DNA extraction and PCR amplification

Total soil and plant tissue DNA were extracted using a FastDNA SPIN Soil kit (MP Biomedicals, Solon, USA). A NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, USA) was utilized to determine the DNA concentration and purity, and 1% agarose gel electrophoresis was used to determine the DNA extraction quality. The primers used to amplify the hypervariable regions V5-V7 of the bacterial 16S rRNA gene were 799F (5′-AACMGGATTAGATACCCKG-3′) and 1193R (5′-ACGTCATCCCCACCTTCC-3′). The primers used to amplify the internal transcribed spacer (ITS) region of ribosomal DNA were ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). The programme for PCR amplification was as follows: initial denaturation at 95 °C for 3 min; 27 cycles of denaturing at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 45 s; a single extension at 72 °C for 10 min; and a final extension at 4 °C. The reaction system included 4 μL 5 × FastPfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL forward primer (5 μM), 0.8 μL reverse primer (5 μM), 0.4 μL FastPfu Polymerase, 0.2 μL BSA, 10 ng template DNA, and ddH₂O up to 20 μL. The reactions were performed in triplicate. The PCR product was extracted from a 2% agarose gel and purified with an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) based on the manufacturer's instructions and quantified using a Quantus Fluorometer (Promega, USA).