Figure 1. Bleeding disease of horse chestnut and biogenesis of a structural barrier after wounding. (A) Typical symptoms of P. syringae pv. aesculi associated bleeding disease observed on the trunk of an A. carnea tree, including bleeding of amber coloured sap and cracking of the bark (arrowheads). Photo taken in Sept. 2008 at N51u 579 260; E5u 349 100. (B) Appearance of surface wounds on A. hippocastanum seedlings mockinoculated (top panel) or inoculated with P. s. pv. aesculi PD5126 (bottom panel) after 3 months. While the seedling bark recovers when mockinoculated by regeneration of periderm, the wounded tissue appears blackened and sunken when inoculated with P. s. pv. aesculi. (C) Immunofluorescent labelling of a transverse section of the wound area sampled directly after inoculation of P. s. pv. aesculi PD4818 (t = 0) indicates
that the bacteria (green) only colonize the outermost cell layer after inoculation. The scale bar indicates 100 mm. (D) A longitudinal section of a mockinoculated wound sampled after 6 days with phloroglucinol-HCl stained lignin (red/purple). A barrier zone composed of several lignified parenchyma cells wide is visible along the wound (arrowhead) along with a broad layer of diffusely lignified cells in the disjointed part (arrow). The scale bar indicates 0.5 mm. (E) Fluorescence microscopic observation on a longitudinal section of a wound sampled 8 days after PD4818 inoculation stained for waxes using Sudan IV. The cells that exhibit lignification, as seen by the yellow/green autofluorescence, also show Sudan IV stained suberin lamellae
that appear in dim red. The arrow reflects the direction of the original inoculation cut. The scale bar indicates 50 mm. (F) Detail of E showing the even deposition of suberin around the plant protoplasts. The scale bar indicates 25 mm. (G) Continuous deposition of suberin by two cells at pit fields (arrowheads). The scale bar indicates 10 mm.
doi:10.1371/journal.pone.0039604.g001