DNA extraction of phytoplasmas
DNA from leaves and phloem scrapings was isolated using a CTAB (cetyltrimethylammonium bromide) extraction method modified from Doyle & Doyle (1990). Due to irregular distribution of ’Ca . P. pyri’ in the top (Seemüller et al. , 1984), in part infection status of pear trees was confirmed by extraction of phloem scrapings from shoots of PD-W inoculated trees, in addition to extraction of leaf tissue from mass flow measurements. Leaves and phloem scrapings were ground in preheated extraction buffer (60 °C, 2.5% (w/v) CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl pH 8.0, 1% (w/v) polyvinylpyrrolidone 40, 0.2% (v/v) 2-mercaptoethanol (with a tissue/buffer ratio 1:10; 0.1 g of tissue in 1 ml buffer) using a homogenizer (BIOREBA AG, Reinach, Switzerland) in extraction bags (BIOREBA AG). Homogenate (1 ml) was transferred into a microcentrifuge tube and incubated at 60 °C for 30 min. An equal volume of chloroform was added, the tube was briefly vortexed and shook for 5 min at room temperature. After a centrifugation step (10,000 ɡ , 6 min at room temperature, Heraeus Fresco 17 Microcentrifuge, Thermo Fisher Scientific, Dreieich, Germany) the aqueous phase was transferred into a new centrifuge tube. For precipitation of nucleic acids an equal volume of isopropanol was added, the tube was inverted and incubated at 4 °C overnight. Precipitate was recovered by centrifugation at 10,000 ɡ for 10 min at room temperature. Supernatant was discarded and the nucleic acid pellet was washed with 70% ethanol (centrifugation step at room temperature, 10,000 ɡ , 10 min), air dried and resuspended in 50 µl high-performance liquid chromatography (HPLC) water (VWR International GmbH, Bruchsal, Germany). Unless explicitly stated elsewhere, laboratory chemicals were purchased from Carl Roth GmbH (Karlsruhe), Bernd Kraft GmbH (Duisburg) and Sigma-Aldrich Chemie GmbH (Taufkirchen), Germany, respectively.