DNA-Seq of rhizospheric soil
Total genomic DNA was extracted from 0.5 g of soil samples using the FastDNA spin kit for soil (MP Biomedicals, Santa Ana, USA) following the manufacturer protocol. The samples extracted DNA were then quantitated via an advanced spectral scanning multimode reader Varioskan Flash (Thermo Fisher Scientific), using the Hoechst 33258 staining dye kit from Biorad under the name of Florescent DNA Quantitation kit (Biorad, Hercules, USA) and following suppliers recommendations. Following this step, the DNA samples were stored at -20 C and then sent to Biomnigene (Besaon, France  http://www.biomnigene.com) to conduct, according to their protocol, the metabarcoding analysis through high-throughput sequencing (Illumina MiSeq technology). Briefly, the 16S V3-V4 DNA regions were PCR-amplified with 16S-341F (5-CCTACGGGNGGCWGCAG-3) and 16S-806R (5- GGACTACHVGGGTWTCTAAT -3) primers. Meanwhile, the ITS1 region was PCR-amplified with ITS1F-Kyo2 (5-TAGAGGAAGTAAAAGTCGTAA-3) and ITS1R (5-TTTCGCTGCGTTCTTCATCG-3) primers. Both 16S and ITS1 primers were merged with Illumina adapters dedicated for hybridization on the flow-cell, sequencing initiation, and indexing. The PCR products were checked, purified, quantified and pooled. Then the pool was sequenced on Illumina MiSeq (2  251-bp paired-end reads). Sequences were demultiplexed with cutadapt 4.0 (with the following specifications: --minimum-length 220 overlap 7 -e 0.15) then imported into the Qiime2 package (2022.2). The primers sequences were then removed from the reads. The sequences were then quality filtered, denoised, merged and clustered with the qiime2 DADA2 module with the default parameters. The taxonomic assignation was performed with the qiime2 classify-sklearn module and the GTDB database (release 202) for the 16S analysis and the UNIGE database (release ver8_99_10.05.2021) for the ITS1 analysis at multiple taxonomic levels: kingdom, phylum, class, order, family, and genus).