Soil microbiome
Total genomic DNA was extracted from samples via the OMEGA Soil DNA Kit (M5635-02, Omega Bio-Tek) according to the manufacturers protocol, followed by storage at 20C. The DNA concentration was quantified via a NanoDrop NC2000 spectrophotometer (Thermo Fisher Scientific), and the integrity was verified via agarose gel electrophoresis.The bacterial 16S rRNA gene (V5V7 region) was amplified with the primers 799F (5-AACMGGATTAGATACCCKG-3) and 1193R (5-ACGTCATCCCCACCTTCC-3). The fungal ITS1 (b) region was targeted via the primers ITS1-F (5-CTTGGTCATTTAGAGGAAGTAA-3) and ITS1-R (5-GCTGCGTTCTTCATCGATGC-3), with sample-specific 7-bp barcodes incorporated for multiplexing. Each 25 L PCR mixture contained 5 Reaction Buffer: 5 L; 5 High GC Buffer: 5 L; dNTPs (2.5 mM): 2 L; forward/reverse primers (10 M): 1 L each; DNA template: 2 L; Q5 High-Fidelity DNA Polymerase (NEB): 0.25 L; ddH2O: 8.75 L. The thermal cycling conditions were as follows: 98C for 5 min; 26 cycles of 98C (30 s), 55C (30 s), and 72C (45 s); and a final extension at 72C for 5 min. Amplicons were purified with Vazyme VAHTSTM DNA Clean Beads, quantified via the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen), and pooled equimolarly. Paired-end sequencing (2  250 bp) was performed on the Illumina MiSeq platform (MiSeq Reagent Kit v3) at Shanghai Personal Biotechnology Co., Ltd. Raw sequences were processed in QIIME2 2019.4[31]: (1) Demultiplexing and primer trimming were performed via Demux and Cutadapt. (2) Quality filtering, denoising, merging, and chimera removal via DADA2. (3) Nonsingleton ASVs were aligned with MAFFT, and a phylogenetic tree was constructed with FastTree2. (4) Taxonomic assignment against the SILVA 138.1 (16S) and UNITE 9.0 (ITS) databases was performed via the classify-sklearn classifier