Histological Examination of Horse Chestnut Infection by Pseudomonas syringae pv. aesculi and Non-Destructive Heat Treatment to Stop Disease Progression Jeroen de Keijzer1, Lambertus A. M. van den Broek2, Tijs Ketelaar1, Andre´ A. M. van Lammeren1* 1 Laboratory of Cell Biology, Wageningen University and Research Centre, Wageningen, The Netherlands, 2 Wageningen UR, Food & Biobased Research, Wageningen, The Netherlands Abstract Since its emergence in Northwest Europe as a pathogen that infects trunks and branches of Aesculus spp. (the horse chestnuts) approximately one decade ago, Pseudomonas syringae pv. aesculi has rapidly established itself as major threat to these trees. Infected trees exhibit extensive necrosis of phloem and cambium, which can ultimately lead to dieback. The events after host entry leading to extensive necrosis are not well documented. In this work, the histopathology of this interaction is investigated and heat-treatment is explored as method to eradicate bacteria associated with established infections. The early wound-repair responses of A. hippocastanum, both in absence and presence of P. s. pv. aesculi, included cell wall lignification by a distinct layer of phloem and cortex parenchyma cells. The same cells also deposited suberin lamellae later on, suggesting this layer functions in compartmentalizing healthy from disrupted tissues. However, monitoring bacterial ingress, its construction appeared inadequate to constrain pathogen spread. Microscopic evaluation of bacterial dispersal in situ using immunolabelling and GFP-tagging of P. s. pv. aesculi, revealed two discriminative types of bacterial colonization. The forefront of lesions was found to contain densely packed bacteria, while necrotic areas housed bacterial aggregates with scattered individuals embedded in an extracellular matrix of bacterial origin containing alginate. The endophytic localization and ability of P. s. pv aesculi to create a protective matrix render it poorly accessible for control agents. To circumvent this, a method based on selective bacterial lethality at 39uC was conceived and successfully tested on A. hippocastanum saplings, providing proof of concept for controlling this disease by heat-treatment. This may be applicable for curing other tree cankers, caused by related phytopathogens. Citation: de Keijzer J, van den Broek LAM, Ketelaar T, van Lammeren AAM (2012) Histological Examination of Horse Chestnut Infection by Pseudomonas syringae pv. aesculi and Non-Destructive Heat Treatment to Stop Disease Progression. PLoS ONE 7(7): e39604. doi:10.1371/journal.pone.0039604 Editor: Zhengguang Zhang, Nanjing Agricultural University, China Received November 2, 2011; Accepted May 23, 2012; Published July 9, 2012 Copyright: 2012 de Keijzer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: A. A. M. van Lammeren was supported by the working group ‘Aesculaap’s, financed by the Dutch Ministry of Agriculture, Nature and Food Quality. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: andre.vanlammeren@wur.nl Introduction Since the last decade necrotic lesions/cankers with gummy exudations are observed on stems and branches of trees in the Aesculus genus, most notably the white horse chestnut (A. hippocastanum). These symptoms are found in Northwest Europe, with reports from Belgium [1], Germany [2], the United Kingdom [3] and The Netherlands [4]. The disease is severe, with a large part of the population exhibiting the typical bleeding symptoms [4,5]. It manifests itself on trunks and branches where it causes necrosis of bark tissues and bleeding of an amber coloured sap, which turns black in later stages (Fig. 1a). Often the vascular cambium becomes necrotic, leading to irregular secondary growth. When necrosis of the phloem is extensive, tree vitality is reduced and girdling lesions ultimately lead to dieback. While Phytophthora species are known to cause bleeding symptoms on horse chestnut [6], this recent outbreak was consistently found to be associated with the Gram-negative bacterium Pseudomonas syringae [1–4]. Intriguingly, the new European isolates were found to show a high degree of genetic similarity to an earlier described Indian P. syringae pathovar that causes leaf spots on A. indica [7], and were therefore accommodated in the same pathovar aesculi [2,3,8]. Indeed the sudden virulence of this horse chestnut pathogen is almost certainly due to its recent arrival in Europe, probably being introduced from India some time shortly preceding the epidemic in Northern Europe [8]. European P. syringae pv. aesculi is found as an epiphyte on leaves, flowers and fruits of the host tree [9]. The bacterium can adopt an endophytic, pathogenic lifestyle in the presence of small wounds, for example leaf scars or superficial injuries caused by human activities [10,11]. Further potential entry points for the bacterium have been shown to include lenticels [11], although spray- inoculation of these sites has been reported unsuccessful, even under lenticel opening conditions [10]. Once an infection is established, it may spread tangentially (in a diffuse manner) or in a predominantly longitudinal direction [10,11]. From the centre of old lesions no or little bacteria can be identified, in contrast to their edges and fresh lesions [5]. This hints at active colonization of new host tissues, but exact routes of bacterial dispersion and sites of multiplication are not known. Wounded plant tissue can lead to desiccation and forms a potential entry point for pathogens. Therefore, it is of PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e39604