Improved method for isolation of DNA from slow-growing basidiomycetes such as Armillaria mellea.

Interest in identification and characterization of fungi is increasing because of the expanding impact of fungi on biotechnological, industrial and medical systems. Fungal taxonomy, traditionally based on comparative morphology, has at times led to confusion when apparently insignificant and continuously variable morphological criteria are used to describe new taxa and to reclassify existing taxa (4). Comparative nucleic acid studies are becoming more widely used in the systematics of fungi, including Basidiomycetes (1–3,6). Existing methods of DNA isolation (7–10) have proved to be unsatisfactory with slow-growing, darkly pigmented Basidiomycetes such as species of Armillaria, Paxillus, Rhizopogon, Collybia, Marasmius and Suillus. Using Armillaria mellea sensu lato as the test species, we have approached the parallel problems of slow growth and poor DNA yields by developing an airlift culture system that decreases the amount of moribund mycelium produced, and by adopting a DNA extraction procedure using cetyltrimethylammonium bromide (CTAB) to remove contaminating pigments and polysaccharides. This isolation method was successfully applied to a broad range of fungi, both laboratory-grown mycelium and basidiocarp samples collected from nature. Fungal cultures were grown at 20°C on modified Melin-Norkrans medium (MMN) containing ammonium tartrate (0.25 g/L), KH2PO4 (0.5 g/L), MgSO4 •7H2O (0.15 g/L), CaCl2 (0.05 g/L), FeCl3 (0.0012 g/L), NaCl (0.025 g/L), glucose (15 g/L), malt extract (3 g/L), yeast extract (0.5 g/L), soy peptone (0.5 g/L), biotin (5 μg/L) and thiamine (100 μg/L) on 1.5% agar. The colonized agar was cut into thin strips and transferred to 300 mL of MMN broth in a 2.8-L Fernbach flask, which was incubated at 25°C for two weeks. The standing cultures were transferred to 2-L Erlenmeyer flasks containing 1.6 L of MMN, which were sparged with air at a flow rate of 300 mL/min for two weeks at 25°C. Mycelia were then harvested by filtration, washed with water, frozen in liquid nitrogen and lyophilized. The dry mycelium was ground to a fine powder (mycelial fragment lengths of 10–100 μm) and used for DNA isolation. To 10 g of powdered mycelium, 100–150 mL of extraction buffer, preheated to 60°C, were added. Extraction buffer consisted of 2% CTAB, 10 mM EDTA, 0.7 M NaCl, 0.05 M Tris-HCl (pH 8.0 at 25°C) and 0.05% β-mercap-