*2.7. Culture-Independent Community Analysis*

Total community DNA was extracted from three groups of surface-sterilized root samples using FastDNA®SPIN for soil kit (MP Biomedicals, Solon, CA, USA) according to the manufacturers' instructions. The purity and concentration of DNA were detected using NanoPhotometer spectrophotometer (Implen, München, Germany) and Qubit 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA). Bacterial DNA pyrosequencing was based on ~460 bp amplicons generated by the PCR primers: 341F (5 -CCTACGGGNGGCWGCAG-3 ) and 805R (5 -GACTACHVGGGTATCTAATCC-3 ) with the barcode spanning the hypervariable regions V3-V4 of the 16S rRNA gene The PCR reaction was carried out in 30 μL reactions with 15 μL of Phusion High-Fidelity PCR Master Mix (New England

Biolabs, Ipswich, MA, USA), 0.2 μM of forward and reverse primers, and about 10 ng template DNA. Thermal cycling conditions were 95 ◦C for 3 min, followed by 25 cycles of 95 ◦C for 30 s, 55 ◦C for 30 s, and 72 ◦C for 30 s, with a final extension at 72 ◦C for 10 min. PCR products was mixed in equidensity ratios. Then, mixture PCR products was purified with GeneJET Gel Extraction Kit (Thermo Scientific, Fermentas, Germany).

Sequencing libraries were generated using NEB Next Ultra DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, Massachusetts, USA) following the manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer and Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, CA, USA). At last, the library was sequenced on an Illumina MiSeq platform and 250 bp paired-end reads.

When the sequencing was finished, we needed to filter the raw data to secure the quality, which mainly included the following steps: (1) Cut the polluted adapter, (2) remove low quality reads, specifically reads with average quality less than 19, based on the Phred algorithm, and (3) remove the reads with N base exceeding 5%.

According to overlap of the clean data, we spliced the paired reads by using the PEAR software [49] to merged sequences. The sequences were then removed Chimeras and clustered into operational taxonomic units (OTUs) by UCLUST [50] based on 97% pairwise identity. Taxonomic classification of the representative sequence for each OTU was done using a RDP classifier or QIIME's closed reference strategy against the 16S rRNA gene database [51].
