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.