Soil DNA extraction, PCR and sequencing

Soil DNA was extracted from 0.5 g of fresh soil using a FastDNA^TM SPIN Kit (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer’s instructions. The concentration and quality of the extracted DNA were quantified with an Eppendorf Biophotometer plus (Eppendorf, Germany), and the DNA was stored at −20 °C.

For each sample, we amplified the V4-V5 region of the bacterial 16S rRNA gene using a broadly conserved primer set (515F-907R) and the fungal ITS sequence (ITS1-ITS2). The primer set 515 F (forward primer: 5′-GTGCCAGCMGCCGCGG-3′) and 907 R (reverse primer: 5′-CCGTCAATTCMTTTRAGTTT-3′ ⁷¹) was used for bacterial ITS sequence amplification, whilst the primer set ITS1 (forward primer: 5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2 (reverse primer: 5′-GCTGCGTTCATCGATGC-3′) was used for fungal ITS sequence amplification⁷². The PCR reactions were carried out in a 20 μL reaction mixture containing 0.8 μL of each primer, 2 μL dNTPs (2.5 mM), 4 μL of 5 × FastPfu Buffer, 0.4 μL of FastPfu polymerase and 10 ng of soil DNA template⁷³. Amplification was initiated at 95 °C for 5 min, followed by 27 (bacteria) or 29 (fungi) cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, followed by a final elongation at 72 °C for 10 min. The PCR products were pooled and visualized on 2% agarose gels, purified using an AxyPrep^TM DNA Gel Extraction Kit (Axygen Biosciences; Union City, CA, USA) according to the manufacturer’s instructions, and quantified using QuantiFluor^TM-ST (Promega, US).

High-throughput sequencing was carried out on the Illumina MiSeq platform by Shanghai Biozeron Bio-pharm Technology Co., Ltd (Shanghai, China). After pyrosequencing, the raw 16S rRNA gene and ITS pyrosequencing data were demultiplexed and quality-filtered using QIIME (version 1.17) based on criteria described in Supplementary Table S1. Finally, using UPARSE (version 7.1 http://drive5.com/uparse/) with a cut-off of 97% similarity, OTUs were clustered, and chimeric sequences were identified and removed using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was analysed using the RDP Classifier (http://rdp.cme.msu.edu/) against the SILVA (SSU123)16S rRNA database with a confidence threshold of 70% ⁷⁴. The ITS sequencing data was classified by using the Unite (Release 6.0 http://unite.ut.ee/index.php).