2.2 Sample collection and DNA extraction
N. tabacum plants were harvested at flowering stage in July 2019 and were separated into leaves, stems, roots, flowers, and seeds. Biomass samples were separately oven-dried (70°C for 48 h) to constant weight. Leaf dry mass per unit area (LMA, g m−2) was calculated as the quotient of the mass and the area of the leaf sample. For microbiome analysis (Ortas, 1997), 24 soil samples were collected (two conditioned soil (continuous cropping soil vs. natural fallow soil) × two treatments (sterilized vs. non-sterilized) × two sample types (bulk soil and rhizosphere) × three replicates). Soil particles not in contact with the N. tabacum root system or other visible plants were collected and defined as bulk soil. In addition, soil remaining on the root segments after a strong shake was considered to be rhizosphere soil. Fresh root (10 g) was added to 90 ml sterilized water, followed by 10 min ultra-sonication and 30 min shaking at 200 rpm. Rhizosphere soil from this suspension was collected by centrifugation at 12 000 × g for 15 min at 25 °C. Both bulk soil and rhizosphere soil were stored at −20 °C until DNA extraction. Each of three biological replicates pooled three individuals. Approximately 250 mg of bulk soil and rhizosphere soil were used for each DNA extraction. DNA extraction was performed with the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. The DNA extract was assessed on a 1% agarose gel, and DNA concentration and purity were determined with a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA).