Remobilization of labelled nitrogen from vegetative tissues to pods of mungbean

Summary Remobilization of¹⁵N from vegetative tissue of mungbean (Vigna radiata (L.) Wilczek) into pods was measured during the reproductive phase of growth. Plant tissue was labelled with¹⁵N during vegetative development. Experiments were conducted in the field at two sites. At one site the soil provided cowpeas with most of their N but at the other site N fixation provided most of the N. Remobilized N from vegetative tissue to pods occurred soon after they began to develop. The quantity of the labelled N ultimately remobilized to the pods amounted to 50% for one cultivar (Tx33) at the high soil N site and 70% at the low N site. For the other cultivar (Tx13) the values were 25% and 30%, respectively. The two cultivars performed very differently with respect to partitioning of N into pods and the rate of N fixation. Even though more N was accumulated in the shoots of the high N fixing cultivar (Tx13) less total N was contained in the pods.     

Estimation of biological nitrogen fixation associated withBrachiaria andPaspalum grasses using15N labelled organic matter and fertilizer

Summary Six pasture grasses,Paspalum notatum cv batatais,P. notatum cv pensacola,Brachiaria radicans, B. ruziziensis, B. decumbens andB. humidicola, were grown in concrete cylinders (60 cm diameter) in the field for 31 months. The soil was amended with either a single addition of¹⁵N labelled organic matter or frequent small (2 kg N. ha^–1) additions of¹⁵N enriched (NH₄)₂SO₄. In the labelled fertilizer treatment soil analysis revealed that there was a very drastic change in¹⁵N enrichment in plant-available nitrogen (NO ₃ ^– +NH ₄ ⁺ ) with depth. The different grass cultivars recovered different quantities of applied labelled N, and evidence was obtained to suggest that the roots exploited the soil to different depths thus obtaining different¹⁵N enrichments in soil derived N. This invalidated the application of the isotope dilution technique to estimate the contribution of nitrogen fixation to the grass cultivars in this treatment. In the labelled organic matter treatment the¹⁵N label in the plant-available N declined at a decreasing rate during the experiment until in the last 12 months the decrease was only from 0.274 to 0.222 atom % excess. There was little change in¹⁵N enrichment of available N with depth, hence it was concluded that although the grasses recovered different quantities of labelled N, they all obtained virtually the same¹⁵N enrichment in soil derived N. Data from the final harvests of this treatment indicated thatB. humidicola andB. decumbens obtained 30 and 40% respectively of their nitrogen from N₂ fixation amounting to an input of 30 and 45 kg N.ha^–1 year^–1 respectively.     

Evaluation of the availability ofAzolla-N and urea-N to rice using15N

Summary The symbiotic association of the water fernAzolla with the blue-green algaAnabaena azollae can fix 30–60 kg N ha^–1 per rice cropping season. The value of this fixed N for rice production, however, is only realized once the N is released from theAzolla biomass and taken up by the rice plants. The availability of N applied asAzolla or as urea was measured in field experiments by two¹⁵N methods. In the first,Azolla caroliniana (Willd.) was labelled with¹⁵N in nutrient solution and incorporated into the soil at a rate of 144 kg N ha^–1. The recovery ofAzolla-N in the above ground parts of rice [Oryza sativa (L) cv. Nucleoryza] was found to be 32% vs. 26% for urea applied at a rate of 100 kg N/ha; there was no significant difference in recovery. In the second, 100 kg N/ha of¹⁵N-urea was applied separately or in combination with either 250 or 330 kg N ha^–1 of unlabelledAzolla. At the higher rate, the recovery ofAzolla-N was significantly greater than that of urea. There was a significant interaction when both N sources were applied together, which resulted in a greater recovery of N from each source in comparison to that source applied separately. Increasing the combined urea andAzolla application rate from 350 kg N ha^–1 to 430 kg N ha^–1 increased the N yield but had no effect on the dry matter yield of rice plants. The additional N taken up at the higher level of N application accumulated to a greater extent in the straw compared to the panicles. Since no assumptions need to be made about the contribution of soil N in the method using¹⁵N-labelledAzolla, this method is preferable to the¹⁵N dilution technique for assessing the availability ofAzolla-N to rice. Pot trials usingAzolla stored at –20°C or following oven-drying showed that both treatments decreased the recovery of N by one third in comparison to freshAzolla.     

Field evaluation of N2-fixation and N-utilization by phaseolus bean varieties determined by15N isotope dilution*

Summary Differences in N₂-fixation byPhaseolus vulgaris bean cultivars were successfully evaluated in the field using¹⁵N isotope dilution technique with a non-fixing test crop of a different species (wheat). The Phaseolus cultivars could have been similarly ranked for N₂-fixation capacity from either seed yield or total nitrogen yield, but the isotope method provided a direct measure of N₂-fixation and made it possible to estimate the proportion of fixed to total nitrogen in the crop and in plant parts. Amounts of nitrogen fixed varied between 24.59 kg N/ha for the 60-day cultivar Goiano precoce to 64.91 kg N/ha for the 90-day cultivar Carioca. The per cent of plant nitrogen due to fixation was 57–68% for the 90-day cultivars and 37% for Goiano precoce (60-day cultivar). Fertilizer utilization was 17–30% of a 20 kg N/ha fertilizer application. 100 kg N/ha fertilizer application decreased N₂-fixation without suppressing it totally. Differences in yield between the highest yielding (Carioca) and the lowest (Moruna) 90-day cultivars were also due apparently to varietal differences in efficiency of conversion of nitrogen to economic matteri.e. seed, as well as to differences in capacity of genotypes for N₂-fixation. The work described here was in part supported by IAEA Research Contract No. RC/2084 UNDP/IAEA Project BRA/78/006     

Interaction between alfalfa cultivars and Rhizobium strains for nitrogen Fixation

Summary Two experiments were conducted in the greenhouse to study the interaction between alfalfa cultivars (Medicago sativa L. and M. falcata L.) and strains of Rhizobium meliloti Dang. for acetylene reduction rate, plant height and dry weights of shoot, root and whole plant. Fifteen alfalfa cultivars were inoculated with 10 strains of Rhizobium in Experiment I. Variance component analysis revealed that more than 30% of the total variance was due to alfalfa cultivars for acetylene reduction rate and 26% was accounted for by Rhizobium strains. More than 36% of the total variation was attributed to the interaction between alfalfa cultivars and Rhizobium strains for this character. Twenty-five host cultivars and 11 Rhizobium strains were included in Experiment II. The results also showed that the interaction of alfalfa cultivars and Rhizobium strains contributed the largest portion of the total variation for dry weights of shoot, root and whole plant and acetylene reduction rate. The results clearly demonstrated that the non-additive effects were the major component of variation for these characters associated with nitrogen fixation in alfalfa. Therefore, an effective way of improving nitrogen fixation in alfalfa is to select for a favourable combination of specific Rhizobium strains and alfalfa cultivars.     

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     

Competition for pulsed resources: an experimental study of establishment and coexistence for an arid-land grass

In arid environments, episodically-pulsed resources are important components of annual water and nutrient supply for plants. This study set out to test whether seedlings have an increased capacity for using pulsed resources, which might then improve establishment when in competition with older individuals. A second aim was to determine whether there is a trade-off in competitive strategies when resources are supplied continuously at low concentrations, or as pulses with pronounced inter-pulse periods. A glasshouse experiment used a target-neighbour design of size-asymmetric competition, with juveniles of Panicum antidotale (blue panicgrass) introduced into contrasting densities of adult plants. Stable isotopes of nitrogen were used for measuring plant resource uptake from pulses, and tolerance to inter-pulse conditions was assessed as the mean residence time (MRT) of nitrogen. A higher root/shoot ratio and finer root system enhanced the capacity of juveniles to use resources when pulsed, rather than when continuously supplied. Higher resource uptake during pulses improved the establishment of juvenile Panicum in mixed cultures with older individuals. However, a trade-off was observed in plant strategies, with juveniles showing a lower MRT for nitrogen, which suggested reduced tolerance to resource deficit during inter-pulse periods. Under field conditions, higher utilization of pulsed resources would lead to the improved seedling establishment of Panicum adjacent to “nurse” plants, whereas mature plants with well-developed roots, exploiting a greater soil volume, maintain more constant resource uptake and retention during inter-pulse periods.     

Interactions between cyanobacterium and fungus during 15N2-incorporation and metabolism in the lichen Peltigera canina

On following N₂-incorporation and subsequent metabolism in the lichen Peltigera canina using ¹⁵N as tracer, it was found, over a 30 min period, that greatest initial labelling was into NH ₄ ⁺ followed by glutamate and the amide-N of glutamine. Labelling of the amino-N of glutamine, aspartate and alanine increased slowly. Pulse-chase experiments using ¹⁵N confirmed this pattern. On inhibiting the GS-GOGAT pathway using l-methionine-dl-sulphoximine and azaserine, ¹⁵N enrichment of glutamate, alanine and aspartate continued although labelling of glutamine was undetectable. From this and enzymic data, NH ₄ ⁺ assimilation in the P. canina thallus appears to proceed via GS-GOGAT in the cyanobacterium and via GDH in the fungus; aminotransferases were present in both partners. The cyanobacterium assimilated 44% of the ¹⁵N₂ fixed; the remainder was liberated almost exclusively as NH ₄ ⁺ and then assimilated by fungal GDH.Abbreviations ADH alanine dehydrogenase - APT aspartate-pyruvate aminotransferase - AOA aminooxyacetate - GDH glutamate dehydrogenase - GOT glutamate-oxaloacetate aminotransferase - GOGAT glutamate synthase - GPT glutamate-pyruvate aminotransferase - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine     

Nutritional effects of non-steady state soil salinity on a salt-tolerant wheat cultivar

Summary Biological nitrogen fixation is considered an important trait of cowpeas (Vigna unguiculata (L.) Walp. var. California Blackeye No. 5) for economical production yet the process does not alone provide the quantity of nitrogen required by the plant for maximum productivity. Two experiments were undertaken to determine the potential of an increase in nodule mass and number of bacteroids resulting in increased nitrogen fixation. Cowpeas were grown in a glasshouse for 7 weeks under conditions forcing near total dependence on biological nitrogen fixation for growth. Nodule mass on the roots was varied by inoculating seeds with various ratios of effective and ineffective rhizobia that could be identified serologically and by the color of nodule formed. The results of both experiments demonstrated a linear relationship between total nodule mass formed by the effective rhizobia and quantity of nitrogen fixed. The regression coefficients were high in both experiments (r=0.99^** and 0.91^**). The relationship between total nitrogen fixed and total number of bacteroids of the effective strain was not consistent. In one experiment the regression coefficient was 0.93^** but in the other experiment it was 0.65^**. From these results it appears that there is good potential for increasing nitrogen fixation in cowpeas by increasing nodule mass. An increase in nodule mass would also result in an increase in the number of bacteroids.     

Comparison of two 15N labelling methods for assessing nitrogen rhizodeposition of pea

Two ¹⁵N labelling methods for assessing net rhizodeposition of nitrogen (N) in pea crop (Pisum sativum L.) were compared in the greenhouse and in the field: the cotton-wick (CW) and the split-root (SR) methods. Rhizodeposition is defined as the organic material lost from roots during their growth through the soil. CW is a method in which ¹⁵N urea was supplied to the plant in pulses via a wick threaded through the stem. In SR, the root system was divided between a hydroponic labelling compartment (LC) containing the labelling nutrient solution (1 or 5 mM ¹⁵NO₃–¹⁵NH₄) and a compartment filled with soil in which the amount of ¹⁵N rhizodeposition was assessed. The percentage of N derived from rhizodeposition (%Ndfr), was used to calculate the amount of N rhizodeposition which was obtained from the ratio of atom % ¹⁵N excess of the soil : atom % ¹⁵N excess of the roots. Above ground parts in the field accumulated markedly more dry matter and N than in the greenhouse, regardless of the labelling method. ¹⁵N enrichments of above ground parts were higher than those of roots recovered from the soil. Results indicated that amount of ¹⁵N applied to plants were lower in SR than in CW. Additionally, LC roots of SR tended to retain large amounts of ¹⁵N. As a consequence, atom % ¹⁵N excess of roots was less than 1% in SR, whereas most values varied from 1% to 4% in CW. However, relationships between enrichments of the soil and of the roots were different in SR and CW. It was not possible to compare the Ndfr:root-N ratio between the two methods, but the ratio of Ndfr:plant-N was found to be 10% higher in SR than in CW. Finally, relative to total plant-N, the total contribution of below ground parts to the N pool of the soil reached 22–25% at maturity for the two methods. From our experiments, we could not conclude that one method is better than the other for estimating either net rhizodeposition of N or the contribution of a pea plant to the soil N pool. However, CW is easier to adapt and monitor under field conditions than SR.     

Effects of plant breeding and selection on yields and nitrogen fixation in soybeans under two soil nitrogen regimes

Summary Soybeans (Glycine max (L.) Merr.) have a high N requirement which is fulfilled by soil N uptake and N₂-fixation. This study was concerned with the effects of past yield selection on N₂-fixation in soybeans.The soybean cultivars, Lincoln, Shelby, and Williams, which represent successive improvements in the Lincoln germplasm, and a non-nodulating control were planted in a soil containing¹⁵N labelled organic matter. Two replications occurred on soil previously cropped to alfalfa and two on soil previously cropped to soybeans. Plants were harvested at five growth stages and leaf area, plant weight, total N, and atom percent¹⁵N were determined. Mature grain was harvested and yield components were also determined, as well as the total N and¹⁵N content.Cultivar differences in total dry matter were only evident at physiological maturity, when Williams contained the greatest dry matter. Williams exhibited the longest period of seed formation and seed fill and also had the highest grain yield which resulted from a larger weight per seed.The N content of the cultivars did not vary until physiological maturity when Williams contained the highest percent N. The quantity of N fixed at physiological maturity was highest for Williams and lowest for Lincoln. Fixed N contained in the harvested grain was greater for Williams than for the other two cultivars. The fraction of the total plant N derived from fixation was not greatly affected by cultivar and all cultivars acquired an average of 50% of their total N through N₂-fixation.Previous cropping history greatly affected the quantity of N fixed and the fraction of the total plant N derived from fixation. Soybeans following soybeans were more dependent upon N₂-fixation than soybeans following alfalfa with the former deriving 65% of the total plant N from fixation and the latter only 32%. These soybean cultivars apparently utilized soil N first and then used N₂-fixation to satisfy their N requirement.The past selection for higher yield has resulted in soybean cultivars with improved capacities to fix atmospheric N₂ and an improved ability to take up available soil N.     

The role of root-associatedKlebsiella pneumoniae in the nitrogen nutrition ofPoa pratensis andTriticum aestivum as estimated by the method of15N isotope dilution

Summary The technique of¹⁵N isotope dilution was used to verify that nitrogen was fixed and transferred to the plant byKlebsiella pneumoniae strain Pp in association withPoa pratensis orTriticum aestivum. Surface sterilized, sprouting seeds were inoculated withK. pneumoniae and grown in sand in modified Leonard jars. Potassium nitrate enriched with¹⁵N was used to provide N concentrations ranging from 10–40 mg Nl^–1 nutrient solution. After 10–18 weeks the shoots and roots were analyzed separately for dry matter, N content, total N, and atom %¹⁵N excess. The acetylene reduction technique was used to test for the presence of N₂-fixing organisms on the roots. The data from¹⁵N isotope dilution demonstrated that up to 33.8% of N in the shoots ofP. pratensis and 15.9% in those ofT. aestivum were derived from associative N₂ fixation byK. pneumoniae. In most experiments the dry matter yield, N content, and total N yield of the shoots ofP. pratensis were increased byK. pneumoniae inoculation, whereas inoculation had no significant effect on the dry matter yield, N content or total N of the shoots ofT. aestivum.     

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.     

Symbiotic N2 fixation in pea and field bean estimated by15N fertilizer dilution in field experiments with barley as a reference crop

Summary The total amount of nitrogen derived from symbiotic nitrogen fixation in two pea and one field bean cultivar, supplied with 50 kg N ha⁻¹ at sowing (‘starter’-N), was estimated to 165, 136, and 186 kg N ha⁻¹, respectively (three-year means). However, estimates varied considerably between the three years. At the full bloom/flat pod growth stage from 30 to 59 per cent of total N₂ fixation had taken place. The proportion of total N derived from N₂ fixation at maturity was higher in seeds than in vegetative plant parts and amounted to 59.5, 51.3 and 66.3 per cent of total above-ground plant N in the two pea cultivars and field bean, respectively (three-year means). The recovery of fertilizer N was 62.2, 70.2, 52.1, and 69.5 per cent in the two pea cultivars, field bean and barley, respectively. Growth analysis indicated that barley did not meet the claims for an ideal reference crop in the¹⁵N fertilizer dilution technique for estimating N₂ fixation in pea and field bean. ‘Starter’-N neither increased the seed yield nor the N content of the grain legumes.     

根瘤菌共生固氮能力的进化模式

根瘤菌-豆科植物共生固氮体系对农业的可持续性发展至关重要,也是研究原核与真核生物互利共生的模式体系之一。长期以来,根瘤菌共生固氮相关研究主要集中在结瘤因子与固氮酶合成及调控等少数关键基因,但仅获得这些关键基因却不能保证细菌获得结瘤固氮能力。随着比较和功能基因组学的快速发展和应用,越来越多的研究发现根瘤菌使用了很多系统发育分支特异的遗传机制与豆科植物建立有效的共生关系,进一步揭示了双方互利共生的复杂性。本综述总结了近年来比较基因组学、遗传学以及实验进化等方面的相关研究进展,在此基础上讨论根瘤菌共生固氮能力的进化模式。     

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.     

An indirect method for estimating 15N isotope fractionation during nitrogen fixation by a legume under field conditions

A new technique is proposed for measuring ¹⁵N isotope fractionation during N fixation that obviates some of the possible disadvantages of existing methods. Accurate calculation of N fixation by legumes using the ¹⁵N natural abundance technique requires a value for the isotopic composition of fixed N as an input. Isotopic fractionation in fixed N in legumes has usually been measured using N- free solution culture but results can vary with Rhizobium strain and growth conditions. The proposed method avoids these problems and can be used as an integral part of a field experiment for evaluating N fixation.The technique is essentially a process of adjusting values of ¹⁵ N for fixed N until % N fixation calculated by the ¹⁵N natural abundance method best matches % N fixation estimated by the ¹⁵N enrichment method. The use of high % N fixation values improves the sensitivity and reliability of the method.A field evaluation of this comparison technique using chickpea (Cicer arietinum L.) provided a ¹⁵N isotope fractionation factor (–2.37) for fixed N close to that obtained by N-free solution culture methods (–2.10). The availability of these two independent techniques allowed mutual corroboration of estimates of ¹⁵N isotope fractionation during N fixation.

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Forests losing large quantities of nitrogen have elevated 15N:14N ratios

Summary Urea (U) and ammonium nitrate (AN) had been applied to a Scots pine (Pinus sylvestris L.) forest in northern Sweden for 18 consecutive years at four doses resulting in total N applications ranging from 0 to 1980 kg ha^–1. The ¹⁵N abundance ( ¹⁵N) of the grass Deschampsia flexuosa (L.) Trin. increased linearly (from –0.7 to 11.0) with application rate in the case of U. The response to AN was in the same direction but smaller. While others have shown that the initial response of nitrogen-limited systems to additions of N is a change of ¹⁵N abundance towards that of added N, this study shows that further and excessive additions leads to a retention of ¹⁵N. Monitoring ¹⁵N abundance over time in dose-response trials of this type thus opens new possibilities to estimate critical loads of N and the point of nitrogen saturation.     

The fate of15N labeled nitrogen applied to mature citrus trees

Summary The efficiency and balance of nitrogen from one year's application was studied in a long-term fertigation experiment. Enriched nitrogen fertilizer, K¹⁵NO₃, was applied to a 22-year-old Shamouti orange tree with a history of high N applications (N₃) and to an N-starved tree (N₁). The distribution of N in the different parts of the trees and in the soil was determined after the experimental trees were excavated. Similar total recovery of the labeled fertilizer N was found in the trees and soil in both treatments (N₁−61.7% N₃−56%). However, the distribution between tree and soil was different. The amount of recovered residual fertilizer in the soil was much larger in the N₃ treatment than in N₁. The highest percentage of fertilizer N was found in the new organs,i.e. fruits, twigs and leaves. The roots and branches took up only 6–14% from the labeled fertilizer. Only 20.9% of the leaf N and 23.4% of the fruit N in the N₃ tree originated in the labeled fertilizer, indicating translocation of N from older parts of the tree to new growth. Evidence was found of storage of N in the wooded branches, while the roots contained a surprisingly small part of labeled fertilizer. Contribution 1599E.     

Fixation of [13N]N2 and transfer of fixed nitrogen in the Anthoceros-Nostoc symbiotic association

The initial product of fixation of [¹³N]N₂ by pure cultures of the reconstituted symbiotic association between Anthoceros punctatus L. and Nostoc sp. strain ac 7801 was ammonium; it accounted for 75% of the total radioactivity recovered in methanolic extracts after 0.5 min and 14% after 10 min of incubation. Glutamine and glutamate were the primary organic products synthesized from [¹³N]N₂ after incubation times of 0.5–10 min. The kinetics of labeling of these two amino acids were characteristic of a precursor (glutamine) and product (glutamate) relationship. Results of inhibition experiments with methionine sulfoximine (MSX) and diazo-oxonorleucine were also consistent with the assimilation of N₂-derived NH ₄ ⁺ by Anthoceros-Nostoc through the sequential activities of glutamine synthetase (EC 6.3.1.2) and glutamate synthase (EC 1.4.7.1), with little or no assimilation by glutamate dehydrogenase (EC 1.3.1.3). Isolated symbiotic Nostoc assimilated exogenous ¹³NH ₄ ⁺ into glutamine and glutamate and their formation was inhibited by MSX, indicating operation of the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway: However, relative to free-living cultures, isolated symbiotic Nostoc assimilated 80% less exogenous ammonium into glutamine and glutamate, implying that symbiotic Nostoc could assimilate only a fraction of N₂-derived NH ₄ ⁺ . This implication was tested by using Anthoceros associations reconstituted with wild-type or MSX-resistant strains of Nostoc incubated with [¹³N]N₂ in the presence of MSX. The results of these experiments indicated that, in situ, symbiotic Nostoc assimilated about 10% of the N₂-derived NH ₄ ⁺ and that NH ₄ ⁺ was made available to Anthoceros tissue where it was apparently assimilated by the GS-GOGAT pathway. Since less than 1% of the fixed N₂ was lost to the suspension medium, it appears that transfer of NH ₄ ⁺ from symbiont to host tissue was very efficient in this extracellular symbiotic association.Abbreviations DON 6-diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-dl-sulfoximine