DNA was extracted from isolates grown on 2% Malt Extract (Difco) and grown at room temperature for 5–7 days. Approximately 50–100 mg of mycelia was harvested, centrifuged to remove excess liquid and transferred to a sterile 2–mL lysing matrix A tube (MP Biomedical; Solon, OH). The tissues were frozen in liquid nitrogen and homogenized for 10 seconds at 4 m/s using a FastPrep-24 5 G benchtop homogenizer (MP Biomedicals; Solon, OH). Wood samples were similarly treated, frozen in liquid nitrogen, but homogenized twice to pulverize the tissue completely. Conversely, for herbarium samples, homogenization speed and time was reduced to avoid shearing of DNA. DNA of samples of H. irregulare infected red pine tissues from Wisconsin was extracted at the Wisconsin Department of Natural Resources in Fitchburg, WI. DNA was extracted using a modified. CTAB extraction protocol with choloroform and ethanol washes [30 (link)].
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