2.3.5. The Soil Enzymatic Activity Detection

Soil enzymatic activity, microbial community composition, and bacterial abundance were analyzed on all refrigerated soil samples. Soil alkaline phosphatase activity was measured using the disodium phenyl phosphate method [28]. Soil sucrase activity was measured using the 3,5-dinitrosalicylic acid method [1]. Soil urease and dehydrogenase activities were determined using phenol-sodium hypochlorite and triphenyl tetrazolium chloride methods, respectively [29]. Microbial community composition was determined using a Gene Amp PCR-System 9700 (Applied Biosystems, Foster City, CA, USA). Briefly, the total DNA was extracted from 0.5 g of soil using a FastDNA Spin Kit for Soil and the FastPrep Instrument (MP Biomedicals, Santa Ana, CA, USA). The DNA quality was assessed on 1% agarose gel, while the quantity of DNA was determined using a Nanodrop-2000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, DE, USA) [30]. Bacterial abundance was quantified following the polymerase chain reaction (PCR) method targeting 515F and 907R (V4-V5

region) primer pairs of 16S rDNA. For microbial community analysis, PCR tests were conducted for each DNA sample, and pooled and purified using a QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA, USA). Approximately equimolar amounts of the PCR products of each sample were combined prior to amplicon sequencing using an Illumina Miseq platform at Shanghai Genesky Biotechnologies (Shanghai, China).
