Bacterial 16S rRNA Gene Sequencing

DNA was extracted from the filters using the DNeasy PowerSoil Extraction kit (Qiagen) following the manufacturer’s protocol. Filters were cut into smaller pieces to facilitate cell disruption during the bead-beating step. Amplicon sequencing of bacterial 16S rRNA genes was carried out targeting the V3-V4 region with the primer combination Bakt_0341F/Bakt_0785R [25 (link)]. PCR amplification, library preparation, and sequencing on an Illumina MiSeq platform using v3 chemistry was performed at LGC (Berlin) as previously described [26 (link)]. Abundances of bacterial 16S rRNA genes were determined by quantitative PCR using Brilliant SYBR Green II Mastermix (Agilent Technologies) on a Mx3000P system (Agilent Technologies) and the primer combination Bakt_0341F [25 (link)] and Bakt_0799R, which is the reverse complement version of the primer 799F [27 (link)] discriminating against chloroplast-derived 16S rRNA genes. For qPCR, we used the following cycling conditions: 10 min at 95 °C, followed by 45 cycles of 30 s at 95 °C, 30 s at 53 °C, and 40 s at 72 °C, and subsequent melting curve analysis. Due to loss of plant material of some samples before determination of leaf dry weight, abundance data are only available for a subset of all samples (see Supplementary Table 2).