With recent methodological advances, molecular markers are increasingly used for semi‐quantitative analyses of fungal communities. The aim to preserve quantitative relationships between genotypes through PCR places new demands on primers to accurately match target sites and provide short amplicons. The internal transcribed spacer (ITS) region of the ribosome encoding genes is a commonly used marker for many fungal groups. Here, we describe three new primers – fITS7, gITS7 and fITS9, which may be used to amplify the fungal ITS2 region by targeting sites in the 5.8S encoding gene. We evaluated the primers and compared their performance with the commonly used ITS1f primer by 454‐sequencing of both artificially assembled templates and field samples. When the entire ITS region was amplified using the ITS1f/ITS4 primer combination, we found strong bias against species with longer amplicons. This problem could be overcome by using the new primers, which produce shorter amplicons and better preserve the quantitative composition of the template. In addition, the new primers yielded more diverse amplicon communities than the ITS1f primer.

SummaryIn northern forests, belowground sequestration of nitrogen (N) in complex organic pools restricts nutrient availability to plants. Oxidative extracellular enzymes produced by ectomycorrhizal fungi may aid plant N acquisition by providing access to N in macromolecular complexes. We test the hypotheses that ectomycorrhizal Cortinarius species produce Mndependent peroxidases, and that the activity of these enzymes declines at elevated concentrations of inorganic N.In a boreal pine forest and a sub-arctic birch forest, Cortinarius DNA was assessed by 454-sequencing of ITS amplicons and related to Mn-peroxidase activity in humus samples withand without previous N amendment. Transcription of Cortinarius Mn-peroxidase genes was investigated in field samples. Phylogenetic analyses of Cortinarius peroxidase amplicons and genome sequences were performed.We found a significant co-localization of high peroxidase activity and DNA from Cortinarius species. Peroxidase activity was reduced by high ammonium concentrations. Amplification of mRNA sequences indicated transcription of Cortinarius Mn-peroxidase genes under field conditions. The Cortinarius glaucopus genome encodes 11 peroxidases -a number comparable to many white-rot wood decomposers.These results support the hypothesis that some ectomycorrhizal fungi -Cortinarius species in particular -may play an important role in decomposition of complex organic matter, linked to their mobilization of organically bound N.

1. Communities of litter saprotrophic and root-associated fungi are vertically separated within boreal forest soil profiles. It is unclear whether this depth partitioning is maintained exclusively by substrate-mediated niche partitioning (i.e. distinct fundamental niches), or by competition for space and resources (i.e. distinct realized niches). Improved understanding of the mechanisms driving spatial partitioning of these fungal guilds is critical, as they modulate carbon and nutrient cycling in different ways. 2.Under field settings, we tested the effects of substrate quality and the local fungal species pool at various depths in determining the potential of saprotrophic and mycorrhizal fungi to colonize and exploit organic matter. Natural substrates of three qualities -fresh or partly decomposed litter or humus -were incubated in the corresponding organic layers of a boreal forest soil profile in a fully factorial design. After one and two growing seasons, fungal community composition in the substrates was determined by 454-pyrosequencing and decomposition was analyzed. 3. Fungal community development during the course of the experiment was determined to similar degrees by vertical location of the substrates (24% of explained variation) and by substrate quality (20%), indicating that interference competition is a strong additional driver of the substrate-dependent depth partitioning of fungal guilds in the system. During the first growing season, litter substrates decomposed slower when colonized by root-associated communities than when colonized by communities of litter saprotrophs, whereas humus was only slightly decomposed by both fungal guilds. During the second season, certain basidiomycetes from both guilds were particularly efficient in localizing and exploiting their native organic substrates although displaced in the vertical profile. This validates that fungal community composition, rather than microclimatic factors, were responsible for observed depth-related differences in decomposer activities during the first season. 4. In conclusion, our results suggest that saprotrophic and root-associated fungal guilds have overlapping fundamental niches with respect to colonization of substrates of different qualities, and that their substrate-dependent depth partitioning in soils of ectomycorrhiza-dominated ecosystems is reinforced by interference competition. Through competitive interactions, mycorrhizal fungi can thus indirectly regulate litter decomposition rates by restraining activities of more efficient litter saprotrophs.

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.