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Bai et al., 2015 Bai Y.

Müller D.B.

Srinivas G.

Garrido-Oter R.

Potthoff E.

Rott M.

Dombrowski N.

Münch P.C.

Spaepen S.

Remus-Emsermann M.

et al. Functional overlap of the Arabidopsis leaf and root microbiota.

Surface-sterilized A. thaliana seeds were sown at a density of four plants per pot (7×7×9 cm) and stratified for 3-4 days. Plants were grown in the greenhouse for 6 weeks under short day conditions (8/16 hr day/night with temperatures of 22°C/18°C and relative humidity of 70%). After harvest, roots were mechanically separated from the adhering soil particles and a defined root segment of 3 cm, beginning 0.5 cm distal from the hypocotyl, was sampled. Soil particles still attached to the roots were removed by gentle tapping. Roots were collected in 15-mL conical tubes containing 5 mL phosphate-buffered saline (PBS)-S buffer (130 mM NaCl, 7 mM NaHPO, 3 mM NaHPO, pH 7.0, 0.02% Silwet L-77) and washed for 15 min at 180 rpm on a shaker. Roots were transferred to a new 15-mL conical tube and the remaining soil particles were centrifuged for 20 min at 15,000 × g. After washing for a second time, roots were transferred to a new 15-mL conical tube and sonicated with ten cycles of a 30 s pulse at 160 W followed by a 30 s interval (Bioruptor Next Gen UCD-300; Diagenode, Belgium). Roots were transferred to a 1.5-mL tube containing 10 mM MgCland mechanically disrupted using sterile 3-mm metal beads and a Precellys24 tissue homogenizer (Bertin, France) by three cycles of 5,000 rpm for 30 s. The supernatant was collected by centrifugation at 1,000 × g for 5 min and serial dilutions were plated onto flour and TWYE media (flour: 6 g/L flour, 0.3 g/L yeast extract, 0.3 g/L sucrose, 0.3 g/L CaCO, 1.8% agar; TWYE: 0.25 g/L yeast extract, 0.5 g/L KHPO, 1.8% agar), including 50 μg/mL benzimidazole to inhibit fungal growth. Plates were incubated for 3-4 days at 28°C. Emerged single colonies were transferred with a sterile pipette tip to 400 μL liquid media in a 96-well format and incubated for up to 7 days at 28°C at 200 rpm. For taxonomic identification, 100 μL of the culture was transferred to a 96-well PCR plate, bacterial cells were heat-disrupted for ten min at 100°C, and cell debris was removed by centrifugation at 3,000 rpm for 10 min. The supernatant was used for PCR amplification of 16S rRNA V5-V8 regions using the primers 799F (5′-AACMGGATTAGATACCCKG-3′) and 1392R (5′-ACGGGCGGTGTGTRC-3′). PCR reactions were performed under sterile conditions using 3 μL of the supernatant in a total volume of 25 μL that contained 1.25 U DFS-Taq DNA Polymerase (Bioron, Germany), 1 × complete reaction buffer, 0.3% BSA, 200 μM of each dNTP, and 400 nM of each primer, using the following PCR program: 94°C for 5 min, 35 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 30 s, followed by 72°C for 5 min. PCR quality was assessed by visualizing 5 μL of the amplicon on a 1% TAE-agarose gel with ethidium bromide. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Germany), DNA concentrations were measured using a Nanodrop spectrophotometer (PeqLab, Germany) and adjusted to 20 ng/μL, and the purified products were subjected to Sanger sequencing. Genomic DNA of each strain was extracted and purified as described previously () and sequenced using a combination of the Illumina HiSeq 2500 (Illumina, USA) and PacBio RS II (Pacific Biosciences, USA) platforms (see below). All sequencing was performed at the Max Planck Genome Center (Cologne, Germany) or at AgBiome (Research Triangle Park, USA).