 1678 kg N ha^-1, of which 1026–1063 kg N ha^-1 was estimated to have been derived from N₂ fixation. At the time of final harvest, 65–73% of the fixed N was present in shoot regrowth of the N₂ fixing shrubs, 9–18% in the roots, 15% in the trunk, and 2–6% in fallen leaves.