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Characterisation and identification of black alder ectomycorrhizas by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS)

K. Pritsch¹, H. Boyle², J.C. Munch³ and F. Buscot¹

¹Institute of Soil Biology, Federal Agricultural Research Centre, Bundesallee 50, D-38116 Braunschweig, Germany, ²State Museum of Natural History Goerlitz, D-02806 Goerlitz, Germany, ³Institute of Soil Ecology, GSF-National Research Centre for Environment and Health GmbH, Neuherberg, Ingolstaedter Landstrasse 1, D-85764 Oberschleissheim, Germany

The New Phytologist (1997) 137: 357-369

Summary
The identity of black alder (Alnus glutinosa (L.) Gaertn.) ectomycorrhizas was investigated using PCR/RFLP analysis of the ITS region from 16 morphotypes sampled at a 60 years old black alder stand. A comparison was made with restriction patterns from sporocarps of 28 mycobionts, of which 16 originated from the same stand, the remaining 12 came from two geographically distant alder stands. Eight of the mycorrhizal types could thus be identified, whereas eight mycorrhizal types remained unidentified. The identified mycorrhizas belonged to the genera Russula, Lactarius, Naucoria, and Cortinarius. Four of the identified ectomycorrhizal types had identical PCR/RFLP profiles to corresponding fruitbodies from all investigated stands with no detectable intraspecific variation, despite the geographical distance of approximately 300 km between the sampling locations. In contrast, intraspecific variation between sporocarps from the different locations was detected in Paxillus rubicundulus, mycorrhizas of which were not found. The diversity of fruiting alder mycobionts at the main experimental plot matched only partially the diversity observed from mycorrhizas by comparing their PCR/RFLP profiles. The results are discussed regarding sampling techniques, PCR/RFLP analyses and ecological aspects.

Materials and Methods

Sampling

The investigations were mainly concentrated at an experimental plot of the Ecosystem Research Centre (University of Kiel) in a black alder (Alnus glutinosa (L.) Gaertn.) stand at Lake Belau (30 km south of Kiel, Schleswig-Holstein, Germany). Monthly sampling over three years (1992-1994) had allowed characterisation of EM morphotypes based upon morphological and anatomical features. In order to identify the morphotypes, sporocarps of potential ectomycorrhizal mycobionts were collected at the experimental plot for one growing season (1994) and excavated together with the root layer beneath them. Sporocarps were additionally collected from two other alder carrs near Braunschweig (Lower Saxony, Germany) (for detailed description of the investigated alder stands and times of sampling, see Boyle, 1996 & Pritsch, 1996; nomenclature of sporocarps following Moser (1983). Root samples were taken from the litter layer to a depth of 5 cm, where roots and litter formed a dense layer. Squares (15 x 15 cm²) of this interwoven layer surrounding sporocarps were taken. Sporocarps and mycorrhizas were examined within two days following sampling. Roots were cleaned cautiously in water and hyphal connections from stipe bases to mycorrhizal tips were traced, when possible. Mycorrhizas were cut off and sorted according to their morphological features. To confirm the morphological typing, several mycorrhizal tips of the respective type were prepared for anatomical characterisation (Pritsch, 1996).

Extraction of DNA
For the DNA extraction from mycorrhizas or sporocarps, fresh or deep frozen (-70 °C) material was used. The total DNA was extracted from small pieces of sporocarp context (1 to 4 mm³) or single mycorrhizal tips according to the procedure described by Henrion et al. (1994a), resuspended in 300 µl Tris-HCl buffer (pH 8) containing 1mM EDTA and stored at 4 °C until use. DNA extraction was replicated at least twice, except when sporocarp material was too sparse.

PCR Conditions
The primers ITS1 and ITS4 (White et al., 1990) used to amplify the ITS region were synthesised by MWG-Biotech (Ebersberg, Germany). For PCR the DNA-extracts from sporocarps and mycorrhizas were diluted with sterile distilled water at ratios of 3-12:1000 (v/v) and 3-12:100 (v/v) respectively. As described by Henrion et al. (1994a) 50 µl of the diluted DNA extracts were gently mixed in 0.5 ml polypropylene tubes with 49.5 ml of the PCR-reactants with final concentrations of 1.5 mM MgCl₂, 200 µm each of dATP, dCTP, dGTP, dTTP, 20 pmol of each primer in buffer for the Taq DNA polymerase supplied by Amersham (Braunschweig, Germany) and covered with two droplets of mineral oil (Sigma). The tubes were immediately placed into an OmniGene HB-TR3 thermocycler. 2.5 U Taq DNA polymerase were added after a denaturation step for 5 min at 94 °C (hotstart). PCR reaction was run in 30 cycles under the following conditions: (i) denaturation of DNA 40 s at 94 °C; (ii) annealing of the primers 40 s, 60 °C; (iii) elongation 40 s, 72 °C. A final cycle of 10 min at 72 °C followed to allow elongation of uncompleted DNA strands. In every series of amplification, controls without DNA served to detect the presence of contaminants in the reagents.

Restriction of DNA and detection of DNA fragments
For RFLP analyses 10 µl of the amplified ITS-PCR products were digested with the restriction endonucleases Alu I, Eco RI, Hae III, Hinf I and Msp I (Boehringer, Mannheim, Germany) following manufacturers recommendations. PCR products were size-fractionated on 1.5% agarose gels (Agarose Ultra PURE, Life Technologies, Inc., Gaitherburg), whereas restriction fragments were separated on 2% agarose gels. The gels were stained with ethidium bromide and photographed under UV-light. X174 DNA, digested with Hae III, was used as size standard for length determination.

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