Fastprep 24 bead beating system

Microbial whole genomic DNA was isolated from AC and DC contents by the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) in combination with the FastPrep-24 bead beating system (MP Biomedicals, Santa Ana, CA, USA) as previously described [31 (link)]. DNA concentration was determined with a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and DNA quality was confirmed with agarose electrophoresis.

Ratios between the oxidized form NAD⁺ and the reduced form NADH were measured using NAD⁺/NADH quantification kit (MAK037, Sigma) with whole cell lysates of wild type S. japonicus cells grown under indicated conditions. Briefly, early exponential cultures were harvested by centrifugation and flash freezing in liquid nitrogen. Cell pellets were then resuspended in NAD⁺/NADH extraction buffer with lysing matrix Y tubes (MP Biomedicals), containing 0.5 mm diameter zirconium oxide beads, before cells were lysed with the Fastprep-24 bead beating system (MP Biomedicals), Cell lysates were then filtered through a 10 kDa protein filter (Millipore) by centrifugation at 12,000 x g, 4 °C. Cleared extracts were processed as per the manufacturer’s instructions.

For the quantification of the T. harzianum strain OMG16 (DSMZ accession no.: 32722) in the maize root endosphere, roots were thoroughly cleaned with a soft brush and water to remove residual soil particles, shortly dried between paper towels and cut into small pieces. Approximately 80 mg fine roots were placed in 2 mL tubes containing 1.0 mm silica spheres including one single 0.25-inch ceramic bead (MP Biomedicals, France) and 400 μL peqGOLD lysis buffer (VWR Peqlab, Germany). Root tissue was homogenized for 3 × 30 s at a speed of 6 m/s in a FastPrep 24 bead-beating system (MP Biomedicals). After each cycle samples were cooled on ice for 1 min. DNA was subsequently extracted utilizing the peqGOLD Fungal DNA Kit (VWR Peqlab), following the manufacturer’s instructions. DNA was eluted in a TE buffer (pH 8.0) and checked on 0.8% TAE agarose gels. DNA concentrations were determined using a Qubit^® 3.0 Fluorometer and the Qubit dsDNA HS Assay Kit according to the instructions of the manufacturer (Thermo Fisher Scientific, Germany). A T. harzianum OMG16-specific primer pair, designed from OMG16 genomic DNA sequences were used for qPCR quantification of T. harzianum OMG16 DNA in the DNA samples according to the method described by Mpanga et al. (2019a) (link).