2.2 Soil sample determination
2.2.1 Sequencing of soil microbial genes
Extraction of DNA: Genomic DNA was isolated from 0.5 g of each soil sample from each sample site using the FastDNA™ SPIN Kit for Soil (MP Biomedicals, CA, USA) and following the manufacturer’s instructions. Extracts of three technical repeats were mixed into a single DNA sample. Extracted genomic DNA was detected by 1% agarose gel electrophoresis. DNA concentration and purity were measured using an ultramicro spectrophotometer (Nano Drop2000, US).
PCR amplification:Based on previous reports, we used the primers 338F-806R for the 16S rRNA of bacteria and the primers SSU0817F-1196R for the 18S gene of fungi. Amplified products were detected by 2% agarose gel electrophoresis, recovered from the gel using the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, CA, USA), washed with Tris-HCl, and verified by 2% agarose gel electrophoresis.
We quantified PCR products using the QuantiFluorTM-ST Fluorometer (Promega Biotech, Beijing, China) and adjusted the samples as required for sequencing. We performed high-throughput sequencing using Illumina Miseq PE300 platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd, China).
2.2.2 Soil microbial carbon and nitrogen determination
Soil microbial biomass C and N were determined via fumigation extraction (Vance et al., 1987). Equivalent amounts of soil were weighed and used for either fumigation or non-fumigation, and the fumigation was conducted for 24 h under vacuum at 25 ℃. After 0.5mol /L K2SO4 extraction, TOC analysis was performed. Soil microbial C and N contents were calculated based on the difference between fumigation and non-fumigation. The calculation formula is as follows.
BC =\(\frac{\left(TOCfumigation\ -\ TOCnonfumigation\right)}{0.45}\)