A nitrogen fertilization field study of carbon-13 and nitrogen-15 transfers in ectomycorrhizas of Pinus sabiniana

Ectomycorrhizal (EM) fungi form relationships with higher plants; plants transfer C to fungi, and fungi transfer nutrients to their host. While evidence indicates that this interaction is largely mutualistic, less is known about how nutrient supply and EM associates may alter C and nutrient exchanges, especially in intact plant-soil-microbe systems in the field. In a dual-labeling experiment with N fertilization, we used C and N stable isotopes to examine in situ transfers in EM pine trees in a Pinus sabiniana woodland in northern California. We added ¹⁵NH₄SO₂ and ¹³CO₂ to track ¹³C transfer from pine needles to EM roots and ¹⁵N transfer from soil to EM roots and pine needles. Transfers of ¹³C and ¹⁵N differed with EM morphotype and with N fertilization. The brown morphotype received the least C per unit of N transferred (5:1); in contrast red and gold morphotypes gained more C and transferred less N (17:1 and 25:1, respectively). N fertilization increased N retention by ectomycorrhizas (EMs) but did not increase N transfer from EMs to pine needles. Therefore N fertilization positively affected both nutrient and C gains by EMs, increasing net C flows and N retention in EMs. Our work on intact and native trees/EM associations thereby extends earlier conclusions based on pot studies with young plants and culturable EM fungi; our results support the concept that EM-host relationships depend on species-level differences as well as responses to soil resources such as N.     

Heterotrophic n(2) fixation and distribution of newly fixed nitrogen in a rice-flooded soil system

Rice (Oryza sativa L.) plants growing in pots of flooded soil were exposed to a ¹⁵N₂-enriched atmosphere for 3 to 13 days in a gas-tight chamber. The floodwater and soil surface were shaded with a black cloth to reduce the activity of phototrophic N₂-fixing micro-organisms. The highest ¹⁵N enrichments were consistently observed in the roots, although the total quantity of ¹⁵N incorporated into the soil was much greater. The rate of ¹⁵N incorporation into roots was much higher at the heading than at the tillering stage of growth. Definite enrichments were also found in the basal node and in the lower outer leaf sheath fractions after 3 days of exposure at the heading stage. Thirteen days was the shortest time period in which definite ¹⁵N enrichment was observed in the leaves and panicle. When plants were exposed to ¹⁵N₂ for 13 days just before heading and then allowed to mature in a normal atmosphere, 11.3% of the total ¹⁵N in the system was found in the panicles, 2.3% in the roots, and 80.7% in the subsurface soil. These results provide direct evidence of heterotrophic N₂ fixation associated with rice roots and the flooded soil and demonstrate that part of the newly fixed N is available to the plant.     

First evidences that the ectomycorrhizal fungus Paxillus involutus mobilizes nitrogen and carbon from saprotrophic fungus necromass

Fungal succession in rotting wood shows a surprising abundance of ectomycorrhizal (EM) fungi during the late decomposition stages. To better understand the links between EM fungi and saprotrophic fungi, we investigated the potential capacities of the EM fungus Paxillus involutus to mobilize nutrients from necromass of Postia placenta, a wood rot fungus, and to transfer these elements to its host tree. In this aim, we used pure cultures of P. involutus in the presence of labelled Postia necromass (¹⁵N/¹³C) as nutrient source, and a monoxenic mycorrhized pine experiment composed of labelled Postia necromass and P. involutus culture in interaction with pine seedlings. The isotopic labelling was measured in both experiments. In pure culture, P. involutus was able to mobilize N, but C as well, from the Postia necromass. In the symbiotic interaction experiment, we measured high ¹⁵N enrichments in all plant and fungal compartments. Interestingly, ¹³C remains mainly in the mycelium and mycorrhizas, demonstrating that the EM fungus transferred essentially N from the necromass to the tree. These observations reveal that fungal organic matter could represent a significant N source for EM fungi and trees, but also a C source for mycorrhizal fungi, including in symbiotic lifestyle.     

Assimilation and translocation of nitrogen and carbon in Curcuma alismatifolia Gagnep

Curcuma or Siam tulip (Curcuma alismatifolia Gagnep.) is an ornamental flowering plant with two underground storage organs, rhizomes and storage roots. Characteristics of N and C assimilation and transport in curcuma were investigated. The plants were treated with ¹⁵NH₄⁺ + ¹⁵NO₃^? and ¹³CO₂ at 10, 13 or 21 weeks after planting. Plants were sampled at several stages up to 32 weeks. The C stored in old storage roots was used rapidly during the first 10 weeks; after which N stored in old rhizomes and old storage roots were used. The daily gain in C depending on photosynthesis was remarkably high between 10 and 21 weeks. However, the daily gain in N was relatively constant throughout the growth period. The ¹⁵N absorbed at 10 weeks was initially accumulated in leaves and roots, but some was transported to flowering organs at 13 weeks. At harvest, 41% of ¹⁵N was recovered in new rhizomes and 17% in new storage roots. After ¹³CO₂ exposure at 10 and 13 weeks, the distribution of ¹³C among organs was relatively constant in subsequent stages. When given ¹³CO₂ at 21 weeks, a large amount of labelled C was recovered in new storage roots and new rhizomes at harvest. Both new rhizomes and new storage roots stored N and C, however, rhizomes played a more important role in supplying N, while storage roots provided C.     

Ectomycorrhizal Communities on the Roots of Two Beech (Fagus sylvatica) Populations from Contrasting Climates Differ in Nitrogen Acquisition in a Common Environment

Beech (Fagus sylvatica), a dominant forest species in Central Europe, competes for nitrogen with soil microbes and suffers from N limitation under dry conditions. We hypothesized that ectomycorrhizal communities and the free-living rhizosphere microbes from beech trees from sites with two contrasting climatic conditions exhibit differences in N acquisition that contribute to differences in host N uptake and are related to differences in host belowground carbon allocation. To test these hypotheses, young trees from the natural regeneration of two genetically similar populations, one from dryer conditions (located in an area with a southwest exposure [SW trees]) and the other from a cooler, moist climate (located in an area with a northeast exposure [NE trees]), were transplanted into a homogeneous substrate in the same environment and labeled with ¹³CO₂ and ¹⁵NH₄⁺. Free-living rhizosphere microbes were characterized by marker genes for the N cycle, but no differences between the rhizospheres of SW or NE trees were found. Lower ¹⁵N enrichment was found in the ectomycorrhizal communities of the NE tree communities than the SW tree communities, whereas no significant differences in ¹⁵N enrichment were observed for nonmycorrhizal root tips of SW and NE trees. Neither the ectomycorrhizal communities nor the nonmycorrhizal root tips originating from NE and SW trees showed differences in ¹³C signatures. Because the level of ¹⁵N accumulation in fine roots and the amount transferred to leaves were lower in NE trees than SW trees, our data support the suggestion that the ectomycorrhizal community influences N transfer to its host and demonstrate that the fungal community from the dry condition was more efficient in N acquisition when environmental constraints were relieved. These findings highlight the importance of adapted ectomycorrhizal communities for forest nutrition in a changing climate.     

Linking aboveground and belowground food webs through carbon and nitrogen stable isotope analyses

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) have been used for more than two decades in analyses of food web structure. The utility of isotope ratio measurements is based on the observation that consumer δ¹³C values are similar (<1‰ difference) to those of their diet, while consumer δ¹⁵N values are about 3‰ higher than those of their diet. The technique has been applied most often to aquatic and aboveground terrestrial food webs. However, few isotope studies have examined terrestrial food web structure that includes both above- and belowground (detrital) components. Here, we review factors that may influence isotopic signatures of terrestrial consumers in above- and belowground systems. In particular, we emphasize variations in δ¹³C and δ¹⁵N in belowground systems, e.g., enrichment of ¹³C and ¹⁵N in soil organic matter (likely related to soil microbial metabolism). These enrichments should be associated with the high ¹³C (~3‰) enrichment in belowground consumers relative to litter and soil organic matter and with the large variation in δ¹⁵N (~6‰) of the consumers. Because such enrichment and variation are much greater than the trophic enrichment generally used to estimate consumer trophic positions, and because many general predators are considered dependent on energy and material flows from belowground, the isotopic variation in belowground systems should be taken into account in δ¹³C and δ¹⁵N analyses of terrestrial food webs. Meanwhile, by measuring the δ¹³C of key predators, the linkage between above- and belowground systems could be estimated based on observed differences in δ¹³C of primary producers, detritivores and predators. Furthermore, radiocarbon (¹⁴C) measurements will allow the direct estimation of the dependence of predators on the belowground systems.     

Arginine bi-directional translocation and breakdown into ornithine along the arbuscular mycorrhizal mycelium

Bi-directional translocation and degradation of Arginine (Arg) along the arbuscular mycorrhizal (AM) fungal mycelium were testified through ¹⁵N and/or ¹³C isotopic labeling. In vitro mycorrhizas of Glomus intraradices and Ri T-DNA-transformed carrot roots were grown in dual compartment Petri dishes. [¹⁵N- and/or¹³C]Arg was supplied to either the fungal compartment or the mycorrhizal compartment or separate dishes containing the uncolonized roots. The levels and labeling of free amino acids (AAs) in the mycorrhizal roots and in the extraradical mycelia(ERM) were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). The ERM of AM fungi exposed in either NH₄ ⁺ or urea as sole external nitrogen source had much higher ¹⁵N enrichment of Arg, compared with those in nitrate or exogenous Arg; however, glycerol supplied as an external carbon source to the ERM had no significant effect on the level of Arg in the ERM. Meanwhile, Arg biosynthesized in the ERM could be translocated intact to the mycorrhizal roots and thereby the level of Arg in the mycorrhizal roots increased to about 20% after culture of ERM in 4 mmol/L NH₄ ⁺ for 6 weeks. Also Arg was found to be bi-directionally transported along the AM fungal mycelium through [U-¹³C]Arg labeling either in the mycorrhizal compartment or in the fungal compartment. Once Arg was translocated to the potential N-limited sites, it would be further degraded into ornithine (Orn) and urea since either [U-¹³C] or [U-¹⁵N/U-¹³C]Orn was apparently shown up in the mycorrhizal root tissues when [U-¹³C] or [U-¹⁵N/U-¹³C]Arg was labeled in the fungal compartment, respectively. Evidently Orn formation indicated the ongoing activities of Arg translocation and degradation through the urea cycle in AM fungal mycelium. Supported by Science and Technology Department of Zhejiang Province (Grant No. 2006C22009).     

Nitrogen supply and cyanide concentration influence the enrichment of nitrogen from cyanide in wheat (Triticum aestivum L.) and sorghum (Sorghum bicolor L.)

Cyanide assimilation by the β‐cyanoalanine pathway produces asparagine, aspartate and ammonium, allowing cyanide to serve as alternate or supplemental source of nitrogen. Experiments with wheat and sorghum examined the enrichment of ¹⁵N from cyanide as a function of external cyanide concentration in the presence or absence of nitrate and/or ammonium. Cyanogenic nitrogen became enriched in plant tissues following exposure to ¹⁵N‐cyanide concentrations from 5 to 200 µm , but when exposure occurred in the absence of nitrate and ammonium, ¹⁵N enrichment increased significantly in sorghum shoots at solution cyanide concentrations of ≥50 µm and in wheat roots at 200 µm cyanide. In an experiment with sorghum using ¹³C¹⁵N, there was also a significant difference in the tissue ¹³C:¹⁵N ratio, suggestive of differential metabolism and transport of carbon and nitrogen under nitrogen‐free conditions. A reciprocal ¹⁵N labelling study using KC¹⁵N and ¹⁵NH₄⁺ and wheat demonstrated an interaction between cyanide and ammonium in roots in which increasing solution ammonium concentrations decreased the enrichment from 100 µm cyanide. In contrast, with increasing solution cyanide concentrations there was an increase in the enrichment from ammonium. The results suggest increased transport and assimilation of cyanide in response to decreased nitrogen supply and perhaps to ammonium supply.     

Stable isotopes reveal contrasting trophic dynamics between host–parasite relationships: A case study of Atlantic salmon (Salmo salar) and parasitic lice (Lepeophtheirus salmonis and Argulus foliaceus)

Across existing fish host–parasite literature, endoparasites were depleted in δ¹⁵N compared to their hosts, while ectoparasitic values demonstrated enrichment, depletion and equivalence relative to their hosts. δ¹³C enrichment varied extensively for both endo- and ectoparasites across taxa and host tissues. In our case study, sea lice (Lepeophtheirus salmonis) were enriched in δ¹⁵N relative to their farmed Atlantic salmon (Salmo salar) hosts, although the value contradicted the average that is currently assumed across the animal kingdom. Common fish lice (Argulus foliaceus) did not show a consistent trend in δ¹⁵N compared to their wild S. salar hosts. Both parasitic species had a range of δ¹³C enrichment patterns relative to their hosts. Farmed and wild S. salar had contrasting δ¹³C and δ¹⁵N, and signals varied across muscle, fin and skin within both groups. L. salmonis and A. foliaceus subsequently had unique δ¹³C and δ¹⁵N, and L. salmonis from opposite US coasts differed in δ¹⁵N. Given the range of enrichment patterns that were exhibited across the literature and in our study system, trophic dynamics from host to parasite do not conform to traditional prey to predator standards. Furthermore, there does not appear to be a universal enrichment pathway for δ¹³C nor δ¹⁵N in parasitic relationships, which emphasizes the need to investigate host–parasite linkages across species.     

The uptake and distribution of 15N2 into the various organs of Typha latifolia L.

Direct evidence of heterotrophic dinitrogen fixation associated with the emergent aquatic angiosperm, Typha latifolia L., was obtained through the exposure of actively growing plants to ¹⁵N₂ gas for 7 days in a gas-tight exposure vessel. Highest enrichments of ¹⁵N were found in roots/rhizomes and leaf bases. Slight enrichments were also found in the leaves due to translocation from the roots, rhizomes and leaf bases. Total fixed ¹⁵N values were 71.8 μg for the plant and 49.1 μg for the soil.Plants growing in silica sand, which received a nutrient solution containing combined nitrogen, exhibited higher enrichments and fixed 86% more ¹⁵N after exposure to ¹⁵N₂ gas than plants which received a nutrient solution lacking combined nitrogen. It is hypothesized that the concentration of combined nitrogen added was insufficient to repress nitrogen fixation and resulted in an increase in nitrogen fixation by associated microorganisms.Propane was used to trace the loss and movement of gases from the ¹⁵N₂ vessel and between the upper leaf chamber and the lower root chamber. Gas was rapidly exchanged between the upper and lower chambers through the leaves and roots of T. latifolia. Further investigations showed that propane moved at a rate of 1223 μmol day⁻¹ from the leaves to the roots and 2652 μmol day⁻¹ from the roots to the leaves. These data demonstrated that gases diffuse rapidly through the plant body of T. latifolia.     

Segregation of nitrogen use between ammonium and nitrate of ectomycorrhizas and beech trees

Here, we characterized nitrogen (N) uptake of beech (Fagus sylvatica) and their associated ectomycorrhizal (EM) communities from NH₄⁺ and NO₃^?. We hypothesized that a proportional fraction of ectomycorrhizal N uptake is transferred to the host, thereby resulting in the same uptake patterns of plants and their associated mycorrhizal communities. ¹⁵N uptake was studied under various field conditions after short‐term and long‐term exposure to a pulse of equimolar NH₄⁺ and NO₃^? concentrations, where one compound was replaced by ¹⁵N. In native EM assemblages, long‐term and short‐term ¹⁵N uptake from NH₄⁺ was higher than that from NO₃^?, regardless of season, water availability and site exposure, whereas in beech long‐term ¹⁵N uptake from NO₃^? was higher than that from NH₄⁺. The transfer rates from the EM to beech were lower for ¹⁵N from NH₄⁺ than from NO₃^?. ¹⁵N content in EM was correlated with ¹⁵N uptake of the host for ¹⁵NH₄⁺, but not for ¹⁵NO₃^?‐derived N. These findings suggest stronger control of the EM assemblage on N provision to the host from NH₄⁺ than from NO₃^?. Different host and EM accumulation patterns for inorganic N will result in complementary resource use, which might be advantageous in forest ecosystems with limited N availability.     

Isotopic enrichment in herbivorous insects: a comparative field-based study of variation

Researchers will be able to use stable isotope analysis to study community structure in an efficient way, without a need for extensive calibrations, if isotopic enrichment values are consistent, or if variation in enrichment values can be predicted. In this study, we generated an experimental data set of δ¹⁵N and δ¹³C enrichment means for 22 terrestrial herbivorous arthropods feeding on 18 different host plants. Mean enrichments observed across a single trophic transfer (plants to herbivores) were −0.53±0.26‰ for δ¹³C (range: −3.47‰ to 1.89‰) and 1.88±0.37‰ for δ¹⁵N (range: −0.20‰ to 6.59‰). The mean δ¹³C enrichment was significantly lower than that reported in recent literature surveys, whereas the mean δ¹⁵N enrichment was not significantly different. The experimental data set provided no support for recent hypotheses advanced to explain variation in enrichment values, including the proposed roles for consumer feeding mode, development type, and diet C:N ratio. A larger data set, formed by combining our experimental data with data from the literature, did suggest possible roles for feeding mode, nitrogen recycling, herbivore life stage, and host plant type. Our results indicate that species enrichment values are variable even in this relatively narrow defined group of organisms and that our ability to predict enrichment values of terrestrial herbivorous arthropods based on physiological, ecological, or taxonomic traits is low. The primary implications are that (1) mean enrichment may have to be measured empirically for each trophic link of interest, rather than relying on estimates from a broad survey of animal taxa and (2) the advantage of using stable isotope analysis to probe animal communities that are recalcitrant to other modes of study will be somewhat diminished as a consequence.Electronic Supplementary Material Supplementary material is available for this article at     

Nitrogen Transfer Within and Between Plants Through Common Mycorrhizal Networks (CMNs)

Mycorrhizae play a critical role in nutrient capture from soils. Arbuscular mycorrhizae (AM) and ectomycorrhizae (EM) are the most important mycorrhizae in agricultural and natural ecosystems. AM and EM fungi use inorganic NH₄ ⁺ and NO₃ ^?, and most EM fungi are capable of using organic nitrogen. The heavier stable isotope ¹⁵N is discriminated against during biogeochemical and biochemical processes. Differences in ¹⁵N (atom%) or δ¹⁵N (‰) provide nitrogen movement information in an experimental system. A range of 20 to 50% of one-way N-transfer has been observed from legumes to nonlegumes. Mycorrhizal fungal mycelia can extend from one plant's roots to another plant's roots to form common mycorrhizal networks (CMNs). Individual species, genera, even families of plants can be interconnected by CMNs. They are capable of facilitating nutrient uptake and flux. Nutrients such as carbon, nitrogen and phosphorus and other elements may then move via either AM or EM networks from plant to plant. Both ¹⁵N labeling and ¹⁵N natural abundance techniques have been employed to trace N movement between plants interconnected by AM or EM networks. Fine mesh (25～45 μm) has been used to separate root systems and allow only hyphal penetration and linkages but no root contact between plants. In many studies, nitrogen from N₂-fixing mycorrhizal plants transferred to non-N₂–fixing mycorrhizal plants (one-way N-transfer). In a few studies, N is also transferred from non-N₂–fixing mycorrhizal plants to N₂-fixing mycorrhizal plants (two-way N-transfer). There is controversy about whether N-transfer is direct through CMNs, or indirect through the soil. The lack of convincing data underlines the need for creative, careful experimental manipulations. Nitrogen is crucial to productivity in most terrestrial ecosystems, and there are potential benefits of management in soil-plant systems to enhance N-transfer. Thus, two-way N-transfer warrants further investigation with many species and under field conditions.     

Nitrogen fixation in Faidherbia albida,Acacia raddiana,Acacia senegal and Acacia seyal estimated using the 15N isotope dilution technique

A pot experiment was conducted in a greenhouse using the ¹⁵N isotope dilution method and two reference plants, Parkia biglobosa and Tamarindus indica to estimate nitrogen fixed in four Acacia species: A raddiana, A. senegal, A. seyal and Faidherbia albida (synonym Acacia albida). For the reference plants, the ¹⁵N enrichments in leaves, stems and roots were similar. With the fixing plants, leaves and stems had similar ¹⁵N enrichments; they were higher than the ¹⁵N enrichment of roots. The amounts of nitrogen fixed at 5 months after planting were similar using either reference plant. Estimates of the percentage of N derived from fixation (%Ndfa) for the above ground parts, in contrast to %Ndfa in roots, were similar to those for the whole plant. However, none of the individual plant parts estimated accurately total N fixed in the whole plant, and excluding the roots resulted in at least 30% underestimation of the amounts of N fixed. Between species, differences in N₂ fixation were observed, both for %Ndfa and total N fixed. For %Ndfa, the best were A. seyal (average, 63%) and A. raddiana (average, 62%), being at least twice the %Ndfa in A. senegal and F. albida. Because of its very high N content, A. seyal was clearly the best in total N fixed, fixing 1.62 g N plant^–1 compared to an average of 0.48 g N plant^–1 for the other Acacia species. Our results show the wide variability existing between Acacia species in terms of both %Ndfa and total N fixed: A. seyal was classified as having a high N₂ fixing potential (NFP) while the other Acacia species had a low NFP.     

Diffusive fractionation complicates isotopic partitioning of autotrophic and heterotrophic sources of soil respiration

Carbon isotope ratios (δ¹³C) of heterotrophic and rhizospheric sources of soil respiration under deciduous trees were evaluated over two growing seasons. Fluxes and δ¹³C of soil respiratory CO₂ on trenched and untrenched plots were calculated from closed chambers, profiles of soil CO₂ mole fraction and δ¹³C and continuous open chambers. δ¹³C of respired CO₂ and bulk carbon were measured from excised leaves and roots and sieved soil cores. Large diel variations (>5‰) in δ¹³C of soil respiration were observed when diel flux variability was large relative to average daily fluxes, independent of trenching. Soil gas transport modelling supported the conclusion that diel surface flux δ¹³C variation was driven by non‐steady state gas transport effects. Active roots were associated with high summertime soil respiration rates and around 1‰ enrichment in the daily average δ¹³C of the soil surface CO₂ flux. Seasonal δ¹³C variability of about 4‰ (most enriched in summer) was observed on all plots and attributed to the heterotrophic CO₂ source.     

Translocation and conservation of organic nitrogen within the coral-zooxanthella symbiotic system of Acropora pulchra, as demonstrated by dual isotope-labeling techniques

Carbon (C) and nitrogen (N) metabolism of the hermatypic coral Acropora pulchra and its symbiotic algae (zooxanthellae) was investigated using ¹³C and ¹⁵N isotope tracers. A. pulchra was incubated in seawater containing ¹³C-labeled bicarbonate and ¹⁵N-labeled nitrate (NO₃⁻) for 24 h (pulse period), and subsequently ¹³C and ¹⁵N isotopic ratios of the host coral and the zooxanthellae were followed in ¹³C- and ¹⁵N-free seawater for 2 weeks (chase period). Under our experimental condition of NO₃⁻ (12 μM), C and N were absorbed by the coral-algal symbiotic system with the C:N ratio of 23 during the pulse period. Taking account of concentration dependence of NO₃⁻ uptake rates determined by a separate experiment, C:N uptake ratios under supposed in situ NO₃⁻ conditions (< 1.0 μM) would be > 3.0 times higher, if the photosynthetic rate did not change. During the pulse period, more than half of the absorbed ¹³C and ¹⁵N appeared in the host fraction in organic forms. ¹³C:¹⁵N ratio at the end of the pulse period was similar between the host and the algal fraction, suggesting that algal photosynthetic products were translocated to the host. It is also implied that C:N ratios of the translocated products change depending on N availability for the zooxanthellae. During the chase period, atom % excess (APE) ¹⁵N of the zooxanthellae constantly declined, while that of the host slightly increased. Consequently, APE ¹⁵N of the both fractions appeared to approach a common steady state value, suggesting that ¹⁵N was recycled within the coral-algal symbiotic system. As for C, > 86% of C photosynthetically fixed by the zooxanthellae accumulated in the host at the end of the pulse period, and had a turnover time of ca. 20 days for the host C pool during the following chase period. C:N ratios of organic matter newly synthesized with NO₃⁻ exponentially declined and converged into 5.7 and 4.5 for the host and the zooxanthellae, respectively. This suggests that organic compounds of high C:N ratios such as lipids and carbohydrates were selectively consumed more rapidly than those of low C:N ratios such as proteins and nucleic acids.     

Increasing abundance of soil fungi is a driver for 15N enrichment in soil profiles along a chronosequence undergoing isostatic rebound in northern Sweden

Soil organic material (SOM) is usually enriched in ¹⁵N in deeper soil layers. This has been explained by discrimination against the heavier isotope during decomposition or by the accumulation of ¹⁵N-enriched microbial biomass versus plant biomass in older SOM. In particular, ectomycorrhizal (EM) fungi have been suggested to accumulate in old SOM since this group is among the most ¹⁵N-enriched components of the microbial community. In the present study we investigated the microbial community in soil samples along a chronosequence (7,800 years) of sites undergoing isostatic rebound in northern Sweden. The composition of the microbial community was analyzed and related to the δ¹⁵N and δ¹³C isotope values of the SOM in soil profiles. A significant change in the composition of the microbial community was found during the first 2,000 years, and this was positively related to an increase in the δ¹⁵N values of the E and B horizons in the mineral soil. The proportion of fungal phospholipid fatty acids increased with time in the chronosequence and was positively related to the ¹⁵N enrichment of the SOM. The increase in δ¹³C in the SOM was much less than the increase in δ¹⁵N, and δ¹³C values in the mineral soil were only weakly related to soil age. The C:N ratio and the pH of the soil were important factors determining the composition of the microbial community. We suggest that the N being transported from the soil to aboveground tissue by EM fungi is a driver for ¹⁵N enrichment of soil profiles.     

Labelling halophilic Archaea using 13C and 15N stable isotopes: a potential tool to investigate haloarchaea consumption by metazoans

The use of stable isotope (SI) labelling and tracing of live diets is currently considered one of the most comprehensive tools to detect their uptake and assimilation by aquatic organisms. These techniques are indeed widely used in nutritional studies to follow the fate of specific microbial dietary components, unraveling trophic interactions. Nevertheless, to the current date our understanding of aquatic trophic relationships has yet to include a whole domain of life, the Archaea. The aim of the present research was, therefore, to describe a halophilic Archaea (haloarchaea) labelling procedure, using the SI ¹³C and ¹⁵N, to enable the application of SI tracing in future studies of haloarchaea consumption by aquatic metazoans. To this end, three ¹³C enriched carbon sources and two ¹⁵N enriched nitrogen sources were tested as potential labels to enrich cells of three haloarchaea strains when supplemented to the culture medium. Our overall results indicate ¹³C-glycerol as the most effective carbon source to achieve an efficient ¹³C enrichment in haloarchaea cells, with Δδ¹³C values above 5000‰ in all tested haloarchaea strains. As for ¹⁵N enriched nitrogen sources, both (¹⁵NH₄)₂SO₄ and ¹⁵NH₄Cl seem to be readily assimilated, also resulting in efficient ¹⁵N enrichment in haloarchaea cells, with Δδ¹⁵N values higher than 20,000‰. We believe that the proposed methodology will allow for the use of SI labelled haloarchaea biomass in feeding tests, potentially providing unambiguous confirmation of the assimilation of haloarchaea biomass by aquatic metazoans.

     

Fine Roots vs. Needles: A Comparison of 13C and 15N Dynamics in a Ponderosa Pine Forest Soil

Plant allocation patterns may affect soil C and N storage due to differences in litter quality and the depth of plant C and N inputs into the soil. We studied the dynamics of dual-labeled (¹³C/¹⁵N) Pinus ponderosa needles and fine roots placed at two soil depths (O and A horizon) in a temperate conifer forest soil during 2 y. Input of C as fine roots resulted in much more C retained in soil (70.5 ± 2.2% of applied) compared with needle C (42.9 ± 1.3% of applied) after 1.5 y. Needles showed faster mass loss, rates of soil ¹³CO₂ efflux, and more ¹⁵N immobilized into microbial biomass than did fine roots. The larger proportion of labile C compounds initially present in needles (17% more needle C was water soluble than in fine roots) likely contributed to its shorter C residence time and greater degree of transformation in the soil. A double exponential decay function best described the rate of ¹³C loss, with a smaller initial pulse of C loss from fine roots (S₁k₁) and a slower decay rate of the recalcitrant C pool for fine roots (0.03 y⁻¹) compared with (0.19 y⁻¹) for needles. Soil ¹³C respiration, representing heterotrophic respiration of litter C, was much more seasonal from the O horizon than from the A. However, offsetting seasonal patterns in ¹³C dynamics in the O horizon resulted in no net effect of soil depth on total ¹³C retention in the soil after 1.5 y for either litter. Almost 90% of applied litter N was retained in the soil after 1.5 y, independent of litter quality or soil depth. Very small amounts of ¹³C or ¹⁵N (<3% of applied) moved to the horizon above or below the placement depth (i.e., O to A or A to O). Our results suggest that plant allocation belowground to fine roots results in more C retained and less N mineralized compared with allocation aboveground to needles, primarily due to litter quality differences.     

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.