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Applied and Environmental Microbiology, January 2003, p. 327-333, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.327-333.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

Renske Landeweert,1* Paula Leeflang,2 Thom W. Kuyper,1 Ellis Hoffland,1,3 Anna Rosling,4 Karel Wernars,2 and Eric Smit2

Subdepartment of Soil Quality, Wageningen University, NL-6700 EC Wageningen,1 Department MGB, National Institute of Public Health and the Environment, NL-3720 BA Bilthoven,2 Laboratory of Soil Science and Geology, Wageningen University, NL-6700 AA, Wageningen, The Netherlands,3 Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden4

Received 18 April 2002/ Accepted 15 October 2002

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.

* Corresponding author. Mailing address: Sub-Dept of Soil Quality, Wageningen University, Box 8005, NL-6700 EC, Wageningen, The Netherlands. Phone: 31 317 48 23 39. Fax: 31 317 48 37 66. E-mail: renske.landeweert{at}wur.nl.

Applied and Environmental Microbiology, January 2003, p. 327-333, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.327-333.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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