Expression of the nitrate transporter nrt2 gene from the symbiotic basidiomycete Hebeloma cylindrosporum is affected by host plant and carbon sources

Although the function of the extramatrical mycelium of ectomycorrhizal fungi is considered essential for the acquisition of nitrogen by forest trees, gene regulation in this fungal compartment is poorly characterized. In this study, the expression of the nitrate transporter gene nrt2 from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was shown to be regulated by plant host and carbon sources. In the presence of a low fructose concentration, nrt2 expression could not be detected in the free-living mycelium but was high in the extramatrical symbiotic mycelium associated to the host plant Pinus pinaster. In the absence of nitrogen or in the presence of nitrate, high sugar concentrations in the medium were able to enhance nrt2 expression. Nevertheless, in the presence of high fructose concentration, high ammonium concentration still completely repressed nrt2 expression indicating that the nitrogen repression overrides sugar stimulation. This is the first report revealing an effect of host plant and of carbon sources on the expression of a fungal nitrate transporter-encoding gene.     

氮沉降对外生菌根真菌的影响

综述了国外氮沉降对外生菌根真菌的影响研究现状 ,主要从菌根形成、形态 (菌丝体、菌根 )变化、子实体生产力和外生菌根真菌群落结构等方面对氮沉降的响应进行了综述 ,并初步探讨了氮饱和的临界负荷。研究表明 ,过量氮沉降会给外生菌根真菌在以下几个方面带来负影响 :(1)影响外生菌根真菌与寄主植物之间的养分分配和循环 ;(2 )降低子实体生产力 ;(3)减少菌丝 ;(4 )降低菌根量及其活力 ;(5 )降低外生菌根真菌丰富度 ;(6 )改变外生菌根真菌群落结构组成 ;(7)降低外生菌根真菌群落功能。还指出了未来该方面研究重点和方向     

Role of nutrient level and defoliation on symbiotic function: experimental evidence by tracing 14C/15N exchange in mycorrhizal birch seedlings

High nutrient availability and defoliation generally reduce ectomycorrhizal colonization levels in trees, but it is not known how this affects the functional aspects of mycorrhizal symbiosis. It was therefore investigated whether (1) defoliation or increasing substrate N availability reduce C allocation from the plant to the fungus and N allocation from the fungus to the plant (symbiotic resource exchange), (2) symbiotic resource exchange depends on relative N and P availability, and (3) fungal N translocation to plant and plant C allocation to fungus are interdependent. Birch (Betula pendula) seedlings were grown in symbiosis with the ectomycorrhizal fungus Paxillus involutus at five times excess N, or at five times excess N and P for 6 weeks. One-half of the plants were defoliated and the plant shoots were allowed to photosynthesize ¹⁴CO₂ while the fungal compartment was exposed to ¹⁴NH₄. After 3 days, the ¹⁴C of plant origin in fungal tissues and ¹⁵N of fungal origin in plant tissues were quantified. Nutrient availability had no observable effect on symbiotic resource exchange in non-defoliated systems. Defoliation reduced symbiotic N acquisition by plants at all levels of nutrient availability, with the reduction being most marked at higher N availability, indicating an increased tendency in the symbiotic system to discontinue resource exchange after defoliation at higher fertility levels. The concentration of ¹⁴C in extramatrical mycelium correlated significantly with the concentration of ¹⁵N in birch shoots. The results support the assumption that N delivery to the host by the mycorrhizal fungus is dependent on C flow from the plant to the fungus, and that exchanges between the partners are reciprocal. No significant reductions in root ¹⁴C content as a response to defoliation were observed, indicating that defoliation specifically reduced allocation to fungus, but not markedly to roots.     

Micronutrient transport in mycorrhizal symbiosis; zinc steals the show

Mycorrhizas are mutually beneficial associations between soil-borne fungi and plant roots. Mycorrhizal fungi provide their host plant with essential nutrients in exchange for sugars and/or lipids. Traditionally, transport and translocation of macronutrients, including nitrogen and phosphorus, throughout the fungal mycelium and towards the host plant are well studied. However, the regulation of nutrient exchange and their contribution in the morphogenesis and development of mycorrhizas remains unclear. In this Opinion, we argue that adding micronutrients in the current models of symbiotic transport is essential to fully understand the establishment, maintenance, and functioning of mycorrhizal associations. Homeostatic mechanisms at the cellular level and the first transport proteins involved have been recently documented for zinc (Zn) in arbuscular mycorrhizal, ectomycorrhizal, and ericoid mycorrhizal fungi. Mycorrhizal plants benefit from an improved Zn status in control conditions and are better protected when environmental Zn availability fluctuates. These recent progresses are paving the way for a better understanding of micronutrient allocation in mycorrhizas. Revising our vision on the role of micronutrients, particularly of Zn, in these interactions will allow a better use of mycorrhizal fungi in sustainable agriculture and forestry, and will increase management practices in waste land, as well as in agricultural and natural ecosystems.     

Zinc toxicity in ectomycorrhizal Pinus sylvestris

Six strains of ectomycorrhizal fungi were compared for their ability to increase zinc tolerance in Pinus sylvestris L. seedlings. Pioneer and late-stage fungi as well as one strain collected from a Zn-polluted site were included in the study. The accumulation of zinc in the host plants was determined at two different sublethal substrate Zn concentrations. The mycobionts varied considerably in their protection of the autobionts against zinc toxicity. Several fungal species reduced zinc accumulation in the pine seedlings. A Thelephora terrestris (Ehrh.) Fr. Strain, however, increased the Zn concentration in its host plants. Specific zinc-retaining capacity of the mycelium and density of the extramatrical mycelium were important features determining the effectiveness of the zinc retention in the fungal symbiont.The experiments were carried out in a growth chamber where plants were cultivated in root observation chambers. The determination of the extent of substrate colonization showed that an elevated zinc concentration in a substrate might be an important stress factor for the growth of an ectomycorrhizal fungus. The growth of the extramatrical mycelium of some species was inhibited at the highest zinc concentration supplied. A determination of the in vitro zinc tolerance of the fungi could not always predict their tolerance as symbionts, where the latter characteristic did not determine the Zn retention capacity of their mycelium.In this paper we also discuss the possibility that on Zn-polluted soils ectomycorrhizal species and strains are selected that are Zn-tolerant and, in addition, are able to protect their own energy source, the autobiont, from toxicity. These mycorrhizal fungi act as a safety net, that can immobilize large amounts of zinc, thus preventing transport to the host plant.     

Plasticity and constraint in growth and protein mineralization of ectomycorrhizal fungi under simulated nitrogen deposition

Ectomycorrhizal fungi allow their host plants access to organic forms of N through enzymatic mineralization of the substrate and enhanced absorption of amino acids and mineral N. The cost to the plant is carbohydrates that support fungal growth and metabolism. Enrichment of soils with mineral N, as through atmospheric deposition, may affect the growth and function of these fungi by direct effects of increased N availability on fungi and indirect effects through reduced plant C allocation to roots. We tested the potential of N enrichment and altered carbohydrate supply to affect the growth and protein mineralization activity of 10 ectomycorrhizal fungi in sterile liquid media. Nitrogen treatments consisted of organic N only vs organic plus mineral N. Carbon treatments consisted of 5 g per liter glucose vs. no glucose added. Fungi differed widely in their growth and mineralization responses to these variables. Seven of 10 fungi had at least 20% reduced growth with reduced carbohydrates. Only 2 of 10 increased growth by 20% or more with increased mineral N. Carbohydrates affected growth more in a purely organic N environment suggesting an energy limitation to mineralization. Protein mineralization activity tended to be depressed by reductions in carbohydrates and increased by increased mineral N. The high sensitivity of fungal growth to carbohydrates suggests important indirect effects of N enrichment via altered C allocation in host trees. Principal Components analysis separated most fungal species along an axis representing a gradient from high protein mineralization efficiency to high intrinsic growth rate. Those fungi with slow growth and efficient mineralization activity corresponded closely to fungi often cited as late successional species, while fungi with high growth rates and low mineralization efficiency are often categorized as early successional. One fungus, Cenococcum geophillum, separated from others on an axis representing strong N dependence in growth. Nitrogen enrichment has the potential to alter the composition and function of the ectomycorrhizal fungus community. Physiological differences among species provide a starting point for predicting community responses and anticipating ecosystem consequences.     

Natural Abundance of <Superscript>15</Superscript>N in Nitrogen-Limited Forests and Tundra Can Estimate Nitrogen Cycling Through Mycorrhizal Fungi: A Review

The hyphae of ectomycorrhizal and ericoid mycorrhizal fungi proliferate in nitrogen (N)-limited forests and tundra where the availability of inorganic N is low; under these conditions the most common fungal species are those capable of protein degradation that can supply their host plants with organic N. Although it is widely understood that these symbiotic fungi supply N to their host plants, the transfer is difficult to quantify in the field. A novel approach uses the natural ¹⁵N:¹⁴N ratios (expressed as δ¹⁵N values) in plants, soils, and mycorrhizal fungi to estimate the fraction of N in symbiotic trees and shrubs that enters through mycorrhizal fungi. This calculation is possible because mycorrhizal fungi discriminate against ¹⁵N when they create compounds for transfer to plants; host plants are depleted in ¹⁵N, whereas mycorrhizal fungi are enriched in ¹⁵N. The amount of carbon (C) supplied to these fungi can be stoichiometrically calculated from the fraction of plant N derived from the symbiosis, the N demand of the plants, the fungal C:N ratio, and the fraction of N retained in the fungi. Up to a third of C allocated belowground, or 20% of net primary production, is used to support ectomycorrhizal fungi. As anthropogenic N inputs increase, the C allocation to fungi decreases and plant δ¹⁵N increases. Careful analyses of δ¹⁵N patterns in systems dominated by ectomycorrhizal and ericoid mycorrhizal symbioses may reveal the ecosystem-scale effects of alterations in the plant–mycorrhizal symbioses caused by shifts in climate and N deposition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Symbiotic control of canopy dominance in subtropical and tropical forests

Subtropical and tropical forests in Asia often comprise canopy dominant trees that form symbioses with ectomycorrhizal fungi, and species-rich understorey trees that form symbioses with arbuscular mycorrhizal fungi. We propose a virtuous phosphorus acquisition hypothesis to explain this distinct structure. The hypothesis is based on (i) seedlings being rapidly colonised by ectomycorrhizal fungi from established mycelial networks that generates positive feedback and resistance to pathogens, (ii) ectomycorrhizal fungi having evolved a suite of morphological, physiological, and molecular traits to enable them to capture phosphorus from a diversity of chemical forms, including organic forms, and (iii) allocation of photosynthate carbon from adult host plants to provide the energy needed to undertake these processes.     

Uptake and transfer of nutrients in ectomycorrhizal associations: interactions between photosynthesis and phosphate nutrition

Energy-dispersive X-ray microanalytical investigations and microautoradiographic studies were carried out to examine whether the uptake and transfer of phosphate (P) by an ectomycorrhizal fungus is affected by the carbohydrate supply of its host plant. For this purpose, non-mycorrhizal seedlings of Pinus sylvestris L. and plants inoculated with the ectomycorrhizal basidiomycete Suillus bovinus (L. ex Fr.) Kuntze were placed in the dark for 7 days in advance of a P supply. The subcellular element distribution and the uptake and distribution of (33)P was analyzed in non-mycorrhizal and mycorrhizal roots of these plants and compared with plants kept constantly under normal light conditions (control plants). The results show that placing non-mycorrhizal plants in the dark in advance of the nutrient supply led to (1) a reduction of the subcellular contents of P, S and K, but to an increase in the cytoplasmic Na content, and (2) an increase of (33)P absorption and translocation to the shoot. It can be assumed that this increased inflow of (33)P in non-mycorrhizal plants was due to P starvation after suppressed photosynthesis and reduced respiration of these plants. The suppression of photosynthesis by an ectomycorrhizal host plant and the resulting lower carbohydrate supply conditions for the ectomycorrhizal fungus led to (1) a decrease of P absorption by the mycobiont, (2) a change of the P allocation in the fungal cell compartments of an ectomycorrhizal root, and (3) a reduction of P transfer to the host plant. However, microautoradiographic studies revealed that, under these conditions, P was also absorbed by the mycorrhizal fungus and translocated via the Hartig net to the host plant. In mycorrhizal roots of plants placed in the dark in advance of the nutrient supply, the cytoplasmic P content of the Hartig net was reduced and, instead, a high number of polyphosphate granules could be detected within the hyphae. The results indicate that the exchange processes between the symbionts in a mycorrhiza are possibly linked and that P uptake and translocation by an ectomycorrhizal fungus is also regulated by the carbohydrate supply of its host plant.     

Development of ectomycorrhizas in model beech–spruce ecosystems on siliceous and calcareous soil: a 4-year experiment with atmospheric CO2 enrichment and nitrogen fertilization

To study responses of forests to global change, model ecosystems consisting of beech and spruce trees were established in open top chambers. The ecosystems were exposed to four conditions for 4 years, each replicated four times: ambient and elevated CO₂, and low and high nitrogen input. At the end of the trial, the trees were 6–8 years old. Each chamber contained two separate compartments with siliceous and calcareous soil. Here, we focus on the development of ectomycorrhizas in the topsoil layer. Ectomycorrhizal fungal biomass associated with the fine roots, estimated by using ergosterol content as a marker, was much higher in the siliceous compared to the richer calcareous soil. Also, in root-free soil samples, the level of ergosterol, indicative of the extraradical mycelium of ectomycorrhizal fungi as well as the mycelium of other fungi, was about six times higher in the siliceous than in the calcareous soil. Conditions of elevated atmospheric CO₂ primarily affected ectomycorrhizas in the calcareous soil. Fungal biomass, calculated per soil volume of the top soil layer, increased significantly, as did the metabolic activity of the ectomycorrhizal fungi, measured as uptake of glucose and synthesis of trehalose. Conditions of nitrogen fertilization affected ectomycorrhizas in the siliceous, nutrient poor soil.     

菌根真菌与植物共生营养交换机制研究进展

菌根是陆地生态系统普遍存在的、由土壤中的菌根真菌侵染宿主植物根系形成的联合共生体.菌根的建立是以共生体双方的营养交换为基础的:菌根真菌从土壤中吸收氮、磷等营养物质并转运给宿主植物,供其生长;作为交换,植物则以脂质或糖的形式向菌根真菌提供其生长所必需的碳水化合物.近年来,菌根真菌与宿主植物间的营养交换机制一直是研究的热点,国内外对菌根真菌介导的植物营养物质吸收和转运机制的研究也取得了巨大进展.本文综述了丛枝和外生两种菌根真菌与宿主植物间营养交换的最新研究进展,尤其是碳、氮、磷等几种重要营养物质的吸收与双向转运机制,以及营养交换在菌根形成中的潜在调控作用,并对目前存在的关键问题和未来研究方向进行了分析和展望,这对菌根模型的建立及菌根效益的优化具有重要意义.     

Biochemical and molecular aspects of C/N interaction in ectomycorrhizal plants: an update

The symbiosis (ectomycorrhiza, ECM) between roots of trees and shrubs of boreal and temperate forest ecosystems and soil fungi is essential for water and nutrient acquisition of the plants. The functionality of ECM is largely dependent on the ability of the host plant to supply photoassimilates to the fungus via the symbiotic interface. Based on sterile in vitro and non-sterile pot experiments, we review data which gives evidence that hexoses are supplied to the fungus by the host plant (mainly glucose and fructose), and that these sugars, at least in part, control development and function of ECM by interfering with fungal gene expression. We further show that any factor which reduces hexose allocation to the host–fungus interface will adversely affect ECM development. As an example, we address the impact of increased supply of nitrogen on the biochemistry of plant–fungus interaction and discuss potential consequences on host performance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ectomycorrhiza: gene expression,metabolism and the wood-wide web

In the ectomycorrhizal symbiosis between fungi and trees, the fungus completely ensheaths the tree roots and takes over water and mineral nutrient supply, while the plant supplies photosynthate. Recent work has focussed on gene expression in the two partners, on the effects of global change and nitrogen deposition rate on the symbiosis, and on the role of mycorrhizal fungi in connecting individual plants to form a 'wood-wide web'.     

Exploitation of pollen by mycorrhizal mycelial systems with special reference to nutrient recycling in boreal forests.

Very large quantities of pollen are released annually by wind-pollinated trees, which dominate northern forest ecosystems. Since pollen is enriched in both nitrogen and phosphorus, this recurrent pulse of deposition constitutes a significant potential source of these elements in what are known to be severely nutrient-limited systems. Here, we demonstrate for the first time, to our knowledge, that an ectomycorrhizal fungus, Paxillus involutus, is able to scavenge effectively for nitrogen and phosphorus in pollen and to return a significant proportion of each nutrient to its autotrophic host, Betula pendula. More than 75 and 96%, respectively, of the nitrogen and phosphorus were removed from pollen in microcosms containing the mycorrhizal fungus, 29 and 25%, respectively, being transferred to the plants. In contrast, in microcosms without the mycorrhizal fungus only 42 and 35%, respectively, of nitrogen and phosphorus were lost from the pollen, presumably as a result of export by saprotrophs, and only 12 and 7%, respectively, were transferred to the plants. We hypothesize that this process of resource recapture, by contributing significantly to the ability of the trees to sustain the necessary annual investment in pollen production, will have a major impact upon their reproductive capabilities and hence 'fitness'.     

Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum: characterization of two di- and tripeptide transporters (HcPTR2A and B)

Constraints on plant growth imposed by low availability of nitrogen are a characteristic feature of ecosystems dominated by ectomycorrhizal plants. Ectomycorrhizal fungi play a key role in the N nutrition of plants, allowing their host plants to access decomposition products of dead plant and animal materials. Ectomycorrhizal plants are thus able to compensate for the low availability of inorganic N in forest ecosystems. The capacity to take up peptides, as well as the transport mechanisms involved, were analysed in the ectomycorrhizal fungus Hebeloma cylindrosporum. The present study demonstrated that H. cylindrosporum mycelium was able to take up di- and tripeptides and use them as sole N source. Two peptide transporters (HcPTR2A and B) were isolated by yeast functional complementation using an H. cylindrosporum cDNA library, and were shown to mediate dipeptide uptake. Uptake capacities and expression regulation of both genes were analysed, indicating that HcPTR2A was involved in the high-efficiency peptide uptake under conditions of limited N availability, whereas HcPTR2B was expressed constitutively.     

Changes in the fungus-specific,soluble-carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius

A simple and convenient culture system has been developed for the analysis of ectomycorrhiza formation under controlled conditions. Rapid and synchronous mycorrhiza synthesis was observed when thin and even layers of Pisolithus tinctorius (Pers.) hyphae were brought at once into contact with the entire root system of 3-month-old Picea abies (L. Karst) plants. Suitable fungal layers were grown on cardboard with limiting glucose supply in the medium to maximize radial growth. The glucose was almost consumed by the time the fungus had spread over the whole cardboard and was ready for inoculation of the roots. At this stage, the fungus contained trehalose and arabitol as the main soluble carbohydrates. A few hours after the assembly of the culture system, contacts between roots and aerial hyphae were observed and a sheath was formed 3 days later, suggesting very rapid ectomycorrhiza formation under these conditions. The pool of soluble carbohydrates of the inoculum, i.e. the extramatrical mycelium, declined after inoculation of the roots and was almost zero after 2 weeks. The supply of carbon by the plant was then sufficient for the fungus to expand the soluble pool efficiently in both the mycorrhizas and the extramatrical mycelium. The kinetics of the carbohydrate pool and the observed differentiation of the short roots to mycorrhizas imply that in our culture system fully functional symbiosis was established no later than 14 days after the plants were inoculated with the fungus.     

Phosphate uptake, transport and transfer by the arbuscular mycorrhizal fungus Glomus intraradices is stimulated by increased carbohydrate availability

* The influence of carbohydrate availability to mycorrhizal roots on uptake, metabolism and translocation of phosphate (P) by the fungus was examined in axenic cultures of transformed carrot (Daucus carota) roots in symbiosis with Glomus intraradices. * 14C-labelled carbohydrates, 33P-phosphate and energy dispersive X-ray microanalysis were used to follow the uptake and transfer of C and P in the arbuscular mycorrhizal (AM) symbiosis. * The uptake of P by the extraradical mycelium (ERM) and its translocation to the mycorrhizal roots was stimulated and the metabolic and spatial distribution of P within the fungus were altered in response to increased carbohydrate availability. Sucrose supply resulted in a decrease of polyphosphates and an increased incorporation into phospholipids and other growth-related P pools and also caused elevated cytoplasmic P levels in the intraradical mycelium (IRM) within the root and higher cytoplasmic P levels in the root cortex. * These findings indicate that the uptake of P by the fungus and its transfer to the host is also stimulated by the transfer of carbon from plant to fungus across the mycorrhizal interface.     

Mycorrhizal types differ in ecophysiology and alter plant nutrition and soil processes

Mycorrhizal fungi benefit plants by improved mineral nutrition and protection against stress, yet information about fundamental differences among mycorrhizal types in fungi and trees and their relative importance in biogeochemical processes is only beginning to accumulate. We critically review and synthesize the ecophysiological differences in ectomycorrhizal, ericoid mycorrhizal and arbuscular mycorrhizal symbioses and the effect of these mycorrhizal types on soil processes from local to global scales. We demonstrate that guilds of mycorrhizal fungi display substantial differences in genome‐encoded capacity for mineral nutrition, particularly acquisition of nitrogen and phosphorus from organic material. Mycorrhizal associations alter the trade‐off between allocation to roots or mycelium, ecophysiological traits such as root exudation, weathering, enzyme production, plant protection, and community assembly as well as response to climate change. Mycorrhizal types exhibit differential effects on ecosystem carbon and nutrient cycling that affect global elemental fluxes and may mediate biome shifts in response to global change. We also note that most studies performed to date have not been properly replicated and collectively suffer from strong geographical sampling bias towards temperate biomes. We advocate that combining carefully replicated field experiments and controlled laboratory experiments with isotope labelling and ‐omics techniques offers great promise towards understanding differences in ecophysiology and ecosystem services among mycorrhizal types.