HISTOLOGICAL PATHOGENESIS OF PSEUDOMONAS SAVASTANOI ON NERIUM OLEANDER

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1 014_JPP729(Saad)_ :25 Pagina 407 Journal of Plant Pathology (2010), 92 (2), Edizioni ETS Pisa, HISTOLOGICAL PATHOGENESIS OF PSEUDOMONAS SAVASTANOI ON NERIUM OLEANDER M. Temsah, L. Hanna and A.T. Saad Faculty of Agricultural and Food Sciences, American University of Beirut, P.O. Box , Beirut, Lebanon SUMMARY The development of the bacterial knot of oleander, caused by the Pseudomonas savastanoi, was studied at different intervals after inoculation to examine anatomical changes in inoculated twig tissues, the movement of bacteria within host tissues, and the formation of defense reactions of the host plant. Pseudomonas savastanoi invades stem tissues of oleander by moving from the inoculation wound into intercellular spaces of parenchyma tissues and systemically further away through the laticifers. Bacterial invasion of oleander tissues causes degradation of primary cell walls of parenchyma cells and laticifers and formation of bacterial cavities followed by hypertrophic and hyperplasic activities. Hyperplasic activities lead to the formation of meristematic cell masses, with differentiation of vascular bundles, which contributes to the development of the knot. The host develops observable microscopic anatomical defense reactions at the different stages of host invasion. Lignin deposits form on the primary cell walls of parenchyma cells and laticifers around bacterial cavities to limit the movement of the pathogen. Tyloses form in the secondary xylem vessels, blocking them and preventing further movement of the bacteria within the vessels. Finally, the formation of impermeable periderm around bacterial cavities, neoformed tissues, xylem elements, and at the surface of the knot, leads to its final decline. Key words: histopathogenesis, anatomy, oleander, bacterial knots. INTRODUCTION The early studies on the histological pathogenesis of Pseudomonas savastanoi on Nerium oleander were carried out in the 1960s by Wilson and coworkers, who studied the stages of knots development, the different Corresponding author: A.T. Saad Fax: tissues involved in their formation, and the bacterial invasion of stem tissues (Wilson, 1965). The purpose of our study was to complement the investigations by Wilson and coworkers, by examining the chronological development of the knots from initiation to full growth, and the formation of host defense reactions. We also compare our results with those of previous studies on other P. savastanoi hosts, namely buckthorn, olive and myrtle (Temsah et al., 2007a, 2007b, 2008). MATERIALS AND METHODS One-year-old oleander seedlings were inoculated with a virulent strain of P. savastanoi, isolated from active knots of naturally infected olive twigs collected from North Lebanon. The isolate was characterized by the LOPAT scheme of tests for grouping green fluorescent pseudomonads (Lelliot and Stead, 1987) to confirm its identity (Saad and Hanna, 2002). A suspension of bacterial inoculum was prepared in sterile water, from 24 h cultures of P. savastanoi growing on nutrient agar (DIFCO), and standardized turbidimetrically to a concentration of 10 7 CFU/ml. Oleander seedlings obtained from rooted cuttings were grown in 30 cm pots. Inoculation of the young oleander twigs was effected according to the procedure reported by Temsah et al. (2007b). The pathological and anatomical features of knot development, on inoculated oleander (Nerium oleander) twigs, were studied using histological techniques and light microscopy. Macroscopic observations on knot development and collection of samples for histological studies were done at 1, 3, 6, 9, 12, 15, 30, and 60 days post inoculation (d.p.i.). Cutting samples from inoculated twigs were fixed, paraffin-embedded, microtomesectioned and stained according to Jensen (1962) and Ruzin (1999), as detailed by Temsah et al. (2007a). Stained sections, 10 µm thickness, were mounted in Permount mounting medium and observed with a light microscope.

014_JPP729(Saad)_407 9-07-2010 12:25 Pagina 408 408 Pathogenesis of P.

2 014_JPP729(Saad)_ :25 Pagina Pathogenesis of P. savastanoi on oleander Journal of Plant Pathology (2010), 92 (2), RESULTS Macroscopic observations on inoculated oleander twigs revealed the appearance of small swellings, at the inoculation point, 9 d.p.i. These swellings grew to become clearly visible knots by 15 d.p.i. Transverse internodal sections of a young healthy oleander twigs between the third and fourth node, consisted of the following tissues from the epidermis into the pith (Fig. 1): (i) a cutinized epidermis of a single layer of cells containing trichomes. As the growth of the stem continues, periderm differentiating below the epidermis is composed of phellogen to the ouside of the phellem or cork and phelloderm towards the interior; (ii) collenchyma consisting of three to four layers of cells; (iii) a cortical parenchyma made up of several layers of cells; (iv) a sclerenchyma or fibers consisting of several masses of cells distributed in the internal cortical parenchyma. The walls of sclerenchyma cells thicken and their lumen decreases with the growth of the stem; (v) vascular tissues composed of primary phloem, a cylinder of secondary phloem, a cylinder of cambium, a cylinder of secondary xylem and primary xylem. Secondary vascular tissues consist of parenchyma tissues traversed by parenchyma rays; (vi) bundles of internal primary phloem that are specific to this host; (vii) non articulated branched laticifers distributed among parenchyma cells of the cortex, phloem and pith. Twenty four hours after inoculation, bacterial inoculum was observed in the intercellular spaces of cortical parenchyma and in the pith, in the case of a deep inoculation wound, degrading the cell walls of adjacent host cells. The occurrence of bacteria in the intercellular spaces of parenchyma tissues was restricted to the area between the inoculation wound and adjacent laticifers. Upon reaching the laticifers, bacteria proliferated in these vessels and invaded other tissues by degrading laticifer walls and adjacent parenchyma cells, thus creating cavities (Fig. 2). The walls of collenchyma and sclerenchyma cells were not degraded. In infected tissues, hypertrophic activity was followed by hyperplasia of parenchyma cells (Fig. 2). Hyperplasic activity started with several cœnocytic divisions, which resulted in cells containing from 2 to 16 nucleated protoplasms. These divisions were periclinal at first, then anticlinal. Irregular deposits of lignin formed on the primary cell walls of degraded cells surrounding the inoculation wound and degraded laticifer cells. These anatomical changes were not visible in stems inoculated with sterile water. On the third and until the ninth d.p.i., bacteria continued to proliferate in the laticifers, with consequent cell wall degradation, as well as hypertrophic and hyperplasic activity in tissue sectors adjacent to the laticifers. On the sixth d.p.i., formation of tyloses took place, which occluded secondary xylem vessels, preventing the movement of bacteria (Fig. 3). In control stems inoculated only with sterile water, mitotic divisions of cells with primary cell walls around the inoculation wound were observed 3 d.p.i (Fig. 4). However, this hyperplasic activity was not preceded by hypertrophy of the cells. The inoculation wound was entirely sealed with neoplastic cell mass and suberized cells on the sixth d.p.i. Twelve d.p.i, masses of meristematic cells started to appear adjacent to bacterial cavities and around infected laticifers (Fig. 5). These meristematic cell clusters Fig. 1. Transverse section of a healthy oleander twig between the third and fourth nodes, from the top, showing cutinized epidermis (e) with trichomes (t); collenchyma (ca); cortical parenchyma (cp) with intercellular spaces (is) and laticifers (l); sclerenchyma or fibers (s); primary phloem (pp); secondary phloem(sp); cambium (c); secondary xylem (sx); primary xylem (px); parenchyma rays (r); internal primary phloem (ip); and pith (p). Bar = 100 µm.

014_JPP729(Saad)_407 9-07-2010 12:25 Pagina 409 Journal of Plant Pathology (2010), 92 (2), 407-413 Temsah et al. 409 Fig. 2.

pericilinal (pc) and anticlinal (ac) divisions; lignin deposits (lg) around laticifers (l) and bacterial cavities (bc). Bar = 10 µm. Fig. 3. Transverse section of an oleander shoot, 6 d.p.i., showing tyloses (ty) in secondary xylem (sx).

3 014_JPP729(Saad)_ :25 Pagina 409 Journal of Plant Pathology (2010), 92 (2), Temsah et al. 409 Fig. 2. Transverse section of an oleander twig, 24 hours after inoculation, showing: cell wall degradation (cd), creating bacterial cavities (bc); hypertrophic (ht) and hyperplasic (hp) activities with pericilinal (pc) and anticlinal (ac) divisions; lignin deposits (lg) around laticifers (l) and bacterial cavities (bc). Bar = 10 µm. Fig. 3. Transverse section of an oleander shoot, 6 d.p.i., showing tyloses (ty) in secondary xylem (sx). Bar = 10 µm. originated from an acceleration of mitotic divisions of parenchyma cells of the cortex and phloem, not preceded by hypertrophic activity. However, parenchyma cells undergoing hypertrophic activity followed by hyperplasia were also present. Xylem elements differentiated within newly formed meristematic cell masses. Fifteen d.p.i., two cortical neoplastic masses of tissues were observed on both sides of the inoculation wounds made in spring, extending to the outside of the inoculation spot (Fig. 6). During their formation, the new neoplastic masses pushed externally, against cortical parenchyma, laticifers, collenchyma and epidermis, inducing flattening of the cells of these tissues (Fig. 6). The cambium produced more secondary xylem. In sum- Fig. 4. Transverse section of an oleander shoot inoculated with sterile water, 6 d.p.i., showing inoculation wound (w), entirely sealed with neoplastic cells (nc) resulting of mitotic division, and by suberized cells (sc). Bar = 100 µm.

014_JPP729(Saad)_407 9-07-2010 12:25 Pagina 410 410 Pathogenesis of P. savastanoi on oleander Journal of Plant Pathology (2010), 92 (2), 407-413 Fig. 5. Transverse section of an oleander shoot, 12 d.

mer inoculations by converse, plants showed additional anatomical modifications 15 d.p.i, consisting of periderm differentiation at the surface of the knot and underneath the flattened tissues and of opening of bacterial cavities to the outside (Fig.

4 014_JPP729(Saad)_ :25 Pagina Pathogenesis of P. savastanoi on oleander Journal of Plant Pathology (2010), 92 (2), Fig. 5. Transverse section of an oleander shoot, 12 d.p.i., showing meristematic cell masses (mc), differentiated xylem elements and bacterial cavities (bc). Bar = 10 µm. mer inoculations by converse, plants showed additional anatomical modifications 15 d.p.i, consisting of periderm differentiation at the surface of the knot and underneath the flattened tissues and of opening of bacterial cavities to the outside (Fig. 7). New masses of neoplastic cells located at the periphery of the knot were surrounded posteriorly by layers of suberized cells (Fig. 8). Numerous vascular bundles arranged in concentric, circular or flattened rings were observed to form during summer inoculation within the knot, while in spring such vascular tissues differentiated at later periods after inoculation. Numerous neoplastic tissue masses covered by periderm developed 30 d.p.i. New periderm tissues were continuously produced after each cracking of preexisting periderm that resulted from the pressure exerted by the developing new neoplastic masses. Sixty d.p.i., periderm differentiated around fibers and enlarged bacterial cavities, thus creating anatomic voids within the knots. The periderm that formed inside the knots and around neoplastic cell masses isolated Fig. 6. Transverse section of an oleander shoot,15 d.p.i., showing two cortical neoplastic masses of tissues (nm) on both sides of the inoculation wound (w). Bar = 200 µm. Insert shows the neoplastic masses pushing against and flattening cells of and cortical parenchyma (cp), collenchyma (ca), laticifers (l), and epidermis (e). Bar = 50 µm.

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. 7. Transverse section of an oleander shoot, 15 d.p.i., showing: A, differentiated periderm (pd) around a bacterial cavity (bc), and B, cracking of the periderm thus C, opening of the bacterial cavity ( ) to the outside of the knot.

5 014_JPP729(Saad)_ :25 Pagina 411 Journal of Plant Pathology (2010), 92 (2), Temsah et al. 411 Fig. 7. Transverse section of an oleander shoot, 15 d.p.i., showing: A, differentiated periderm (pd) around a bacterial cavity (bc), and B, cracking of the periderm thus C, opening of the bacterial cavity ( ) to the outside of the knot. Bar = 10 µm. these tissues and provoked cracking, deterioration and disintegration of the tumors (Fig. 9). DISCUSSION Our comparative studies on the pathogenesis of Pseudomonas savastanoi to buckthorn, myrtle, olive (Temsah et al., 2007a, 2007b, 2008) and oleander (this study) have revealed histological differences among the different hosts, reflecting variations in pathogen advance, knot development and host defense reactions. In oleander stems, tissue invasion and rate of colonization by the pathogen, cell wall degradation and hypertrophic and hyperplasic activity, were more rapid than in other hosts. Likewise, defense mechanisms were different and developed faster. of primary cell walls, because of the high enzyme production (Wilson, 1965). Hypertrophic activity in parenchyma cells was preceded by a coenocytic phase in oleander, where neoplastic cells contained up to 16 nuclei in a single cell. Octonuclear cell division was observed in neoplasic cells of olive (Temsah et al., 2008), whereas diplonuclear and quadronuclear cell divisions were observed in neoplastic cells of buckthorn (Temsah et al., 2007a). Knot initials formed because of the hypertrophic and hyperplasic activity of parenchyma cells consequent to the action of auxins (Saad and Hanna, 2002) and cytokinins (Surico et al., 1985), known to be produced by P. savastanoi. The in vitro production of IAA Path of pathogen invasion in Nerium oleander. In oleander, P. savastanoi moved from the inoculation wound into the intercellular spaces of the cortical parenchyma, degrading the cell walls of adjacent cells. Once the inoculum attained the laticifers, invasion proceeded through these vessels where bacteria proliferated and moved systemically. By contrast, in buckthorn, myrtle and olive, invasion occurred only through the intercellular spaces of cortical and pith parenchyma tissues of the stem Cell wall degradation, hypertrophy and hyperplasia. Cell wall degradation occurred within the first 24 h after inoculation of oleander. In contrast to this early occurrence, cell wall degradation occurred 3 d.p.i. in olive, 6 d.p.i. in buckthorn, and 9 d.p.i. in myrtle. P. savastanoi invaded oleander tissues through degradation and lysis Fig. 8. Transverse section of an oleander shoot, 15 d.p.i., showing neoplastic masses (nm) surrounded posteriorly by suberized cells (sc). Bar = 50 µm.

014_JPP729(Saad)_407 9-07-2010 12:25 Pagina 412 412 Pathogenesis of P. savastanoi on oleander Journal of Plant Pathology (2010), 92 (2), 407-413 Fig. 9. Transverse section of an oleander shoot, 60 d.

6 014_JPP729(Saad)_ :25 Pagina Pathogenesis of P. savastanoi on oleander Journal of Plant Pathology (2010), 92 (2), Fig. 9. Transverse section of an oleander shoot, 60 d.p.i., showing the periderm (pd) tissues at the surface of the knot, surrounding the neoplastic masses (nm) and bacterial cavities inside the knots resulting in anatomic voids (av). Bar = 200 µm. was also reported for P. savastanoi isolates from oleander (Wilson, 1965) and olive (Surico et al., 1985). Hypertrophy and hyperplasic activities and degradation of cell walls were not observed to occur in oleander collenchyma tissues, which lack intercellular spaces and laticifers. Plant tissues involved in the development of the knot. In the early stages of disease development, all parenchymatous cells of the cortex, phloem, xylem and pith of oleander stem tissues, in contact with bacterial inoculum, underwent hypertrophic and hyperplasic activities. In advanced stages of knot development, 15 d.p.i., the knot was formed, principally from the proliferation of meristematic cells of parenchyma, cortex and phloem. The hypertrophic and hyperplasic activities induced by P. savastanoi varied with the stages of knot development. During the first 12 d.p.i., both auxins and cytokinins were probably produced at the same time so as to favor the simultaneous development of both activities. At 15 d.p.i. only hyperplasic activity, differentiation of vascular bundles and periderm continued to take place in the developing knot, whose rate of development varied with the season at which inoculation was made. Following warm summer inoculations, a faster rhythm of cell division and knot growth was registered as compared with inoculations made during the cool spring season. In oleander, fully grown knots continued to develop as a result of the hyperplasic activity of parenchyma cells of the cortex and phloem. Buckthorn knots were formed by hyperplasic activities of parenchyma cells of the cortex, parenchyma rays, and or pith cells (Temsah et al., 2007a), In myrtle, knot formation was due to hyperplasic activity of parenchyma cells of the cortex and primary xylem parenchyma (Temsah et al., 2007b). In olive, knots developed by hyperplasic activities of parenchyma cells of the cortex (phelloderm, cortical parenchyma), and phloem parenchyma (Temsah et al., 2008). Histology of host resistance. Oleander reacted to infection with various types of histological defense structures to limit the progress of bacterial invasion and knot growth. In the early stages of disease development, lignin was deposited on the degrading primary cell walls of cells surrounding bacterial cavities and on the walls of degrading laticifers. Tyloses in xylem vessels developed 6 d.p.i obstructing bacterial colonization and movement. The role of tyloses in the prevention of the intrusion and spread of bacteria into xylem vessels was reported by Kozlowski and Pallardy (1997). Lignin de-

7 014_JPP729(Saad)_ :25 Pagina 413 Journal of Plant Pathology (2010), 92 (2), Temsah et al. 413 posits were observed around bacterial cavities and infected laticifers in the early stages of disease development, whereas in olive and myrtle lignin was deposited only on the walls of cells surrounding bacterial cavities (Temsah et al., 2007b, 2008). In the advanced stages of knot development, periderm tissues differentiated internally around bacterial cavities, fibers, vascular bundles, neoplastic masses, and at the surface of the knots, allowing the liberation of bacteria to the outside of the knot. Periderm differentiated around bacterial cavities also in buckthorn (Temsah et al., 2007a). The periderm contributes to the decline of the knots at the peak of their developmental stages. It is differentiated also around bacterial cavities creating anatomic voids inside the knot. Impermeable periderm tissues surrounding neoplastic masses lead to depletion of water and nutrients, thus evoking the deterioration of the knots. REFERENCES Jensen W.A., Botanical Histochemistry: Principles and Practice. W.H. Freeman, San Francisco, CA, USA. Kozlowski T.T., Pallardy S.G., Physiology of Woody Plants. 2 nd ed. Academic Press, San Diego, CA, USA. Lelliot R.A., Stead D.E., Methods for the Diagnosis of Bacterial Diseases of Plants. Blackwell Scientific, Oxford, UK. Ruzin S. E., Plant Microtechnique and Microscopy. Oxford University Press, Oxford, UK. Saad A.T., Hanna L., Two new hosts of Pseudomonas savastanoi and variability in strains isolated from different hosts. Phytopathology 92: S71. Surico G., Iacobellis N.S., Sisto A., Studies on the role of indole-3-acetic acid and cytokinins in the formation of knots on olive and oleander plants by Pseudomonas syringae pv. savastanoi. Physiological Plant Pathology 26: Temsah M., Hanna L., Saad A.T., 2007a. Anatomical observations of Pseudomonas savastanoi on Rhamnus alaternus. Forest Pathology 37: Temsah M., Hanna L., Saad A.T., 2007b. Histological pathogenesis of Pseudomonas savastanoi on Myrtus communis. Journal of Plant Pathology 89: Temsah M., Hanna L., Saad A.T., Anatomical pathogenesis of Pseudomonas savastanoi on olive and genesis of knots. Journal of Plant Pathology 90: Wilson E.E., Pathological histogenesis in oleander tumors induced by Pseudomonas savastanoi. Phytopathology 55: Received July 30, 2009 Accepted October 31 st, 2009

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ANATOMICAL PATHOGENESIS OF PSEUDOMONAS SAVASTANOI ON OLIVE AND GENESIS OF KNOTS

008_JPP_143RP_225_colore 21-07-2008 10:37 Pagina 225 Journal of Plant Pathology (2008), 90 (2), 225-232 Edizioni ETS Pisa, 2008 225 ANATOMICAL PATHOGENESIS OF PSEUDOMONAS SAVASTANOI ON OLIVE AND GENESIS