The surface of all herbal materials was cleaned with 75% ethanol to avoid fungal DNA contamination. About 60 mg of the materials were cut into pieces, added with 10% polyvinylpyrrolidone (PVP), and then ground with a FastPrep bead mill (Retsch MM400, Germany). The total DNA was extracted with a Plant Genome DNA Kit (Tiangen Biotech Co., China) in accordance with the manufacturer's instructions. The ITS2 sequences were amplified using universal primers ITS-S2F (5′-ATGCGATACTTGGTGTGAAT-3′) and ITS-S3R (5′-GACGCTTCTCCAGACTACAAT-3′) as previously described (Chen et al., 2010 (link)). Polymerase chain reaction (PCR) amplification was performed in a 25 μL reaction mixture containing 12.5 μL of 2 × PCR Master Mix (Aidlab Biotechnologies Co., Ltd.), 1.0 μL of each primer (2.5 μM), 2 μL (about 30 ng) of DNA templates, and filled with double-distilled water. The reactions were performed with the following thermal program: 94°C for 5 min and 40 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 45 s, followed by 72°C for 10 min. The PCR products were sequenced by the Major Engineering laboratory of the Chinese Academy of Agricultural Sciences (Beijing, China).
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