Soil microbial DNA sequencing
DNA was extracted from soil samples using the MOBIO PowerSoil® DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, USA). DNA quality, concentration and purification were checked on 1% agarose gel electrophoresis and NanoDrop One UV-Vis Spectrophotometer (Thermo Scientific, Wilmington, USA). The V4 hypervariable region of the 16S rRNA for bacteria and second internal transcribed spacer (ITS2) region of the rRNA operon for fungi were amplified using primer combinations 515F/806R and ITS3F/ITS4R respectively. Polymerase chain reaction (PCR) were conducted using BioRad S1000 (Bio-Rad Laboratory, CA), with a 50 μl mixture containing 25 μl of 2 × Premix Taq, 10 mM of each primer, 60 ng DNA and nuclease-free water. The PCR amplification had an initial denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C (30 s), annealing at 52 °C (30 s), extension at 72 °C (30 s), and a final extension at 72 °C (10 min). PCR products were extracted and purified using E.Z.N.A.® Gel Extraction Kit (Omega, USA). Amplicon libraries were prepared using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, USA) followed the manufacturer’s protocol, and PE250 sequencing was performed on NovaSeq 6000 Sequencing System (Illumina, San Diego, USA).
Operational taxonomic units (OTUs) were categorized at 97% sequence similarity using the UPARSE package, and taxonomic assignment for bacteria and fungi was performed using Silva and Unite database respectively. Singleton sequences, chloroplast or mitochondria (16S amplicon) sequences and sequences that could not assign to the kingdom level were removed from the final OTU table. Each sample was rarefied to the same number of reads as the smallest sample (52190 and 29940 reads for bacteria and fungi respectively) for further analyses. Alpha diversity for soil bacteria and fungi was measured using the exponential Shannon-Weiner index based on the rarefied OTU tables. Fungal OTUs were assigned to arbuscular mycorrhizal fungi (AMF) and putative plant pathogens using FUNGuild (Nguyen et al. 2016) and further checked based on literature (Roncero et al. 2003; Wu et al. 2008; Mendes et al. 2013).