16s rRNA sequencing of stigma microbiota
Genomic DNA was extracted from stigma tissue using commercial kit Genomic Mini AX Bacteria+ (A&A Biotechnology, Poland) with additional step of mechanical lysis in FastPrep-24 (MP Biomedicals, United States) with zirconia beads. DNA was purified using Anty-Inhibitor Kit (A&A Biotechnology, Poland) and DNA quantity was measured using Qubit 4 Fluorometer. The presence of bacterial DNA in the tested sample was confirmed with Real-Time PCR carried out in the Mx3000P thermocycler (Stratagene, United States) using SYBR Green dye as fluorochrome and universal starters (Ferris et al. 1996). The V3-V4 16S rRNA and ITS amplicon libraries were prepared in accordance with 16S Metagenomic Sequencing Library Guidelines Preparation Part # 15044223 Rev. B, including two-step PCR with Herculase II Fusion DNA Plymerase Nextera XT Index Kit V2 kit. The quality of the library has been checked according to the Illumina qPCR Quantification Protocol Guide using 2200 TapeStation System (Agilent, United States).  Finally, sequencing was performed on Illumina MiSeq PE300 platform.