Determination of symptoms and leaf morphology
Next to the fundamental observation and assessment of the known symptoms
following a phytoplasma infection the impact on the morphology of apple,
pear and peach leaves was investigated with the determination of the
length and maximum width of the leaf lamina. Photographs (Canon EOS
760D, Canon Deutschland GmbH, Krefeld, Germany) were taken to visualize
characteristic symptoms at whole plant and leaf level.
Microscopic analyses of theplant vascular morphology
and callose deposition
Cross sections of the midribs were done in the middle (halfway from the
base to the tip) of each leaf. Therefore, pieces of about 1 x 1 cm were
fixed in 2.5% (w/v) glutaraldehyde, 2% (v/v) paraformaldehyde in 0.1 M
sodium-potassium phosphate buffer (pH 7.4, Merck KGaA, Darmstadt,
Germany). Sections were cut at a thickness of 20 µm with a cryostat
(Leica JUNG CM3000, Leica Microsystems, Wetzlar, Germany) at a chamber
temperature of -26 °C and a specimen head temperature of -23 °C. Pieces
were bound to a specimen disc by embedding them in plant tissue freezing
medium (Jung, Leica Microsystems, Wetzlar, Germany) and frozen at the
quick freeze shelf for 10 min prior sectioning. Each cross section was
stained for at least 30 min with 0.1% aniline blue solution (Sigma
Aldrich, St. Louis, Missouri, USA) to visualize callose deposition at
sieve plates.
Each cross section was imaged using an AXIO Imager.M2 (Zeiss Microscopy
GmbH, Jena, Germany) equipped with a 10x objective (N-Achroplan 10x/0.3)
and a 40x objective (W N-Achroplan 40x/0.75). The bright field and
fluorescence images were recorded with a colour camera (AXIOCAM 503
colour Zeiss, Jena, Germany) by use of a DAPI (EM 445/50 nm) filter.
Each digital image of infected and healthy cultivars was analysed with
the determination of (1) the diameter of midribs, (2) the area of the
vascular bundle, (3) the xylem area, (4) the phloem area and (5) the
area of 10 sieve elements per section using the ZEN®software (Zeiss, Jena, Germany). The digital images were processed with
the ZEN® software and edited with
Adobe® PhotoShop to optimize brightness, contrast and
colouring. The intensity of aniline blue fluorescence was measured using
the ZEN® software by analysing the whole phloem area
as region of interest (ROI) and ROIs of healthy and infected plants were
comparatively evaluated.
Determination of the phloem
mass flow velocity
The phloem mass flow rate was measured with the phloem mobile
fluorochrome 5,6-carboxyfluorescein diacetate (CFDA) dye (ThermoFisher
Scientific, Waltham, Massachusetts, USA). CFDA permeates the plasma
membrane in the non-fluorescent acetate form and is cleaved by cytosolic
enzymes producing membrane impermeant fluorescent carboxyfluorescein
(CF) (handbook from Molecular Probes, Eugene, OR, USA). CF is trapped
inside SEs transported by mass flow in the sieve tubes. A stock solution
was prepared by solubilisation of 1 mg CFDA in 1 ml DMSO. A working
solution of 1 μl stock solution in 1 ml buffer solution (containing 2
mol m-3 KCl, 1 mol m-3CaCl2, 1 mol m-3MgCl2, 50 mol m-3 mannitol, and 2.5
mol m-3 MES/NaOH buffer, pH 5.7) was applied at a cut
leaf tip. After an inoculation period of 1 to 2 h at room temperature
each leaf was removed from the plant. Immediately cross sections of the
mid ribs were made by hand with a sharp and fresh razor blade in one
centimeter intervals from the basal side of the leaf. Sections were
covered with distilled H2O, a cover glass and examined
for appearance of fluorescence emitted from CF (emission 510-580 nm)
with an inverted fluorescence microscope (AxioVert S100, Carl Zeiss,
Jena, Germany). The transport velocity was calculated by dividing the
measured distance the CF moved in the sieve elements from the
application side towards the leaf base with the exact inoculation time
(from dipping one leaf tip into CFDA to removing of the specific leaf
from the plant).