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).