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Traditional strict separation of fungi into ecological niches as mutualist, par- asite or saprotroph is increasingly called into question. Sequences of assumed saprotrophs have been amplified from plant root interiors, and several saprotrophic genera can invade and interact with host plants in labo- ratory growth experiments. However, it is uncertain if root invasion by sapro- trophic fungi is a widespread phenomenon and if laboratory interactions mirror field conditions. Here, we focused on the widespread and speciose saprotrophic genus Mycena and performed (1) a systematic survey of their occurrences (in ITS1/ITS2 datasets) in mycorrhizal roots of 10 plant spe- cies, and (2) an analysis of natural abundances of 13C/15N stable isotope signatures of Mycena basidiocarps from five field locations to examine their trophic status. We found that Mycena was the only saprotrophic genus con- sistently found in 9 out of 10 plant host roots, with no indication that the host roots were senescent or otherwise vulnerable. Furthermore, Mycena basi- diocarps displayed isotopic signatures consistent with published 13C/15N profiles of both saprotrophic and mutualistic lifestyles, supporting earlier laboratory-based studies. We argue that Mycena are widespread latent invaders of healthy plant roots and that Mycena species may form a spec- trum of interactions besides saprotrophy also in the field.

Plant–soil feedbacks regulate plant productivity and diversity, but potential mechanisms underpinning such feedbacks, such as the allocation of recent plant assimilate, remain largely untested especially for plants forming tripartite symbioses. We tested how soils from under alder Alnus glutinosa and beneath other species of the same and different families affected alder growth and nutrition, and colonization of roots by nitrogen-fixing Frankia bacteria and ectomycorrhizal fungi. We also measured how the soil environment affected carbon capture and allocation by pulse labelling seedlings with 13CO 2. We then tested for linkages between foliar nutrient stoichiometry and carbon capture and allocation and soil origin using statistical modelling approaches. Performance of alder and nitrogen nutrition were best on home and birch Betula pendula soils (both Betulaceae), whereas performance on Douglas fir Pseudotsuga menziesii (Pinaceae) soil was poor. Plants growing in P. menziesii soil were virtually devoid of Frankia and ectomycorrhizas, and the natural abundance 15N signatures of leaves were more enriched indicating distinct nitrogen acquisition pathways. Seedlings in these soils also had smaller 13C fixation and root allocation rates, leading to smaller 13C respiration rates by microbes. Statistical models showed that the best predictors of foliar N concentration were 13C allocation rates to fine roots and net CO 2 exchange from the mesocosms. The best predictors for foliar phosphorus concentration were net CO 2 exchange from the mesocosms and soil origin; seedlings in home soils tended to have greater foliar phosphorus compared to birch soils while seedlings from Douglas fir soils were no different from the other treatments. Foliar phosphorus concentration was not correlated with plant available or total soil phosphorus for any of the soils. Home soils also resulted in distinct ectomycorrhizal communities on seedlings roots, which could be responsible for greater foliar phosphorus concentration. Our findings show how the association of alder with nitrogen-fixing Frankia relieved nitrogen limitation in the seedling triggering a performance feedback loop. We propose that relief of nitrogen limitation likely increases plant phosphorus demand, which may promote the formation of ectomycorrhizas in nutrient-deficient soils. The formation of tripartite symbioses therefore generates positive plant–soil feedbacks, which enables plants to acquire mineral nutrients otherwise inaccessible in trade for carbon. A free Plain Language Summary can be found within the Supporting Information of this article.

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.

Shrubs are the dominant fuel for wildfires in heathland systems. Despite this, there are relatively few studies which explore the processes of flame spread in shrub fuels. A series of laboratory flame spread experiments are used to identify the relationships between the fuel moisture content of the fuel components present in typical UK upland heath and the resulting fire spread dynamics. Measurements of energy release, flame spread rate and mass loss are made to characterise the burning of 2 m x 0.75 m fuel beds and heat flux measurements are made to record the magnitude of the propagating flux. Fuel moisture thresholds for fine and coarse fuel types required for fire spread are identified, and the magnitude of the in-bed heat fluxes are reported. From the observations, it is suggested that the leading edge of the flame front is driven by the moisture content of the fine dead material suspended in the heather canopy while the trailing edge is dominated by the burning of coarser fuels which supports the burning of fine green fuels. These findings allow further targeted experimental study and can be used to aid in determining fire effects.

Non-native plant pests and pathogens are increasing exponentially, causing extirpation of foundation species. The impact of large-scale declines in a single host on associated biodiversity is widely documented. However, the impact of multiple host loss on biodiversity and whether these impacts are multiplicative has not been assessed. Ecological theory suggests that systems with greater functional redundancy (alternative hosts) will be more resilient to the loss of sympatric hosts. We test this theory and show its importance in relation to pest/pathogen impact assessments. We assessed the potential impact on biodiversity of the loss of two widely occurring sympatric European tree species, Fraxinus excelsior and Quercus petraea/robur, both of which are currently threatened by a range of pests and pathogens. At the UK scale, the total number of associated species at risk of extirpation from plant diseases affecting these two sympatric hosts is greater than the sum of the associated species at risk from declines in either host alone. F. excelsior hosts 45 obligate species (species only found on that host) and Q. petraea/robur hosts 326. However, a decline in both these trees would impact 512 associated species, across multiple taxon groups, a 38% increase. Assessments at a local scale, 24 mixed F. excelsior–Q. petraea/robur woodlands revealed that these impacts may be even greater due to a lack of functional redundancy. Only 21% of sites were able to provide functional redundancy for F. excelsior and Q. petraea/robur associated species which can use other tree species. In most woodlands, the tree species required to provide functional redundancy were not present, although the site conditions were often suitable for them to grow. Synthesis. Understanding of functional redundancy should be applied to assessments of pests/pathogens impact on biodiversity. In risk assessments, higher impact scores should be given to pests/pathogens affecting hosts occurring with other host plant species already impacted by pests/pathogens. Current pest/pathogen risk assessment approaches that ignore the cumulative, cascading effects shown in this study may allow an insidious, mostly overlooked, driver of biodiversity loss to continue. Biotechnology and Biological Sciences Research Council, Grant/Award Number: BB/N022831/1; Rural and Environment Science and Analytical Services Division. info:eu-repo/semantics/publishedVersion

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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.

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.

AIM: Polar and alpine ecosystems appear to be particularly sensitive to increasing temperatures and the altered precipitation patterns linked to climate change. However, little is currently known about how these environmental drivers may affect edaphic organisms within these ecosystems. In this study, we examined communities of plant root‐associated fungi (RAF) over large biogeographical scales and along climatic gradients in the North Atlantic region in order to gain insights into the potential effects of climate variability on these communities. We also investigated whether selected fungal traits were associated with particular climates. LOCATIONS: Austria, Scotland, Mainland Norway, Iceland, Jan Mayen and Svalbard. TAXA: Root fungi associated with the ectomycorrhizal and herbaceous plant Bistorta vivipara. METHODS: DNA metabarcoding of the ITS1 region was used to characterize the RAF of 302 whole plant root systems, which were analysed by means of ordination methods and linear modelling. Fungal spore length, width, volume and shape, as well as mycelial exploration type (ET) of ectomycorrhizal (ECM) basidiomycetes were summarized at a community level. RESULTS: The RAF communities exhibited strong biogeographical structuring, and both compositional variation as well as fungal species richness correlated with annual temperature and precipitation. In accordance with general island biogeography theory, the least species‐rich RAF communities were found on Jan Mayen, a remote and small island in the North Atlantic Ocean. Fungal spores tended to be more elongated with increasing latitude. We also observed a climate effect on which mycelial ET was dominating among the ectomycorrhizal fungi. MAIN CONCLUSIONS: Both geographical and environmental variables were important for shaping root‐associated fungal communities at a North Atlantic scale, including the High Arctic. Fungal OTU richness followed general biogeographical patterns and decreased with decreasing size and/or increasing isolation of the host plant population. The probability of possessing more elongated spores increases with latitude, which may be explained by a selection for greater dispersal capacity among more isolated host plant populations in the Arctic.

Recent molecular studies have identified substantial fungal diversity in indoor environments. Fungi and fungal particles have been linked to a range of potentially unwanted effects in the built environment, including asthma, decay of building materials, and food spoilage. The study of the built mycobiome is hampered by a number of constraints, one of which is the poor state of the metadata annotation of fungal DNA sequences from the built environment in public databases. In order to enable precise interrogation of such data – for example, “retrieve all fungal sequences recovered from bathrooms” – a workshop was organized at the University of Gothenburg (May 23-24, 2016) to annotate public fungal barcode (ITS) sequences according to the MIxS-Built Environment annotation standard (http://gensc.org/mixs/). The 36 participants assembled a total of 45,488 data points from the published literature, including the addition of 8,430 instances of countries of collection from a total of 83 countries, 5,801 instances of building types, and 3,876 instances of surface-air contaminants. The results were implemented in the UNITE database for molecular identification of fungi (http://unite.ut.ee) and were shared with other online resources. Data obtained from human/animal pathogenic fungi will furthermore be verified on culture based metadata for subsequent inclusion in the ISHAM-ITS database (http://its.mycologylab.org).

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.

UNITE (https://unite.ut.ee; Nilsson et al. 2018) is an international community of scientists and citizen scientists established in 2001. The ambition of UNITE is to develop: 1) datasets and tools for robust and reproducible molecular identification; 2) Persistent Identifiers based system for the communicating fungal species. Datasets of the nuclear ribosomal internal transcribed spacer (ITS) region, form the basis for UNITE. The current version includes nearly 1 million public fungal ITS sequences. Datasets are curated and annotated by community members. During the past 15 years, they made more than 275 000 improvements. In the complete absence of Latin names for species, UNITE offers a unique system where species hypotheses (SH) are provided with Digital Object Identifiers (DOIs). The current version 8 of UNITE offers more than 800 000 DOI-based SHs. One such SH DOI page is shown in Fig. 1. These DOI identifiers are also incorporated into the taxonomic backbone, making communication of taxa seamless in both directions. DOI identifiers of species hypotheses are also used by GBIF (Global Biodiversity Information Facility) in order to publish high-throughput sequencing taxon occurrence data in their data portal. UNITE serves as a data provider for a range of metabarcoding software pipelines and regularly exchanges data with all major fungal sequence databases and other community resources. Recent improvements include ITS-based species hypotheses for all eukaryotes and aggregation of full-length, high-quality ITS sequences generated by the PacBio Sequel system (https://www.pacb.com/products-and-services/sequel-system) from diverse material samples.

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.

The suitability of alternative tree species to replace species that are either threatened by pests/disease or at risk from climate change is commonly assessed by their ability to grow in a predicted future climate, their resistance to disease and their production potential. The ecological implications of a change in tree species are seldom considered. Here, we develop and test 3 methods to assess the ecological suitability of alternative trees. We use as our case study the systematic search for an alternative tree species to Fraxinus excelsior (currently declining throughout Europe due to Hymenoscyphus fraxineus). Those trees assessed as most similar to F. excelsior in selected ecosystem functions (decomposition, leaf litter and soil chemistry) (Method A) were least similar when assessed by the number of ash-associated species that also use them (Method B) and vice versa. Method C simultaneously assessed ecosystem functions and species use, allowing trade-offs between supporting ecosystem function and species use to be identified. Using Method C to develop hypothetical scenarios of different tree species mixtures showed that prioritising ecosystem function and then increasing the mixture of tree species to support the greatest number of ash-associated species possible, results in a mixture of trees more ecologically similar to F. excelsior than by simply mixing tree species together to support the greatest number of ash-associated species. We conclude that establishing alternative tree species results in changes in both ecosystem function and species supported and have developed a general method to assess suitability that simultaneously integrates both ecosystem function and the ‘number of species supported’.

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.

The changing nature of the pollution climate in the second half of the 20th Century has resulted in the increased importance of ozone and nitrogen pollutants and a decreased importance of sulphur dioxide. However, there have been few studies of the effects of any dry deposited gas on individual bryophyte species or on bryophyterich plant communities. In those studies which have been made, there is evidence that well hydrated bryophytes may not be particularly sensitive to ozone or NOx at realistic atmospheric concentrations. Ombrotrophic bryophytes are largely dependent on wet deposition as a source of nitrogen. A good example of the potential latent problem is the increase in total tissue nitrogen concentration in ombrotrophic bryophytes. There is ample evidence to suggest that this is an inevitable and widespread response to enhanced atmospheric nitrogen deposition. The responses of bryophytes to wet atmospheric nitrogen deposition can be examined most readily through studies of nitrate assimilation in near pristine environments.

In alpine ecosystems, nitrogen (N) deposition has been linked to plant community composition change, including loss of bryophytes and increase of graminoids. Since bryophyte growth is stimulated by increased N availability, it has been hypothesized that loss of bryophyte cover is driven by enhanced decomposition. As bryophyte mats are a significant carbon (C) store, their loss may impact C storage in these ecosystems. We used an N deposition gradient across 15 sites in the UK to examine effects of N deposition on bryophyte litter quality, decomposition and C and N stocks in Racomitrium moss-sedge heath. Increasing N deposition reduced C : N in bryophyte litter, which in turn enhanced decomposition. Soil N stocks increased significantly in response to increased N deposition, and soil C : N declined. However, depletion of the bryophyte mat and its replacement by graminoids under high N deposition was not associated with a change in total ecosystem C stocks. We conclude that decomposition processes in Racomitrium heath are very sensitive to N deposition and provide a mechanism by which N deposition drives depletion of the bryophyte mat. Nitrogen deposition did not measurably alter C stocks, but changes in soil N stocks and C : N suggest the ecosystem is becoming N saturated.

Kõljalg, Urmas et al., The nuclear ribosomal internal transcribed spacer (ITS) region is the formal fungal barcode and in most cases the marker of choice for the exploration of fungal diversity in environmental samples. Two problems are particularly acute in the pursuit of satisfactory taxonomic assignment of newly generated ITS sequences: (i) the lack of an inclusive, reliable public reference data set and (ii) the lack of means to refer to fungal species, for which no Latin name is available in a standardized stable way. Here, we report on progress in these regards through further development of the UNITE database (http://unite.ut.ee) for molecular identification of fungi. All fungal species represented by at least two ITS sequences in the international nucleotide sequence databases are now given a unique, stable name of the accession number type (e.g. Hymenoscyphus pseudoalbidus|GU586904|SH133781.05FU), and their taxonomic and ecological annotations were corrected as far as possible through a distributed, third-party annotation effort. We introduce the term ‘species hypothesis’ (SH) for the taxa discovered in clustering on different similarity thresholds (97–99%). An automatically or manually designated sequence is chosen to represent each such SH. These reference sequences are released (http://unite.ut.ee/repository.php) for use by the scientific community in, for example, local sequence similarity searches and in the QIIME pipeline. The system and the data will be updated automatically as the number of public fungal ITS sequences grows. We invite everybody in the position to improve the annotation or metadata associated with their particular fungal lineages of expertise to do so through the new Web-based sequence management system in UNITE., The North European Forest Mycologists network is acknowledged for support. Urmas Kõljalg and Kessy Abarenkov are supported by the Estonian Research Council grant no 8235.

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.

The death of native trees caused by non-native pathogens is a global problem. An assessment of the potential ecological and conservation impacts of any tree disease should identify: (1) ecosystem functions associated with the tree species; (2) which species use the tree and how; (3) the suitability of alternative tree species to replace the threatened tree species; and (4) potential management options to mitigate or reduce the impact of the disease. We assess the potential ecological impact of Hymenoscyphus pseudoalbidus (ash dieback) on Fraxinus excelsior in the UK. 953 species were identified as associated with F. excelsior trees: 12 birds, 28 mammals, 58 bryophytes, 68 fungi, 239 invertebrates, 548 lichens. Forty-four ‘obligate’ species were identified: 11 fungi, 29 invertebrates and 4 lichens; and 62 ‘highly associated’ species. Off-setting the loss of ash with ‘alternative tree species’ may be one ‘solution’ to the biodiversity threat. No single alternative tree species can act as host for all ash-associated species but Quercus robur / petraea can host 69%. In an assessment of ecosystem function, when compared to other European deciduous tree species, F. excelsior interacts with the environment in a unique way, particularly in relation to nutrient cycling. Exploration of different management scenarios in response to ash dieback indicated that management which did not remove infected F. excelsior trees was the best for ‘obligate’ and ‘highly associated’ species. The results highlight wide-ranging ecological implications of ash dieback of relevance to other invasive pests and pathogens that are threatening the integrity of other tree species and woodland ecosystems.

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.

Earthworms have an important role in ‘bioturbation’—the mixing of soil due to biological processes. Quantification of earthworm bioturbation relies on estimating earthworm egestion rates which in turn depend on two parameters: the gut content of the worms and the gut transit time (GTT). Gut content can be determined relatively easily, but determining GTT is problematic. The present study aimed at estimating daily soil egestion rates of Aporrectodea caliginosa and Lumbricus terrestris, refining the most common approach for estimating GTT by using fungal spores as natural markers in ingested soil. This approach avoids the use of artificial markers that may adversely affect the earthworms. Gut transit time was estimated by tracking the passage of marked soil through the gut by the appearance of the spores in the egested faeces. Gut transit time was estimated to be 9.6 ± 0.3 h for A. caliginosa and 11.6 ± 0.5 h for L. terrestris. Gut content averaged 465 ± 40(± standard error (SE)) mg dw g−1 dw worm for A. caliginosa and 265 ± 80 mg dw g−1 dw worm for L. terrestris. From these values, daily egestion rates of 1.16 and 0.66 g dw faeces g−1 dw worm d−1 were calculated for A. caliginosa and L. terrestris, respectively. Both values compare well to literature values for each species. The presented method for GTT estimation is inexpensive, rapid and easy to evaluate, with spores being a good alternative to existing markers.

Ectomycorrhizal fungi commonly associate with the roots of forest trees where they enhance nutrient and water uptake, promote seedling establishment and have an important role in forest nutrient cycling. Predicting the response of ectomycorrhizal fungi to environmental change is an important step to maintaining forest productivity in the future. These predictions are currently limited by an incomplete understanding of the relative significance of environmental drivers in determining the community composition of ectomycorrhizal (ECM) fungi at large spatial scales. To identify patterns of community composition in ECM fungi along regional scale gradients of climate and nitrogen deposition in Scotland, fungal communities were analysed from 15 seminatural Scots pine (Pinus sylvestris L.) forests. Fungal taxa were identified by sequencing of the ITS rDNA region using fungal-specific primers. Nonmetric multidimensional scaling was used to assess the significance of 16 climatic, pollutant and edaphic variables on community composition. Vector fitting showed that there was a strong influence of rainfall and soil moisture on community composition at the species level, and a smaller impact of temperature on the abundance of ectomycorrhizal exploration types. Nitrogen deposition was also found to be important in determining community composition, but only when the forest experiencing the highest deposition (9.8 kg N ha(-1) yr(-1) ) was included in the analysis. This finding supports previously published critical load estimates for ectomycorrhizal fungi of 5-10 kg N ha(-1) yr(-1) . This work demonstrates that both climate and nitrogen deposition can drive gradients of fungal community composition at a regional scale.

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.

Large forest areas of South-East Asia, are dominated by the Dipterocarpaceae tree family, which contains many important timber species. Unlike many other tropical trees, Dipterocarpaceae rely on ectomycorrhizal (ECM) root symbiosis for their mineral nutrition. This study aims to document the richness and community composition of ECM fungi in a dry deciduous forest in Thailand. Combining morphological and molecular identification methods revealed 69 species of ECM fungi that belong to 17 phylogenetic lineages. The /russula-lactarius, /tomentella-thelephora, /sordariales, /sebacina and /cantharellus lineages were the most species-rich. The fungal richness is comparable to other tropical rain forest sites, but the phylogenetic community structure has elements of both tropical and temperate ecosystems. Unlike tropical rain forests, the Cenococcum geophilum complex was one of the most frequent fungal taxa that had a relatively high ITS genetic diversity over the small sampling area. This study provides the first snapshot insight into the fungal community of dry dipterocarp forests. However, it is necessary to broaden the spatial and temporal scales of sampling to improve our understanding of the below-ground relations of dry and humid tropical forests.

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 Escherichia coli RecBCD helicase–nuclease, a paradigm of complex protein machines, initiates homologous genetic recombination and the repair of broken DNA. Starting at a duplex end, RecBCD unwinds DNA with its fast RecD helicase and slower RecB helicase on complementary strands. Upon encountering a Chi hot spot (5′-GCTGGTGG-3′), the enzyme produces a new 3′ single-strand end and loads RecA protein onto it, but how Chi regulates RecBCD is unknown. We report a new class of mutant RecBCD enzymes that cut DNA at novel positions that depend on the DNA substrate length and that are strictly correlated with the RecB:RecD helicase rates. We conclude that in the mutant enzymes when RecD reaches the DNA end, it signals RecB’s nuclease domain to cut the DNA. As predicted by this interpretation, the mutant enzymes cut closer to the entry point on DNA when unwinding is blocked by another RecBCD molecule traveling in the opposite direction. Furthermore, when RecD is slowed by a mutation altering its ATPase site such that RecB reaches the DNA end before RecD does, the length-dependent cuts are abolished. These observations lead us to hypothesize that, in wild-type RecBCD enzyme, Chi is recognized by RecC, which then signals RecD to stop, which in turn signals RecB to cut the DNA and load RecA. We discuss support for this “signal cascade” hypothesis and tests of it. Intersubunit signaling may regulate other complex protein machines.

A recent analysis of the Xerocomus subtomentosus complex in Europe using rDNA-ITS sequence data distinguished four taxa in Europe. Two of these corresponded to the established taxa X. subtomentosus and X. ferrugineus, and a new taxon, X. chrysonemus, was described. The fourth taxon was noted but left undescribed owing to lack of material. Here, we describe this taxon as X. silwoodensis sp. nov. X. silwoodensis is a rare but widespread taxon known from single sites in Italy and Spain, and three in the UK. The features of X. silwoodensis basidiomes are very similar to other members of the complex but the pileus colours tend to show richer red-brown tones and the stipe often radicates deeply into the substrate. The taxon also exhibits a strong preference for associating with Populus species, whereas the other taxa are associated with either Quercus (X. chrysonemus) or generalists on broadleaved hosts (X. subtomentosus) or conifers and broadleaved trees (X. ferrugineus). Microscopically, the spore characteristics of X. silwoodensis are similar to the recently described X. chrysonemus, but differ significantly from both X. subtomentosus and X. ferrugineus. X. silwoodensis is probably overlooked due to the resemblance to other taxa within the complex. The present study on the identification and description of X. silwoodensis should reduce the confusion associated with the identification of taxa within this species complex and lead to a more accurate assessment of the geographic distribution and conservation needs of the taxa.

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.

Identification of species within the boletoid genus Xerocomus has relied heavily upon the macromorphological features of the basidiomes. However, the phenotypic plasticity of these features has resulted in considerable confusion over the delimitation of taxa. In this study, we examined collections attributed to the X. subtomentosus complex in Europe using morphological and rDNA-ITS sequence data. In total, 45 European collections from a wide range of geographical areas and ecological conditions were included in the study. In spite of detecting considerable genetic variation, even within individual basidiomes of X. subtomentosus, molecular data, spore size, flesh colour, and the colour of the basal mycelium allow for the recognition of four distinct taxa: two correspond to X. subtomentosus (13 collections) and X. ferrugineus (20); one X. chrysonemus sp. nov. (10), to date only found in the UK, is described as new; and the existence of another taxon (two; Italy and UK) is noted but left undescribed owing to lack of material. Eight collections from North America were also included in the study, from which two taxa with a close affinity to X. ferrugineus were recognised.

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.

Identification of ectomycorrhizal (ECM) fungi is often achieved through comparisons of ribosomal DNA internal transcribed spacer (ITS) sequences with accessioned sequences deposited in public databases. A major problem encountered is that annotation of the sequences in these databases is not always complete or trustworthy. In order to overcome this deficiency, we report on UNITE, an open-access database. • UNITE comprises well annotated fungal ITS sequences from well defined herbarium specimens that include full herbarium reference identification data, collector/source and ecological data. At present UNITE contains 758 ITS sequences from 455 species and 67 genera of ECM fungi. • UNITE can be searched by taxon name, via sequence similarity using blastn, and via phylogenetic sequence identification using galaxie. Following implementation, galaxie performs a phylogenetic analysis of the query sequence after alignment either to pre-existing generic alignments, or to matches retrieved from a blast search on the UNITE data. It should be noted that the current version of UNITE is dedicated to the reliable identification of ECM fungi. • The UNITE database is accessible through the URL http://unite.zbi.ee

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.