Transverse hand sections cut from the petiole were put in distilled water and stained with toluidine blue as described by Villodron41. Also, leaf sections were prepared as explained by Ruzin et al.42. In brief, small pieces of leaves were fixed with FAA (formalin: glacial acetic acid:ethyl alcohol: 5:5:90). Fixation was followed by an ethanol dilution series and subsequent stepwise exchange of ethanol with ‘Histoclear’ (xylem substitute, National Diagnostics GA, USA). Samples were then embedded in paraffin and cut with an RM2245 microtome (Leica Biosystems, Germany) into 20-μm thick sections. Furthermore, sections were stained using safranin-fast green and examined with a Leica DMLB microscope (Leica Microsystems, Germany) to observe the tissue morphology. The microscope was equipped with a DS-Fi1 camera (Nikon Instruments Inc., Japan).

SEM observations of the leaf surface and attachment of the bacteria to the abaxial leaf surface of different cultivars were studied 3 h post application of 10 µl of bacterial suspension (108 CFU/ml, OD600 = 0.1) onto the leaf surface. The leaves were allowed to dry for 3 h before fixation of the samples in 70% ethanol overnight, followed by dehydration with 90%, 95%, and 100% ethanol, each for an hour. Finally, samples were dried on K850 critical point dried and coated with gold–palladium alloy on mini sputter coater, following the manufacturer’s instructions (Quorum Technology Ltd., UK). The samples were observed under SEM, Jeol JSM 5410 (JEOL Inc, MA, USA). Leaves for SEM were taken from three different plants of each cultivar, with ten replicates each. Images are representative of bacterial colonization patterns on each of the calla lily cultivars/hybrids.

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