P. syringae in Aesculus, Infection and Control 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 mock- inoculated (top panel) or inoculated with P. s. pv. aesculi PD5126 (bottom panel) after 3 months. While the seedling bark recovers when mock- inoculated 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 mock- inoculated 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 importance for plants to separate healthy from disrupted tissues. Generally, wounds or infection sites in tree barks can be sealed off by de novo periderm (consisting of phellem, phellogen and phelloderm) formation, which is considered a non-specific defence response [12]. Formation of a new periderm layer near infection courts in Aesculus bark has been observed, but appeared ineffective in preventing (further) bacterial invasion [4,11]. However, before the development of a new periderm, cell wall reinforcing components are deposited by existing cells near wounds in many tree species [13–16]. In this context, lignin and suberin are found as widespread components of initial wound-repair and may function in preventing water loss and further tissue collapse [13]. For horse chestnut the timing and nature of such structural cell wall reinforcements in a wound boundary zone have not been described and its possible role in preventing the development of bleeding disease is unknown. Understanding of bacterial behaviour and localization within horse chestnut tissues might provide insights in the sudden virulence of this novel horse chestnut pathogen and may be of value for comprehension of the infection process of other, related pathogens. In this study the early infection process of P. syringae pv. aesculi is visualized in artificially inoculated A. hippocastanum seedlings using immunohistochemistry and constitutive, plasmid- borne GFP expression. The early structural defence response is characterised using various histochemical stains, and its relation to bacterial spread is evaluated. Lesion edges were observed to discern potential routes of bacterial migration through host tissues. Additionally, an approach encompassing selective, heat-based control of the causal bacteria is explored. Results To investigate the early events after P. s. pv. aesculi comes in contact with internal host tissues, bacteria were applied to small wounds inflicted on the stems of A. hippocastanum seedlings. The method of wounding did not cause irrecoverable damage, since mock inoculated wounds showed generation of a new periderm confluent with the existing one within 3 months (Fig. 1B upper panel). Inoculated wounds did not exhibit such a recovery (Fig. 1B lower panel). Microscopic analysis of the inoculation site directly after application of the bacteria (t = 0) revealed that the pathogen only colonized the ruptured cells on the surface of the wound (Fig. 1C), assuring that the inoculation method itself did not bring the bacteria in deeper tissues. PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e39604