Dry Grinding at Near-Ambient Temperatures for Extracting DNA from Rust and Other Fungal Spores

Methods for extracting PCR-amplifiable DNA from the spores of rusts and other plant pathogenic, obligately parasitic fungi are hindered by small cell size and thick spore walls that make uniform disruption or digestion difficult and by the abundant pigments, oils, protein, and RNA that can interfere with the subsequent purification and PCR amplification (6,14). For some rusts for which abundant urediniospores (U spores) are obtainable, masses of U spores can be germinated in a monolayer on the surface of buffers and then ground for DNA extraction by methods used for fungal hyphae (9–13,15,16). However, the miniprep isolation of DNA directly from ungerminated spores is a more reasonable approach for the study of most obligately parasitic fungi. One miniprep method (3,7) is widely used for RAPDbased studies of population diversity (5,7,8) and marker linkage (4) in pine rusts. This method grinds mixtures of aeciospores (A spores), diatomaceous earth, and SDS buffer in microcentrifuge tubes using individual plastic minipestles but results in significant DNA shearing (14). Comparisons with four additional methods of disrupting A spores of pine rust fungi (Cronartium species) with three methods of DNA extraction/purification identified the highest yields (100–700 ng DNA/ 10–30 mg sample) and lowest shearing (approximately 23 kb) when dry spores were ground with dry ice using a mortar and pestle (14). Two new near-ambient temperature dry grinding miniprep protocols are described for small quantities of rust spores that appear to yield high molecular weight DNA in greater quantities than previously reported for other miniprep (2,14) and most macroprep (1, 6,12,15, except 10) protocols. The preferred method simultaneously grinds up to 12 samples in individual tubes in a reciprocal action shaker (FastPrep® FP120 Machine; Qbiogene, Carlsbad, CA, USA) and the other improves methods for minipestles but is more labor-intensive and has lower yields than the shaker method. Both methods demonstrate that DNA in desiccated spores undergoes minimal degradation during spore wall disruption at near-ambient grinding temperatures and that the presence of buffers during disruption increases DNA shearing. Protocols in Table 1 have been optimized for 10 mg quantities of U and A spores of the white pine blister rust fungus C. ribicola plus acid-washed diatomaceous earth (Sigma, St. Louis, MO, USA). Spores were desiccated for seven days over silica gel at 4°C (approximately 0% relative humidity) and mixed well with 25 mg acid-washed diatomaceous earth before dry grinding using either method. The tubes of the grinding matrix for the shaker contained 1 g 0.8-mm diameter zircon spheres, plus a single 2-mm zircon sphere. Formerly prepackaged as “Orange-Capped Lysing Matrix” (Bio101, Vista, CA, USA), tubes of this matrix are currently obtained by adding one 2mm sphere from either Lysing Matrix H or I to a tube of Lysing Matrix K (Qbiogene), or by special purchase from Qbiogene. Minipestles were Pellet Pestles® obtained from KontesKimble (Vineland, NJ, USA). SDSBME incubation buffer (3) was added to the desiccated spores after disruption by either method. Subsequent incubaBenchmarks