Ectomycorrhizal transfer of amino acid-nitrogen to the alpine sedge Kobresia myosuroides

Previous work in the Colorado alpine ecosystem has shown that amino acids are a potentially important N source for the sedge, Kobresia myosuroides . This plant is the only known sedge to harbour associations with ectomycorrhizal fungi. The aim of the present work was to test the hypothesis that these ectomycorrhizas transfer N from amino acids in the soil solution to the host plant, and thereby have an important role in the N nutrition of this species. We used a two-chamber system (rhizoboxes) in which K. myosuroides plants were separated from a soil chamber by nylon mesh that allowed fungal hyphae, but not plant roots, to cross it. Injections of [¹⁵N, 2-¹³C]glycine were made into the soil chamber. The hyphal crossings on half of the rhizoboxes were regularly disrupted to control for leakage of label across the barrier. Plants in the intact rhizoboxes showed significantly higher ¹⁵N enrichment than those in controls, and mycorrhizal root tips were significantly more enriched than bulk roots. The mycorrhizas transferred an average of 1.3% of the added ¹⁵N label to plants, a figure comparable to those obtained in previous studies in which plant roots were directly exposed to label. We conclude that fungal associations have an important role in the N nutrition of K. myosuroides by transferring N from amino acids to their hosts.     

Nitrogen assimilation in Lolium perenne colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum

To investigate nitrogen assimilation in Lolium perenne L. colonized by the arbuscular mycorrhizal (AM) fungus Glomus fasciculatum (Thax. sensu Gerd.), nitrate uptake, key enzyme activities, and ¹⁵N incorporation into free amino acids were measured. After a 4-h labelling period with [¹⁵N]nitrate, ¹⁵N content was higher in roots and shoots of AM-plants than in those of control plants. Glutamine synthetase (GS) and nitrate reductase (NR) activities were increased in shoots of AM-plants, but not in roots. More label was incorporated into amino acids in shoots of AM plants. Glutamine, glutamate, alanine and γ-aminobutyric acid were the major sinks for ¹⁵N in roots and shoots of control and AM plants. Interactions between mycorrhizal colonization, phosphate and nitrate nutrition and NR activity were investigated in plants which received different amounts of phosphate or nitrate. In shoots of control plants, NR activity was not stimulated by high levels of phosphate nutrition but was stimulated by high levels of nitrate. At 4 m M nitrate in the nutrient solution, NR activity was similar in control and AM plants. We concluded that mycorrhizal effects on nitrate assimilation are not mediated via improved phosphate nutrition, but could be due to improved nitrogen uptake and translocation.     

Improvement of the 15N dilution method for estimation of absorption of NOx by plants supplied with 15N-labelled fertilizer

To develop further the methods for estimation of NOx absorption by plants supplied with ¹⁵N-labelled fertilizer, we proposed a new calculation method, total N fixed method (TNF), and compared with the ¹⁵N dilution method and the classical mass balance method (MB).
Hydroponically grown soybean plants were supplied with ¹⁵N-labelled nitrate and exposed to 200–250 nl l⁻¹ NO₂ for 7 d. The proportions of the N derived from NO₂ to total N in exposed plants were estimated by the three methods.
The reported rates of NO₂ absorption by several plant species, estimated by the ¹⁵N dilution method, were recalculated using the TNF method. The results of the two methods were compared and showed that: (1) The ¹⁵N dilution method overestimated the content of NO₂-N in exposed plants compared with the MB method whilst the TNF method produced estimations of NO₂-N closer to those by the MB method when the plants were supplied with 5 m M nitrate. (2) The differences in estimations between the MB method and either the ¹⁵N dilution method or the TNF method increased with decreasing supply of ¹⁵N-labelled nitrate to roots.     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.     

Effect of plant nitrogen status on the contribution of arbuscular mycorrhizal hyphae to plant nitrogen uptake

The contribution of hyphae of Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 107) to the acquisition of mineral nitrogen by Triticum aestivum L. cv. Hano (wheat) was tested under conditions of low P and high N (+N−P) or low N (−N−P). Mycorrhizal colonisation increased the shoot dry weight and plant tissue concentrations of P and cations. However, N tissue concentrations of mycorrhizal plants were not increased, although nitrate reductase activities were significantly higher (in vivo activity) in +N−P mycorrhizal compared to non-mycorrhizal roots. Severe plant N deficiency reduced the percentage root length colonised (but not the percentage viable colonisation), hyphal length, total 15 N uptake by hyphae and dry weight of mycorrhizal plants. Although mycorrhizal colonisation did not affect the overall plant N status, hyphae transported 1% (−N−P) and 7% (+N−P) of the 15 N-labelled NH₄NO₃ to mycorrhizal plants over 48 h. The higher rate of hyphal N uptake was apparently related to the more extensive hyphal growth at the higher level of plant N supply. However, the hyphal N supply was not sufficiently high to sustain adequate N nutrition of the plants supplied with very low amounts of N to the roots. Conversely, a sufficient N supply to the roots was important for the development of an extensive mycelium.     

Global patterns of foliar nitrogen isotopes and their relationships with climate, mycorrhizal fungi, foliar nutrient concentrations, and nitrogen availability

Ratios of nitrogen (N) isotopes in leaves could elucidate underlying patterns of N cycling across ecological gradients. To better understand global-scale patterns of N cycling, we compiled data on foliar N isotope ratios (δ¹⁵N), foliar N concentrations, mycorrhizal type and climate for over 11 000 plants worldwide. Arbuscular mycorrhizal, ectomycorrhizal, and ericoid mycorrhizal plants were depleted in foliar δ¹⁵N by 2‰, 3.2‰, 5.9‰, respectively, relative to nonmycorrhizal plants. Foliar δ¹⁵N increased with decreasing mean annual precipitation and with increasing mean annual temperature (MAT) across sites with MAT ≥ −0.5°C, but was invariant with MAT across sites with MAT < −0.5°C. In independent landscape-level to regional-level studies, foliar δ¹⁵N increased with increasing N availability; at the global scale, foliar δ¹⁵N increased with increasing foliar N concentrations and decreasing foliar phosphorus (P) concentrations. Together, these results suggest that warm, dry ecosystems have the highest N availability, while plants with high N concentrations, on average, occupy sites with higher N availability than plants with low N concentrations. Global-scale comparisons of other components of the N cycle are still required for better mechanistic understanding of the determinants of variation in foliar δ¹⁵N and ultimately global patterns in N cycling.     

Nitrogen and phosphorus acquisition by the mycelium of the ectomycorrhizal fungus Paxillus involutus and its effect on host nutrition

The contribution of the extramatrical mycelium to N and P nutrition of mycorrhizal Norway spruce ( Picea abies (L.) Karst.) was investigated. Seedlings either inoculated with Paxillus involutus (Batsch) Fr. or non-mycorrhizal were grown in a two compartment sand culture system where hyphae were separated from roots by a 45 μm nylon net. Nutrient solution of the hyphal compartment contained either 1.8 m m NH₄⁺ and 0.18 m m H₂PO₄⁻ or no N and P. Aluminium added to the hyphal compartment as a tracer of mass flow was not detected in the plant compartment, indicating that measurements of N and P transfer by the mycelium were not biased by solute movement across the nylon net.
The addition of N and P to the hyphal compartment markedly increased dry weight, N and P concentration and N and P content of mycorrhizal plants. Calculating uptake from the difference in input and output of nutrient in solution confirmed a hyphal contribution of 73% and 76% to total N and P uptake, respectively. Hyphal growth was increased at the site of nutrient solution input.     

Transfer of nitrogen from a tropical legume tree to an associated fodder grass via root exudation and common mycelial networks

Symbiotic dinitrogen fixation by legume trees represents a substantial N input in agroforestry systems, which may benefit the associated crops. Applying ¹⁵N labelling, we studied N transfer via common mycelial networks (CMN) and root exudation from the legume tree Gliricidia sepium to the associated fodder grass Dichantium aristatum . The plants were grown in greenhouse in shared pots in full interaction (treatment FI) or with their root systems separated with a fine mesh that allowed N transfer via CMN only (treatment MY). Tree root exudation was measured separately with hydroponics. Nitrogen transfer estimates were based on the isotopic signature of N ( δ ¹⁵N) transferred from the donor. We obtained a range for estimates by calculating transfer with δ ¹⁵N of tree roots and exudates. Nitrogen transfer was 3.7–14.0 and 0.7–2.5% of grass total N in treatments FI and MY, respectively. Root δ ¹⁵N gave the lower and exudate δ ¹⁵N the higher estimates. Transfer in FI probably occurred mainly via root exudation. Transfer in MY correlated negatively with grass root N concentration, implying that it was driven by source-sink relationships between the plants. The range of transfer estimates, depending on source δ ¹⁵N applied, indicates the need of understanding the transfer mechanisms as a basis for reliable estimates.     

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR⁻) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH₄¹⁵NO₃ medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas ¹⁵N enrichment values indicated that 13–27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

Use of different plant parts to study N2 fixation with 15N techniques in field-grown red clover (Trifolium pratense)

Red clover, Trifolium pratense L., is the dominant forage legume in Sweden and is usually harvested twice per year, once in June and once in August. Two ¹⁵N-based methods –¹⁵N isotopic dilution (ID) and ¹⁵N natural abundance (NA) – were used to study N₂ fixation from spring until first harvest in late June, from first to second harvest in late August, and from second harvest until first frost in autumn in Umeå, Sweden. The material studied comprized three neighbouring fields carrying a first year ley, a second year ley and a third year ley. For the ¹⁵N ID method, small amounts of highly enriched ¹⁵N-nitrate were added to experimental plots. The non-legumes in the plots, essentially Phleum pratense L. together with Festuca pratensis L., served as reference plants for both the ID and ¹⁵N NA measurements. Dry matter, N and ¹⁵N were separately analysed in leaves (laminae), stems (including petioles), stubble and roots. The proportion of N derived from air (pNdfa) was then calculated for each plant part and for whole plants. Estimates of the proportion of N derived from N₂ fixation (pNdfa) were always very high, usually ≥0.8. Generally, estimates of pNdfa obtained by the ID and NA methods were similar, but the ID method gave higher estimates of pNdfa than the NA method when the highest N₂ fixation levels were recorded, at the August harvest. Regression analyses suggest that estimates of pNdfa in leaves could provide useful indications of pNdfa in shoots and whole T. pratense plants, thus avoiding the need for time-consuming root analyses.     

Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata ssp. ambigua

The influence of three organic compounds and bakers' dry yeast on growth of external mycelium and phosphorus uptake of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith (BEG 87) was examined. Two experiments were carried out in compartmentalized growth systems with root-free sand or soil compartments. The sand and soil in the root-free compartments were left untreated or uniformly mixed with one of the following substrates (0.5 mg g⁻¹ soil): bakers' dry yeast, bovine serum albumin, starch or cellulose. Effects of the organic substrates on biomass and hyphal length density of the arbuscular mycorrhizal fungus were examined by using specific fatty acid signatures in combination with direct microscopy. Micro-organisms other than the arbuscular mycorrhizal fungus were measured by fatty acid signatures, and radioactive ³³P labelling of the root-free soil was used to determine arbuscular mycorrhizal hyphal phosphorus uptake. In general, hyphal growth of G. intraradices was enhanced by yeast and bovine serum albumin, whereas the carbon sources, starch and cellulose, depressed fungal growth. By analysing the fatty acid 16:1ω5 from phospholipids (indicating mycelium) and neutral lipids (indicating storage structures) it was shown that increased fungal growth due to yeast was mainly in vegetative hyphae and less in storage structures. Arbuscular mycorrhizal hyphal phosphorus uptake was decreased by cellulose, but unaffected by the other substrates compared with the control. This means that both growth and phosphorus transport by the arbuscular mycorrhizal fungus were decreased under cellulose treatment. However, the composition of the microbial community varied under different substrate conditions indicating a possible interactive component with arbuscular mycorrhizal hyphal growth and phosphorus uptake.     

An arbuscular mycorrhizal inoculum enhances root proliferation in, but not nitrogen capture from, nutrient-rich patches in soil

Most work on root proliferation to a localized nutrient supply has ignored the possible role of mycorrhizal fungi, despite their key role in nutrient acquisition. Interactions between roots of Plantago lanceolata , an added arbuscular mycorrhiza (AM) inoculum and nitrogen capture from an organic patch ( Lolium perenne shoot material) dual-labelled with ¹⁵N and ¹³C were investigated, to determine whether root proliferation and nitrogen (N) capture was affected by the presence of AM fungi. Decomposition of the organic patch in the presence and absence of roots peaked in all treatments at day 3, as shown by the amounts of ¹³CO₂ detected in the soil atmosphere. Plant N concentrations were higher in the treatments with added inoculum 10 d after patch addition, but thereafter did not differ among treatments. Plant phosphorus concentrations at the end of the experiment were depressed by the addition of the organic residue in the absence of mycorrhizal inoculum. Although uninoculated plants were also colonized by mycorrhizal fungi, colonization was enhanced at all times by the added inoculum. Addition of the AM inoculum increased root production, observed in situ by the use of minirhizotron tubes, most pronouncedly within the organic patch zone. Patch N capture by the end of the experiment was c . 7.5% and was not significantly different as a result of adding an AM inoculum. Furthermore, no ¹³C enrichments were detected in the plant material in any of the treatments showing that intact organic compounds were not taken up. Thus, although the added AM fungal inoculum benefited P. lanceolata seedlings in terms of P concentrations of tissues it did not increase total N capture or affect the form in which N was captured by P. lanceolata roots.     

Plant growth-altering effects of Azospirillum brasilense and Bacillus C–11–25 on two wheat cultivars

The effect of Azospirillum brasilense Cd, Bacillus C–11–25, indole acetic acid, gibberellic acid and cytokinin on plant growth characteristics of two wheat ( Triticum aestivum L. emend Thell) cultivars was studied under laboratory and greenhouse conditions. Responses of wheat plants to bacterial inoculation were similar to those caused by the addition of gibberellic acid in growth pouches. Chester and Fielder wheat varieties differed in responses to the bacteria and hormone additions. When added to growth pouches, bacterial culture filtrates and dead bacterial cells caused plant growth responses similar to those caused by the addition of live cells. Bacteria and hormone additions resulted in increased permeability of Fielder wheat to ¹⁵Nlabelled nitrate, and decreased nitrate permeability of Chester wheat. Bacterial inoculation of soil in pots caused ¹⁵N isotope dilution in Fielder but not in Chester wheat. Hormone addition to pots caused isotope dilution in Chester wheat. It appeared that genetic differences between cultivars affected plant growth responses. The accuracy of estimates of N₂ fixation by associative bacteria based on ¹⁵N isotope dilution calculations may be reduced if control plants differ in plant response to these bacteria.     

Sources of proline-nitrogen in water-stressed soybean (Glycine max). II. Fate of 15N-labelled protein

The absorption of nitrate, protein metabolism and the source of nitrogen for proline synthesis were studied in soybean ( Glycine max L. cv. Akisengoku) with ¹⁵N tracer technique under water stress conditions. The absorption of nitrate was sensitive to water stress and the flow of nitrate into the leaves completely ceased under severe stress conditions. Net protein loss from the water-stressed leaves was attributable to both a decrease in synthetic activity and a stimulation of protein degradation. Proline and asparagine accumulated extensively in the severely water-stressed plant tissues, especially in the younger green leaves. Fifty four % of the loss of leaf protein-¹⁵N during the stress period was balanced by a gain in ¹⁵N in the free amino acids, 41% being found in proline and asparagine. The increase in ¹⁵N content of the free proline was 3 times greater than the decrease in ¹⁵N content of the protein-bound proline in the leaf. The results indicate that the accumulation of proline in response to water stress was caused by enhanced synthesis and that the nitrogen source for this proline is the leaf protein. The possible association of these findings with stress tolerance is discussed.     

Competition for tracer 15N in tussock tundra ecosystems

The objectives of this study were to assess the roles of plant species, time, and site on competition for tracer ¹⁵N (without carrier) in tussock tundra ecosystems. Six experimental sites were located in northern Alaska. After one year across the experimental sites, the recovery of ¹⁵N by litter (11.3–16.3%) and mosses (5.4–16.4%) was significantly greater than for aboveground vascular plants (2.6-5.0%). ¹⁵N recoveries by tundra vascular plants (2.6–5.0%) were low when compared to forest trees (9–25%) which suggest that competition for nitrogen is particularly severe in these cold-dominated tundra ecosystems. There were no significant differences among sites in ¹⁵N recoveries by vascular plants, by mosses, or by litter. There was a statistically significant decline in ¹⁵N recovery with time for Vaccinium vitis-idaea and Eriophorum vaginatum between the second and third year. The shallow rooted Vaccinium vitis-ideae was more highly labeled than the deep rooted Eriophorum vaginatum . Nearness to the source of the applied ¹⁵N played a critical role in competition for surface applied nitrogen.     

Suppression of ammonium uptake by nitrogen supply and its relief during nitrogen limitation

Barley ( Hordeum vulgare , cv. Triumph), wheat ( Triticum aestivum , cv. Kleiber) and oat ( Avena sativa , cv. Tarok) were grown until day 20 in nitrate-containing solutions or in nitrogen-free solutions for periods up to 8 days immediately prior to day 20. They then were exposed for 4 h to complete, nitrate-free solutions containing 0.5 or 2.0 mM ammonium (98 atom%¹⁵N). In all 3 species in 2 experiments, net ammonium uptake was low in plants grown continuously in nitrate, and increased 3 to 4-fold with increasing nitrogen deprivation. Charge balance during net ammonium uptake was largely maintained by the sum of net potassium and net proton efflux. Variations in root ammonium concentration at the time of exposure to the ammonium solutions revealed no consistent pattern with net ammonium uptake, implying that a product of ammonium assimilation may serve as a negative effector for the uptake process. In nitrogen-replete plants, and in those deprived of nitrogen for 2 days, the amounts of endogenous ¹⁴N-ammonium recovered in the ambient ¹⁵N-ammonium solution during the 4-h uptake period were greater than the initial amounts of ¹⁴N-ammonium present in the root tissue. Significant generation of ¹⁴N-ammonium from endogenous organic nitrogen sources was thus evident in all 3 species.     

Use of 15N stable isotope to quantify nitrogen transfer between mycorrhizal plants

Aims Mycorrhizas (fungal roots) play vital roles in plant nutrient acquisition, performance and productivity in terrestrial ecosystems. Arbuscular mycorrhizas (AM) and ectomycorrhizas (EM) are mostly important since soil nutrients, including NH₄⁺, NO₃^? and phosphorus, are translocated from mycorrhizal fungi to plants. Individual species, genera and even families of plants could be interconnected by mycorrhizal mycelia to form common mycorrhizal networks (CMNs). The function of CMNs is to provide pathways for movement or transfer of nutrients from one plant to another. In the past four decades, both ¹⁵N external labeling or enrichment (usually expressed as atom%) and ¹⁵N naturally occurring abundance (δ¹⁵N, ‰) techniques have been employed to trace the direction and magnitude of N transfer between plants, with their own advantages and limitations.     

Anaerobic nitrate reduction to ammonium in two strains isolated from coastal marine sediment: A dissimilatory pathway

Abstract: A total of 28 nitrate-reducing bacteria were isolated from marine sediment (Mediterranean coast of France) in which dissimilatory reduction of nitrate to ammonium (DRNA) was estimated as 80% of the overall nitrate consumption. Thirteen isolates were considered as denitrifiers and ten as dissimilatory ammonium producers. ¹⁵N ammonium production from ¹⁵N nitrate by an Enterobacter sp. and a Vibrio sp., the predominant bacteria involved in nitrate ammonification in marine sediment, was characterized in pure culture studies. For both strains studied, nitrate-limited culture (1 mM) produced ammonium as the main product of nitrate reduction (> 90%) while in the presence of 10 mM nitrate, nitrite was accumulated in the spent media and ammonia production was less efficient. Concomitantly with the dissimilation of nitrate to nitrite and ammonium the molar yield of growth on glucose increased. Metabolic products of glucose were investigated under different growth conditions. Under anaerobic conditions without nitrate, ethanol was formed as the main product; in the presence of nitrate, ethanol disappeared and acetate increased concomitantly with an increased amount of ammonium. These results indicate that nitrite reduction to ammonium allows NAD regeneration and ATP synthesis through acetate formation, instead of ethanol formation which was favoured in the absence of nitrate.