Search

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.

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.

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.

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.

• The natural abundance of 13C (δ13C) and 15N (δ15N) of saprotrophic and ectomycorrhizal (ECM) fungi has been investigated on a number of occasions, but the significance of observed differences within and between the two trophic groups remains unclear. • Here, we examine the influence of taxonomy, site, host and time upon isotopic data from 135 fungal species collected at two forest sites in Sweden. • Mean δ13C and δ15N values differed significantly between ECM and saprotrophic fungi, with only a small degree of overlap even at the species level. Among ECM fungi, intraspecific variation in δ15N was low compared with interspecific and intergeneric variation. Significant variation due to site, year and host association was found. • At broad scales a number of factors clearly influence δ13C and δ15N values making interpretation problematic. We suggest that values are essentially site-specific within the two trophic groups, but that species-level patterns exist potentially reflecting ecophysiological attributes of species. The species is therefore highlighted as the taxonomic level at which most information may be obtained from fungal δ13C and δ15N data.

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 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.

Swedish foresters are placing increasing reliance in burning of forestland and green tree retention, in order to enhance biodiversity in the Swedish boreal forests. However, much remains to be learned about how to optimise nature conservation goals by different logging and burning procedures. We monitored the survival of ectomycorrhizal (EM) fungi as mycorrhizas, at a clear-cut, a seed tree stand and an uncut stand of Scots pine in central Sweden, with and without burning at two levels of fire severity. The abundance of mycorrhizas and the EM fungal diversity declined with increased logging intensity and with increased depth of burn. Deep burning fires in combination with logging or fire-caused tree mortality can kill much of the existing EM community. Logging intensity, fire intensity and fire severity are all factors that can be manipulated, thus changing the effects on EM fungi and other soil biota.

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.

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.

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.

The (137)Cs activities in soil profiles and in the mycelia of four ectomycorrhizal fungi were studied in a Swedish forest in an attempt to understand the mechanisms governing the transfer and retention of (137)Cs in forest soil. The biomass of four species of fungi was determined and estimated to be 16 g m(-2) in a peat soil and 47-189 g m(-2) in non-peat soil to the depth of 10 cm. The vertical distribution was rather homogeneous for two species (Tylospora spp. and Piloderma fallax) and very superficial for Hydnellum peckii. Most of the (137)Cs activity in mycelium of non-peat soils was found in the upper 5 cm. Transfer factors were quite high even for those species producing resupinate sporocarps. In the peat soil only approximately 0.3% of the total (137)Cs inventory in soil was found in the fungal mycelium. The corresponding values for non-peat soil were 1.3, 1.8 and 1.9%.

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 .