Histological examination of horse chestnut infection by Pseudomonas syringae pv. aesculi and non-destructive heat treatment to stop disease progression

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 39 °C 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.

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 N51° 57′ 26″; E5° 34′ 10″. (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 µm. (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 µm. (F) Detail of E showing the even deposition of suberin around the plant protoplasts. The scale bar indicates 25 µm. (G) Continuous deposition of suberin by two cells at pit fields (arrowheads). The scale bar indicates 10 µm.

Figure 2. The infection process of P. syringae pv. aesculi and bacterial behaviour in planta.

The infection process up to 18 weeks after inoculation visualized by immunocytochemically labelling of P. syringae pv. aesculi. Arrows in A, C, K, L, M, N and P indicate the longitudinal direction. Scale bars indicate 100 µm in all micrographs except B and C where 10 µm is indicated. (A) Six days after inoculation, when the lining of lignified and suberized cells is prominent, clusters of bacteria are observed in deeper parenchyma tissues (arrowheads) by immunolabelling. Furthermore, bacteria remain in the original inoculation cut (arrows). The image shown is taken from a longitudinal section. (B) A bacterial accumulation in a cortex parenchyma cell observed in a longitudinal section of an infection site sampled 14 days after inoculation. Besides the large cluster, several individual bacteria are scattered in intercellular spaces. (C) A longitudinal section of phloem tissue 6 days post inoculation showing the decoration of bark fibres by P. s. pv. aesculi seemingly following the vertical orientation of these elements. (D, E) Close-up and micrograph of transversal section of a mock-inoculated site on a horse chestnut plant 7 weeks after inoculation. The asterisk points to site of wounding during inoculation. (F, G, H) Close up and transversal sections of inoculation sites 15 weeks after inoculation. Note partially restored development of secondary xylem despite infection. Asterisks point to site of wounding during inoculation. (I, J) Transversal sections of cortex in mock-inoculated (I) and infected (J) horse chestnut 12 days after inoculation. Note ablated plant cells filled with bacteria in J. (K) Clusters of bacteria in a longitudinal section of the xylem close to the inoculation site, 4 weeks after inoculation. (L–N) Longitudinal sections of the cambium region with bacteria in intercellular spaces of phloem parenchyma (L), ray parenchyma (M) and along phloem fibres (N), 3 weeks after inoculation. (O) P. s. pv. aesculi in intercellular spaces observed in a transversal section of the cambial zone at 4 weeks after inoculation. (P) Bacteria decorating a bundle of phloem fibres which is surrounded by newly formed periderm (*) and degenerating parenchyma 4 weeks after inoculation. (Q) Bacteria colonizing necrotic bark tissue at 10 weeks after inoculation. (R-T) Transversal sections of enclosures surrounded by periderm with bacteria ex planta at 4, 10 and 18 weeks after inoculation, respectively.

Figure 3. Behaviour of GFP-expressing P. syringae in planta.

(A) An A. hippocastanum seedling inoculated with strain PD4818-pMP4655 photographed after 2 months shows vertical lesion expansion (up to 6 cm in length) and tissue necrosis after removal of the outer tissues of the bark. The inoculation site is indicated with an arrowhead. Note the bending of the infection around the lower node. The scale bar indicates 1 cm. (B) Side view of a longitudinally split piece of stem from a PD4818-pMP4655 inoculated seedling removed 1–2 cm from the inoculation site 4 weeks after inoculation. Necrosis and discolouration is visible in the cambial and phloem areas. (C) A longitudinal section of an infection border in bark tissue after inoculation with PD4818-pMP4655 sampled 4 weeks after inoculation. A parabolic shaped zone with elevated cell wall autofluorescence is discernible (arrowheads) with a P. s. pv. aesculi colony located in the tip of the infected zone, at about 1 cm away from the inoculation site. The boxed area is shown in detail in D. The scale bar indicates 25 µm. (D) Numerous bacteria are clustered near the forefront of an advancing lesion in parenchyma 4 weeks after inoculation. The bacteria observed here are relatively small and densely packed. The scale bar indicates 10 µm. (E) Within the necrotic area near the original site of inoculation several clusters of bacteria are found that occupy the cavities left by dead cells. Image taken from a longitudinal section of a 6 week old infection. The scale bar indicates 25 µm. (F) A large aggregate of PD4818-pMP4655 located in necrotic tissue of a 4 week old lesion. Individual bacteria were spaced apart and did not undergo any type of motility. The scale bar indicates 25 µm. (G) Detail of a PD4818-pMP4655 cluster in necrotic tissue of a 4 week old infection, emphasizing the spacing between individual bacteria. The scale bar indicates 10 µm. (H) Immunodetection of wild-type PD4818 in the necrotic tissue of a 10 week old infection showing a similar spatial arrangement of bacteria to that shown in G. The scale bar indicates 10 µm.

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 µm. (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 µm. (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 µm.