Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)     

Disproportionate abundance between ectomycorrhizal root tips and their associated mycelia

Extensive knowledge of various ectomycorrhizal fungal communities has been obtained over the past 10 years based on molecular identification of the fungi colonizing fine roots. In contrast, only limited information exists about the species composition of ectomycorrhizal hyphae in soil. This study compared the ectomycorrhizal external mycelial community with the adjacent root-tip community in a Danish beech forest. Sand-filled in-growth mesh bags were used to trap external mycelia by incubating the mesh bags in the soil for 70 days. The adjacent ectomycorrhizal root-tip communities were recorded at the times of insertion and retrieval of the mesh bags. Ectomycorrhizal fungi were identified by sequencing the internal transcribed spacer region. In total, 20, 31 and 24 ectomycorrhizal species were recorded from the two root-tip harvests and from the mesh bags, respectively. Boletoid species were significantly more frequent as mycelia than as root tips, while russuloid and Cortinarius species appeared to be less dominant as mycelia than as root tips. Tomentella species were equally frequent as root tips and as mycelia. These discrepancies between the root-tip and the mycelial view of the ectomycorrhizal fungal community are discussed within the framework of ectomycorrrhizal exploration types.     

Water fluxes within beech stands in complex terrain

We investigated the water balances of two beech stands (Fagus sylvatica L.) on opposite slopes (NE, SW) of a narrow valley near Tuttlingen in the southern Swabian Jura, a low mountain range in Southwest Germany. Our analysis combines results from continuous measurements of forest meteorological variables significant to the forest water balance, stand transpiration (ST) estimates from sap flow measurements, and model simulations of microclimate and water fluxes. Two different forest hydrological models (DNDC and BROOK90) were tested for their suitability to represent the particular sites. The investigation covers the years 2001–2007. Central aims were (1) to evaluate meteorological simulations of variables below the forest canopy, (2) to evaluate ST, (3) to quantify annual water fluxes for both beech stands using the evaluated hydrological models, and (4) to analyse the model simulations with regard to assumptions inherent in the respective model. Overall, both models were very well able to reproduce the observed dynamics of the soil water content in the uppermost 30 cm. However, the degree of fit depended on the year and season. The comparison of experimentally determined ST within the beech stand on the NE-slope during the growing season of 2007 with simulated transpiration did not yield a reliable statistical relationship. The simulation of water fluxes for the beech stand on the NE- and SW-slopes showed similar results for vegetation-related fluxes with both models, but different with respect to runoff and percolation flows. Overall, the higher evaporation demand on the warmer SW-slope did not lead to a significantly increased drought stress for the vegetation but was reflected mainly in decreased water loss from the system. This finding is discussed with regard to potential climate change and its impact on beech growth.     

Acid and calcareous soils affect nitrogen nutrition and organic nitrogen uptake by beech seedlings (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) under drought,and their ectomycorrhizal community structure

Aims

The role of different soil types for beech productivity and drought sensitivity is unknown. The aim of this experimental study was to compare mycorrhizal diversity between acid sandy and calcareous soils and to investigate how this diversity affects tree performance, nitrogen uptake and use efficiency (NUE).

Methods

Beech trees were germinated and grown in five different soil types (pH 3.8 to 6.7). One-and-a-half-year-old plants were exposed for 6 weeks to sufficient or low soil humidity. Tree biomass, root tip mycorrhizal colonization and community structure, root tip mortality, leaf area, photosynthesis, nitrogen concentrations, NUE and short-term ¹⁵N uptake from glutamine were determined.

Results

Soil type did not affect photosynthesis or biomass formation, with one exception in calcareous soil, where root mortality was higher than in the other soil types. Beech in acid soils showed lower mycorrhizal colonization, higher nitrogen tissue concentrations, and lower NUE than those in calcareous soils. Drought had no effect on nitrogen concentrations or NUE but caused reductions in mycorrhizal colonization. Mycorrhizal species richness correlated with nitrogen uptake and NUE. Nitrogen uptake was more sensitive to drought in calcareous soils than in acid soils.

Conclusions

Beech may be more drought-susceptible on calcareous sites because of stronger decrease of organic nitrogen uptake than on acid soils.

     

Limited transfer of nitrogen between wood decomposing and ectomycorrhizal mycelia when studied in the field

Transfer of ¹⁵N between interacting mycelia of a wood-decomposing fungus (Hypholoma fasciculare) and an ectomycorrhizal fungus (Tomentellopsis submollis) was studied in a mature beech (Fagus sylvatica) forest. The amount of ¹⁵N transferred from the wood decomposer to the ectomycorrhizal fungus was compared to the amount of ¹⁵N released from the wood-decomposing mycelia into the soil solution as ¹⁵N-NH₄. The study was performed in peat-filled plastic containers placed in forest soil in the field. The wood-decomposing mycelium was growing from an inoculated wood piece and the ectomycorrhizal mycelium from an introduced root from a mature tree. The containers were harvested after 41 weeks when physical contact between the two foraging mycelia was established. At harvest, ¹⁵N content was analyzed in the peat (total N and ¹⁵NH₄ ⁺) and in the mycorrhizal roots. A limited amount of ¹⁵N was transferred to the ectomycorrhizal fungus and this transfer could be explained by ¹⁵NH₄ ⁺ released from the wood-decomposing fungus without involving any antagonistic interactions between the two mycelia. Using our approach, it was possible to study nutritional interactions between basidiomycete mycelia under field conditions and this and earlier studies suggest that the outcomes of such interactions are highly species-specific and depend on environmental conditions such as resource availability.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

Carbon fluxes, nitrogen cycling, and soil microbial communities in adjacent urban, native and agricultural ecosystems

Urban ecosystems are expanding globally, and assessing the ecological consequences of urbanization is critical to understanding the biology of local and global change related to land use. We measured carbon (C) fluxes, nitrogen (N) cycling, and soil microbial community structure in a replicated (n=3) field experiment comparing urban lawns to corn, wheat–fallow, and unmanaged shortgrass steppe ecosystems in northern Colorado. The urban and corn sites were irrigated and fertilized. Wheat and shortgrass steppe sites were not fertilized or irrigated. Aboveground net primary productivity (ANPP) in urban ecosystems (383±11 C m^?2 yr^?1) was four to five times greater than wheat or shortgrass steppe but significantly less than corn (537±44 C m^?2 yr^?1). Soil respiration (2777±273 g C m^?2 yr^?1) and total belowground C allocation (2602±269 g C m^?2 yr^?1) in urban ecosystems were both 2.5 to five times greater than any other land‐use type. We estimate that for a large (1578 km²) portion of Larimer County, Colorado, urban lawns occupying 6.4% of the land area account for up to 30% of regional ANPP and 24% of regional soil respiration from land‐use types that we sampled. The rate of N cycling from urban lawn mower clippings to the soil surface was comparable with the rate of N export in harvested corn (both ～12–15 g N m^?2 yr^?1). A one‐time measurement of microbial community structure via phospholipid fatty acid analysis suggested that land‐use type had a large impact on microbial biomass and a small impact on the relative abundance of broad taxonomic groups of microorganisms. Our data are consistent with several other studies suggesting that urbanization of arid and semiarid ecosystems leads to enhanced C cycling rates that alter regional C budgets.     

Methanol and other VOC fluxes from a Danish beech forest during late springtime

In-canopy mixing ratio gradients and above-canopy fluxes of several volatile organic compounds (VOCs) were measured using a commercial proton transfer reaction mass spectrometer (PTR-MS) in a European beech (Fagus sylvatica) forest in Denmark. Fluxes of methanol were bidirectional: Emission occurred during both day and night with highest fluxes (0.2 mg C m⁻² h⁻¹) during a warm period; deposition occurred dominantly at daytime. Confirming previous branch-level measurements on beech, the forest’s monoterpene emissions (0–0.5 mg C m⁻² h⁻¹), and in-canopy mixing ratios showed a diurnal cycle consistent with light-dependent emissions; a result contrasting temperature-only driven emissions of most conifer species. Also emitted was acetone, but only at ambient temperatures exceeding 20°C. Slow deposition dominated at lower temperatures. Our in-canopy gradient measurements contrast with earlier results from tropical and pine forest ecosystems in that they did not show this beech ecosystem to be a strong sink for oxygenated VOCs (OVOCs). Instead, their gradients were flat and only small deposition velocities (<0.2 cm s⁻¹) were observed to the onsite soil. However, as methanol soil uptake was consistent and possibly related to soil moisture, more measurements are needed to evaluate its soil sink strength. In turn, as canopy scale fluxes are net fluxes with stomatal emissions from photosynthesizing leaves potentially affecting non-stomatal oxygenated VOC uptake, only independent, controlled laboratory experiments may be successful in separating gross fluxes.     

Interactions between mammals and ectomycorrhizal fungi

Many ectomycorrhizal (ECM) fungi produce fruit-bodies below ground and rely on animals, especially mammals, for dispersal of spores. Mammals may therefore play an important role in the maintenance of mycorrhizal symbiosis and biodiversity of ECM fungi in many forest ecosystems. Given the pivotal role played by mycorrhizal fungi In the nutrition of their plant hosts and, possibly, in the determination of plant community structure, the ecological significance of mycophagous mammals may extend to the productivity and diversity of plant communities. Mycologists and mammalogists have been aware of the interaction between their study organisms for many years, but recent research has produced new insights Into the evolution of mammal-vectored spore dispersal among ECM fungi, the ecological importance of mycophagy to small mammals, and the effectiveness of mammals as spore-dispersal agents.     

Carbon and nitrogen dynamics in acid detergent fibre lignins of beech (Fagus sylvatica L.) during the growth phase

To study the incorporation of carbon and nitrogen in different plant fractions, 3‐year‐old‐beech (Fagus sylvatica L.) seedlings were exposed in microcosms to a dual‐labelling experiment employing ¹³C and ¹⁵N throughout one season. Leaves, stems, coarse and fine roots were harvested 6, 12 and 18 weeks after bud break (June to September) and used to isolate acid‐detergent fibre lignins (ADF lignin) for the determination of carbon and nitrogen and their isotope ratios. Lignin concentrations were also determined with the thioglycolic acid method. The highest lignin concentrations were found in fine roots. ADF lignins of all tissues analysed, especially those of leaves, also contained significant concentrations of nitrogen. This suggests that lignin‐bound proteins constitute an important cell wall fraction and shows that the ADF method is not suitable to determine genuine lignin. ADF lignin should be re‐named as ligno‐protein fraction. Whole‐leaf biomass was composed of 50 to 70% newly assimilated carbon and about 7% newly assimilated nitrogen; net changes in the isotope ratios were not observed during the experimental period. In the other tissues analysed, the fraction of new carbon and nitrogen was initially low and increased significantly during the time‐course of the experiment, whereas the total tissue concentrations of carbon remained almost unaffected and nitrogen declined. At the end of the experiment, the whole‐tissue biomass and ADF lignins of fine roots contained about 65 and 50% new carbon and about 50 and 40% new nitrogen, respectively. These results indicate that significant metabolic activity was related to the formation of structural biopolymers after leaf growth, especially below‐ground and that this activity also led to a substantial binding of nitrogen to structural compounds.     

Carbon fluxes in mature peach leaves

The turnover and transport of sugars are described in peach (Prunus persica L. Batsch), a species exporting both sucrose and sorbitol. Apparent export rate was slower in peach leaves than in leaves of herbaceous species. Sorbitol was the major soluble end product of photosynthesis and the major soluble carbohydrate in the leaf (higher than sucrose). Carbon fluxes were described using ¹⁴C labeling, radioactivity loss curves, and compartmental analysis during the second half of the photoperiod when chemical steady state was reached for soluble carbohydrates. The measured specific radioactivity of sucrose was typical of a primary product. The delayed decrease in specific radioactivity of sorbitol indicated that part of it was secondarily synthesized. Sucrose is proposed to be the carbon source for the delayed synthesis of sorbitol in the light. The sorbitol to sucrose ratio was higher in the petiole than in the leaf tissues. In phloem sap, obtained using stylectomy of aphids and collected from the main stem between source leaves and apex, this ratio was lower than in the petiole, suggesting a preferential sorbitol demand by sinks.     

Relationships between fungal uptake of ammonium, fungal growth and nitrogen availability in ectomycorrhizal Pinus sylvestris seedlings

 Nitrogen deposition and intentional forest fertilisation with nitrogen are known to affect the species composition of ectomycorrhizal fungal communities. To learn more about the mechanisms responsible for these effects, the relations between fungal growth, nitrogen uptake and nitrogen availability were studied in ectomycorrhizal fungi in axenic cultures and in symbiosis with pine seedlings. Effects of different levels of inorganic nitrogen (NH₄) on the mycelial growth of four isolates of Paxillus involutus and two isolates of Suillus bovinus were assessed. With pine seedlings, fungal uptake of ¹⁵N-labelled NH₄ was studied in short-term incubation experiments (72 h) in microcosms and in long-term incubation experiments (3 months) in pot cultures. For P. involutus growing in symbiosis with pine seedlings, isolates with higher NH₄ uptake were affected more negatively at high levels of nitrogen availability than isolates with lower uptake. More NH₄ was allocated to shoots of seedlings colonised by a high-uptake isolate, indicating transfer of a larger fraction of assimilated NH₄ to the host than with isolates showing lower NH₄ uptake rates. Thus low rates of N uptake and N transfer to the host may enable EM fungi avoid stress induced by elevated levels of nitrogen. Seedlings colonised by S. bovinus transferred a larger fraction of the ¹⁵N label to the shoots than seedlings colonised by P. involutus. Seedling shoot growth probably constituted a greater carbon sink in pot cultures than in microcosms, since the mycelial growth of P. involutus was more sensitive to high NH₄ in pots. There was no homology in mycelial growth rate between pure culture and growth in symbiosis, but N uptake in pure culture corresponded to that during growth in symbiosis. No relationship was found between deposition of antropogenic nitrogen at the sites of origin of the P. involutus isolates and their mycelial growth or uptake of inorganic nitrogen. Accepted: 18 September 1998     

Carbon fluxes in coniferous and deciduous forest soils

Aims

Our aims were to identify responsible factors for the site-to-site variability in soil CO₂ efflux and to assess the sources of soil CO₂ of different forest types on a regional scale.

Methods

Soil CO₂ effluxes were measured over 1–4 years in four coniferous and three deciduous forests of Bavaria, Germany, and related to climate, soil properties and forest productivity. Total belowground carbon allocation (TBCA) was assessed using soil CO₂ effluxes and aboveground litterfall. Additionally, CO₂ production of organic layers was examined over 10 months under constant conditions in an incubation experiment.

Results

Annual soil CO₂ effluxes were not different among the forest sites, but predicted effluxes at a given temperature of 10°C revealed some significant differences and correlated with the phosphorous stock of the organic layers. The incubation study indicated 50% faster decomposition of organic layers from deciduous than from coniferous forests. TBCA related to soil CO₂ efflux was smaller in the deciduous than in the coniferous forests. The ratio of TBCA to soil CO₂ efflux was positively correlated with the C stock of organic layers.

Conclusions

Our results suggest that marked differences in site characteristics have little impact on soil CO₂ effluxes at the regional scale, but the contribution of soil CO₂ sources varies among the forest types.     

Rapid nitrogen transfer from ectomycorrhizal pines to adjacent ectomycorrhizal and arbuscular mycorrhizal plants in a California oak woodland

Nitrogen transfer among plants in a California oak woodland was examined in a pulse-labeling study using 15N. The study was designed to examine N movement among plants that were mycorrhizal with ectomycorrhizas (EM), arbuscular mycorrhizas (AM), or both. Isotopically enriched N (K15NO3-) was applied to gray pine (Pinus sabiniana) foliage (donor) and traced to neighboring gray pine, blue oak (Quercus douglasii), buckbrush (Ceanothus cuneatus) and herbaceous annuals (Cynosurus echinatus, Torilis arvensis and Trifolium hirtum). After 2 wk, needles of 15N-treated pines and foliage from nearby annuals were similarly enriched, but little 15N had appeared in nontreated (receiver) pine needles, oak leaves or buckbrush foliage. After 4 wk foliar and root samples from pine, oak, buckbrush and annuals were significantly 15N-enriched, regardless of the type of mycorrhizal association. The rate of transfer during the first and second 2-wk periods was similar, and suggests that 15N could continue to be mobilized over longer times.     

Modification of water flows and nitrogen fluxes by shelterbelts

The introduction of shelterbelts (mid-field trees linear structures) in a uniform agricultural landscape composed mainly of cultivated fields is one of the best tools for managing heat balance and water regime in the landscape. Evapotranspiration rates, surface runoff and percolation of water across a soil profile are controlled by shelterbelts. The heat transfers from cultivated fields to shelterbelts by advection processes enhance evapotranspiration rates from trees. Depth of groundwater table and thermal conditions have important bearing on the uptake of groundwater by trees. The studies showed that in the Turew agricultural landscape, located near Poznan, Poland, shelterbelts use for evapotranspiration 40 percent more water than cultivated fields. For air heating cultivated fields use 280 percent of solar energy more than shelterbelts. The shelterbelts limit N-NO₃⁻ spreading with groundwater very effectively. Concentrations of inflowing nitrates with groundwater dropped to 2.3–24.4 percent of the input from fields. In contrast to nitrate ions the ammonium behave entirely different. Often concentration of ammonium increased in groundwater under shelterbelts. Higher concentrations of N-NH₄⁺ under shelterbelts than under field indicated that ammonium ions were released during decomposition of organic matter. This supposition was directly proved by estimates of very high rates of urease activity which enzyme converts urea nitrogen into NH₃ in the final stages of protein decomposition. It was estimated that N₂O fluxes from soil of shelterbelts are much smaller than from cultivated fields. The balance of nitrogen inputs and outputs in a shelterbelt indicated that the internal recycling of this element is of crucial importance for the controlling efficiency of nitrogen compounds spreading in an environment.     

Competition for nitrogen between Pinus sylvestris and ectomycorrhizal fungi generates potential for negative feedback under elevated CO2

We investigated fungal species-specific responses of ectomycorrhizal (ECM) Scots pine (Pinus sylvestris) seedlings on growth and nutrient acquisition together with mycelial development under ambient and elevated CO₂. Each seedling was associated with one of the following ECM species: Hebeloma cylindrosporum, Laccaria bicolor, Suillus bovinus, S. luteus, Piloderma croceum, Paxillus involutus, Boletus badius, or non-mycorrhizal, under ambient, and elevated CO₂ (350 or 700 μl l⁻¹ CO₂); each treatment contained six replicates. The trial lasted 156 days. During the final 28 days, the seedlings were labeled with ¹⁴CO₂. We measured hyphal length, plant biomass, ¹⁴C allocation, and plant nitrogen and phosphorus concentration. Almost all parameters were significantly affected by fungal species and/or CO₂. There were very few significant interactions. Elevated CO₂ decreased shoot-to-root ratio, most strongly so in species with the largest extraradical mycelium. Under elevated CO₂, ECM root growth increased significantly more than hyphal growth. Extraradical hyphal length was significantly negatively correlated with shoot biomass, shoot N content, and total plant N uptake. Root dry weight was significantly negatively correlated with root N and P concentration. Fungal sink strength for N strongly affected plant growth through N immobilization. Mycorrhizal fungal-induced progressive nitrogen limitation (PNL) has the potential to generate negative feedback with plant growth under elevated CO₂. Responsible Editor: Herbert Johannes Kronzucker