2.3.2 Characterization of soil microorganisms
The treatments in this experiment were labeled as LDN (low-density non-rhizosphere soil), LDR (low-density rhizosphere soil), HDN (high-density non-rhizosphere soil), and HDR (high-density rhizosphere soil). Soil DNA was extracted from 0.3 g of sieved (1 mm) soil using a Power Soil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA). The extracted genomic DNA was assessed by 1% w/w agarose gel electrophoresis and stored at -80°C (Rodrigues et al., 2013).
PCR amplification of the V3-V4 region of bacterial 16S rDNA was conducted using the universal primers 341F (5’-CCTAYGGGRBGCASCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). All PCR reactions were carried out in 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 2 μM of each forward and reverse primers, and about 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s. Final elongation was done at 72°C for 5 min. Each sample was measured in three technical repetitions.
The same volume of 1X loading buffer (containing SYB green) and PCR products were mixed and electrophoresed on 2% w/v agarose gel for detection. The mixed PCR products were purified using a Qiagen Gel Extraction Kit (Qiagen, Dusseldorf, Germany). Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) following manufacturer’s recommendations, and index codes were added. The library quality was assessed on a Qubit@ 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA) and an Agilent Bioanalyzer 2100. The library was sequenced on an Illumina NovaSeq platform, and 250 bp paired-end reads were generated (Li et al., 2020; Shao et al., 2018).
Based on the 16S rDNA PCR amplification, a linear discriminant (LDA) effect size (LEfSe) analysis was conducted to identify taxa with significant differences in abundance among the treatments and to construct the relevant cladograms (Segata et al., 2011).
To estimate alpha diversity, the OTU table was rarified, and three metrics were calculated: Chao 1 index to estimate the abundance, the observed OTUs, and Shannon index to estimate diversity (Vishnivetskaya et al., 2011).