2.5. Polymerase chain reaction (PCR) amplification
The copy numbers of fungi and bacteria were measured by PCR amplification following Xia et al. (2011). DNA was extracted from 0.5 g soil using the NucleoSpin Soil DNA extraction kit (Macherey-Nagel, Duren, Germany). DNA extracts were stored at -20℃. The 16s rRNA gene, a molecular marker for bacteria, was amplified using 515F/907R (forward 5’-GTGCCAGCMGCCGCGG-3’; reverse 5’-CCGTCAATTCMTTTRAGT-3’). PCRs were performed in 20 μl containing 1 μl of TB Green Fast qPCR Mix, 2.5 μl of each primer (10 μM), 1 μl of DNA template and 20 μl of molecular biology quality water. The amplifications were performed in a CFX-96 (Bio-Rad) following the thermal program: (1) 95°C for 5 min; (2) 40 cycles at 95°C for 45 s, 51°C for 45 s and 72°C for 60 s; and (3) 72°C for 5 min.
The ITS gene, a molecular marker for fungi, was amplified using ITS1F/ITS2R (forward 5’-CTTGGTCATTTAGAGGAAGTAA-3’; reverse 5’-GCTGCGTTCTTCATCGATGC-3’). PCRs were performed in 20 μl containing 1 μl of TB Green Fast qPCR Mix, 2.5 μl of each primer (10 μM), 1 μl of DNA template and 20 μl of molecular biology quality water. The amplifications were performed in a CFX-96 (Bio-Rad) following the thermal program: (1) 95°C for 5 min; (2) 40 cycles at 95°C for 45 s, 55°C for 45 s and 72°C for 60 s; and (3) 72°C for 5 min. The F:B values were calculated using the ratio of the fungal-to-bacterial gene copy numbers. qPCR cannot provide an estimation of the F:B biomass ratio because different taxa contain an unknown number of copies of the rDNA operon in their genomes. But it provides information about the differences in the relative abundance of fungi and bacteria across soil samples (Fierer et al., 2005).