DNA extraction of phytoplasmas
DNA from leaves and phloem scrabbings was isolated using a CTAB (cetyltrimethylammonium bromideextraction 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 scrabbings from shoots of PD inoculated trees, in addition to extraction of leaf tissue from mass flow measurements. Leaves and phloem scrabbings 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 (10000 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 10000 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, 10000 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.