Nan Hui, Rauni Strömmer, Teija Koivula, Martin Romantschuk, Communities of ectomycorrhizal fungi in a Pb contaminated pine forest site Nan Hui, Rauni Strömmer, Teija Koivula, Martin Romantschuk, Dept. of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, FI-15140 Lahti, Finland

Introduction Hälvälä an old shotgun shooting range located at a pine forest site, located in southern Finland, is used as the study area.

Study areas

Contamination Level mg/kg

Clean Polluted

Mycorrhiza A mycorrhiza is a symbiotic association between a fungus and the roots of a plant. root

Types of Mycorrhizas The two most common types ectomycorrhizas Basidiomycota and Ascomycota endomycorrhizas (Arbuscular mycorrhizas) associated with the roots of herbaceous plants other mycorrhizas

Pine root tips and Ectomycorrhizal fungi 1mm

The main point of the study Find the Ectomycorrhizal fungi species difference between Clean and Lead Polluted area. Find the species which are particularly Lead tolerable. Find the species which are Lead sensitive.

Method and material Amplifying certain area Total DNA extraction Amplifying certain area of Fungal Ribosomal DNA by PCR Aligning the sequence with the genebank database sequence the PCR product

DNA Extraction from pine root tips Root tips isolation FastDNA SPIN Kit for soil modified the original protocol

PCR amplification Fungal ribosomal RNA gene Primer selection ITS1 ITS2 Fun18f ITS1 ITS2 18S 5.8S 28S ITS4

Sequencing and Bioinformation In Biocenter Viikki, Helsinki Staden Package EMBL A Nucleotide Sequence Database http://www.ebi.ac.uk/embl/ UNITE A molecular database for the identification of ectomycorrhizal fungi http://unite.ut.ee/

Result Altogether 351 sequences were distributed into 37 different groups. These groups were aligned to EMBL-EBI database and UNITE database.

The phylogenetic trees are made by neighborjoining method (PHYLIP) The phylogenetic trees are made by neighborjoining method (PHYLIP). Dermocystidium salmonis is used as an outgroup. The tree colored according to the distribution of species in the three environmental conditions. Clean: gray; Medium: magenta; Polluted(III): red; Both in Medium and polluted:Yellow The uncolored four species could be found in all the three environmental conditions.

Fig.1 Species tree in the clean area

Frequency of the species in different study areas Clean Medium Polluted

Common species in all areas Lactarius rufus Piloderma fallax Piloderma reticulatum Piloderma croceum Species found only in clean area Tylospora asterophora Tylospora fibrillosa Russula decolorans Mortierella verticillata* Russula betularum Species found only in polluted area Tomentella stuposa Suillus luteus Cortinarius armeniacus Nolanea conferenda Thelephora terrestris Amanita spissa

Conclusion Clear difference is found between clean and polluted area. In clean area, all the species are recognizable by genebank database. In polluted area, some unknown novel species are found and given a number.

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