Biological weathering is a function of biotic energy expenditure. Growth and metabolism of organisms generates acids and chelators, selectively absorbs nutrient ions, and applies turgor pressure and other physical forces which, in concert, chemically and physically alter minerals. In unsaturated soil environments, plant roots normally form symbiotic mycorrhizal associations with fungi. The plants provide photosynthate-carbohydrate-energy to the fungi in return for nutrients absorbed from the soil and released from minerals. In ectomycorrhiza, one of the two major types of mycorrhiza of trees, roots are sheathed in fungus, and 15—30% of the net photosynthate of the plants passes through these fungi into the soil and virtually all of the water and nutrients taken up by the plants are supplied through the fungi. Here we show that ectomycorrhizal fungi actively forage for minerals and act as biosensors that discriminate between different grain sizes (53—90 μm, 500—1000 μm) and different minerals (apatite, biotite, quartz) to favour grains with a high surface-area to volume ratio and minerals with the highest P content. Growth and carbon allocation of the fungi is preferentially directed to intensively interact with these selected minerals to maximize resource foraging.

Boreal forest trees influence mineral weathering rates via exudation and uptake processes. Most trees in the boreal forest live in symbiosis with ectomycorrhizal (EcM) fungi that sheath most of the root tips and form the main interface between the tree and the soil. Current weathering models do not take into account the nature and scale of fungal-mineral interactions. Here we show for the first time grain-scale effects of EcM fungi in symbiosis with a host plant on mineral weathering under sterile conditions. EcM fungi actively direct their growth and energy flow towards mineral grains containing essential nutrient elements for the tree and fungus.

Fine roots of forest trees form together with certain soil fungi symbiotic structures (ectomycorrhizas), where fungal hyphae are in intimate contact with plant cells. Due to root cell degeneration, plant DNA is released and could be taken up by the fungus. The possibility that horizontal gene transfer might result in a risk for the environment should be evaluated before a massive release of genetically engineered trees into nature occurs, even though only a few convincing examples of horizontal gene transfer are known. Transgenic poplars containing a construct of the Streptomyces hygroscopicus bar gene under the control of the Cochliobolus heterostrophus GPD (glyceraldehyde-3-phosphate dehydrogenase) promoter were generated by Agrobacterium-mediated transformation. The functionality of this construct in the ectomycorrhizal model fungus Amanita muscaria was previously verified by protoplast-based fungal transformation. 35 000 ectomycorrhizas, formed between transgenic poplars and non-transgenic A. muscaria hyphae, were isolated and transferred to selective agar plates. Putative herbicide-resistant fungal colonies were obtained after the first round of selection. However, none of these colonies survived a transfer onto fresh selection medium, nor did they contain the bar gene, indicating that no horizontal gene transfer from poplar to A. muscaria occurred during symbiosis under axenic conditions. However, since ectomycorrhizas are associated under natural conditions with viruses, bacteria and other fungi, these additional associations should be evaluated in future.

Most mycorrhizas are ‘balanced’ mutualistic associations in which the fungus and plant exchange commodities required for their growth and survival. Myco-heterotrophic plants have ‘exploitative’ mycorrhizas where transfer processes apparently benefit only plants. Exploitative associations are symbiotic (in the broad sense), but are not mutualistic. A new definition of mycorrhizas that encompasses all types of these associations while excluding other plant-fungus interactions is provided. This definition recognises the importance of nutrient transfer at an interface resulting from synchronised plant-fungus development. The diversity of interactions between mycorrhizal fungi and plants is considered. Mycorrhizal fungi also function as endophytes, necrotrophs and antagonists of host or non-host plants, with roles that vary during the lifespan of their associations. It is recommended that mycorrhizal associations are defined and classified primarily by anatomical criteria regulated by the host plant. A revised classification scheme for types and categories of mycorrhizal associations defined by these criteria is proposed. The main categories of vesicular-arbuscular mycorrhizal associations (VAM) are ‘linear’ or ‘coiling’, and of ectomycorrhizal associations (ECM) are ‘epidermal’ or ‘cortical’. Subcategories of coiling VAM and epidermal ECM occur in certain host plants. Fungus-controlled features result in ‘morphotypes’ within categories of VAM and ECM. Arbutoid and monotropoid associations should be considered subcategories of epidermal ECM and ectendomycorrhizas should be relegated to an ECM morphotype. Both arbuscules and vesicles define mycorrhizas formed by glomeromycotan fungi. A new classification scheme for categories, subcategories and morphotypes of mycorrhizal associations is provided.

The ecology and forest management of tropical montane cloud forests of the Neotropics have attracted little scientific attention so far, and understanding of the ecosystem is still fragmentary (Churchill et al. 1995). The montane forests of southern Ecuador are an outstanding biodiversity hotspot for vascular plants (Barthlott et al. 1996), including a multitude of tree species (Madsen & Øllgard 1994). However, identification of species, studies of phenology, productivity, seed production and growth of seedlings have only just begun (Homeier & Breckle 2002). None of the trees has been investigated with respect to their mycorrhizal status, although it is well established that mycorrhizal symbioses are not only important for survival of trees in nutrient-poor habitats (Read 1991) and play a key role for nutrient cycling and nutrient retention in the humus layers (Medina & Cuevas 1993, Rilling et al. 2001), but also have an important impact on the composition of plant communities (Allen et al. 1995, Kottke 2002, van der Heijden et al. 1998).

Dominance of primary rain forests by the ectomycorrhizal, leguminous canopy tree Dicymbe corymbosa (Caesalpiniaceae) was investigated in the Pakaraima Mountains of western Guyana. In five 1-ha forest inventory plots in the Upper Ireng and Upper Potaro River drainages basal areas of D. corymbosa ranged from 38.4-52.8 m2 ha-1 (63-85% of total) among all trees ≥10 cm diameter at breast height (dbh), values in the upper range for tropical moist forests worldwide. The high basal areas for D. corymbosa were due to the prevalence of large (>150 cm dbh), multi-stemmed individuals. Stem densities in Dicymbe plots ranged from 276-433 ha-1, with D. corymbosa contributing 24.6-59.8%. In three 1-ha mixed forest plots adjacent to the Dicymbe plots, D. corymbosa was absent. In the mixed forests, stem densities were higher (480-585 ha-1), basal areas were lower (36.7-39.8 m2 ha-1), species diversity was higher, and canopy tree species were more equitably distributed than in the Dicymbe plots. Tree community composition was not qualitatively different between Dicymbe and mixed forests. In the Dicymbe plots, mean sapling and seedling densities of D. corymbosa were significantly higher than most other canopy species, indicating persistent monodominance. Edaphic variation did not account for variation in forest composition. Life history traits are discussed which may contribute to clumping in D. corymbosa, including coppicing and mast-fruiting, and the potential role of litter-trapping physiognomy and ectomycorrhiza-mediated nutrient dynamics in promoting monodominance is noted.

There has been conflicting debate over many years regarding the trophic status of Matsutake. Here we examined the morphology, structure and ultrastructure of Pinus densiflora roots collected from a Tricholoma matsutake Shiro within a pure Japanese red pine stand. Molecular investigations (PCR–RFLP analyses) indicated that T. matsutake was the highly dominant fungus within both the Shiro and the colonized root tips, suggesting that reported root morphology modifications can be attributed to T. matsutake infection. The external morphology of Matsutake-colonized roots is consistent with previous descriptions. The presence of extraradical mycelium, mantle, and intracortical Hartig net hyphae indicates clearly that T. matsutake forms an ectomycorrhizal association with P. densiflora in naturally occurring Shiros. The elucidation, for the first time, of the Hartig net ultrastructure at the host–fungus interface provides further and convincing evidence of a conventional ectomycorrhizal association. The progressive blackening, observed from base to tip in dominant mycorrhizal types, due to increased deposition of polyphenol and subsequent necrosis, appears to be a result of infection. However, the presence of highly nucleated vascular tissue indicates the viability of the vascular cylinder in these roots bearing necrotic cortices. Such a preponderance of black necrotic cortical tissues among colonized roots may reflect some atypical behaviour of T. matsutake.

A cultivation method was developed to enable exposure of ectomycorrhizal plants with intact extramatrical mycelium to solutions containing different concentrations of aluminium or heavy metals. Pinus sylvestris seedlings colonized by Suillus variegatus (two isolates), Rhizopogon roseolus or Paxillus involutus (two isolates) were used. Seedlings were transferred to Petri dishes containing glass beads and exposed to elevated concentrations of Al, Cd, Cu, or Ni in two ways: immediately following transfer; and after allowing mycorrhizal seedlings to develop an extraradical mycelium that colonized the interface between the upper surface of the beads and the metal- containing solution. Production of organic acids in mycorrhizal and non-mycorrhizal systems was measured by withdrawing samples from the solution and analyzing by HPLC. In most experiments, levels of oxalic acid were significantly higher in mycorrhizal treatments than in non-mycorrhizal controls. The measured levels of organic acids were variable, but the results obtained suggest that production of oxalic acid is stimulated by exposure to elevated Al in mycorrhizal seedlings colonized by S. variegatus and R. roseolus. Elevated Al concentrations also increased oxalic acid production by non-mycorrhizal seedlings significantly in two of four Al experiments performed, but the measured concentrations were significantly lower than in corresponding mycorrhizal treatments in both cases. Malonic acid was found in the culture solution of non-mycorrhizal and P. involutus- colonized seedlings, but only trace amounts were found in S. variegatus or R. roseolus-infected seedlings. Citric, shikimic, lactic, acetic, propionic, fumaric, formic, iso-butyric and butyric acid were found in variable concentrations. Production of oxalic acid by seedlings colonized by S. variegatus BL or P. involutus was not stimulated by exposure to 0.44 μM Cd or 17 μM Ni. Exposure to 0.157 mM Cu in two separate experiments using P. involutus 87.017 and two strains of S. variegatus (BL and I59) appeared to stimulate production of oxalic acid irrespective of mycorrhizal status or species.

Ectomycorrhizal seedlings of Scots pine (Pinus sylvestris) inoculated with the nitrotolerant Laccaria bicolor and the nitrophobic Suillus bovinus were exposed to ambient (350 μl l−1) and elevated (700 μl l−1) [CO2]. After 79 d the seedlings were labelled for 28 d with 14CO2, after which they were harvested. 14C was determined in shoots, roots plus mycorrhizas, soil, and below-ground respiration; nitrogen was determined in shoots and roots. Total net 14C uptake increased under elevated [CO2]. The extra carbon did not increase the shoot mass but was translocated to the roots and resulted in a decreased shoot-to-root ratio in the Suillus-inoculated seedlings. Laccaria-inoculated seedlings did not incorporate the additional carbon in root or fungal tissue but only increased below-ground respiration. S. bovinus acquired or transferred nitrogen better than L. bicolor and enabled the seedlings to perform better with regard to net carbon uptake under elevated [CO2]. This resulted in nitrogen concentrations in shoots of Suillus-inoculated seedlings that were twice as high as in Laccaria-inoculated seedlings, irrespective of [CO2]. The higher nitrogen concentration in the shoots resulted in a doubling of the 14C uptake per unit shoot mass. Our results suggest that the ability of ectomycorrhizal Scots pine seedlings to respond positively to elevated atmospheric [CO2] is strongly fungal-species specific.

The effects of mycorrhiza formation in combination with elevated CO2 concentrations on carbon metabolism of Norway spruce (Picea abies) seedlings and aspen (Populus tremula × Populus tremuloides) plantlets were analysed. Plants were inoculated for 6 wk with the ectomycorrhizal fungi Amanita muscaria and Paxillus involutus (aspen only) in an axenic Petri-dish culture at 350 and 700 μl l−1 CO2 partial pressure. After mycorrhiza formation, a stimulation of net assimilation rate was accompanied by decreased activities of sucrose synthase, an increased activation state of sucrose-phosphate synthase, decreased fructose-2,6-bisphosphate and starch, and slightly elevated glucose-6-phosphate contents in source leaves of both host species, independent of CO2 concentration. Exposure to elevated CO2 generally resulted in higher net assimilation rates, increased starch as well as decreased fructose-2,6-bisphosphate (aspen only) content in source leaves of both mycorrhizal and nonmycorrhizal plants. Our data indicate only slightly improved carbon utilization by mycorrhizal plants at elevated CO2. They demonstrate however, that both factors which modulate the sink-source properties of plants increase the capacity for sucrose synthesis in source leaves mainly by allosteric enzyme regulation.

Seedlings of the myco-heterotrophic orchid Corallorhiza trifida which had been germinated in the field in mesh bags developed hyphal links and mycorrhizas with Betula pendula and Salix repens, but not with Pinus sylvestris, when transplanted into soil microcosms. The fungus connecting the myco-heterotroph to Betula and Salix formed endomycorrhiza in the orchid with typical pelotons, but formed ectomycorrhizas with the autotrophs. The orchid plants, when linked to Betula and Salix by fungal hyphae, gained 6–14% in weight over 25–28 wk. In microcosms supporting P. sylvestris, and in control microcosms which lacked autotrophs, the Corallorhiza plants lost 13% of their weight over the same period. In the course of the 28-wk experimental period new Corallorhiza seedlings, in addition to those added as part of the experiment, appeared in the microcosms containing Salix and Betula but not in the Pinus microcosms. Shoots of Betula and Salix plants grown in association with Corallorhiza were fed with 14CO2, and the movement of the isotope was subsequently traced by a combination of digital autoradiography and tissue oxidation. Direct transfer of C from both autotrophs to the myco-heterotroph occurred in all cases where the associates had become connected by a shared fungal symbiont. Orchid seedlings lacking these hyphal connections, introduced to the microcosms as controls immediately before isotope feeding, failed to assimilate significant amounts of C. The results provide the first experimental confirmation that growth of Corallorhiza trifida can be sustained by supply of C received directly from an autotrophic partner through linked fungal mycelia.

The mycobionts of Piceirhiza bicolorata, a distinct ectomycorrhizal morphotype of conifers and hardwoods, have been identified by internal transcribed spacer 1 (ITS1) nuclear ribosomal DNA (rDNA) sequence comparison of the fungi involved. Samples of Piceirhiza bicolorata were obtained from seedlings of Picea abies, Pinus sylvestris, Betula pubescens, Populus tremula, Quercus robur and Salix phylicifolia. In an initial screening, the fungus amplified with universal ITS primers from ectomycorrhizal root samples of P. bicolorata shared approx. 95% ITS1 sequence identity with the ericoid mycorrhizal fungus Hymenoscyphus ericae. A total of 77 out of 88 (= 87.5%) DNA samples (i.e. 52/56 root samples and 25/32 axenic culture isolates) of P. bicolorata were successfully amplified with a taxon-selective primer designed for exclusive amplification of H. ericae-like strains. Forty-seven amplicons were sequenced, yielding 15 different ITS1 genotypes that differed by 1–14 nucleotide character state changes. An inferred ITS1 phylogeny (maximum parsimony) showed that a single major evolutionary lineage of P. bicolorata embraced the historically important H. ericae isolates in a 100% bootstrap-supported clade. The 15 P. bicolorata genotypes were positioned in four subclades, roughly corresponding to morphological groups of P. bicolorata isolates observed in axenic culture. Culture isolates of H. ericae and P. bicolorata share some common morphological features including slow, dense growth and formation of short aerial hyphal aggregates. Our results suggest that members of the H. ericae aggregate participate in the formation of the distinct ectomycorrhizal morphotype P. bicolorata. This opposes the widely accepted discrimination of ericoid and ectomycorrhizal mycobionts of the boreal forest ecosystem. The high prevalence of the P. bicolorata morphotype on pioneer seedlings of P. sylvestris, B. pubescens and S. phylicifolia at a copper mine spoil was remarkable. Hypotheses of possible nutrient mobilization and detoxification potentials of the fungal associates of P. bicolorata are discussed. We hypothesize that ericoid and ectomycorrhizal plants may share mycobionts of the H. ericae aggregate.

The inter- and intracellular distribution of phosphate in non-mycorrhizal and ectomycorrhizal roots of Pinus sylvestris was analysed by energy-dispersive X-ray spectroscopy after cryofixation, freeze-drying, pressure infiltration with resin and dry cutting of the material. The results show (1) that in non-mycorrhizal roots the distribution of phosphate across the root cross section is relatively homogenous, (2) that the intracellular distribution of phosphate depends on the external supply, and (3) that the phosphate absorption can be increased by a supply of NH4. Under phosphate starvation, the phosphate is mainly localized in the cell cytoplasm, whereas under high external phosphate this element accumulates in the vacuoles of cortical cells. It can be inferred that at low external conditions phosphate is mainly located in the cytoplasm of cells to protect metabolism from deficiency, that phosphate is translocated to the shoot via symplastic and apoplastic pathways, and that especially the vacuolar pool size is regulated by the external phosphate supply. The nutrition of the host plant under phosphate starvation was improved by a mycorrhizal infection with Suillus bovinus. However, this effect might not be the same for all ectomycorrhizal infections and supply conditions. With high external phosphate an ectomycorrhizal infection with S. bovinus had no effect on intracellular phosphate within the roots, the shoot contents of mycorrhizal plants were less than those of non-mycorrhizal seedlings and translocation rate across the mycorrhizal interface appeared to be independent of the availability of phosphate. Intracellular phosphate in fungal cells of mycorrhizal roots was independent of the external supply and seemed to be regulated by the formation of polyphosphates. A supply of (NH4)2HPO4, which led to an increase in cytoplasmic phosphate levels in the hyphae of the Hartig net of the Paxillus involutus mycorrhiza also led to higher phosphate in the plant cell compartments. Therefore, it can be inferred that the translocation of phosphate from fungus to host plant across the mycorrhizal interface is regulated by the phosphate concentration in the cytoplasm of the Hartig net and by the efflux rate into the interfacial apoplast.

The apoplasmic permeability of ectomycorrhizal roots of intact Eucalyptus pilularis seedlings infected with Pisolithus tinctorius on aseptic agar plates was examined using the nonbinding fluorochrome 8-hydroxypyrene-1,3,6-trisulphonate and lanthanum ions in conjunction with anhydrous freeze substitution and dry sectioning. Most mycorrhizas formed in the air above the agar surface, and in these the sheath rapidly became nonwettable and impermeable to the fluorochrome but was nevertheless permeable to lanthanum ions. In a few mycorrhizas which developed in contact with the agar the sheath remained permeable to both tracers when fully developed. This increased hydrophobicity of the sheath in mycorrhizas in the air above the agar surface might be explained by deposition of hydrophobins, but nevertheless it still allows an apoplasmic pathway for radial movement of ions. Regardless of their sheath permeation both apoplasmic tracers were always found throughout the Hartig net and were arrested at the Casparian bands and suberin lamellae of the exodermis. It is concluded that the fluorochrome must have moved longitudinally along the Hartig net which is a region of higher permeability than the sheath. Casparian bands in the exodermis of ectomycorrhizal roots have similar properties to those in nonmycorrhizal roots in excluding solutes and their exclusion of lanthanum ions indicates that they are not permeable to ions. The data do not support the concept of a totally sealed apoplasmic exchange compartment, but the differential permeability suggests that the sheath might allow radial transfer of ions but block loss of sugars and organic molecules of similar size.

Clones of ectomycorrhizal fungi can colonize new areas through production of vegetative mycelium or spore dispersal, but the relative importance of these processes in nature is not known. In this study, sporocarps of an ectomycorrhizal fungus, Suillus grevillei, were mapped and sampled from a Larix kaempferi stand at the foot of Mt Fuji. DNA was extracted directly from each sporocarp, and DNA polymorphism was analysed by polymerase chain reaction (PCR) amplification of inter-simple sequence repeat (ISSR) regions primed by (GTG)5, (GCC)5 and (GACA)4. Different sensitivities to detect polymorphism were found among the three primers, with (GACA)4 showing the highest sensitivity. Forty seven sporocarps were analysed by the three ISSR primers and divided into 34 genets based on combination of PCR fingerprints. In the population 28 genets were represented by individual sporocarps. In most cases, sporocarps grown in aggregation (within a circle of 50 cm diameter) showed some different ISSR band patterns. These results suggest that genets of S. grevillei at the test site are relatively small. The genetic similarities between the 34 genets were also calculated and similarity groups were determined by the criterion that all similarity F values of genets within a group were not <80%. In general, the genets within a similarity group located close to each other. The results of multiple different but highly related genets in a small area suggest that the population of S. grevillei in this stand is not spread and maintained by clonal mycelium extension but is reproduced by spore dispersal.

Interactions between saprotrophic and ectomycorrhizal fungi have been largely ignored, although their mycelia often share the same microsites. The mycelial systems show general similarity to each other and, although the enzymatic potential of the saprotrophic fungi is generally considered to be higher, the importance of organic nutrient sources to ectomycorrhizal fungi is now widely accepted. In the experiments described here, nutritional interactions involving transfer of elements from one mycelium to the other have been monitored dynamically using radioactive tracers and a non-destructive electronic autoradiography system. Microcosms were used in which mycelial systems of the ectomycorrhizal fungi Suillus variegatus and Paxillus involutus, extending from Pinus sylvestris host plants, were confronted with mycelia of the saprotroph Hypholoma fasciculare extending from wood blocks. The fungi showed a clear morphological confrontation response. The mycorrhizal mycelium often formed dense patches over the Hypholoma mycelia. Up to 25% of the 32P present in the Hypholoma mycelium was captured by the mycorrhizal fungi and translocated to the plant host within 30 d. The transfer of 32P to the saprotroph from labelled mycorrhizal mycelium was one to two orders of magnitude lower. The significance of this transfer as a ‘short cut’ in nutrient cycling is discussed.

Short-term phosphate uptake rates were measured on intact ectomycorrhizal and non-mycorrhizal Pinus sylvestris seedlings using a new, non-destructive method. Uptake was quantified in semihydroponics from the depletion of Pi in a nutrient solution percolating through plant containers. Plants were grown for 1 or 2 months after inoculation at a low relative nutrient addition rate of 3% d−1 and under P limitation. Four ectomycorrhizal fungi were studied: Paxillus involutus, Suillus luteus, Suillus bovinus and Thelephora terrestris. The Pi-uptake capacity of mycorrhizal plants increased sharply in the month after inoculation. The increase was dependent on the development of the mycobionts. A positive correlation was found between the Pi-uptake rates of the seedlings and the active fungal biomass in the substrate as measured by the ergosterol assay. The highest Pi-uptake rates were found in seedlings associated with fungi producing abundant external mycelia. At an external Pi concentration of 10 μM, mycorrhizal seedlings reached uptake rates that were 2.5 (T. terrestris) to 8.7 (P. involutus) times higher than those of non-mycorrhizal plants. The increased uptake rates did not result in an increased transfer of nutrients to the plant tissues. Nutrient depletion was ultimately similar between mycorrhizal and non-mycorrhizal plants in the semihydroponic system. Net Pi absorption followed Michaelis–Menten kinetics: uptake rates declined with decreasing Pi concentrations in the nutrient solution. This reduction was most pronounced in non- mycorrhizal seedlings and plants colonized by T. terrestris. The results confirm that there is considerable heterogeneity in affinity for Pi uptake among the different mycobionts. It is concluded that the external mycelia of ectomycorrhizal fungi strongly influence the Pi-uptake capacity of the pine seedlings, and that some mycobionts are well equipped to compete with other soil microorganisms for Pi present at low concentrations in soil solution.

The use of biofuels has been proposed as one possible substitute for fossil fuels, which contribute substantially to the increase in [CO2] in the atmosphere. However, increased harvesting of forest residues for biofuel might affect the availability of base cations, P and N, as well as the development, community dynamics and function of ectomycorrhizas. This in turn might influence nutrient uptake and tree growth. In this study we investigated the effects of repeated forest residue harvesting on ectomycorrhizal species colonizing spruce roots in the humus layer of a 35-yr-old forest. Harvesting significantly decreased the thickness of the humus layer as well as decreasing the numbers of ectomycorrhizal root tips both per metre root length and per unit humus volume. Changes in mycorrhizal community structure were studied by ITS typing with the use of PCR–RFLP analysis. In total, 19 different ITS types were found on two different sampling occasions (autumn and spring); 11 of these were common to both samplings. Nine of the ITS types were identified to at least the genus level by comparison with RFLP patterns of identified fruiting bodies or axenic cultures. Five species, Cortinarius sp. 2, Thelephora terrestris (Ehrenb.) Fr., Lactarius theiogalus (Bull.[ratio ]Fr.) S. F. Gray s.st. Neuhoff, Tylospora fibrillosa Donk and Tö-96-12, occurred on over 5% of the total sampled root tips. Together these five types colonized 63% of the mycorrhizas screened. A similarity index assessment showed no shift in mycorrhizal community structure as a result of harvesting. Our findings suggest that the repeated removal of forest residues might have a strong effect on the quantity and development of ectomycorrhizal roots in the organic horizon, but little effect on the species composition of the community.

An experiment was performed to find out whether ectomycorrhizal (ECM) fungi alter the nitrogen (N) isotope composition, δ15N, of N during the transport of N from the soil through the fungus into the plant. Non- mycorrhizal seedlings of Pinus sylvestris were compared with seedlings inoculated with either of three ECM fungi, Paxillus involutus, Suillus bovinus and S. variegatus. Plants were raised in sand in pots supplied with a nutrient solution with N given as either NH4+ or NO3−. Fractionation against 15N was observed with both N sources; it decreased with increasing plant N uptake, and was larger when NH4+ was the source. At high ratios of Nuptake/Nsupplied there was no (NO3−), or little (NH4+), fractionation. There seemed to be no difference in fractionation between ECM and non-mycorrhizal plants, but fungal rhizomorphs were sometimes enriched in 15N (up to 5‰ at most) relative to plant material; they were also enriched relative to the N source. However, this enrichment of the fungal material was calculated to cause only a marginal decrease (−0.1‰ in P. involutus) in δ15N of the N passing from the substrate through the fungus to the host, which is explained by the small size of the fungal N pool relative to the total N of the plant, i.e. the high efficiency of transfer. We conclude that the relatively high 15N abundance observed in ECM fungal species should be a function of fungal physiology in the ECM symbiosis, rather than a reflection of the isotopic signature of the N source(s) used. This experiment also shows that the δ15N of plant N is a good approximation of δ15N of the available N source(s), provided that N is limiting growth.

Many soil fungi colonize the roots of pines to form symbiotic organs known as ectomycorrhizas. Dichotomous branching of short lateral roots and the formation of coralloid organs are diagnostic of ectomycorrhizas in many pine species, although the regulation of these changes in root morphology is not well understood. We used axenic root cultures of six pine species to examine the role of auxin, cytokinin, ethylene and nutrients in the regulation of root architecture. Surprisingly, extensive dichotomous and coralloid branching of lateral roots occurred spontaneously in Pinus taeda, P. halepensis and P. muricata. In P. sylvestris, P. ponderosa and P. nigra, treatment with auxin transport inhibitors (ATIs), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the ethylene-releasing compound 2-chloroethylphosphonic acid (CEPA or ethephon) induced extensive dichotomous branching and coralloid organ formation. Formation of both spontaneous and ATI-induced coralloid structures was blocked by treatment with an ethylene synthesis inhibitor L-α-(2-aminoethoxyvinyl)glycine; this inhibition was reversed by either ACC or CEPA. In addition, the induction of this unique morphogenetic pattern in pine root cultures was regulated by nutrient levels. The morphology and anatomical organization of the chemically induced dichotomous and coralloid structures, as well as the regulation of their formation by nutrient levels, show a striking similarity to those of ectomycorrhizas.