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.