The occurrence of matrix embedded bacteria within the host tissues combined with lesion expansion that takes place mostly underneath the periderm (Fig. 3B), imply that once endophytically established, the pathogen is poorly accessible from the outside. This could limit the effectiveness of disease control by agents that require physical contact. Indeed, established cankers in fruit trees caused by P. syringae appear notoriously difficult to control [28]. Thus, for management of P. s. pv. aesculi it may be relevant to target other parts of the (largely unknown) disease cycle or establish measures that can penetrate to reach internal bacterial populations. To this end, a heat-based control method was conceived and tested on A. hippocastanum saplings. Pioneering experiments to test the efficacy of heat-treatment of whole plants indicated that P. s. pv. aesculi associated with active lesions is rendered inactive after a two day exposure to 39uC, which is tolerated by A. hippocastanum. Hence, thermal treatment may be a feasible, non-invasive control method employable for disease management on a larger scale. A potential issue with applying heat-treatment under field conditions could lie in differences in temperature sensitivity that may occur among various isolates, as is reported for Erwinia amylovora [44]. However, the extremely high genetic homogeneity among UK P. s. pv. aesculi isolates [8] implies that variation in heat-resistance among bacterial strains is minimal, suggesting heat-treatment of A. hippo- castanum may be a widely applicable method. To date, heat-based phytopathogen control has proven to be a useful method to manage various post-harvest diseases [45,46]. The results presented in this study demonstrate that heat- treatment can also be used on growing, vegetative plant organs as a control strategy to inactivate P. s. pv. aesculi. Such treatment could be practiced on seeds, saplings and affected aerial organs of trees, as will be tested in future studies. For mature trees complicating factors like secondary infection by opportunistic pathogens or reinfection should, however, be considered. None- theless, heat-treatment could well shift the balance in plant pathogen interaction in favour of the host, enabling sufficient recovery and revitalization in an otherwise lethal infection. In addition, the high potential for repelling this particular disease might mean that this strategy can be expanded to control similar diseases in other tree species caused by related bacterial pathogens. Materials and Methods Bacterial Strains, Culture Conditions and Plasmids Pseudomonas syringae pv. aesculi strains (listed in table 1) were grown in Lysogeny Broth (LB; [47]) at 28uC and 180 rpm. E. coli strains (Table 1) were routinely cultured in LB at 37uC and 225 rpm. Plasmid pMP4655 (obtained from the Department of Molecular Microbiology, Leiden University; Table 1) was introduced in E.coli DH5a and GM119 by electroporation. Antibiotics were added in the following concentrations unless stated otherwise: tetracycline, 40 mg/mL; carbenicillin, 100 mg/ mL and kanamycin, 50 mg/mL. Strains were stored at 280uC for long-term conservation. Transformation of P. s. pv. aesculi by Triparental Conjugation The two E. coli donor strains harbouring pMP4655, helper strain cel40 containing pRK2013 and recipient P. s. pv. aesculi strains were grown until late log phase. Antibiotics present in the initial culture medium were first removed by collecting cells at 1,0006g for 5 min and resuspending them in their original culture volume of LB. All cultures were pelleted again and resuspended in 3% of their original culture volume of LB. Aliquots of 30 mL were P. syringae in Aesculus, Infection and Control mixed in a 1:1:2.5 and 1:1:5 (helper:donor:recipient) ratio and transferred to sterile, 20 mm ø BA85 nitrocellulose filters with 0.45 mm pore size (Schleicher & Schuell), which were incubated overnight on LB plates. The cells were collected from the filters in 1 mL PBS and plated on LB plates containing carbenicillin and 15 mg/mL tetracycline. Well grown colonies were serially diluted in PBS and restreaked on plates containing carbenicillin and 30 mg/mL tetracycline to yield pure P. s. pv. aesculi transformants. Both E. coli donorstrains appeared equally potent for conjugal transfer of pMP4655. Plant Cultivation and Inoculation For the analysis of the early defence response and bacterial invasion upon inoculation, experiments were conducted with one and two-year old horse chestnut (A. hippocastanum) seedlings. Plants were maintained at room temperature under natural light in containers with 5 L potting soil and watered weekly. Experiments were repeated three times. Eight plants in total were inoculated at various sites on the stem for sampling in time, two inoculations at opposite sides of the stem. At each sample moment (see ‘Sampling, fixation and sectioning’) one set of opposite inoculation sites was removed. Plants were either mock-inoculated as a control (3 plants in total) or inoculated with P. s. pv. aesculi (5 plants in total). For artificial infection of the plants a superficial wound was inflicted on a surface sterilized part of the stem by making a small (2–3 mm), diagonal cut with a downward angle using a sterile razorblade. Care was taken not to cut deeper than the cambial zone. These wounds were inoculated by pipetting 5 mL of sterile PBS or 5 mL PBS containing 108 cfu/mL P. s. pv. aesculi PD4818 into the wound fissure. For the analysis of bacterial spread on the long term, 20 plants were inoculated at 6 sites per plant (including 5 mock inoculated plants). At each sample moment 4 plants were harvested (one of them mock inoculated) and of each plant one sample was selected randomly for analysis. Infection experiments with GFP expressing bacteria (isolate PD4818 harbouring pMP4655) were performed on three separate occasions in climate cells using 6 two-year old horse chestnut seedlings maintained at 21uC, 70–90% RH and a 16 h light, 8 h dark cycle. Plants were sampled after 4 and 6 weeks to examine in planta behaviour of bacteria, and monitored over a period of 8 weeks. Sampling, Fixation and Sectioning For the analysis of the early stages of the infection process samples of stem were removed from the inoculated seedlings around the original inoculation site at 0, 1, 2, 3, 4, 6, 8 and 14 days after inoculation. For investigation of later stages, samples were taken at 2, 3, 4, 10 and 18 weeks after inoculation. The sampled pieces of stem were fixed in 4% w/v paraformaldehyde, 0.05% v/v glutaraldehyde and 0.01% v/v Triton X-100 in 0.1 M phosphate buffer (pH 7.2) by vacuum infiltration (10 kPa). Infection experiments with GFP-expressing bacteria were sampled at 4 and 6 weeks after inoculation and two inoculation sites were examined per time-point. When preparing samples with GFP- expressing bacteria, the fixation step was omitted. Transverse and longitudinal sections of ,20 mm thick were made with a Reichert sledge microtome. Fixed samples were stored in 0.1 M phosphate buffer (pH 7.2) containing 0.02% w/v NaN3 at 4uC. Staining and Immunolabelling To stain lignin, sections were incubated in 10% w/v phloro- glucinol in ethanol for 20 min and subsequently in 20% v/v HCl for 3 min. Sections were subsequently washed twice for 30 seconds PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e39604