1. Field Sampling

Experimental site and field trial description

This study was performed in the framework of the long-term “DOK” farming system trial (bioDynamic, bioOrganic, Konventionell, Mäder et al. (2002) in Switzerland (Therwil, in the vicinity of Basel), as a part of ERA-Net Biodiversa project “SOILCLIM” (http://www.biodiversa.org/976). In four replicated winter wheat (Triticum aestivum) plots (each plot 5 X 20m) of two farming systems, i.e. CONMIN (conventional system with mineral fertilization) and BIODYN (biodynamic) three treatments were established in early spring 2017, resulting in 24 plots. The three treatments were: a) a rainout-shelter for a precipitation reduction and simulation of summer drought conditions (R), b) a control treatment for roof artefacts (RC), and unmanipulated ambient control without any rainout-shelter (C).

Soil sampling and DNA extraction

Bulk soil samples were collected in April (corresponds to T1; 4 weeks) and in June (corresponds to T3; 13 weeks). A total of 48 bulk soil samples (2 systems X 3 treatments X 2 sampling times X 4 blocks), 24 samples per sampling time were collected. Samples were sieved (2 mm sieve) to remove stones and roots and stored at -20°C. Total genomic DNA was extracted from 500 mg of the composite sample with the NucleoSpin Soil kit from Macherey-Nagel (Germany) according to the manufacturer´s instructions.

PCR conditions and sequencing

The 1.5kbp fragment, flanking parts of SSU, LSU and complete ITS region, was amplified in two sequential steps. Firstly, a target region was amplified using the PCR primers (SSUmCf/LSUmBr) and in the second step, wobble primers with padding sequences and sample-specific barcodes were attached. Barcoded DNA samples were pooled together, DNA libraries prepared with SMRTbell™ Template Prep Kit 1.0-SPv3, and sequenced on 4 Sequel SMRT cells.