Soil microbial community composition
We extracted microbial DNA from the 90 soil samples using PowerSoil Pro
DNA extraction kits (QIAGEN) following the manufacturer’s protocol.
However, we were unable to collect leaf microbes at the end of the
experiment because washing leaves of herbaceous species often damages
plant tissues which could bias estimates of plant biomass. We also
extracted microbial DNA from leaf and soil inoculum collected in the
field as well as background soil substrate filled in the pots at the
beginning of the experiment.
We amplified the ITS2 rDNA region to determine the composition of fungal
communities using a two-step nested PCR. The first step was conducted
using a mixture of SSUmAf (SSUmAf1 and SSUmAf2) as forward primers and a
mixture of LASmAr (LASmAr1-4) as reverse primers. The use of this primer
set allowed us to amplify Glomeromycota in order to enhance the
identification of arbuscular mycorrhizal fungi, which is a major
beneficial microbial group associated with herbaceous plants . We
prepared triplicate 25 μL PCR reagent with 5 μL 5× Phusion HF Buffer
(Thermo Fisher Scientific: Frederick, Maryland, USA), 0.5 μL dNTPs (10
mM), 0.75 μL DMSO, 0.5 μL primer mix (10 μM each primer, 2 forward and 4
reverse primers), 0.25 μL Phusion Hot Start II polymerase (2 U/μL)
(Thermo Fisher Scientific), 2 µL DNA template and 15.5 µL
molecular-grade H2O. PCR reactions were performed using the following
condition: 30 s initial denaturation at 98°C, followed by 40 cycles of
10 s at 98°C, 30 s at 60°C, and 1 min at 72°C, with a final 10-minute
elongation at 72°C. After that, we conducted the second step PCR using
ITS7o and ITS4 as forward and reverse primers respectively . The PCR
reagent was prepared with the same components as the first step and
running with the following condition: 30 s initial denaturation at 98°C,
followed by 31 cycles of 10 s at 98°C, 30 s at 49°C, and 30 s at 72°C,
with a final 10-minute elongation at 72°C.
The V5-V6 region of bacteria 16S rRNA genes was amplified using primers
799F and 1115R to determine the composition of bacterial communities. We
used the same PCR reagents as described above and ran the PCR with the
following conditions: 30 s initial denaturation at 98°C, followed by 35
cycles of 15 s at 98°C, 30 s at 64°C, and 30 s at 72°C, with a final
10-minute elongation at 72°C. The PCR products of both ITS2 and 16S
amplicons were normalized using a SequalPrep Normalization kit (Thermo
Fisher Scientific), then pooled and purified using AMPure (Beckman
Coulter Life Sciences: Brea, California, USA) to avoid contaminants.
After that, we prepared the DNA library by mixing equimolar
concentrations of DNA for each sample and sequenced the DNA on an
Illumina MiSeq using Illumina MiSeq reagent kit v3 (Illumina: Hayward,
California, USA)