2.2 Soil physical, chemical, and microbial analyses
Soil pH was determined with the slurry method: air-dried soil was shaken in water (soil: water [m/v] = 1:2.5) for 2 min, allowed to stand for 30 min, and pH was measured in the supernatant with an electrode. For nitrate measurement, 10 g fresh soil was extracted into 50 mL of 2 M KCl (aq) with shaking for 1 h at 25 °C, followed by filtration; and, the filtrate was analyzed on a Continuous Flow Analyzer (Technicon Corporation, Oregon, U.S.A.). SOC content was determined by the wet oxidation method using potassium dichromate in acid medium followed by redox titration and TN using the Kjeldahl method.
The relative abundances of C compound classes were determined in air-dried soils using Fourier-transformed infrared spectroscopy (attenuated total reflection Fourier-transformed infrared spectroscopy), with 64 scans by a Thermo-Scientific (Waltham, MA, USA), Nicolet iS50 FT-IR and peak assignments were made following Parikh (Parikh et al., 2014; Qin et al., 2021). DOM extraction and fluorescence excitation-emission (EEM) analysis were conducted with reference to previous studies (Huang et al., 2020).
Briefly, 5 g of air-dried soil samples were placed in 50-mL centrifuge tubes and mixed with 25 mL distilled water with a soil-to-water ratio of 1:2.5 (w/v). The mixture was then by a shaker at 25℃ at 200 rpm for 12 hours in the dark. All of the extracts were centrifuged at 3000×g for 10 min and filtered through prewashed 0.45 μM cellulose acetate filters. The concentration of DOM was determined by the TOC tester (Germany multi N/C 3100). Fluorescence EEM was measured with a Hitachi spectrophotometer (Hitachi, Tokyo, Japan) at ambient temperature, across the excitation wavelengths 220-550 nm with an interval of 5 nm and the emission wavelengths 250-550 nm with an interval of 5 nm and scanning speed at 3000 nm min-1. The filtered DOM samples were transferred into brown glass sample bottles to prevent photodecomposition and stored in a refrigerator at 4 °C. All analyses were conducted within 24 h after extraction.
The humification index (HIX) describes the DOM humification degree, with a higher value corresponding to lower H/C ratios and more ring structure. The HIX was determined using interpolated values for excitation at 254 nm, as the area under the emission peak from 435 to 480 nm was divided by the area under the peak from 330 to 345 nm (Ohno, 2002). The fluorescence index (FI), calculated as em 470 nm/em 520 nm at ex 370 nm, is an indicator of precursor material to the humic portion of OM, with higher values indicating microbe-dominated origins (FI ≥ 1.8) and lower values indicating plant-dominated (FI ≤ 1.2) (T. Liu et al., 2021; McKnight et al., 2001).
According to the instructions of the FastDNA SPIN Kit for Soil (Q-BIOgene, Carlsbad, USA), we extracted microbial DNA. And then, we detected the concentration and purity of DNA using an automatic microplate reader (BioTek ELX 800, USA). The primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Mori et al., 2014) were used to amplify the V3–V4 hypervariable region of the bacterial 16S rRNA gene. The primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) (Adams et al., 2013) were used to amplify the fungal ITS1 region. The PCR amplification steps were as following: pre-denaturation at 94 °C for 5 min, followed by 30 cycles of deformation at 95 °C for 30 s, annealing at 58 °C for 20 s, elongation at 72 °C for 6 s, and a final extension at 72 °C for 7 min. The obtained PCR product was further purified and quantified using QuantiFluor™-ST (Promega, USA) according to the manufacturer’s procedure. Finally, all the gene sequencing was performed on an Illumina HiSeq PE150 system (Illumina Corporation, USA) by Biomarker Technologies Co, LTD.
Primer sequences were trimmed after the raw sequences were de-noised, sorted, and separated using Trimmomatic (version 0.33). The remaining sequences were filtered for redundancy, and all unique sequences for each sample were then clustered into operational taxonomic units (OTUs) at similarities of 97%. Sequences with putative chimeras and ambiguous bases were discarded. The taxonomic identify (species level) of representative sequences for each OTU was determined using the Silva reference database (http://www.arb-silva.de) for the 16S rRNA genes and the Unite reference database (http://unite.ut.ee/index.php) for the ITS using the RDP naïve Bayesian classifier with the BLAST tool in QIIME (http://qiime.org/index.html).