Characterization of Botrytis cinerea Colonization in Resistant and Susceptible Grape Leaf Tissues

To characterize the colonization of “Pingli-5” (HR, Highly Resistant) and “Red Globe” (HS, Highly Susceptible) by B. cinerea, 2–3 cm² leaf pieces were collected at 4, 8, 12, 18, 24, 36, 48, 72, and 96 hpi, fixed, and decolorized in ethanol/trichloromethane (3:1, v/v) containing 0.15% (w/v) trichloroacetic acid, before clearing in saturated chloral hydrate, and were then stored in 20% glycerol. Samples were subsequently stained with aniline blue solution (for staining fungal tissues a blue color) and examined with an Olympus BX-51 microscope (Olympus Corporation, Japan). For each sample, fungal germination, and infection percentages were examined. For scanning electron microscopy (SEM), leaf tissues were cut into small pieces (0.5–1 cm²), fixed in 4% (v/v) glutaraldehyde in phosphate buffer (0.1 M, pH 6.8) for 12 h at 4°C, and rinsed in the same buffer four times for 10–15 min. After dehydration in a graded ethanol series (30, 50, 70, 80, 90, 100%, v/v), the samples were then critical-point dried, coated with gold in a sputter coater, and examined with a JEOL FESEM S-4800 scanning electron microscope at 15 kV (Cheng et al., 2012 (link)).