At each site five randomized samples of 5 kg each were taken from an area of 400 m2 from the A horizon (0–10 cm depth) and mixed. Soils were sampled on April, 11th 2006 and immediately stored at 4°C Y-27632 purchase until further analysis. Soils were homogenised, sieved (<2 mm) and kept at 4°C before

processing. DNA extraction and PCR DNA was extracted in triplicate from each soil (1 g fresh weight per extraction) using the Ultra Clean Soil DNA Isolation Kit (MoBio) according to the manufacturer’s instructions and further purified with the QIAquick PCR Purification Kit (Qiagen). Fungal ITS-region and partial LSU were GSK3235025 mouse amplified with ITS1F (Gardes and Bruns 1993), which is specific for fungi, and the universal eukaryotic primer TW13 (Taylor and Bruns 1999). The resulting PCR products ranged from 1.1 to 1.8 kb in size. The LSU region serves for higher order identification of fungi without homologous ITS reference sequences in

public databases. PCRs contained GoTaq Green Master Mix (Promega), 1 μM of each primer, 0.5 mg/ml BSA and 0.5 μl soil DNA in a total volume of 20 μl. PCRs were run in triplicate on a T3 Thermocycler (Biometra). The following thermocycling program was used: 95°C for mTOR tumor 2′30″ (1 cycle); 94°C for 30″–54°C for 30″–72°C for 1′30″ (30 cycles); and 72°C for 5′ (1 cycle). The nine replicate PCR products for each soil (three DNAs for each soil times three replicas for each DNA) were pooled before ligation to minimize effects from spatial heterogeneity and variability during PCR amplification (Schwarzenbach et al. 2007). For each soil a clone library (96 independent clones each) of ITS/LSU-PCR-products was constructed in plasmid pTZ57R/T (Fermentas) according to manufacturer’s instructions. Insert PCR products (ITS1F/TW13) from individual clones were directly subjected to RFLP analyses. The reaction was performed with the restriction endonuclease BsuRI (Fermentas, isoschizomere of HaeIII) for 2 h at 37°C and the fragments were separated on a 3% high

resolution agarose gel. Initially Carbohydrate up to 4 randomly selected clones that produced an identical pattern were sequenced (Big Dye Terminator v3.1, Cycle Sequencing Kit, ABI) using the primers ITS1F, ITS3 (White et al. 1990) and TW13. Sequencing reactions were purified over Sephadex-G50 in microtiterplates and separated on a DNA sequencer (ABI 3100 genetic analyzer, Pop69, BDv3.1) at the Department of Applied Genetics und Cell Biology, University of Natural Resources and Applied Life Sciences, Vienna (Austria). Where sequencing of more than one representative of one RFLP-pattern resulted in sequences with less than 97% identity in the ITS region or less than 99% identity in the LSU region (see cut-off values for species delineation below), all clones from the particular pattern were sequenced. General molecular genetic manipulations were carried out according to Sambrook and Russell (2001).