Biomass production and nitrogen utilization by Alnus incana when grown on N2 or NH 4 + made available at the same rate

A single clone of Alnus incana (L.) Moench was grown in a controlled-environment chamber. The plants were either inoculated with Frankia and fixed atmospheric nitrogen or were left uninoculated but received ammonium at the same rate as the first group fixed their nitrogen. Nitrogen fixation was calculated from frequenct measurements of acetylene reduction and hydrogen evolution. The diurnal variation of acetylene reduction was also taken into account. The relative efficiency of nitrogenase could be used in the calculations of fixed nitrogen since the Frankia used did not show any detectable hydrogenase activity. Alders fixing nitrogen developed more biomass, longer shoots, larger leaf areas and contained more nitrogen than alders receiving ammonium. In one experiment, almost all ammonium given to the non-nodulated alders was taken up and 15% of the nitrogen taken up was excreted. In the other experiment, 34% of the ammonium was left in the nutrient solution and 8% of the nitrogen taken up was excreted. Alders inoculated with Frankia did not excrete any detectable amount of nitrogen. It seems that the energy demand for nitrogen fixation is not so high that biomass production in alders is retarded. The symbiotic system of A. incana and Frankia seems to be more efficient in utilizing its nitrogen than non-symbiotic A. incana receiving ammonium.     

Effects of elevated oxygen tensions on acetylene reduction in Alnus incana-Frankia symbioses

An open flow-through gas system was used to determine the effect of C₂H₂ and elevated O₂ on acetylene reduction activity (ARA) and respiration of the intact, potted root system of Alnus incana (L.) Moench in symbiosis with Frankia Avcll or with a local source of Frankia . Both symbiotic systems responded to C₂H₂ by an immediate plateau range in ARA. The Plateau in ARA was in some cases followed by a decline of less extent than reported for many legumes. A concurrent decline in net respiration of the root system was on average 8% of the CO₂ efflux prior to C₂H₂ introduction.
Respiration of the root systems in both symbioses responded to elevated oxygen levels in the 10 kPa C₂H₂ atmosphere by an increase of up to 17% of the net respiration prior to C₂H₂ introduction in 21 kPa O₂. In contrast, the elevated oxygen levels resulted in an immediate drop in ARA followed by a minor increase to a stable level lower than that at the preceding, lower oxygen tension. The symbiosis with the local Frankia had lost all ARA when the partial pressure of O₂ exceeded 50 kPa, whereas the symbiosis with Avcll still had some activity at 80 kPa O₂. This difference in tolerance of elevated O₂ clearly shows that the oxygen exclusion mechanisms may be controlled by the microsymbiont in Alnus-Frankia symbioses. The symbiotic systems recovered ARA to a similar extent when returned from elevated O₂ levels to 21 kPa O₂.     

Primary metabolism in N2-fixing Alnus incana-Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

The primary nitrogen metabolism of the N₂-fixing root nodule symbiosis Alnus incana (L.)–Frankia was investigated by ³¹P and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. Perfusion of root nodules in a pulse–chase approach with ¹⁵N- or ¹⁴N-labeled NH₄⁺ revealed the presence of the amino acids alanine (Ala), -amino butyric acid, glutamine (Gln), glutamic acid (Glu), citrulline (Cit) and arginine (Arg). Labeling kinetics of the Gln amide-N and -amino acids suggested that the glutamine synthetase (GS; EC 6.3.1.2)–glutamate synthase (GOGAT; EC 1.4.1.13) pathway was active. Inhibition of the GS-catalyzed reaction by methionine sulphoximine abolished incorporation of ¹⁵N. Cit was labeled in all three N positions but most rapidly in the position, consistent with carbamoyl phosphate as the precursor to which Gln could be the amino donor catalyzed by carbamoyl phosphate synthase (CPS; EC 6.3.5.5). Ala biosynthesis occurred consistent with a flux of N in the sequence Gln–Glu–Ala. ³¹P NMR spectroscopy in vivo and of extracts revealed several metabolites and was used in connection with the ¹⁵N pulse–chase experiment to assess general metabolic status. Stable concentrations of ATP and UDP-glucose during extended perfusions showed that the overall root nodule metabolism appeared undisturbed throughout the experiments. The metabolic pathways suggested by the NMR results were confirmed by high activities of the enzymes GS, NADH-GOGAT and ornithine carbamoyltransferase (OCT; EC 2.1.3.3). We conclude that the primary pathway of NH₄⁺ assimilation in A. incana root nodules occurs through the GS–GOGAT pathway. Biosynthesis of Cit through GS–CPS–OCT is important and is a link between the first amino acid Gln and this final transport and storage form of nitrogen.Abbreviations AlaDH l-Alanine dehydrogenase - Cit Citrulline - CPS Carbamoyl phosphate synthase - GABA -Amino butyric acid - GOGAT Glutamate synthase - GS Glutamine synthetase - MDH Malate dehydrogenase - MSO Methionine sulphoximine - NMR Nuclear magnetic resonance - OCT Ornithine carbamoyltransferase - PEPC Phosphoenolpyruvate decarboxylase - UDPGlc Uridine 5-diphosphoglucose     

Uptake and evolution of H2 and reduction of C2H2 by root nodules and nodule homogenates ofAlnus glutinosa

Summary In the growing season no net H₂ evolution is detected when root nodules ofAlnus glutinosa are incubated in air or in argon containing 20% O₂. Due to the hydrogenase activity, N₂-fixing root nodules consume added H₂ at a rate of about 1.4 moles H₂.g fresh nodule^–1.h^–1. The uptake of H₂ is only found in summer. At the end of the season, in autumn, nodules evolve significant quantities of H₂ although the nodules still continue to fix nitrogen. In-vitro studies with fractionated homogenates of summer-harvested nodules show that the recovery of the hydrogenase is high when using methylene-blue or phenazine metasulfate as electron acceptors. No hydrogenase activity is detected in homogenates of autumn-harvested nodules.The hydrogenase is localised in the microsymbiont.     

Acetylene reduction byDaviesia mimosoides under Eucalyptus

Summary Daviesia mimosoides is a common understorey legume in Eucalyptus forests of the Brindabella Range in southeastern Australia, capable of fixing atmospheric nitrogen. Rates of N fixation were measured by the acetylene-reduction technique over a growing season in the field. Pot trials under controlled conditions were also carried out to elucidate effects of soil moisture, temperature, and light. Average rates in the field varied from about 1–5 μ mol C₂H₄/g/h (wet weight of nodule), but rates up to 14 μ mol C₂H₄/g/h were measured in optimum controlled conditions. Annual N-fixation rates approximate 4.5–7.0 kg/ha. In pot trials, rate of acetylene reduction decreased with soil moisture to about−10 MPa tension, with a marked depression at about−6 MPa, but within the normal field range of soil moisture there was little correlation of moisture with average acetylene reduction rate. Rates were similar in the temperature range of 20–30°C, but were depressed by either low or high temperature (<10 or >30°C). Diurnal fluctuations in acetylene reduction rates were not correlated with solar radiation, but rates were limited by high mid-day temperatures.     

Field measurement of symbiotic nitrogen fixation in an established lucerne ley using15N and an acetylene reduction method

Summary Lucerne is an important forage legume in the south and south-east of Sweden on well-drained soils. However, data is lacking on the apparent amount of nitrogen derived through N₂ fixation by field-grown lucerne. This report provides basic information on the subject. The experiment was performed in a lucerne ley grown 40 km north of Uppsala. The input of nitrogen through fixation to the above-ground plant material of an established lucerne (Medicago sativa L.) ley was estimate by¹⁵N methodology during two successive years. The amount of fixed N was 242 kg N ha^–1 in 1982 and 319 kg N ha^–1 in 1983. The proportion of N derived from the atmosphere (%Ndfa) was 70% and 80% for the two years respectively. The first harvest in both years contained a lower proportion fixed N. Both N₂ fixation and dry matter production were enhanced during the second year, particularly in the first harvest. The Ndfa was 61% in the first harvest in 1982, compared to 72% Ndfa during the same period in 1983. This demonstrates the strong influence of environment on both dry matter production and N₂ fixation capacity of the lucerne.In addition anin situ acetylene reduction assay was used in 1982 to measure the seasonal distribution of the N₂ fixation and in 1983 to study the effect of soil moisture on the N₂ fixation process. The seasonal pattern showed great dependence on physiological development and harvest pattern of the lucerne ley. The maximum rate of N₂ fixation occurred at the bud or early flower stage of growth and was followed by a rapid decline as flowering proceeded. After harvest the nitrogenase activity markedly decreased and remained low during at least two weeks until regrowth of new shoots began. Irrigation doubled the nitrogenase activity of the lucerne in late summer 1983, when soil moisture content in the top soil was near wilting point. No changes in nitrogenase activity did occur in response to watering earlier during the summer, when the soil matric potential was around –0.30 MPa.     

Nodulation and N2 fixation byInga jinicuil,a woody legume in coffee plantations

Summary Nodule biomass and yearly C₂H₂ reduction rates are reported forInga jinicuil, a leguminous tree used for shade in Mexican coffee plantations. Annual fixation by this species approximates 35 kg ha^–1; which, when compared to nitrogen additions from fertilizers, represents an important nitrogen input to the coffee ecosystem.     

The response of field grownPhaseolus vulgaris to Rhizobium inoculation and the quantification of N2 fixation using15N

Summary A field experiment was performed to assess the effects of Rhizobium inoculation and nitrogen fertilizer (100 kg N ha^–1) on four cultivars of Phaseolus beans; Carioca, Negro Argel, Venezuela 350 and Rio Tibagi. In the inoculated treatment 2.5 kg N ha^–1 of¹⁵N labelled fertilizer was added in order to apply the isotope dilution technique to quantify the contribution of N₂ fixation to the nutrition of these cultivars.Nodulation of all cultivars in the uninoculated treatments was poor, but the cultivars Carioca and Negro Argel were well nodulated when inoculated. Even when inoculated, nodulation of the cultivars Venezuela 350 and Rio Tibagi was poor and these cultivars showed little response to inoculation in terms of nitrogen accumulation or grain yield. The estimates of the contribution of N₂ fixation estimated using the isotope dilution technique, for the Carioca and Negro Argel cultivars, amounted to 31.7 and 18.4 kg N ha^–1 respectively. These two cultivars produced 991 and 883 kg ha^–1 of grain, respectively, when inoculated and 663 and 620 kg ha^–1 with the addition of 100 kg N ha^–1 of N fertilizer. The response to nitrogen was particularly poor due to high leaching losses in the very sandy soil at the experimental site.The Venezuela 350 and Rio Tibagi cultivars only responded to N fertilizer and not to inoculation with Rhizobium which stresses the great importance of selecting plant cultivars for nitrogen fixation in the field.     

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.     

Denitrification and nitrogen fixation by Azospirillum

A model system is described where Azospirillum and germinated wheat seeds were grown in association for a week and then assayed for nitrogen fixation (C₂H₂-reduction) and denitrification (N₂O-formation) activities. The association performed C₂H₂-reduction and N₂O-formation under microaerobic conditions. Both activities were measurable after already 3–5 h of incubation with substantial rates and were strictly dependent on the presence of both plants and bacteria. During the week of the growth of the association, the bacteria had lived exclusively from the carbon compounds supplied by the roots of the plants. C₂H₂-reduction activity by the association was more or less the same with all the Azospirillum brasilense strains, but lower with A. lipoferum and with the A. amazonense strains tested. Two nitrogenase negative mutants of Azospirillum brasilense showed virtually no activity in the association. C₂H₂-reduction activity was strongly dependent on the growth temperature of the association. Denitrification (N₂O-formation) was high also at higher temperatures and at pH-values in the medium around 7.8 but not at neutrality and was strictly dependent on nitrate. The Azospirillum strain used strongly determined the rate of the N₂O-formation in the association. It is suggested that Azospirillum may be beneficial to crops particularly under tropical conditions.Dedicated to Professor Dr. Gerhart Drews, Freiburg, on the occasion of his 60th birthday     

Misconceptions and practical problems in the use of 15N soil enrichment techniques for estimating N2 fixation

The ¹⁵N methods are potentially accurate for measuring N₂ fixation in plants. The only problem with those methods is, how to ensure that the ¹⁵N/¹⁴N ratio in the plant accurately reflects the integrated ¹⁵N/¹⁴N ratio (R) in soil which is variable in time and with soil depth. However, the consequences of using an inappropriate reference plant vary with the level of N₂ fixation and the conditions under which the study was made. For example, the errors introduced into the values of N₂ fixation are higher at low levels of fixation, and decrease with increasing rates of fixation. At very high N₂ fixation rates, the errors are often insignificant. Also, the magnitude of error is proportional to the rate of decline of the ¹⁵N/¹⁴N ratio with time. Since N₂ fixation in most plants would be expected to below 60%, the question of how to select a good reference plant is still pertinent. In this paper, we have discussed some of the criteria to adopt in selecting reference plants, e.g. how to ensure that the reference plant is not fixing N₂, is absorbing most of its N from the same zone as the fixing plant, and in the same pattern with time, etc. In addition, we have discussed ¹⁵N labelling materials and methods that are likely to minimize any errors even when the fixing and reference plants don't match well in certain important criteria. The use of slow release ¹⁵N fertilizer or ¹⁵N labelled plant materials results in slow changes in the ¹⁵N/¹⁴N ratio of soil, and is strongly recommended. Where ¹⁵N inorganic fertilizers are used, the application of the fertilizer in small splits at various intervals is recommended over a one-time application. The problem with the reference crop, which has sometimes discouraged potential users of the ¹⁵N methods, is surmountable, as discussed in this paper.     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.     

15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed ¹⁵N₂ and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to ¹⁵N₂, showed that highest initial ¹⁵N₂-labelling was into NH ₄ ⁺ . After 12 min little further increase in the NH ₄ ⁺ label occurred while that in the amide group of glutamine and in glutamate continued to increase. The ¹⁵N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of ¹⁵N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total ¹⁵N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which ¹⁴N₂ was added to cephalodia previously exposed to ¹⁵N₂, showed that the NH ₄ ⁺ pool rapidly became depleted of ¹⁵N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The ¹⁵N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the ¹⁵N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.Abbreviations MSX L-methionine-SR-sulphoximine - AOA amino-oxyacetate - HEPES N-2-hydroxymethylpiperazine-N-2-ethane sulphonic acid - Tris tris-(hydroxymethyl) methylamine - GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase - GPT glutamate-pyruvate aminotransferase - APT aspartate-pyruvate aminotransferase - ADH alanine dehydrogenase - GOT glutamate-oxaloacetate aminotransferase     

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.     

A comparison between the acetylene reduction method,the isotope dilution method and the total nitrogen difference method for measuring nitrogen fixation in lucerne (Medicago sativa L.)

Summary In view of the increasing need for the exact estimation of the input of nitrogen in agroecosystems, an application of the acetylene-reduction technique was developed. The technique, consisting of a plastic bag incubation system, using propane as an internal standard of the apparent volume, made it possible to carry out repeated incubations on the same plant system. The evaluated technique included studies of the diffusion of ethylene and propane in a soil column, as well as studies of the optimal substrate concentration and grade of purity of the substrate. In addition, the conversion factor between amounts of reduced acetylene as compared wtih reduced dinitrogen was determined by¹⁵N₂ incubations to be 4.41. The developedin situ acetylene-reduction technique was compared with an isotope dilution method and a total nitrogen difference method. By comparing the derived total nitrogen fixation values from each with the value derived from the acetylene-reduction method; it was shown that the values differed significantly. The acetylene-reduction method gave the highest nitrogen fixation values, the isotope dilution the lowest values and the total nitrogen difference method was intermediate. No statistical significant difference existed between the two different reference crops used in the isotope dilution method.     

15N natural abundance in plants of the Amazon River floodplain and potential atmospheric N2 fixation

Summary The¹⁵N natural abundance values of various Amazon floodplain (várzea) plants was investigated. Samples of young leaf tissues were collected during three different periods of the river hydrography (low water, mid rising water and high water) and during one period in the Madeira River (high water). A large variation of¹⁵N abundance was observed, both among the different plant types and between the different flood stages. This variation probably, reflected, in part, the highly variable nature of the floodplain, sometimes dry and oxygenated and at other times inundated and anaerobic and, in part, changes in plant nitrogen metabolism. Comparison of the nitrogen isotopic composition of leguminous plants with that of non-leguminous plants showed that, on average, the¹⁵N abundance was lower in the legumes than non-legumes, suggesting active N-fixation. Also, the¹⁵N natural abundance in aquatic grasses of the generaPaspalum, was in general, lower than the¹⁵N abundance of aquatic grasses of the generaEchinochloa. As both of these grasses grow in the same general habitat, it appears thatPaspalum grasses may also be nitrogen fixers.     

Effects of nickel on Frankia and its symbiosis with Alnus glutinosa (L.) Gaertn

The tolerance of nickel by Frankia in culture and in symbiosis with Alnus was determined. Yield of three Frankia strains was not affected significantly by 2.25 mM nickel when cultured in propionate medium containing hydolysed casein as nitrogen source. Yield of two strains in medium without combined nitrogen, and thus reliant on fixed nitrogen, was stimulated markedly by the same nickel concentration. Utilisation of nickel for synthesis of uptake hydrogenases is presumed to be the cause of enhanced nitrogenase activity.Although growth was reduced, treatment of 2-month-old seedlings with 0.025 mM nickel for 4 weeks did not affect nodulation significantly while nitrogenase activity was doubled. Nodulation and nitrogenase activity of seedlings receiving 0.075 mM nickel were inhibited markedly, while 0.5 mM nickel was lethal to all seedlings after 4 weeks of treatment. A few small, ineffective nodules were initiated early on some of the latter seedlings, suggesting that effects of nickel on host plant processes rather than Frankia are the primary cause of inhibition of nodulation. This interpretation is supported by the retention of substantial nitrogenase activity in 10-month-old plants 1 day after the treatment with 0.59 mM nickel, when the nickel content of roots and nodules was already maximal. No nitrogenase activity was detected after 3 days, by which time the leaves were almost completely necrotic. Over a 4 day period, most nickel was retained in the roots and nodules. Supplying histidine simultaneously at concentrations equal to, or in excess of, nickel prevented wilting and leaf necrosis, but did not increase translocation of nickel to the shoot.     

Heterotrophic nitrogen fixation (acetylene reduction) associated to flooded rice: A modified measurement technique in the field

A modifiedin situ technique for measuring heterotrophic nitrogen fixing (acetylene reducing) activity associated to rice is proposed. Ethylene evolution rates measured in opaque cylinders covering the stems of rice plants which have been cut 10 cm over the water level were found independent of the diurnal cycle. Cutting of the leaves resulted in decreased variation between plants and suppression of the acceleration of ethylene evolution rate after 12 h incubation as compared to intact plants. In both systems ethylene evolved was swept by a current of methane and the molar ratio between methane and ethylene was stabilized after 12 h. Methane evolution rates remained stable during 12 h and more than 24 h in whole plants and cut plants respectively. It is suggested that alteration in the active gas transport system after 12 h incubation under 10% acetylene may lead to erroneous evaluation of the actual ethylene production in the root's environment. The average values of ethylene evolution rates by cut plants between 12 and 24 h of incubation may be used for comparative studies of nitrogen fixing activity associated to flooded rice.     

Seasonal variation in rates of heterotrophic nitrogen fixation (acetylene reduction) in Zostera noltii meadows and uncolonised sediments of the Bassin d'Arcachon, south-west France

Nitrogen fixation (acetylene reduction) rates were measured over an annual cycle in meadows of the seagrass Z. noltii and uncolonised sediments of the Bassin d'Arcachon, south-west France, using both slurry and whole core techniques. Measured rates using the slurry technique in Z. noltii colonised sediments were consistently higher than those determined in isolated cores. This was probably due to the release of labile organic carbon sources during preparation of the slurries. Thus, in colonised sediments the whole core technique may provide a more accurate estimate of in situ activity. Acetylene reduction rates measured by the whole core technique in colonised sediments were 1.8 to 4-fold greater, dependent upon the season, in the light compared with those measured in the dark, indicating that organic carbon released by the plant roots during photosynthesis was an important factor regulating nitrogen fixation. In contrast acetylene reduction rates in uncolonised sediments were independent of light.Addition of sodium molybdate, a specific inhibitor of sulphate reduction inhibited acetylene reduction activity in Z. noltii colonised sediments by > 80% as measured by both slurry and whole core techniques irrespective of the light regime, throughout the year inferring that sulphate reducing bacteria (SRB) were the dominant component of the nitrogen fixing microflora. A mutualistic relationship between Z. noltii and nitrogen fixing SRB in the rhizosphere, based on the exchange of organic carbon and fixed nitrogen is proposed. In uncolonised sediments sodium molybdate initially severely inhibited acetylene reduction rates, but the level of this inhibition declined over the course of the year. These data indicate that the nitrogen fixing SRB associated with the Zostera roots and rhizomes were progressively replaced by an aerobic population of nitrogen fixers associated with the decomposition of this recalcitrant high C:N ratio organic matter.Acetylene and sulphate reduction rates in the seagrass beds showed distinct summer maxima which correlated with a reduced availability of NH ₄ ⁺ in the sediment and the growth cycle of Z. noltii in the Bassin. Overall, these data indicate that acetylene reduction (nitrogen fixation) activity in the rhizosphere of Z. noltii was regulated both by release of organic carbon from the plant roots and maintenance of low ammonium concentrations in the root zone due to efficient ammonium assimilation.Nitrogen fixation rates determined from acetylene reduction rates measured by the whole core technique ranged from 0.1 to 7.3 mg N m^–2 d^–1 in the Z. noltii beds and between 0.02 and 3.7 mg N m^–2 d^–1 in uncolonised sediments, dependent upon the season. Nitrogen fixation in the rhizosphere of Z. noltii was calculated to contribute between 0.4 and 1.1 g N m^–2 y^–1 or between 6.3 and 12% of the annual fixed nitrogen requirement of the plants. Heterotrophic nitrogen fixation therefore represents a substantial local input of fixed nitrogen to the sediments of this shallow coastal lagoon and contributes to the overall productivity of Z. noltii in this ecosystem.     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.