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).