Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

Abstract

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (>99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had >98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.Ectomycorrhizal (EM) fungi are major components of the soil fungal community in most boreal and temperate forests and several (sub)tropical forests. The EM fungi efficiently take up water and organic as well as inorganic nutrients from the soil and translocate these to colonized tree roots, receiving host carbohydrates in return. The extraradical mycelium is the part of the EM association actively involved in uptake of nutrients and water. Species-specific variations in EM extraradical mycelial structure and activity enable single EM fungal species to exploit specific resources (30). The concept of ecological specialization among EM fungi is now generally accepted (14) and underlines the proposed importance of EM fungal species diversity in soil ecosystems (2,7,21). To gain more insights into the functional roles of different EM fungal species and understand their functional diversity, it is essential to obtain information about ecologically relevant features of the symbiosis, in particular the spatial distribution of EM extraradical mycelium.Until the advent of molecular techniques, it had been impossible to acquire data on the mycelial distribution of individual fungal species in soil. Most EM diversity studies are currently based on EM root tip inventories. Morphotype techniques combined with molecular techniques enable identification of EM fungi on root tips extracted from soil and provide information on EM fungal species diversity on a fraction of the tree root system. When using molecular techniques for identification of fungal material on roots, the distribution of extraradical mycelium in the bulk s...

Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

“…However, Russula sp. and Lactarius sp., both having smooth hyphal mantles and lacking large amounts of emanating hyphae (Agerer 2001), have been detected in several molecular studies on soil fungal diversity (Chen and Cairney 2002;Dickie et al 2002;Landeweert et al 2003;Smit et al 2003).…”

“…In contrast to the detection of fungal pathogens, accurate detection of EM mycelium in soil involves total removal of (fragments of) EM root tips. Separation of root tips from soil samples is often done by sieving the samples (Guidot et al 2002;Landeweert et al 2003) or by the removal of root tips with a pair of forceps under a dissecting microscope (Chen and Cairney 2002;Dickie et al 2002;Landeweert et al 2003). In some cases complete hyphal strands and mycelium have been extracted from soil (Zhou et al 2001;H.…”

“…Recently, molecular methods based on total soil DNA extracts were used to detect and identify EM mycelium in natural soil (Chen and Cairney 2002;Dickie et al 2002;Landeweert et al 2003). These molecular methods enable the identification of fungi in soil without the extraction or cultivation of the organisms themselves.…”

“…In addition, the method used to extract root tips from the soil that will be used to detect mycelium should be chosen after careful consideration. Other studies have removed root tips from soil samples by visually checking for the presence of root tips under the dissecting microscope or by sieving the soil before extracting total soil DNA (Chen and Cairney 2002;Dickie et al 2002;Landeweert et al 2003). Several sieve mesh sizes have been used to separate soil from EM root tips but it is unknown whether and how these different sieve mesh sizes influence the outcome of the study.…”

Rock-eating mycorrhizas: their role in plant nutrition and biogeochemical cycles

“…Because EcM weathering is expected to take place at the hyphal tips by exudation of LMWOA, it was necessary to identify and quantify hyphae of EcM fungi in bulk soil as well. Landeweert et al (2003a) successfully extracted basidiomycete DNA from the bulk soil, using soil samples from the same soil columns and soil horizons as in the accompanying root tip study by Rosling et al (2003) described above. The amounts of DNA decreased with soil depth.…”