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).