Herbaceous rhizosphere microbial diversity and community structure along the altitudinal gradient are regulated by climatic and geographical factors and soil ecological stoichiometry

Rhizosphere soil moisture (SM) was measured by constant weight after drying. The rhizosphere soil pH was measured by an electrode pH meter (Leici, PHS25, China). Electrical conductivity (EC) was determined by the leaching electrode method (Leici, DDS-11A, China). The water/soil ratio was 2.5:1. Soil organic carbon (SOC) was quantified by the dichromate oxidation method. Total nitrogen (TN) was quantified using the Kjeldahl digestion method. Total phosphorus (TP) was quantified using the molybdenum antimony anticolorimetry method.
For all rhizosphere soil samples, Illumina-based absolute quantification sequencing was used to measure microbial diversity, as detailed below: the DNA from each soil sample was extracted using the FastDNA® SPIN Kit for Soil DNA Extraction (MP Biomedicals, Santa Ana, CA, USA). The extracted DNA was purified using the DNeasy PowerClean Pro Cleanup Kit (Qiagen, Valencia, CA, USA). Polymerase chain reaction (PCR) was performed using the ABI 2720 Thermal Cycler (Thermo Fisher Scientific, USA). The amplification products were identified using agarose gel electrophoresis, and three parallel PCR amplification products of each soil sample were purified and recovered using the Agencourt AMPure XP system gel recovery kit (Beckman Coulter, Brea, CA, USA). Purified amplicons were blended in equivalent measures, and sequencing was performed on the Illumina NovaSeq platform at Genesky Biotechnologies, Inc. (Shanghai, China).