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For successful colonization of host roots, ectomycorrhizal (EM) fungi must overcome host defense systems, and defensive phenotypes have previously been shown to affect the community composition of EM fungi associated with hosts. Secondary metabolites, such as terpenes, form a core part of these defense systems, but it is not yet understood whether variation in these constitutive defenses can result in variation in the colonization of hosts by specific fungal species.We planted seedlings from twelve maternal families of Scots pine (Pinus sylvestris) of known terpene genotype reciprocally in the field in each of six sites. After 3 months, we characterized the mycorrhizal fungal community of each seedling using a combination of morphological categorization and molecular barcoding, and assessed the terpene chemodiversity for a subset of the seedlings. We examined whether parental genotype or terpene chemodiversity affected the diversity or composition of a seedling's mycorrhizal community.While we found that terpene chemodiversity was highly heritable, we found no evidence that parental defensive genotype or a seedling's terpene chemodiversity affected associations with EM fungi. Instead, we found that the location of seedlings, both within and among sites, was the only determinant of the diversity and makeup of EM communities.These results show that while EM community composition varies within Scotland at both large and small scales, variation in constitutive defensive compounds does not determine the EM communities of closely cohabiting pine seedlings. Patchy distributions of EM fungi at small scales may render any genetic variation in associations with different species unrealizable in field conditions. The case for selection on traits mediating associations with specific fungal species may thus be overstated, at least in seedlings., Host defense genotype has previously been shown to affect associations with ectomycorrhizal (EM) fungi, but the extent to which secondary metabolites such as terpenes affect these associations is unknown. We conducted a reciprocal transplant field experiment to test the effect of varying terpene genotype on the EM community composition of individual seedlings and found that the only the location of a seedling affects community composition. Other processes affecting EM community composition at small scales, such as environmental preference and interspecific interactions, may thus preclude the expression of any effect of genotype in wild conditions.

The root‐associated habit has evolved on numerous occasions in different fungal lineages, suggesting a strong evolutionary pressure for saprotrophic fungi to switch to symbiotic associations with plants. Species within the ubiquitous, saprotrophic genus Mycena are frequently major components in molecular studies of root‐associated fungal communities, suggesting that an evaluation of their trophic status is warranted. Here, we report on interactions between a range of Mycena species and the plant Betula pendula. - In all, 17 Mycena species were inoculated onto B. pendula seedlings. Physical interactions between hyphae and fine roots were examined using differential staining and fluorescence microscopy. Physiological interactions were investigated using 14C and 32P to show potential transfer between symbionts. - All Mycena species associated closely with fine roots, showing hyphal penetration into the roots, which in some cases were intracellular. Seven species formed mantle‐like structures around root tips, but none formed a Hartig net. Mycena pura and Mycena galopus both enhanced seedling growth, with M. pura showing significant transfer of 32P to the seedlings. - Our results support the view that several Mycena species can associate closely with plant roots and some may potentially occupy a transitional state between saprotrophy and biotrophy.

SummaryRecently, several saprotrophic genera have been found to invade/interact with plant roots in laboratory growth experiments, and this coincides with reports of abundant saprotrophic fungal sequences in plant roots. However, it is uncertain if this reflects field phenomena, and if reports on coincidentally amplified saprotrophs are simply coincidental.We investigated root invasion by presumed saprotrophic fungi by focusing on the large genus Mycena in 1) a systematic analysis of the occurrence of saprotrophic fungi in new and previously published ITS1/ITS2 datasets generated from roots of 10 mycorrhizal plant species, and 2) we analysed natural abundances of 13C/15N stable isotope signatures of fungal/plant communities from five comparable field locations to examine the trophic status of Mycena species.Mycena was the only saprotrophic genus consistently found in 9 of 10 plant host roots, with high within-host variation in Mycena sequence proportions (0-80%) recovered. Mycena carpophores displayed isotopic signatures consistent with published 13C/15N profiles of both saprotrophic or mutualistic lifestyles, with considerable intraspecific variation, resembling the patterns seen in growth experiments. These results indicate that multiple Mycena species opportunistically invade the roots of a range of plant species, possibly forming a spectrum of interactions. This potentially challenges our general understanding of fungal ecology.Originality significance statementThis is the first study to apply a dual approach of systematic metabarcoding of plant roots and stable isotope signatures on dried field material to the large and common saprotrophic fungal genus Mycena. This is significant as it shows that members of this genus, normally not expected to be found inside plant roots at all, are in fact associated eith multiple plant hosts. The study furthermore shows that species in this genus may occupy different ecological roles in the field besides being saprotrophic. That a large and common fungal genus known to be a quantitatively important litter decayer can be an opportunistic root invader and interact with host plants is of interest to all mycologists and ecologists working on plant-fungus/microb symbiosis.

The mechanisms regulating community composition and local dominance of trees in species-rich forests are poorly resolved, but the importance of interactions with soil microbes is increasingly acknowledged. Here, we show that tree seedlings that interact via root-associated fungal hyphae with soils beneath neighbouring adult trees grow faster and have greater survival than seedlings that are isolated from external fungal mycelia, but these effects are observed for species possessing ectomycorrhizas (ECM) and not arbuscular mycorrhizal (AM) fungi. Moreover, survival of naturally-regenerating AM seedlings over ten years is negatively related to the density of surrounding conspecific plants, while survival of ECM tree seedlings displays positive density dependence over this interval, and AM seedling roots contain greater abundance of pathogenic fungi than roots of ECM seedlings. Our findings show that neighbourhood interactions mediated by beneficial and pathogenic soil fungi regulate plant demography and community structure in hyperdiverse forests., Associations with mycorrhizal fungi can affect the outcome of plant competition in complex ways. Here the authors use a decade-long field survey and two hyphal exclusion experiments to reveal a critical role of underground fungal networks in facilitating seedling growth and fitness of ectomycorrhizal plants but not arbuscular mycorrhizal plants.

Stipitate hydnoid fungi are considered to be rare in the United Kingdom based on infrequent and localised observations of fruit bodies. Here, we investigate whether the production of stipitate hydnoid fruit bodies is related to weather conditions using a 14 year standardised survey of 11 species associated with Scots pine. Fruit body production was highly variable over time and asynchronous between species. Relationships with climatic predictors were variable between species, however both overall abundance and species richness of stipitate hydnoid fruit bodies were related to rainfall. These results suggest that climatic conditions in the preceding months can influence the likelihood of observing stipitate hydnoid fruit bodies, but that a large part of variation in fruiting of these taxa remains unexplained.

We characterised the fungi isolated from necrotic lesions observed on roots of the noxious weed Jacobaea vulgaris and assessed their host range. ITS–rDNA sequence data identified twenty-one isolates of Fusarium and TEF-1α sequencing separated these isolates into four clades namely Fusarium avenaceum / Fusarium acuminatum , Fusarium redolens , Fusarium culmorum / Fusarium cerealis and Fusarium solani . Representatives of each clade were tested for virulence against ragwort seedlings. All isolates in the F. avenaceum / F. acuminatum clade caused visible disease symptoms. Host range of three isolates was tested using six pasture grass species along with Trifolium pratense and Trifolium repens . No disease symptoms were detected on the grasses. All three isolates, however, caused disease symptoms on T. repens , and two isolates also attacked T. pratense. The results demonstrate that several species of Fusarium can colonise ragwort roots but only isolates from one clade cause significant disease symptoms on ragwort seedlings.

Partitioning of soil phosphorus (P) pools has been proposed as a key mechanism maintaining plant diversity, but experimental support is lacking. Here, we provided different chemical forms of P to 15 tree species with contrasting root symbiotic relationships to investigate plant P acquisition in both tropical and subtropical forests. Both ectomycorrhizal (ECM) and arbuscular mycorrhizal (AM) trees responded positively to addition of inorganic P, but strikingly, ECM trees acquired more P from a complex organic form (phytic acid). Most ECM tree species and all AM tree species also showed some capacity to take up simple organic P (monophosphate). Mycorrhizal colonisation was negatively correlated with soil extractable P concentration, suggesting that mycorrhizal fungi may regulate organic P acquisition among tree species. Our results support the hypothesis that ECM and AM plants partition soil P sources, which may play an ecologically important role in promoting species coexistence in tropical and subtropical forests.

Summary The interaction between the ash dieback fungus, Hymenoscyphus fraxineus, and two commonly grown members of the Oleaceae (Forsythia × intermedia ‘Lynwood’ and Ligustrum vulgare) was investigated. Shoots were inoculated with three isolates of H. fraxineus and harvested after 2 and 10 weeks. DNA was extracted from tissue collected at the point of inoculation, as well as 15 mm below and above (distal), and analysed using fungal-specific ITS primers and specific primers for H. fraxineus. The fungus was frequently detected in tissue at the point of inoculation, but rarely in distal samples. There was no evidence that H. fraxineus was pathogenic on either of the two oleaceous species over the duration of the experiment.

Typically, Mycena species are viewed as saprotrophic fungi. However, numerous detections of Mycena spp. in the roots of green plants suggest that a continuum from saprotrophy to biotrophy could exist. In particular, mycenoid species have repeatedly been found in Ericaceae plant roots. Our study asked whether (1) Mycena species are commonly found in the roots of green Ericaceae plants; (2) Mycena sequences are limited to a single group/lineage within the genus; and (3) a Mycena sp. can behave as a beneficial root associate with a typical ericoid mycorrhizal plant (Vaccinium corymbosum), regardless of how much external labile carbon is available. We detected Mycena sequences in roots of all sampled Ericaceae plants. Our Mycena sequences clustered in four different groups distributed across the Mycena genus. Only one group could be assigned with confidence to a named species (M. galopus). Our Mycena sequences clustered with other Mycena sequences detected in roots of ericoid mycorrhizal plant species collected throughout Europe, America, and Australia. An isolate of M. galopus promoted growth of V. corymbosum seedlings in vitro regardless of external carbon supply in the media. Seedlings inoculated with M. galopus grew as well as those inoculated with the ericoid mycorrhizal fungus Rhizoscyphus ericae. Surprisingly, this M. galopus isolate colonized Vaccinium roots and formed distinctive peg-like structures. Our results suggest that Mycena species might operate along a saprotroph–symbiotic continuum with a range of ericoid mycorrhizal plant species. We discuss our results in terms of fungal partner recruitment by Ericaceae plants.

In-growth bags are increasingly used to study extramatrical mycelium (EMM) of ectomycorrhizal fungi in forest soils. In this paper we tested whether bag size and presence of soil fauna in bags influence the production, isotopic composition, carbon (C) and nitrogen (N) content of the EMM. Cylindrical in-growth mesh bags (2- or 5-cm-diameter; with or without openings – (1 or 2 mm), allowing faunal colonization or not) were harvested 37, 48, 81 and 283 days after installation in July and the EMM biomass was determined from elemental analyses of the extractable amount of mycelia. The occurrence of openings allowed animals to invade the bags but this did not affect the amount of EMM. We suggest further studies in this matter since the number of animals was low and variable. In the first harvest, mycelial biomass C was three times greater in 2-cm than in 5-cm-bags. After 81 days, mycelial biomass C was 54% greater in the 2-cm (54 kg ha −1 ) than in the 5-cm bags (35 kg ha −1 ). While total mycelial C did not change over winter, N content increased suggesting a role for the EMM in the storage of N from autumn to spring. The δ 13 C and δ 15 N of the EMM changed between the first three harvests. We hypothesize these changes to be mainly driven by changes in plant C and N sinks. The relation between the isotopic composition of sporocarp exploration type, plant roots and EMM is discussed.

A major gap in our understanding of biodiversity–ecosystem function relationships concerns the role of intra- and interspecific diversity of mycorrhizal fungi, which are critical for plant fitness, biogeochemical cycling and other processes. Here, we test the hypothesis that the identity and richness of ectomycorrhizal (ECM) fungi at the intra- and interspecific levels affect ecosystem multifunctionality by regulating plant and fungal productivity, soil CO2 efflux and nutrient retention. Microcosms containing Scots pine (Pinus sylvestris) seedlings colonized by different ECM fungal isolates, in monocultures and mixtures, enabled us to test for both intra- and interspecific identity and richness effects, and transgressive overyielding. Intra- and interspecific identity had modest but significant effects on plant and fungal productivity and nutrient retention, but no effect on CO2 efflux. Intraspecific richness increased plant root productivity and ECM root tips but decreased hyphal length, whereas interspecific richness had no effects. Interspecific mixtures outperformed the most productive monocultures in only 10% of the cases, compared with 42% for the intraspecific mixtures. Both intra- and interspecific identity and richness of ECM fungi regulate ecosystem multifunctionality, but their effects on the direction and magnitude of individual variables differ. Transgressive overyielding suggests that positive niche complementarity effects are driving some of the responses to intraspecific richness.

Willows (Salix spp.) are mycorrhizal tree species sometimes cultivated as short rotation coppice (SRC) on arable sites for energy purposes; they are also among the earliest plants colonising primary successional sites in natural stands. The objective of this study was to analyse the degree of colonisation and diversity of ectomycorrhizal (EM) communities on willows grown as SRC in arable soils and their adjacent natural or naturalized stands. Arable sites usually lack ectomycorrhizal host plants before the establishment of SRC, and adjacent natural or naturalized willow stands were hypothesized to be a leading source of ectomycorrhizal inoculum for the SRC. Three test sites including SRC stands (Salix viminalis, Salix dasyclados, and Salix schwerinii) and adjacent natural or naturalized (Salix caprea, Salix fragilis, and Salix × mollissima) stands in central Sweden were investigated on EM colonisation and morphotypes, and the fungal partners of 36 of the total 49 EM fungi morphotypes were identified using molecular tools. The frequency of mycorrhizas in the natural/naturalized stands was higher (two sites) or lower (one site) than in the corresponding cultivated stands. Correspondence analysis revealed that some EM taxa (e.g. Agaricales) were mostly associated with cultivated willows, while others (e.g. Thelephorales) were mostly found in natural/naturalized stands. In conclusion, we found strong effects of sites and willow genotype on EM fungi formation, but poor correspondence between the EM fungi abundance and diversity in SRC and their adjacent natural/naturalized stands. The underlying mechanism might be selective promotion of some EM fungi species by more effective spore dispersal.

Hypocreopsis rhododendri is a rare fungus that grows on woody stems in hyperoceanic climax scrub on the west coasts of Britain, Ireland, and France. Knowledge of the distribution and abundance of the fungus is based entirely on sporocarp records; it does not account for any occurrence as vegetative mycelia. To address this issue, a H. rhododendri-specific polymerase chain reaction (PCR) was developed and used to assay Corylus avellana (hazel) stems for the presence of H. rhododendri mycelia. The primers ITSHrF and ITSHrR were designed within the internal transcribed spacer 2 region, and their specificity to H. rhododendri was established by their failure to amplify DNA extracted from 14 other Hypocreaceae species. The sensitivity of the assay was demonstrated by amplifying DNA extracted from 4 mg C. avellana wood spiked with 0.0013 % H. rhododendri mycelium. Samples of wood and bark were then taken from around and directly underneath 11 H. rhododendri sporocarps and assayed for the presence of H. rhododendri. PCR products were obtained from a third of the surface bark samples, but only one faint product was obtained from 70 samples taken from beneath the outer bark. The results support the view that H. rhododendri does not form mycelia within stems. We suggest that H. rhododendri is not a saprotrophic fungus, but instead appears to be a parasitic on the wood decay fungus Hymenochaete corrugata, with which it always occurs. Evidence that tissue of H. corrugata is present within the sporocarps of H. rhododendri is discussed.

Porcini (Boletus section Boletus: Boletaceae: Boletineae: Boletales) are a conspicuous group of wild, edible mushrooms characterized by fleshy fruiting bodies with a poroid hymenophore that is ‘‘stuffed’’ with white hyphae when young. Their reported distribution is with ectomycorrhizal plants throughout the Northern Hemisphere. Little progress has been made on the systematics of this group using modern molecular phylogenetic tools because sampling has been limited primarily to European species and the genes employed were insufficient to resolve the phylogeny. We examined the evolutionary history of porcini by using a global geographic sampling of most known species, new discoveries from little explored areas, and multiple genes. We used 78 sequences from the fast-evolving nuclear internal transcribed spacers and are able to recognize 18 reciprocally monophyletic species. To address whether or not porcini form a monophyletic group, we compiled a broadly sampled dataset of 41 taxa, including other members of the Boletineae, and used separate and combined phylogenetic analysis of sequences from the nuclear large subunit ribosomal DNA, the largest subunit of RNA polymerase II, and the mitochondrial ATPase subunit six gene. Contrary to previous studies, our separate and combined phylogenetic analyses support the monophyly of porcini. We also report the discovery of two taxa that expand the known distribution of porcini to Australia and Thailand and have ancient phylogenetic connections to the rest of the group. A relaxed molecular clock analysis with these new taxa dates the origin of porcini to between 42 and 54 million years ago, coinciding with the initial diversification of angiosperms, during the Eocene epoch when the climate was warm and humid. These results reveal an unexpected diversity, distribution, and ancient origin of a group of commercially valuable mushrooms that may provide an economic incentive for conservation and support the hypothesis of a tropical origin of the ectomycorrhizal symbiosis.

The plant intermediate wintergreen (Pyrola media, Ericaceae) is in need of conservation action in Scotland. Although widespread, it is locally distributed in dwarf shrub heath and more commonly in Scots pine (Pinus sylvestris) woodlands. A recent study on the mycorrhizal status of Pyrola suggested that they associate with a restricted range of ectomycorrhizal (ECM) fungi. Here, we examined the hypothesis that specialisation by P. media for fungi usually associated with Scots pine is a factor in promoting its occurrence in this habitat. The fungal community associated with the roots of P. media growing in a Scots pine forest was determined by morphotyping, polymerase chain reaction, cloning and sequencing. Molecular identification found 49 taxa representing ecto- and ericoid mycorrhizal fungi, dark septate endophytes, saprotrophs, and fungi of unknown trophic status. The majority of the taxa (67.4%) were Basidiomycota, with 24.4% known to be ECM fungi specific to Pinus sp. or conifers. However, a wide range of other mycorrhizal fungi with varying degrees of host specificity were also found, including taxa usually associated with deciduous hosts. In conclusion, the broad range of mycorrhizal fungi recovered from the roots of P. media suggests that specialization is not a major factor in determining its distribution.

Fungal peroxidases (ClassII) have a key role in degrading recalcitrant polyphenolic compounds in boreal forest wood, litter and humus. To date, their occurrence and activity have mainly been studied in a small number of white-rot wood decomposers. However, peroxidase activity is commonly measured in boreal forest humus and mineral soils, in which ectomycorrhizal fungi predominate. Here, we used degenerate PCR primers to investigate whether peroxidase-encoding genes are present in the genomes of a wide phylogenetic range of ectomycorrhizal taxa. Cloning and sequencing of PCR products showed that ectomycorrhizal fungi from several different genera possess peroxidase genes. The new sequences represent four major homobasidiomycete lineages, but the majority is derived from Cortinarius, Russula and Lactarius. These genera are ecologically important, but consist mainly of non-culturable species from which little ecophysiological information is available. The amplified sequences contain conserved active sites, both for folding and substrate oxidation. In some Cortinarius spp., there is evidence for gene duplications during the evolution of the genus. ClassII peroxidases seem to be an ancient and a common feature of most homobasidiomycetes, including ectomycorrhizal fungi. Production of extracellular peroxidases may provide ectomycorrhizal fungi with access to nitrogen sequestered in complex polyphenolic sources.

In nutrient poor environments, such as boreal forests, many of the most important interactions between plants take place belowground. Here, we report the results of two approaches to obtain estimates of the lateral spread of tree roots. In a Pinus sylvestris forest and a Picea abies dominated forest, lateral root spread was estimated by analysing the distribution of ectomycorrhizal sporocarps on tree-girdled and adjacent non-girdled plots and by a 15N uptake experiment, where the soil in circular 1 m2 plots was labelled and the uptake by surrounding trees was studied. Both methods gave similar estimates of lateral root spread, i.e. between 4 and 5 m, and both studies indicated overlapping of root systems. Up to 11 trees took up tracer from a 1 m2 labelled area, although not all trees close to the 15N-labelled area took up tracer. The study clearly showed the dependence of ectomycorrhizal fungi on photosynthate and indicated a high potential for belowground competition between trees due to substantially overlapping root systems.

The aim of a 3-year study was to investigate whether inoculation of Pinus sylvestris L. and Picea abies (L.) Karst. seedlings with mycorrhizas of Cenococcum geophilum Fr., Piceirhiza bicolorata, and Hebeloma crustuliniforme (Bull.) Quel. has any impact on: 1) survival and growth of outplanted seedlings on abandoned agricultural land, and 2) subsequent mycorrhizal community development. For inoculation, the root system of each plant was wrapped in a filter paper containing mycelium, overlaid with damp peat-sand mixture and wrapped in a paper towel. In total, 8,000 pine and 8,000 spruce seedlings were planted on 4-ha of poor sandy soil in randomized blocks. Already after the first year natural mycorrhizal infections prevailed in the inoculated root systems, and introduced mycorrhizas were seldom found. Yet, the seedlings that had been pre-inoculated with C. geophilum and the P. bicolorata during the whole 3-year period showed significantly higher survival and growth as compared to controls. Moreover, the independent colonization of roots by C. geophilum and the P. bicolorata from natural sources was also observed. A diverse mycorrhizal community was detected over two growing seasons in all treatments, showing low impact of inoculation on subsequent fungal community development. A total of 19 additional ectomycorrhizal morphotypes was observed, which clustered into two well-separated groups, according to host tree species (pine and spruce). In conclusion, the results showed limited ability to increase tree survival and growth, and to manipulate the mycorrhizal community even by extensive pre-inoculations, indicating that fungal community formation in root systems is governed mainly by environmental factors.

In northern forest ecosystems, most soil nitrogen (N) is in organic form and forest trees are largely dependent on ectomycorrhizal (ECM) fungi and their degradative abilities for N uptake. The ability of ECM fungi to acquire N from organic substrates should, therefore, be a widespread trait given its ecological importance. However, little is known about the degradative abilities of most ECM fungi as they remain untested due to problems of isolation or extremely slow growth in pure culture. In this paper, we present data on extracellular protease activity of 32 species of ECM fungi, most of which have not previously been cultured. Milk powder plates and zymograms were compared for detecting protease activity in these intractable species. In total, 29/32 of the species produced extracellular protease activity, but detection was method dependent. Growth on milk powder plates detected protease activity in 28 of 32 species, while zymograms only detected proteases in Amanita muscaria, Russula chloroides, Lactarius deterrimus and Lactarius quieticolor. The study supports the hypothesis that protease excretion is a widespread physiological trait in ECM fungi and that this ability is of considerable significance for nitrogen uptake in forest ecosystems.

Soil abiotic factors are considered to be important in determining the distribution of ectomycorrhizal (ECM) fungal species; however, there are few field data to support this. Here, we relate ECM species distributions to changes in soil chemistry along a short (90-m), natural nutrient gradient. The ECM community was characterized, using morphological and molecular techniques, in soil samples collected at 10-m intervals. There were pronounced changes in ECM fungal community structure along the transect, with many taxa showing discrete distributions. Although there was a change of host from Pinus to Picea along the gradient, host-specific fungi did not account for the observed change in community structure. Ordination analyses showed that community structure was strongly correlated with soil characteristics, in particular extractable ammonium and base saturation. However, autocorrelation among soil parameters makes it difficult to isolate the effects of individual parameters. The distinctive changes in soil and vegetation along the transect used in this study provided an exceptional opportunity to examine the local-scale impact of natural spatial heterogeneity on an ECM fungal community.

Fungi colonizing decayed roots of Pinus sylvestris and Picea abies seedlings were assessed by pure-culture isolation and direct sequencing of DNA extracted from roots collected from three environments: bare-root forest nurseries; afforested clear-cuts; and abandoned farmland. Pure-culture isolation from 1500 roots collected from 480 seedlings (240 of each tree species) yielded 1110 isolates which, based on mycelial morphology and ITS rDNA sequencing, were found to represent 87 distinct taxa. Direct ITS rDNA sequencing from decayed sections of 140 roots (70 of each tree species) yielded 160 sequences representing 58 taxa. Direct sequencing revealed a significantly higher fungal diversity per root segment than pure-culture isolation. Overall, a total of 131 taxa were found, 92 of which (70·2%) were identified at least to genus level. Only 14 taxa (10·7%) were detected by both methods, while 73 (55·7%) were detected exclusively by isolation and 44 (33·6%), exclusively by sequencing. The pathogens Fusarium oxysporum (25·6%) and Nectria radicicola (14·9%) were the most common isolates. In contrast, direct sequencing most frequently detected endophyte Phialocephala fortinii (33·1%) and Chalara sp. NS234A2 (10·0%). There were no significant differences in species richness between roots from the different environments, but there was a marked effect on fungal community structure. The results demonstrate that different fungi inhabit decayed roots of conifer seedlings in different environments, and that pure-culture isolation and direct sequencing are complementary methods that are both necessary for a complete description of the fungal communities colonizing diseased conifer roots.

Summary • While it is accepted that many ectomycorrhizal fungi can assimilate organic substrates and facilitate transfer of their elemental components to plants, the fate of the carbon contained in these materials remains uncertain. Here we investigate the compartmentation of carbon and nitrogen in ectomycorrhizal seedlings of Pinus sylvestris fed with double-labelled (15N and 13C) glycine as their sole N source. • Using isotope ratio mass spectrometry, the quantities of N and C derived from this glycine were determined in sequentially harvested samples of mycorrhizas, roots and shoots. • Whereas considerable quantities of 15N were observed in the mycorrhizal tips, roots and shoots, comparable amounts of 13C were observed only in mycorrhizal tips and roots. • It is clearly important to resolve the role of compound specificity as a factor determining the extent of amino-acid C transfer from roots to shoots. However, from the standpoint of the C budget of the whole plant, wherever heterotrophically acquired C is available as an energy source it will reduce demands on photosynthetically fixed sources of the element.

Summary • The genetic potential of ectomycorrhizal fungi to produce N-acetylhexosaminidases was investigated here. N-acetylhexosaminidases are enzymes that cleave monosaccharides from oligomers of N-acetylhexosamines and play an important role in the degradation of chitin. • Degenerate PCR-primers were designed against genes coding for N-acetylhexosaminidases in basidiomycetes. PCR was performed with DNA templates extracted from sporocarps of 26 ectomycorrhizal fungal species and two saprotrophs. • PCR-products were obtained from 18 species representing 12 genera distributed throughout the basidiomycete phylogeny. Sequencing confirmed that the products were homologous with N-acetylhexosaminidase genes from plants, animals and other fungi. Some species yielded two PCR-products representing isoenzymes. • Chitin constitutes a potentially important nitrogen source in soil. Our results demonstrate that a wide range of ectomycorrhizal fungi have the genetic potential to produce N-acetylhexosaminidases, and the expression of this potential would enable them to exploit polymers of amino sugars as a source of nitrogen for themselves and their host plants.

Two willow clones, Salix viminalis L. and S. dasyclados Wimm., growing in separate blocks in mineral soil in one short-rotation forestry (SRF) plantation in Estonia, were analysed to compare the frequency, structure and species composition of ectomycorrhizal (EM) colonisation on their fine roots. Salix dasyclados had significantly greater EM colonisation on root tips (94%) than S. viminalis (75%). The development of EM and associated fungal structures in fine roots (Hartig net, mantle, Hartig net and mantle combined, surface hyphae, endophyte) was similar for both Salix clones. Nine groups of ectomycorrhizal tips were distinguished on the basis of morphological characterisation. Due to high variability among samples, no significant differences between the Salix clones could be found with respect to the abundance of these morphological groups. In total, nine fungal taxa were identified as fungal associates of the two Salix clones. Identification was based upon amplifying and sequencing the rDNA-ITS region of the fungal associates of the root tips. Phialophora finlandia was the most common fungal associate. Morphological groups with well-defined features (e.g., Tomentella sp.) showed limited taxonomic variation, while groups with ill-defined features contained wider taxonomic variation, including unrelated species (e.g., Laccaria sp., Hebeloma helodes). EM fungal diversity was low and largely comprised species considered as pioneer or early stage fungi. A tendency towards higher EM levels with higher N and organic matter concentrations, and low pH, P and K concentrations were observed when the mean levels of EM colonisation were compared with soil variables. It is suggested that EM colonisation level may be primarily determined by the tree species but that local soil nutrient status also influences colonisation. The quality of root litter produced by the Salix clones is known to differ and this may have a role in determining EM colonisation.

Effects of elevated atmospheric carbon dioxide (CO2) levels on the production and spread of ectomycorrhizal fungal mycelium from colonised Scots pine roots were investigated. Pinus sylvestris (L.) Karst. seedlings inoculated with either Hebeloma crustuliniforme (Bull:Fr.) Quel. or Paxillus involutus (Fr.) Fr. were grown at either ambient (350 ppm) or elevated (700 ppm) levels of CO2. Mycelial production was measured after 6 weeks in pots, and mycelial spread from inoculated seedlings was studied after 4 months growth in perlite in shallow boxes containing uncolonised bait seedlings. Plant and fungal biomass were analysed, as well as carbon and nitrogen content of seedling shoots. Mycelial biomass production by H. crustuliniforme was significantly greater under elevated CO2 (up to a 3-fold increase was observed). Significantly lower concentrations and total amounts of N were found in plants exposed to elevated CO2.

Summary • Studies of ectomycorrhizal fungal communities in forest soils are usually restricted to the uppermost organic horizons. Boreal forest podzols are highly stratified and little is known about the vertical distribution of ectomycorrhizal communities in the underlying mineral horizons. • Ectomycorrhizal root tips were sampled from seven horizons in three continuous columns of a 52-cm deep podzol profile. Root tips were sorted into morphological groups and the colonising fungi identified by sequencing of the rDNA ITS region. The vertical distribution of mycorrhizal taxa was examined. • A relationship between ectomycorrhizal species composition and soil horizon was found. Tomentellopsis submollis, three Piloderma species and Dermocybe spp. were found predominantly in the upper horizons while Suillus luteus, Lactarius utilis and three undescribed Piloderma species were associated with the mineral horizons. • Two thirds of the root tips were found in the mineral soil and half of the taxa were restricted to the mineral horizons. The results highlight the need to include the mineral soil in order to gain a more accurate representation of the ectomycorrhizal community.

Summary • Ectomycorrhizal (ECM) fungi were screened for laccase-like genes by polymerase chain reaction (PCR) using primers for white rot fungal laccase genes, and expression of the genes was examined by reverse transcriptase polymerase chain reaction (RT-PCR) for Piloderma byssinum in axenic culture under different nutrient conditions. • Laccase-like genes were present in Rhizopogon roseolus along with several Russulales and Atheliaceae taxa, and showed strong nucleotide sequence similarity to laccase genes in white rot fungi. Multiple laccase-like genes were only identified in Piloderma spp. • Laccase-like genes were expressed in Piloderma spp., with transcript levels some six times higher under high nitrogen conditions in P. byssinum than when nitrogen availability was lower. • The potential roles of laccases in nutrient mobilization and/or differentiation of multihyphal ECM fungal structures are discussed.

The effect of lime and ash additions upon the below ground ectomycorrhizal communities was studied at two spruce forest sites, Horrod and Hasslov, in Southern Sweden. At Hasslov, fifteen years after the addition of 8.75 ton ha-1 of dolomite, the species richness of the mycorrhizal community was unchanged. However, the liming resulted in an almost total replacement of species normally found under the site conditions, with only three morphotypes from a total of 24 distinguished being common to both the control and limed plots. At Horrod, four years after the application of 3.25 ton ha-1 crushed lime, there was a significant reduction in the number of species recorded. This was not a result of a reduction in root tips sampled, since there was no significant treatment effect upon root tip abundance. Instead, it appeared that the less common species present in the control plots had been lost following liming. At the same site, the addition of 4.28 ton ha-1 of wood ash had little effect upon the mycorrhizal community as a whole, with only small changes recorded in the abundance of a small number of species. This study highlights the urgent need for additional data on the temporal and load response of ectomycorrhizal fungi to the addition of lime. This information is vital in view of the proposed widespread use of lime as a counter measure to soil acidification.

The growth of three strains of Cantharellus cibarius on liquid media containing ammonium, nitrate and bovine serum albumin (BSA) in different combinations was determined. The most readily utilisable source of N was ammonium. BSA utilisation was limited compared with media containing ammonium. Growth on nitrate was also poor, suggesting a limited capacity of C. cibarius to metabolise this nitrogen source. There was some indication of considerable intraspecific variation within C. cibarius in the utilisation of nitrogen sources. Possible links between atmospheric nitrogen deposition and the observed decrease of sporocarp formation by C. cibarius in Europe are discussed. We highlight the potential ecological significance of bacteria associated with C. cibarius which may circumvent the need for fungal extracellular enzymes to access complex nitrogen sources.

Summary • Fungal strains of the ‘Hymenoscyphus ericae aggregate’ (Helotiales, Ascomycota) were tested for their ability to form ecto-(ECM) and ericoid (ERM) mycorrhizas. • Twelve fungal test isolates were inoculated in vitro onto sterile ectomycorrhizal and ericoid host plants. • Five isolates of ectomycorrhizal origin formed ECM, three isolates of ericoid origin formed ERM, but none of the tested isolates was able to form both ECM and ERM. Confirmed ectomycorrhizal strains formed the characteristic black-and-hyaline mantles of the Piceirhiza bicolorata morphotype. This morphology represented an intermediate stage, where mantle-colour changed from light brown to black. The progressive darkening appeared to result from a gradual acropetal melanization of the outer mantle layer. • The synthesis results confirm that the ability to form both ecto- and ericoid mycorrhizal symbioses has evolved within the H. ericae aggregate. In addition, a phylogeny based on ITS1–5.8S–ITS2 rDNA sequences showed that the aggregate includes the ect(endo)mycorrhizal dark septate fungus Phialophora finlandia, grouping with four of the ECM-forming strains. The H. ericae aggregate therefore contains a number of closely related taxa with wide ecological attributes.

Summary Changes in species richness and distributions of ectomycorrhizal (ECM) fungal communities along altitudinal gradients have been attributed to changes in both host distributions and abiotic variables. However, few studies have considered altitudinal relationships of ECM fungi associated with a single host to identify the role of abiotic drivers. To address this, ECM fungal communities associated with one host were assessed along five altitudinal transects in Scotland. Roots of Scots pine (Pinus sylvestris) were collected from sites between 300 and 550–600 m altitude, and ECM fungal communities were identified by 454 pyrosequencing of the fungal internal transcribed spacer (ITS) region. Soil moisture, temperature, pH, carbon : nitrogen (C : N) ratio and organic matter content were measured as potential predictors of fungal species richness and community composition. Altitude did not affect species richness of ECM fungal communities, but strongly influenced fungal community composition. Shifts in community composition along the altitudinal gradient were most clearly related to changes in soil moisture and temperature. Our results show that a 300 m altitudinal gradient produced distinct shifts in ECM fungal communities associated with a single host, and that this pattern was strongly related to climatic variables. This finding suggests significant climatic niche partitioning among ECM fungal species.

Summary • Changes in below-ground ectomycorrhizal (ECM) community structure in response to elevated CO2 and balanced nutrient addition were investigated in a 37-yr-old Picea abies forest. •Trees in whole-tree chambers were exposed to factorial combinations of ambient/elevated CO2 (700 ppm) and fertilization (+/–). ECM fungal community structure was determined in 1997 and 2000 using a combination of morphotyping and molecular analyses. Samples were taken both from chambers and from reference trees receiving the same fertilization treatments but without chambers. •Significant effects on ECM community structure were found in response to elevated CO2. Neither elevated CO2 nor fertilization altered species richness; however, there was considerable variation among samples, which may have masked treatment effects on individual species. After 3 yr, the effects of elevated CO2 on community composition were of the same magnitude as those seen after 15 yr of fertilization treatment. •Our results show that increasing atmospheric CO2 concentrations affect the community structure of root symbionts colonizing forest trees. The potential effects of altered ECM community structure on allocation and turnover of carbon and nutrients within forest ecosystems are discussed.

Summary • Genes for ligninolytic enzymes, normally associated with white-rot fungi, are shown to be widespread in a broad taxonomic range of ectomycorrhizal (ECM) fungi. • ECM fungi were screened for lignin peroxidase (LiP) and manganese peroxidase (MnP) genes by PCR using primers specific for known isozymes in the white-rot fungus Phanerochaete chrysosporium, with DNA sequencing used to confirm the identity of the amplified fragments. • Genes for LiPs were detected in ECM fungi representing the orders Agaricales, Aphyllophorales, Boletales, Cantharellales, Hymenochaetales, Sclerodermatales, Stereales and Thelephorales. MnP genes were detected in only Cortinarius rotundisporus and three ECM Stereales taxa. • The presence of genes for decomposer activities supports putative evolutionary relationships between ECM and saprotrophic fungi. Expression of the lignolytic genes may facilitate ECM fungal access to nutrients associated with dead plant material in soil and potentially a supplementary carbon supply. Strict functional boundaries between ECM and decomposer fungi may be less clear-cut than previously thought.

The respiratory activities of plant roots, of their mycorrhizal fungi and of the free-living microbial heterotrophs (decomposers) in soils are significant components of the global carbon balance, but their relative contributions remain uncertain. To separate mycorrhizal root respiration from heterotrophic respiration in aboreal pine forest, we conducted a large-scale tree-girdling experiment, comprising 9 plots each containing about 120 trees. Tree-girdling involves stripping the stem bark to the depth of the current xylem at breast height terminating the supply of current photosynthates to roots and their mycorrhizal fungi without physically disturbing the delicate root-microbe-soil system. Here we report that girdling reduced soil respiration within 1-2 months by about 54% relative to respiration on ungirdled control plots, and that decreases of up to 37% were detected within 5 days. These values clearly show that the flux of current assimilates to roots is a key driver of soil respiration; they are conservative estimates of root respiration, however, because girdling increased the use of starch reserves in the roots. Our results indicate that models of soil respiration should incorporate measures of photosynthesis and of seasonal patterns of photosynthate allocation to roots.

Resupinate thelephoroid fungi (hereafter called tomentelloid fungi) have a world-wide distribution and comprise approximately 70 basidiomycete species with inconspicuous, resupinate sporocarps. It is only recently that their ability to form ectomycorrhizas (EM) has been realized, so their distribution, abundance and significance as mycobionts in forest ecosystems is still largely unexplored. In order to provide baseline data for future ecological studies of tomentelloid fungi, we explored their presence and abundance in nine Swedish boreal forests in which the EM communities had been analysed. Phylogenetic analyses were used to compare the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) sequence data obtained from mycobionts on single ectomycorrhizal tips with that obtained from sporocarps of identified tomentelloid fungi. Five species of Tomentella and one species of Pseudotomentella were identified as ectomycorrhizal fungi. The symbiotic nature of Tomentella bryophila, T. stuposa, T. badia and T. atramentaria is demonstrated for the first time. T. stuposa and Pseudotomentella tristis were the most commonly encountered tomentelloid fungi, with the other species, including T. sublilacina, only being recorded from single stands. Overall, tomentelloid fungi were found in five of the studies, colonizing between 1 and 8% of the mycorrhizal root tips. Two of the five sites supported several tomentelloid species. Tomentelloid fungi appear to be relatively common ectomycorrhizal symbionts with a wide distribution in Swedish coniferous forests. The results are in accordance with accumulating data that fungal species which lack conspicuous sporocarps may be of considerable importance in EM communities.

Studies of effects of fertilizer treatment on ectomycorrhizal fungal community structure have predominantly been based on large, single additions of nitrogen. Studies involving chronic additions of nutrients in combination with irrigation are much less common. We used morphotyping to study effects of balanced additions of a nutrient solution on ectomycorrhizal fungal community structure in a 36-year-old stand of Picea abies (L.) Karst. Despite high variability among individual samples, principal components analysis revealed a clear shift in community structure in response to fertilization. Irrigated plots receiving only water did not differ significantly from untreated control plots. Mycorrhizal root tips colonized by Cenococcum geophilum Fr. were significantly more common in fertilized plots than in control plots. Possible responses by other ectomycorrhizal species were masked by high variability. Over sixty morphotypes were distinguished, but there was no measurable effect of either fertilizer or irrigation treatment on morphotype richness or total number of root tips.

Natural abundances of 15N and N concentrations of 34 fruit bodies from 24 species of ectomycorrhizal and saprophytic fungi were measured in a temperate Central European mixed forest stand. The fungi of the two life forms are known to be capable of utilizing different types of N sources (organic N compounds from the humus, inorganic N from the soil and N from litter or wood) differing by their 15N natural abundance values. Based on the two life forms and the three different N sources, four functional groups of fungi were distinguished: (1) ectomycorrhizal fungi capable of utilizing organic N from the humus; (2) ectomycorrhizal fungi known to depend on inorganic N compounds in the soil; (3) saprophytes capable of utilizing organic N from the humus; and (4) saprophytes utilizing N from dead wood or litter. Large differences were found between species in the δ15N values (−3.0 to 3.3‰) and in the N concentrations (0.84 to 6.61 mmol eq N g dw−1) of the fruit bodies. In most cases fungi were more enriched in 15N than their respective bulk N source was. Fungi living in humus, and presumably having access to organic N compounds (groups 1 and 3), were significantly more enriched in 15N than fungi which are known to depend on inorganic N (e.g. Laccaria, group 2), or fungi living on litter or wood (group 4), irrespective of whether they were ectomycorrhizal or saprophytic species. Fungi living in humus had significantly higher N concentrations than fungi living on litter or wood.

We studied the role of taxonomical and functional ectomycorrhizal (ECM) fungal diversity in root formation and nutrient uptake by Norway spruce (Picea abies) seedlings with fast- and slow-growing phenotypes. Seedlings were grown with an increasing ECM fungal diversity gradient from one to four species and sampled before aboveground growth differences between the two phenotypes were apparent. ECM fungal colonization patterns were determined and functional diversity was assayed via measurements of potential enzyme activities of eight exoenzymes probably involved in nutrient mobilization. Phenotypes did not vary in their receptiveness to different ECM fungal species. However, seedlings of slow-growing phenotypes had higher fine-root density and thus more condensed root systems than fast-growing seedlings, but the potential enzyme activities of ectomycorrhizas did not differ qualitatively or quantitatively. ECM species richness increased host nutrient acquisition potential by diversifying the exoenzyme palette. Needle nitrogen content correlated positively with high chitinase activity of ectomycorrhizas. Rather than fast- and slow-growing phenotypes exhibiting differing receptiveness to ECM fungi, our results suggest that distinctions in fine-root structuring and in the belowground growth strategy already apparent at early stages of seedling development may explain later growth differences between fast- and slow-growing families.

A greenhouse experiment was used to study the effects of host genotype on short root formation and ectomycorrhizal (ECM) fungal community structure in Norway spruce (Picea abies (L.) Karst.). Rooted cuttings representing 55 clones were inoculated with a mix of vegetative hyphae of five ECM fungal species (Laccaria sp., Amphinema byssoides, Piloderma sp., Cadophora finlandia, Paxillus involutus). After one growing season, the ECM fungal community structure was determined by amplifying the fungal internal transcribed spacer (ITS) of ribosomal DNA directly from ECM root tips. Restriction profiles of obtained amplicons were then compared to those of the inoculated strains. Spruce clones differed in their ECM fungal community composition; we found a statistically significant clone-specific effect on ECM fungal diversity and dominating fungal species. Nevertheless, the broad sense heritabilities of the levels of Laccaria sp., Piloderma sp. and A. byssoides colonisations as well as the ECM fungal community structure were low (H 2 = 0.04−0.11), owing to the high within-clone variation. As nitrogen concentration of needles correlated negatively with ECM fungal richness, our results imply that in the experimental conditions nutrient acquisition of young trees may benefit from colonisation with only one or two ECM fungal species. The heritability of short root density was moderate (H 2 = 0.41) and highest among all the measured shoot and root growth characteristics of Norway spruce cuttings. We suggest that the genetic component determining root growth and short root formation is significant for the performance of young trees in natural environments as these traits drive the formation of the below-ground symbiotic interactions.

A colorimetric method was developed to permit semi-quantitative measurement of substrate acidification by different ectomycorrhizal and one saprotrophic fungus growing on media containing one of five different minerals. Overall, substrate acidification differed between fungal species and the degree of variation in acidification in response to different minerals was highly species-dependent. Mycena galopus and Cortinarius glaucopus produced the least biomass of all tested species and produced the highest amount of acidification per unit mycelial density. Substrate acidification by C. glaucopus was inversely related to mycelial density, with particularly high acidification at low mycelial density on medium enriched with tri-calcium phosphate. Substrate acidification by M. galopus was constant irrespective of mycelial density and varied only according to mineral treatment, with higher substrate acidification on tri-calcium phosphate compared to the other minerals.

Background Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32) into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi. Results We detected 9 lineages of GH32 genes in fungi, 4 of which we describe for the first time. GH32 gene number in fungal genomes ranged from 0–12. Ancestral state reconstruction of GH32 gene abundance showed a strong correlation with nutritional mode, and gene family expansion was observed in several clades of pathogenic filamentous Ascomycota species. GH32 gene number was negatively correlated with animal pathogenicity and positively correlated with plant biotrophy, with the notable exception of mycorrhizal taxa. Few mycorrhizal species were found to have GH32 genes as compared to other guilds of plant-associated fungi, such as pathogens, endophytes and lichen-forming fungi. GH32 genes were also more prevalent in the Ascomycota than in the Basidiomycota. Conclusion We found a strong signature of both ecological strategy and phylogeny on GH32 gene number in fungi. These data suggest that plant biotrophic fungi exhibit a wide range of ability to access plant-synthesized sucrose. Endophytic fungi are more similar to plant pathogens in their possession of GH32 genes, whereas most genomes of mycorrhizal taxa lack GH32 genes. Reliance on plant GH32 enzyme activity for C acquisition in these symbionts supports earlier predictions of possible plant control over C allocation in the mycorrhizal symbiosis.

Ectomycorrhizal (ECM) fungi are often considered to be most prevalent under conditions where organic sources of N predominate. However, ECM fungi are increasingly exposed to nitrate from anthropogenic sources. Currently, the ability of ECM fungi to metabolize this nitrate is poorly understood. Here, growth was examined among 106 isolates, representing 68 species, of ECM fungi on nitrate as the sole N source. In addition, the occurrence of genes coding for the nitrate reductase enzyme (nar gene) in a broad range of ectomycorrhizal fungi was investigated. All isolates grew on nitrate, but there was a strong taxonomic signature in the biomass production, with the Russulaceae and Amanita showing the lowest growth. Thirty-five partial nar sequences were obtained from 43 tested strains comprising 31 species and 10 genera. These taxa represent three out of the four clades of the Agaricales within which ECM fungi occur. No nar sequences were recovered from the Russulaceae and Amanita, but Southern hybridization showed that the genes were present. The results demonstrate that the ability to utilize nitrate as an N source is widespread in ECM fungi, even in those fungi from boreal forests where the supply of nitrate may be very low.

Mycorrhizal fungi are exposed to all or many of the environmental stresses that other fungi may experience. These include extremes of temperature and pH, anoxia, water stress, physical fragmentation, toxic metals and other pollutants, as well as anthropogenic stresses arising from applications of fertilisers, lime and wood ash. Fungi can respond to these stresses by altering their morphology, modifying their external environment or adapting their internal metabolism, although the degree of phenotypic plasticity may vary. Since mycorrhizal fungi obtain their carbon from autotrophic host plants, the fungi may also be exposed to stress through changes in carbon allocation from hosts. The refinement and application of molecular identification methods has led to the realisation that the degree of host specificity shown by some mycorrhizal fungi may be higher than assumed in the past. This implies that the availability of compatible roots will influence fungal survival and changes in the species composition of plant communities. Restricting the supply of assimilates from compatible host roots may thus limit the growth of certain fungi. Other types of biotic stress may arise from interactions with competing mycorrhizal symbionts, other fungi, soil animals or bacteria. These different types of stress are reviewed from physiological, ecological and evolutionary perspectives in an attempt to identify potentially fruitful lines of future research.

Fungi colonising root tips of Pinus sylvestris and Picea abies grown under four different seedling cultivation systems were assessed by morphotyping, direct sequencing and isolation methods. Roots were morphotyped using two approaches: (1) 10% of the whole root system from 30 seedlings of each species and (2) 20 randomly selected tips per plant from 300 seedlings of each species. The first approach yielded 15 morphotypes, the second yielded 27, including 18 new morphotypes. The overall community consisted of 33 morphotypes. The level of mycorrhizal colonisation of roots determined by each approach was about 50%. The cultivation system had a marked effect on the level of mycorrhizal colonisation. In pine, the highest level of colonisation (48%) was observed in bare-root systems, while in spruce, colonisation was highest in polyethylene rolls (71%). Direct internal transcribed spacer ribosomal DNA sequencing and isolation detected a total of 93 fungal taxa, including 27 mycorrhizal. A total of 71 (76.3%) fungi were identified at least to a genus level. The overlap between the two methods was low. Only 13 (13.9%) of taxa were both sequenced and isolated, 47 (50.5%) were detected exclusively by sequencing and 33 (35.5%) exclusively by isolation. All isolated mycorrhizal fungi were also detected by direct sequencing. Characteristic mycorrhizas were Phialophora finlandia, Amphinema byssoides, Rhizopogon rubescens, Suillus luteus and Thelephora terrestris. There was a moderate similarity in mycorrhizal communities between pine and spruce and among different cultivation systems.

Fungi play crucial roles in the biogeochemistry of terrestrial ecosystems, most notably as saprophytes decomposing organic matter and as mycorrhizal fungi enhancing plant nutrient uptake. However, a recurrent problem in fungal ecology is to establish the trophic status of species in the field. Our interpretations and conclusions are too often based on extrapolations from laboratory microcosm experiments or on anecdotal field evidence. Here, we used natural variations in stable carbon isotope ratios (δ 13 C) as an approach to distinguish between fungal decomposers and symbiotic mycorrhizal fungal species in the rich sporocarp flora (our sample contains 135 species) of temperate forests. We also demonstrated that host-specific mycorrhizal fungi that receive C from overstorey or understorey tree species differ in their δ 13 C. The many promiscuous mycorrhizal fungi, associated with and connecting several tree hosts, were calculated to receive 57–100% of their C from overstorey trees. Thus, overstorey trees also support, partly or wholly, the nutrient-absorbing mycelia of their alleged competitors, the understorey trees.

Ectomycorrhizas (ECM) are dual organs formed between the terminal feeder roots of many plant species and certain soil fungi. The species richness and taxonomic diversity of ECM symbionts is impressive: ca. 7-10,000 fungal and ca. 8,000 plant species may be capable of forming ECM. The latter are the dominant components of forest and woodland ecosystems over much of the earth's surface. The obligate nature of the symbiosis for ECM fungi has been brought into question by reports that some species produce sporocarps under field conditions in the absence of a host plant. We suggest that there is no unequivocal evidence to support this. The spread of tree roots is often underestimated and small, overlooked hosts such as dwarf Salix spp or sedges may explain the appearance of ECM sporocarps in vegetation apparently devoid of ECM hosts. Compared to plant material, the sporocarps of ECM fungi contain high concentrations of N and P. We show that it would take between 3 and 14 million mycorrhizal tips, or 1800 km of hyphae, to supply the N in one sporocarp of Boletus edulis . The mantle formed by the fungus over the root tip is the likely site of storage for the N and P required for sporocarp production, and we discuss the chemical and structural mechanisms developed on mantles by ECM fungi to defend this resource against fungivory.

Isolation of Russula aeruginea and its mycorrhizal synthesis with Sitka spruce is reported. The mycorrhizas are described and compared with those previously reported in the genus Russula .