Inhibition of nitrogen fixation by nitrite inAnabaena variabilis

Addition of nitrite to rapidly growing, nitrogen-fixing filaments ofAnabaena variabilis caused an immediate drop in nitrogenase activity. This was followed by a transient induction of nitrite reductase, recovery of nitrogen fixation and cyanobacterial growth. The experiments with isolated heterocysts and a partially purified nitrogenase preparation from heterocysts showed that nitrite primarily exerted its inhibitory effect by inactivating nitrogenase irreversibly, rather than interfering with photosynthetic energy conservation.Abbreviations ATCC American type culture collection - Chl chlorophyll - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - Tes 2-{[2 hydroxy-1,1-bis(hydroxymethyl)ethyl] amino} ethane sulfonic acid     

Planktonic nitrogen fixation in Lake Malawi/Nyasa

Nitrogen (N₂) fixation has been identified as possibly an important source of “new” nitrogen (N) to the epilimnion of Lake Malawi but studies in 1999–2000 and 2002 (September–December) estimated that the contribution of N₂-fixation by heterocystous Anabaena filaments to the N budget of the lake’s epilimnion is only 3–4% of total N input. N₂-fixation rates in Lake Malawi, as estimated by the acetylene reduction assay were higher during the stratified season (September–March) than during the rest of the year. Planktonic N₂-fixation in Lake Malawi can be monitored by measuring heterocyst biovolume concentrations because a significant correlation (r ² = 0.945, P < 0.0001) was observed between the two parameters. Heterocyst density cannot be used to estimate N₂-fixation because heterocyst sizes in the lake change continuously as at least two Anabaena species with different heterocyst dimensions are present. During September–October 2002, a species similar to A. maxima, contained larger heterocysts (16.34 ± 2.46 and 13.25 ± 1.89 μm in cross and apical section dimensions, respectively). In November–December of 2002, A. discoidea dominated and had smaller sized heterocysts (8.92 ± 1.13 and 7.25 ± 0.95 μm in cross and apical section dimensions). Since planktonic N₂-fixation is higher near shore than offshore, its contribution to the N budget in the littoral zone where high densities of grazing benthic fish occur may be more critical to maintaining the high rates of benthic algal productivity observed.     

Endophytic nitrogen fixation in sugarcane: present knowledge and future applications

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N₂-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N₂-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and ¹⁵N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N₂-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N₂ fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N₂-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.     

Algal nitrogen fixation on temperate arable fields

Summary N₂-fixation by algae on the Broadbalk continuous wheat experiment was measured over a two year period using the acetylene reduction technique. The plots studied receive spring fertilizer treatments including farmyard manure and combinations of nitrochalk and Na, P, K and Mg which have remained much the same since the experiment started in 1843.Nitrogen applied at 196 kg ha^–1 in spring suppressed algal N₂-fixation until late in the season but at lower levels (48 kg N ha^–1) the denser plant canopy increased both surface moisture and fixation. Herbicide treatment decreased fixation on plots of moderate nutritional status early in the season but had little effect on unfertilised plots where weed cover was sparse. On plots where weed and crop cover was very dense herbicide treatment increased fixation in August.Algal N₂-ase activity, assayed by C₂H₂ reduction, continued throughout the night at a rate which averaged 33% of the midday value. Laboratory experiments indicate that dark fixation is very temperature sensitive and this value may represent a maximum. Algal crust in the field dried to 4.5–6.8% H₂O content became active 3 1/2 h after rewetting and reached a steady state after 7 h which represented only 6–22% of that at the previous maximum suggesting that many cells had been killed.In a year with average rainfall algae on plots receiving 48 kg N ha^–1 were estimated to fix 25–28 kg N ha^–1 and plots without fertiliser 13–19 kg N ha^–1. Algal fixation appeared to make a substantial contribution to the continuing fertility of unfertilised plots.     

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.     

Effect of salinity and inoculation on growth,nitrogen fixation and nutrient uptake ofVigna radiata (L.) Wilczek

This study reports the effect of salinity and inoculation on growth, ion uptake and nitrogen fixation byVigna radiata. A soil EC_e level of 7.5 dS m⁻¹ was quite detrimental causing about 60% decline in dry matter and grain yield of mungbean plants whereas a soil EC_e level of 10.0 dS m⁻¹ was almost toxic. In contrast most of the studied strains of Rhizobium were salt tolerant. Nevertheless, nodulation, nitrogen fixation and total nitrogen concentration in the plant was drastically affected at high salt concentration. A noticeable decline in acetylene reduction activity occurred when salinity level increased to 7.5 dS m⁻¹.     

Growth and nitrogen fixation ofAeschynomene under water stressed conditions

Summary Studies on the tolerance ofAeschynomene americana L. to periods of flooding or soil moisture deficit were conducted in an attempt to elucidate nitrogen fixation as affected by soil moisture. Nitrogenase activity was not reduced significantly in pot-grown Aeschynomene plants subjected to flooding in greenhouse conditions. After 20 days of withholding water from the soil, nitrogenase activities of the drought-stressed plants were much lower than those of either the well-watered or flooded plants. Leaf water potentials were similar in flooded and control plants; however, the droughted plants had leaf water potentials that were 4 bars lower than those of the control plants. Aeschynomene plants were tolerant to long-term periods of flooding, but exhibited a reduction in nitrogenase activity and leaf water status when subjected to soil moisture deficits.     

Fructose supports glycogen accumulation,heterocysts differentiation,N2 fixation and growth of the isolated cyanobiont Anabaena azollae

Cells of the cyanobiont Anabaena azollae isolated from the water fern Azolla filiculoides were found to take up and utilize fructose in the light for mixotrophic growth. Fructose was favored by the cyanobiont as a substrate over sucrose and glucose. Cell growth in the presence of 8 mM fructose led to glycogen accumulation in the cells which approached 20% of the cell dry weight within 2 to 3 days, followed by reduction of glycogen content during the fourth day. Glucose-6-phosphate dehydrogenase activity was increased 5–6-fold in the fructose grown cells from the third day of growth onwards. The frequency of heterocysts in fructose-grown cells increased from 6 to 18%, and acetylene reduction by nitrogenase was increased 3-fold in the presence of fructose as compared with control cells, with maximum values observed between the third and fifth day of mixotrophic growth. Fructose-supported growth yielded a 2–4-fold increase in cell dry weight over controls.It is suggested that fructose-supported development and growth of the cyanobiont in batch cultures may resemble its mixotrophic growth and development in situ in the leaf cavity of the host fern Azolla.Abbreviation G6PDH glucose-6-phosphate dehydrogenase     

The influence of temperature and nitrate on vegetative growth and nitrogen accumulation by nodulated soybeans

Summary Inoculated soybeans [Glycine max (L.) Merrill] were grown in controlled environments to evaluate the relationship between temperature and applied NO₃−N on growth rates, N accumulation, and acetylene reduction activity during the vegetative growth stage. Soybeans were grown at day/night temperatures of 22/18 and 26/22°C in sand culture with daily applications of 21.4 mM (high) and 2.1 mM (low) NO₃−N in a complete nutrient solution for durations of 14, 21, and 42 days after emergence and with an N-free solution. Dry matter and N accumulation were greater at 26/22 than 22/18°C. In general, both increased as the level and duration of applied NO₃−N was increased. These increases were attributable to an abbreviation in the interval between emergence and onset of rapid growth. The presence and assimilation of NO₃−N, even at the high level, did not inhibit development of functional nodules. Neither mass nor acetylene reduction activity of nodules was reduced by high NO₃−N; however, the root mass was increased by NO₃−N more than the nodule mass. There was an interaction between temperature and NO₃−N on specific nodule activity as measured by acetylene reduction. The specific nodule activity was unaffected by NO₃−N at 22/18°C, but at 26/22°C the specific activity was lower in the absence of NO₃−N than when NO₃−N was present. Apparently, rapid early growth at 26/22°C depleted cotyledonary reserves of N before nodules became active and, thereafter, the plants were unable to develop adequate leaf area to support nodule development and functioning. This result has implications in N fertilization of late-planted soybeans. Paper number 6637 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina, 27650. The research was supported in part by a grant from the North Carolina Soybean Producers Association and by USDA-SEA-CR grant 701-15-26.     

Effects of sulphur nutrition on growth and nitrogen fixation of pea (Pisum sativum L.)

A S-deficient soil was used in pot experiments to investigate the effects of S addition on growth and N₂-fixation in pea (Pisum sativum L.). Addition of 100 mg S pot⁻¹ increased seed yield by more than 2-fold. Numbers of pods formed were the most sensitive yield component affected by S deficiency. Sulphur addition also increased the concentration of N in leaves and stems, and the total content of N in the shoots. The amounts of N fixed by pea were determined at four growth stages from stem elongation to maturity, using the ¹⁵N dilution technique. Sulphur addition doubled the amount of N fixed at all growth stages. In contrast, leaf chlorophyll content and shoot dry weight were increased significantly by S addition only after the flowering and pod fill stage, respectively. Pea roots were found to have high concentrations of S, reaching approximately 10 mg g⁻¹ dry weight and being 2.6–4.4 times the S concentration in the shoots under S-sufficient conditions. These results suggest that roots/nodules of pea have a high demand for S, and that N₂-fixation is very sensitive to S deficiency. The effects of S deficiency on pea growth were likely to be caused by the shortage of N, due to decreased N₂-fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of plant genotype and nitrogen fertilizer on symbiotic nitrogen fixation by soybean cultivars

Summary Isotopic as well as non-isotopic methods were used to assess symbiotic nitrogen fixation within eight soybean [Glycine max (L.) Merr.] cultivars grown at 20 and 100 kg N/ha levels of nitrogen fertilizer under field conditions.The¹⁵N methodology revealed large differences between soybean cultivars in their abilities to support nitrogen fixation. In almost all cases, the application of 100 kg N/ha resulted in lower N₂ fixed in soybean than at 20 kg N/ha in the first year of the study. However, N₂ fixed in one cultivar, Dunadja, was not significantly affected by the higher rate of N fertilizer application. These results were confirmed by measurements of acetylene reduction activity, nodule dry weight and N₂ fixed as measured by the difference method. Further proof of differences in N₂ fixed within soybean cultivars and the ability of Dunadja to fix similar amounts of N₂ at 20 and 100 kg N/ha was obtained during a second year experiment. Dunadja yield was affected by N fertilizer and produced larger yield at 100 kg N/ha than at 20 kg N/ha. This type of cultivar could be particularly useful in situations where soil N levels are high or where there is need to apply high amounts of N fertilizer.The present study reveals the great variability between legume germplasms in the ability to fix N₂ at different inorganic N levels, and also the potential that exists in breeding for nitrogen fixation associative traits. The¹⁵N methodology offers a unique tool to evaluate germplasms directly in the field for their N₂ fixation abilities at different N fertilizer levels.     

Effect of varying periods of inoculation ofAzolla pinnata R. Brown on its growth,nitrogen fixation and response to rice crop

Summary Inoculation of water fernAzolla pinnata R. Brown (Bangkok isolate) at the rate of 500kg fresh weight ha⁻¹ in rice fields at weekly intervals after planting in addition to 30 kg N ha⁻¹ as urea showed a decrease in its growth and N₂-fixation with delay in application. Use of Azolla up to 3 weeks after planting (WAP) during wet and 4 WAP during dry season produced significantly more grain yield than 30 kg N ha⁻¹, whereas its application upto one WAP produced more grain yield than 60 kg N ha⁻¹. Grain yield with Azolla applied at the time of planting was similar to that of 60 kg N treatment during the wet season. Higher grain yields in zero and one WAP Azolla treatments resulted due to increase in both number of panicles m⁻² and number of grains/panicle while the subsequent Azolla inoculations increased grain yield mainly by producing more number of grains/panicle. Dry matter and total N yields at maturity of rice crop were more with Azolla application upto 3 WAP during wet and 2 WAP during dry season while the reduction in sterility (%) was observed upto one WAP over 30 kg N ha⁻¹ during both seasons. Number of tillers m⁻² and dry matter production at maximum tillering and flowering were more than 30 kg N ha⁻¹ with the use of Azolla upto one WAP. Increased grain N yield was observed with the use of Azolla upto 4 WAP during two seasons whereas straw N yield increased upto one WAP during wet and 2 WAP during dry season.     

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     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.     

The effects of immobilization on the biochemical,physiological and morphological features of Anabaena azollae

Anabaena azollae, a presumptive isolate from Azolla filiculoides, was immobilized in polyurethane foam, hydrophilic polyvinyl foam and alginate. When viewed by low-temperature scanning electron microscopy a thick mucilage layer covered the surface of both cells and matrix; this closely resembles the mode of attachment of the symbiont Anabaena in the Azolla leaf cavity. The heterocyst frequency of the immobilized A. azollae doubled relative to free-living cells and reached a level of 14–17%. Immobilization induced increases in both hydrogen production via nitrogenase or hydrogenase and in the rates and stabilization of acetylene reduction (N₂-fixation). Ammonia production by immobilized cells with L-methionine-D,L-sulfoximine (MSX) is greater than that of freeliving cells. Immobilized cells without MSX were, however, able to excrete ammonium at lower rates thus emulating the characteristic of the symbiotic cyanobacteria (A. azollae) in the leaf cavity of Azolla.Abbreviations Chl chlorophyll - GS glutamine synthetase - MSX L-methionine-D,L-sulfoximine - SEM scanning electron microscopy - PU polyurethane - PV polyvinyl     

Enrichment and isolation of nitrogen fixing hydrogen bacteria

An enrichment method for nitrogen fixing hydrogen bacteria is described. The procedure invariably resulted in the isolation of yellow-pigmented coryneform bacterial strains assigned to Corynebacterium autotrophicum. The procedure included a serial transfer in an ammonium-free mineral liquid medium under an atmosphere of 10% hydrogen, 5% oxygen, 10% carbon dioxide and 75% nitrogen, followed by a short alkali treatment and by streaking on nutrient broth-succinate agar. The ability to fix nitrogen was confirmed by the acetylene reduction test and by ¹⁵N₂ incorporation.     

Application of 15N natural abundance technique for evaluating biological nitrogen fixation in oil palm ecotypes at nursery stage in pot experiments and at mature plantation sites

A range of different species of diazotrophic bacteria has been found in tissues and the rhizosphere of oil palm plants, suggesting a potential to benefit from biological nitrogen fixation (BNF). A few studies have confirmed that plantlets at nursery stage can benefit significantly from BNF after inoculation with Azospirillum spp. but no data are available regarding the benefit from naturally-occurring diazotrophic bacteria in oil palm. The results described here were derived from two pot trials laid out under controlled conditions with plantlets from two important regions for palm oil production in Brazil, as well as from different field sites of mature oil palm plantations. The ¹⁵N natural abundance technique was employed to estimate plant dependence on BNF (%Ndfa) by the different ecotypes grown in soil and previously characterized as hosting diazotrophic bacteria. From both pot trials it was possible to identify some ecotypes of high potential for N₂-fixation that reached in some cases approximately 50%Ndfa. However, the accuracy of measurement still needs to be improved using more suitable reference plants for pot experiments. Values of δ ¹⁵N signals from oil palm and reference plants in the field were inconclusive concerning any benefit from BNF to oil palm, owing to apparently high temporal and spatial variability of δ ¹⁵N of the plant-available N in the heterogeneous soil matrix for the different palm and reference plant tested.     

Interactions ofRhizobium japonicum strains in soybean nodules and their contribution to nitrogen fixation

Summary Rhizobium japonicum strain 8-0 Str^R applied as inoculum to Clark 63 soybeans formed small ineffective nodules which had very low nitrogenase activity compared to nodules formed by two effective strains, 110 Tet^R and 138 Kan^R. Mean numbers of cells per milligram of nodule tissue for plants up to 34 days old were 7.7×10⁶ for 8-0 Str^R, 4.1×10⁸ for 110 Tet^R and 7.6×10⁸ for 138 Kan^R. Cell counts per unit mass of nodule were independent of plant age for strains 110 Tet^R and 138 Kan^R, however, for strain 8-0 Str^R, 25 and 34 days old plants had fewer viable cells per nodule mass than 18 day old plants. When a mixture of two effective strains was used, the nodules of individual plants were predominantly caused by either 110 Tet^R or 138 Kan^R. In one experiment the predominance was random, but in another, strain 110 Tet^R clearly dominated. Strain 138 Kan^R was absent in some nodules on 18 day old plants, and in others, less than 10² cells per nodule were found. When strains 8-0 Str^R and 138 Kan^R were used as mixed inoculum, most of the nodules had strain 8-0 Str^R but strain 138 Kan^R was detected in many nodules and was generally evident in the largest nodules. Nitrogenase activity by many individual nodules was low except for nodules which had cells of 138 Kan^R. Nitrogenase activity by whole root systems of these plants was relatively high and similar to plants that had only nodules of strain 138 Kan^R. Similar relationships were observed for a mixed inoculum of 8-0 Str^R and 110 Tet^R. In general, mixed inoculations resulted in nodules with a particular strain being dominant for each individual plant. Double infections within individual nodules were not uncommon and such nodules often had disproportionate numbers of cells of two competingR. japonicum strains.Contribution from the Laboratory of Soil Microbiology, Department of Agronomy, Missouri Agricultural Experiment Station. Missouri Journal Series Number 7967.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Utilisation du15N2 pour estimer la fixation d'azote et sa repartition chez les legumineuses

Resumé Les auteurs proposent une méthode d'utilisation du¹⁵N₂ pour l'étude de la fixation d'azote dans les associations complexes légumineuses-Rhizobium cultivées sur sol. La procédure consiste, à marquer l'atmosphère du sol à l'aide de¹⁵N₂ et à calculer la quantité d'azote total fixé pendant cette période. Les premiers résultats obtenus sur des haricots et des trèfles démontrent qu'après seulement 7 h d'incubation, des quantités significatives de¹⁵N sont mesurées dans les plantes permettant ainsi de déterminer précisément la valeur du rapport C₂H₄/N₂ qui a été établi entre 2,6 et 3,1 dans les conditions de l'expérience. Sur de longues périodes, ces mêmes quantités se sont avérées suffisantes pour suivre la dynamique de l'azote des nodules vers les organes reproducteurs de plants de haricots.

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Use of¹⁵N to estimate dinitrogen fixation and distribution in legumes

Summary A method for use of¹⁵N₂ in the study of dinitrogen fixation by complex legumes-Rhizobium associations grown on soil is proposed. The procedure consists in labelling the soil atmosphere with¹⁵N₂ during short periods of time, measuring¹⁵N enrichment in the plants and calculating the total nitrogen fixed during this period. The first results obtained with bean and clover plants demonstrate that after only 7 h of incubation, significative amounts of¹⁵N are measured in the plants to allow precise determination of C₂H₄/N₂ ratio which ranged between 2.6 and 3.1 in this experiment. Over longer periods of time, such amounts are meaningfull to follow the pattern of N dynamic from the nodules to the reproductive organs of bean plants.