Soil Fungal Community DNA Profiling

According to the manufacturer's instructions, sample DNA was extracted from 0.5 g of fresh soil using FastDNA Spin Kit for Soil (MP Biomedicals, LLC., Solon, OH, USA). The DNA integrity and concentrations were tested through electrophoresis and a NanoDrop®ND 2000 UV vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and stored at −20 °C. The ITS1 region was amplified using primer ITS 5 F (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and primer ITS 2 R (5′-GCTGCGTTCTTCATCGATGC-3′) (White et al., 1990 (link)). PCR mixtures (20 μl) contained 10 μl × TransStart Top Green qPCR SuperMix, 1 mM of each primer, 10 ng of ten-fold diluted DNA template, and 7.0–8.6 μl of Milli-Q water. The PCR thermal cycling conditions were as follows: 5 min at 95°C for the initial denaturation; 40 cycles of 5 s at 95 °C and 30 s at 58°C; and the final step of 40 s at 72 °C. The reactions were followed by melting curve analysis with temperatures increasing from 50 to 99 °C. As previously described by Kim et al. (2020 (link)), standard curves were generated. Products were pooled in equal amounts. Library preparation and sequencing were performed using the Illumina MiSeq300 platform (Illumina, San Diego, CA, USA).