The first study site was located in the Font Roja Natural Park, in the district of l’Alcoià (Alicante, SE Spain). The second site is the Sierra Mariola, a mountainous formation located between the provinces of Valencia and Alicante (Belda et al., 2009). Soil samples and small fragments of the roots of some L. abortivum individuals were taken from different sampling sites (Figure 1. and Suppl. Table S1 [pdf on line]). Each of the sampling points were defined by at least 10 random plots (200 m²) and vegetation was sampled according to the methodology, based on transects, called “quadrat technique” (Grant, 1981). The measure of total plant cover was also taken on the methodology of Braun-Blanquet (1965). Point samples were marked with a Trimble® GPS unit, using a spatial resolution of 1:5000. Georeferenced points were exported to ArcView® format (*.shp), to edit a localization map of the sampling points (Figure 1).

For the isolation of total DNA from core soils, 5 g of core soil were suspended in 5 ml of T.E. 1X (10 mMTris-HCl, 1 mM EDTA; pH 7.5). This soil suspension was sonicated for 10 min at 4 °C and centrifuged for 1 min at 2,500 rpm. Supernatants were recovered and this process was repeated twice, each time obtaining supernatants, which were pooled and centrifuged for 30 min at 11,000 rpm at 4 °C. After centrifugation, the supernatant was discarded and we added 2 ml of CTAB-PVP lysis buffer (2% CTAB, 20 mM EDTA, 100 mMTris-HCl, 4 mM NaCl and 2% Polyvinylpyrrolidone) with 2 μl of 1:10 dilution protein K (Fluka) to the pellet obtained. The lysis mixture was incubated for 1 h at 65 °C. After incubation, the samples were sequentially extracted with an equal volume of phenol, chloroform and isoamylalcohol (1:1:24 V/V), centrifuged for 10 min at 14,000 rpm, the supernatant was extracted with an equal volume of chloroform, centrifuged for 10 min at 14,000 rpm, and precipitated with 2.5 V of 100% ethanol. The pellet was washed with 70% ethanol, air dried, and finally dissolved in 50 μl of nuclease free water.

CTAB-PVP based extraction method was used for the isolation of genomic DNA from 100 mg basidiocarps of Russula spp. and from 500 mg orchid roots. Frozen tissues were crushed in a mortar, using liquid nitrogen and suspended in the lysis buffer and then incubated for 1 h at 65 °C. Samples were subjected to phenol-chloroform extraction and isopropanol precipitation. The pellet obtained was washed with cold 70% ethanol, air dried and dissolved in 200 µl of TNE buffer (10 mMTris-HCl, 0.1 MNaCl, 1 mM EDTA, pH 7.5). Extracts were then treated with 1 µl of RNaseA (Sigma, www.sigmaaldrich.com) incubated at 37 °C for 30 min, extracted with phenol-chloroform and finally precipitated with ethanol. The pellet was washed with cold 70% ethanol, air dried and finally dissolved in 50 µl of TE buffer. DNA from soil, roots and fungi was purified using a Geneclean spin kit (Q-Biogene Inc., Carlsbad, California).

The primer design for specificity detection of Russula spp. was performed with PRISE (Fu et al., 2008). We used an ITS region of R. delica (Genbank acc. number: AF345250) as seed sequence. The hit table was generated using this seed sequence as a query in the BLAST server (http://www.ncbi.nlm.nih.gov/blast/), obtaining a 500 sequences for generation Hit table. The PRISE software generated 100 primer pairs, and we selected the primer pairs RusPrise1F-RusPrise1R (5´- CACCCCTTTGTGCATCAC -3´ - 5´- CTTCATCGATGCGAGAGC -3´) because it was satisfied the criteria of having a Tm greater than 55 °C, a length of 18 mer and 100% sequence similarity with seeds sequence. The position of the selected primers in the ribosomal region is shown in Suppl. Figure S1 [pdf on line].

We performed a gradient PCR using DNA from Russula basidiocarps, which was carried out using 5 μl of 1:10 dilution of DNA, and a mixture of DNA as a control from soil fungus: Beauveria bassiana, Pochonia chlamydosporia and Pochonia rubescens (50 ng each). PCR reactions contained, in a volume of 40 µl, the four dNTPs at 0.2 mM each (Fermentas, www.thermoscientificbio.com/fermentas/), two primers at 20 pmol each, 2 mM MgCl₂ and 1 U of GoTaq (Promega, www.promega.es). Reactions were started with a denaturation step at 94 °C for 5 min, followed by 35 cycles each with a gradient of annealing temperature (40, 42, 44, 47 and 50) for 20 s, and elongation at 72 °C for 30 s and 94 °C for 30 s, and finally by an extension step at 72 °C for 5 min.

To detect Russula spp. in roots from orchids and in different soils we used 1 μl of purified DNA from each sample with the RusPrise1F-RusPrise1R and ITS1F-ITS4 primers. PCR conditions and product detection were described above, except for the melting temperature (Tm) which was fixed at 55 °C. PCR products from basidocarps, Limodorum roots and soil from Font Roja were sequenced by automated DNA sequencing at Macrogen sequence service (Macrogen Inc., Southern Korea). Comparative sequence analyses were performed using the BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and were deposited in the GenBank (JF415931-JF415933).