Determination of symptoms and leaf morphology
Next to the general 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 document
characteristic symptoms at whole plant and leaf level.
Microscopic analyses of theplant vascular morphology
and callose deposition
Cross sections of the midribs were created 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 into plant tissue
freezing medium (Jung, Leica Microsystems, Wetzlar, Germany) and then
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
ZEN® software and edited with Adobe®PhotoShop to optimize brightness, contrast and colouring. The intensity
of aniline blue fluorescence was measured using 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
esterases generating membrane-impermeant fluorescent carboxyfluorescein
(CF) (handbook from Molecular Probes, Eugene, OR, USA). CF is trapped
inside SEs and 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
centimetre 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), by
the means of an inverted fluorescence microscope (AxioVert S100, Carl
Zeiss, Jena, Germany). The transport velocity was calculated by dividing
the measured distance the CF moved within the sieve elements from the
application side towards the leaf base by the exact inoculation time
(from dipping one leaf tip into CFDA to removing of the specific leaf
from the plant).