P. syringae in Aesculus, Infection and Control Figure 4. A fibrillar matrix produced in vitro by P. syringae pv. aesculi enwraps aggregates of immobilized bacteria. (A) After incubation in minimal medium a fibrillar material covering clustered bacterial cells is observed by scanning electron microscopy (left-hand panel). Such material is not encountered after incubation in LB, wherein only the polar flagella are observed extracellularly (right-hand panel). The insets show the bacterial cells and the surrounding material in more detail. Note that due to the completely dehydrated state of the specimen, the here presented image likely does not depict the true spatial lay-out of a bacterial cluster. The scale bars indicate 1 mm. (B) A typical cluster observed in a PD4818-pMP4655 culture grown in minimal medium visualized by confocal laser scanning microscopy. GFP-expressing bacteria appear in green, while addition of propidium iodide (PI) brightly stained dead individuals red and led to a mild staining of the amorphous material enwrapping the bacteria within a cluster. The bottom and right panels are computed images of the two orthogonal planes along the z-axis indicated by the dark grey arrows. The light grey arrows indicate the position of the optical plane shown in the top left panel. Note that single bacteria are spaced from neighbouring cells in 3 dimensions. The scale bars indicate 10 mm. (C) Three 10 second interval frames taken from a time-lapse movie of the edge of a PD4818-pMP4655 cluster (depicted in the DIC image) were differentially colour coded and merged to reveal displacement of individual bacteria over time. Several motile bacteria freely moved on the left appearing in a single colour in the merged image, while bacteria within the cluster remained at a fixed location as revealed by their white colour in the merged image. Additionally, oscillatory motion of 2 bacteria is discernable in the cluster margin (arrowhead), indicated by the slight colour shift. The scale bar indicates 5 mm. doi:10.1371/journal.pone.0039604.g004 (HPAEC) was used for further identification of the sugar composition of the exopolysaccharide. Under stress conditions the typical degradation products of alginate (guluronic acid and mannuronic acid) were observed as two neighbouring peaks (Fig. S2), although the relative presence of guluronic acid is lower than observed for the alginate derived from brown algae used as control. Taking into account the presence of these two peaks it is concluded that alginate was the predominant polysaccharide in the extracellular material produced by P. s. pv. aesculi under salt stress. Previous studies point to the possible additional presence of glucuronic acid [22] which elutes approximately at the same time as mannuronic acid. In addition, rhamnose and glucose, although at lower concentra- tions than alginate, were identified (Fig. S2). Minor traces of an unidentified sugar were also present. Alginate is often found to be produced by plant pathogenic Pseudomonas spp. when associated with their host [23–25]. Its secretion aids in retaining water and is thereby thought to contribute to bacterial colonization and symptom development [26]. The ability of P. s. pv. aesculi to produce alginate was further supported by the presence of genes associated with its biosynthesis and regulation thereof as screened in silico. Sequence data of P. s. pv. aesculi strain 2250 [8] indicated that this strain has a full repertoire of genes encoding alginate biosynthesis and regulatory proteins with close homology to these of P. s. pv. tomato DC3000 (Table S1). Furthermore, most of the genes predicted to function in biosynthesis were found to be arranged in the canonical alginate biosynthesis operon structure [27]. Heat-treatment as a Feasible Control Method for Horse Chestnut Bleeding Disease Once established, bacterial cankers on trees are notoriously difficult to control, largely due to the site of bacterial proliferation that is poorly accessible from the outside [28]. As a method to inactivate the horse chestnut pathogen during its endophytic lifestyle, it was hypothesized that high-temperature treatment may prove adequate. Initial experiments using liquid cultures of P. s. pv. aesculi pointed out that a 24 hour incubation at 39uC consistently yielded no culturable bacteria (data not shown). Then, to assess the feasibility of such a heat-treatment method in situ, the survival of P. s. pv. aesculi PD4818-pMP4655 was monitored after infected plants were incubated at 39uC for 48 hours. From infected areas of heat-treated plants no viable bacteria were re-isolated, while all untreated lesions readily yielded P. s. pv. aesculi PD4818-pMP4655 (Table 2). The heat-treated plants showed no visible injury, except occasional damage of leaf edges. These observations indicate that a 2 day incubation at 39uC is sufficient to inactivate bacteria associated with active lesions, while minimally affecting the host plant, thus providing proof of concept for heat-treatment as a high-potential control method to stop horse chestnut bleeding disease progression. Discussion The recent emergence of P. s. pv. aesculi as a pathogen that infects trunks and branches of Aesculus spp. has severely affected the vitality of the population of these trees, which are highly regarded for their ornamental value. Although genomic data has PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e39604