Vol. 55, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1989, p. 533-5380099-2240/89/020533-06$02.00/0Copyright ©) 1989, American Society for Microbiology

Scanning Electron Microscopy of Invasion of Apple Leaves andBlossoms by Pseudomonas syringae pv. syringae

E. LUCIENNE MANSVELT' AND M. J. HATTINGH2*

Fruit and Fruit Technology Research Institute' and Department of Plant Pathology,2 University of Stellenbosch,Stellenbosch 7600, South Africa

Received 1 July 1988/Accepted 14 November 1988

Scanning electron microscopy indicated that Pseudomonas syringae pv. syringae L795 entered leaves throughstomata and multiplied in the substomatal chambers. Strain L195 applied to blossoms colonized stigmas andalso occurred in intercellular spaces of styles. Nonpathogenic strain L796 failed to colonize blossoms. This studysuggests that inoculum of pathogenic P. syringae pv. syringae builds up on apple leaves and blossoms.

Pseudomonas syringae pv. syringae van Hall causes bac-terial blister bark of apple (Maluis domestica Borkh.) inSouth Africa (9). The pathogen frequently multiplies onapple leaves and blossoms during the growing season with-out causing lesions (13). However, it is uncertain where onthese plant surfaces P. syringae pv. syringae multiplies andif underlying tissue is invaded. Scanning electron micros-copy of other host-pathogen systems has shown that popu-lations of bacteria on leaves colonize substomatal cavities (1,2, 6, 14-16, 18, 19, 24, 25) or trichomes (3, 11, 21), whereaspopulations on blossoms occur on stigmas (4, 6, 12, 20, 23)and the hypanthium (6, 12, 20). This paper reports theecological niche of P. syringae pv. syringae on apple leavesand blossoms and subsequent entry of tissue.The three strains of P. syringae pv. syringae used were

characterized in a previous study (17): strains L195 and L795are pathogenic on shoots of apple, cherry, pear, and plum,whereas L796 is nonpathogenic. Stock cultures were main-tained at room temperature on slants of nutrient agar (DifcoLaboratories, Detroit, Mich.) supplemented with 20% glyc-erol. Inoculum suspensions of the strains were preparedfrom cultures grown overnight on King medium B (7) at 26°Cand suspended in sterile distilled water to 106 CFU/ml aspreviously described (11). Concentrations were confirmedby dilution plating.

Potted Oregon Spur apple trees were used. Bacterialsuspensions were applied to leaves or blossoms by gentlespraying with an atomizer until runoff. Controls weresprayed with sterile distilled water. Leaves on vigorousshoots (20 cm) on 1-year-old trees were treated with P.syringae pv. syringae L795. Individual shoots were coveredwith moist plastic bags for 24 h before leaves were sprayed.Trees were then left uncovered in a greenhouse kept atapproximately 24°C. Blossom inoculations were performedon 3-year-old trees that were removed from cold storage andallowed to break dormancy in the greenhouse. Separateflower clusters in full bloom were sprayed with P. syringaepv. syringae L195 or L796.Leaf tissue (5-mm2 squares) was sampled at 45 min and at

3, 6, 9, and 12 days after spraying. Intact blossoms weresampled after 1 and 2 days. Petals of blossoms were removedand discarded, and remaining floral parts were separated bycutting off stamens and filaments at the sites of attachmentwith a sterile blade (12). Sections were fixed in a 5%glutaraldehyde solution, dehydrated in an alcohol series, and

* Corresponding author.

dried in a critical point drier under CO2 (11). Samples weremounted on stainless steel stubs. Some leaf samples werefractured through mesophyll tissue in the plane of the leafsurface as described by Sigee and Al-Issa (22). Segments offloral parts or longitudinal sections cut with a new razorblade were mounted. Specimens were gold-coated and ex-amined with an ISI 10-nm scanning electron microscope(International Scientific Instruments, Santa Clara, Calif.) at15 kV.

Leaves. Forty-five minutes after application of pathogenicstrain L795, bacterial cells were randomly dispersed over theleaf surface (Fig. 1A). After 3 days, small aggregates oc-curred near or over stomata. In fractured tissue, bacteriawere also seen inside the substomatal chamber, where theyhad clustered at or near the pore (Fig. 1B).

Six days after spraying, larger masses of bacteria (Fig. 1C)were associated with many of the stomata. Bacteria withinthe substomatal cavity were embedded in a dense layer (Fig.1D). Few bacteria were seen in the intercellular spaces of thespongy parenchyma of the mesophyll. Bacteria were other-wise unevenly distributed over the leaf surface and occurredsparsely at the base of trichomes (Fig. 1E). However, P.syringae pv. syringae L795 failed to invade trichomes onapple leaves, although the same strain is capable of coloniz-ing trichomes on pear leaves extensively (11).We assume that substomatal chambers were directly col-

onized by P. syringae pv. syringae entering through sto-mata. The pathogen might also have reached some chambersafter having moved from colonized pockets through theintercellular spaces of the spongy parenchyma. However,intercellular spread appears to have been fairly restricted,and bacteria were not seen beyond the outer cell layers ofthe spongy parenchyma. In contrast, P. syringae pv. mors-prunorum entering stomata on cherry leaves spreads fromthe mesophyll, invades the xylem of minor veins, and thenmigrates to other regions in the leaf blade and petiole (17).Furthermore, P. syringae pv. syringae introduced into pet-ioles on plum trees spreads through the xylem to leaves,where it is extruded through stomata (18).Nine days after spraying, fibrillar material was observed

on the stomata (Fig. 1F). Bacteria inside the substomatalchamber were embedded in similar material (Fig. 2A). Ag-gregates of bacteria were sparsely dispersed in the intercel-lular spaces of the spongy parenchyma (Fig. 2B). Twelvedays after spraying, only a few bacterial cells could bedistinguished on the leaf surface or associated with fibrillarmaterial present on stomata or in the substomatal chamber.

533

on May 24, 2018 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

FIG. 1. Scanning electron micrographs of apple leaves treated with pathogenic strain L795 of P. syringae pv. syringae. (A) Bacteria onabaxial leaf surface 45 min after application. (B) Bacteria inside substomatal cavity clustered on the paracytic stoma after 3 days. (C to E)Six days after application. Bacteria are massed at a stoma (C) and are embedded in a slimelike substance in the substomatal cavity (D), andsingle cells are present at the base of a trichome (E). (F) Fibrillar material on stoma after 9 days. Bars, 5 p.m.

534 NOTES

on May 24, 2018 by guest

http://aem.asm

.org/D

ownloaded from

NOTES 535

FIG. 2. Scanning electron micrographs of apple leaves and blossoms treated, respectively, with pathogenic strains L795 and L195 of P.syringae pv. syringae. (A and B) Nine days after treatment of leaves. Bacteria are embedded in fibrillar material inside the substomatal cavity(A) and in intercellular spaces of the spongy parenchyma of the mesophyll (B). (C to E) One day after treatment of blossoms. (C) Bacterialcells on stigmatic papillae and on surfaces of underlying cells (arrow). (D) Invaded papilla. Note the absence of bacteria from intercellularspaces. (E) Bacteria at penetration site of pollen tube (arrow). (F) Mass of bacterial cells on papillae 2 days after application. Bars, 5 ,um.

VOL. 55, 1989

on May 24, 2018 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

FIG. 3. Scanning electron micrographs of apple blossoms 2 days after applying pathogenic strain L195 (A to E) and 1 day after applyingnonpathogenic strain L796 (F) of P. syringae pv. syringae. (A) Bacteria (arrows) within collapsed regions of the cuticular layer of stigmaticcells beneath the protruding papillae. (B) Longitudinal section showing bacteria in the intercellular spaces of stigmatic tissue. (C to E) Bacteriaassociated with stylar tissue. Masses of bacteria in intercellular spaces (C) were confined to the outer cell layers of the style (D) but alsooccurred externally on the surface of the longitudinal groove of the conduplicate style (E). (F) Single bacterial cells on surface of papillae.Bars, 5 ~tm.

536 NOTES

on May 24, 2018 by guest

http://aem.asm

.org/D

ownloaded from

VOL. 55, 1989

Bacteria were not seen on the surfaces of control leavessprayed with water or in cross sections or longitudinalsections of these leaves. No lesions developed on leavessprayed with the bacterial suspension.

P. syringae pv. syringae is not known to cause lesions onapple leaves. Nevertheless, the abundant presence of thepathogen in substomatal chambers and in intercellularspaces of the spongy parenchyma of the mesophyll hasepidemiological implications. Apart from being sheltered inleaf tissue, P. syringae pv. syringae was also extrudedthrough stomata after having multiplied extensively in sub-stomatal chambers. Inoculum in orchards might be replen-ished in this way.

Blossoms. One day after application of pathogenic strainL195, bacteria were noted on the surfaces of papillae andunderlying cells of the stigma (Fig. 2C) but were not seenwithin tissues (Fig. 2D) or on other blossom parts. Bacteriawere occasionally found where a pollen germ tube hadentered the stigma (Fig. 2E).Two days after application of strain L195, masses of

bacteria were present on papillae and underlying cells (Fig.2F). Bacterial aggregates were also prominent within col-lapsed regions in the cuticular layer of underlying cells (Fig.3A). Longitudinal sections revealed large numbers of bacte-ria in the intercellular spaces of stigmatic tissue (Fig. 3B) andstylar tissue (Fig. 3C). Bacteria within styles appeared tohave been confined to the intercellular spaces of cellsimmediately below the epidermis (Fig. 3D). Bacteria werealso present in the longitudinal groove of the conduplicatestyle (Fig. 3E).

Failure of pathogenic P. syringae pv. syringae to causenecrosis of apple blossoms agrees with the results obtainedwith treated leaves of this host. Stigmatic papillae werepreferentially colonized, and the pathogen spread intercellu-larly through the style. In contrast, P. syringae pv. syringaecolonizing stigmas of pear blossoms causes blast (12). Er-winia amylovora brings about blossom necrosis of differentponp fruits in the same way (6, 20, 23).The pathogen caused less extensive damage on apple than

on pear stigmas and styles (10). On apple blossoms, discol-oration and browning of stigmas associated with the naturaldegeneration that follows anthesis might have maskedexpression of disease symptoms. Likewise, the physicalappearance of stigmas and styles in orchards is not corre-lated with the presence of E. amylovora, although degener-ation on control blossoms might be evident only later (23).One day after application of nonpathogenic strain L796, a

few scattered bacterial cells were seen on papillae (Fig. 3F).Bacteria were even more difficult to find after 2 days. Nonewere seen in the intercellular spaces of the style. Bacteriawere not seen on any part of control blossoms sprayed withwater.

In view of the ability of saprophytic Erwinia herbicola toexploit this niche (4), we are unsure why nonpathogenic P.syringae pv. syringae L796 failed to multiply on the stig-matic surface. Klement and Goodman (8) suggested thatavirulent bacteria elicit a hypersensitive reaction in appletissue. However, strain L796 does not cause a hypersensi-tive reaction in tobacco leaf tissue (E. L. Mansvelt, unpub-lished data), and we found no evidence of tissue collapse,associated with the reaction, on apple blossoms.We found no proof that P. syringae pv. syringae invaded

nectariferous tissue of the hypanthium of apple blossoms asit does on pear blossoms (12). The tight circular arrangementof the stamens and abundant stylar trichomes associatedwith apple blossoms might have prevented bacterial cells

from contacting the surface of this tissue. Under fieldconditions, E. amylovora is washed by rainwater fromstigmas to hypanthia, where infection occurs (23), but we donot know if this applies to P. syringae pv. syringae. An earlyreport (20) suggested that E. amylovora breaches the thinwalls of papillae and spreads intercellularly to the recepta-cle. We found that intercellular spread of P. syringae pv.syringae from stigmas downward was limited to the periph-ery of the style. The core cells of the style are smaller andmore compact, and the minute intercellular spaces appar-ently restrict bacterial migration in this region. The presenceof bacteria on the surface of the groove of the conduplicatestyle indicates that the pathogen might have been extrudedfrom heavily colonized underlying tissue.

In conclusion, our investigation shows that large popula-tions of pathogenic P. syringae pv. syringae can be associ-ated with macroscopically symptomless apple leaves andblossoms. Pathogenic strains of P. syringae pv. syringaegain entry into Oregon Spur apple trees through stomata onleaves and stigmas on blossoms. The subsequent presence oflarge numbers of bacteria inside substomatal cavities and onstigmas suggests that these niches are preferred sites ofcolonization on apple trees. Occupation of protected sitesmight explain how bacterial populations survive dry, ad-verse conditions on leaf surfaces (5), why large proportionsof resident populations of P. syringae pv. syringae ondeciduous fruit trees are not killed by copper sprays (25),and why leaves carrying these populations cannot be surfacedisinfested (15).

We thank C. R. Swart for assistance with scanning electronmicroscopy and P. S. Knox-Davies for reading the manuscript.

LITERATURE CITED1. Bashan, Y., E. Sharon, Y. Okon, and Y. Henis. 1981. Scanning

electron and light microscopy of infection and symptom devel-opment in tomato leaves infected with Pseudomonas tomato.Physiol. Plant Pathol. 19:139-144.

2. Gitaitis, R. D., D. A. Samuelson, and J. 0. Strandberg. 1981.Scanning electron microscopy of the ingress and establishmentof Pseudomonas alboprecipitans in sweet corn leaves. Phyto-pathology 71:171-175.

3. Haas, J. H., and J. Rotem. 1976. Pseudomonas lachrymansadsorption, survival, and infectivity following precision inocu-lation of leaves. Phytopathology 66:992-997.

4. Hattingh, M. J., S. V. Beer, and E. W. Lawson. 1986. Scanningelectron microscopy of apple blossoms colonized by Erwiniaamylovora and E. herbicola. Phytopathology 76:900-904.

5. Hirano, S. S., and C. D. Upper. 1983. Ecology and epidemiologyof foliar bacterial plant pathogens. Annu. Rev. Phytopathol.21:243-269.

6. Huang, J.-S. 1986. Ultrastructure of bacterial penetration inplants. Annu. Rev. Phytopathol. 24:141-157.

7. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two simplemedia for the demonstration of pyocyanin and fluorescein. J.Lab. Clin. Med. 44:301-307.

8. Klement, Z., and R. N. Goodman. 1966. Hypersensitive reactioninduced in apple shoots by an avirulent form of Erwinia amylo-vora. Acta Phytopathol. Acad. Sci. Hung. 1:177-184.

9. Mansvelt, E. L., and M. J. Hattingh. 1986. Bacterial blister barkand blight of fruit spurs of apple in South Africa caused byPseudomonas syringae pv. syringae. Plant Dis. 70:403-405.

10. Mansvelt, E. L., and M. J. Hattingh. 1986. Pear blossom blast inSouth Africa caused by Pseudomonas syringae pv. syringae.Plant Pathol. 35:337-343.

11. Mansvelt, E. L., and M. J. Hattingh. 1987. Scanning electronmicroscopy of colonization of pear leaves by Pseudomonassyringae pv. syringae. Can. J. Bot. 65:2517-2522.

12. Mansvelt, E. L., and M. J. Hattingh. 1987. Scanning electronmicroscopy of pear blossom invasion by Pseudomonas syringae

NOTES 537

on May 24, 2018 by guest

http://aem.asm

.org/D

ownloaded from

538 NOTES APPL. ENVIRON. MICROBIOL.

pv. syringae. Can. J. Bot. 65:2523-2529.13. Mansvelt, E. L., and M. J. Hattingh. 1988. Resident populations

ofPseudomonas syringae pv. syringae on leaves, blossoms, andfruits of apple and pear trees. J. Phytopathol. 121:135-142.

14. Mew, T. W., I. C. Mew, and J. S. Huang. 1984. Scanningelectron microscopy of virulent and avirulent strains of Xantho-monas campestris pv. oryzae on rice leaves. Phytopathology74:635-641.

15. Miles, W. G., R. H. Daines, and J. W. Rue. 1977. Presympto-matic egress ofXanthomonas pruni from infected peach leaves.Phytopathology 67:895-897.

16. Roos, I. M. M., and M. J. Hattingh. 1983. Scanning electronmicroscopy of Pseudomonas syringae pv. morsprunorum onsweet cherry leaves. Phytopathol. Z. 108:18-25.

17. Roos, I. M. M., and M. J. Hattingh. 1987. Pathogenicity andnumerical analysis of phenotypic features of Pseudomonassyringae strains isolated from deciduous fruit trees. Phytopa-thology 77:900-908.

18. Roos, I. M. M., and M. J. Hattingh. 1987. Systemic invasion ofcherry leaves and petioles by Pseudomonas syringae pv. mor-

sprunorum. Phytopathology 77:1246-1252.

19. Roos, I. M. M., and M. J. Hattingh. 1987. Systemic invasion ofplum leaves and shoots by Pseudomonas syringae pv. syringaeintroduced into petioles. Phytopathology 77:1253-1257.

20. Rosen, H. R. 1936. Mode of penetration and of progressiveinvasion of fire-blight bacteria into apple and pear blossoms.Arkansas Agric. Exp. Stn. Bull. 331.

21. Schneider, R. W., and R. G. Grogan. 1977. Tomato leaf tri-chomes, a habitat for resident populations of Pseudomonastomato. Phytopathology 67:898-902.

22. Sigee, D. C., and A. N. Al-Issa. The hypersensitive reaction intobacco leaf tissue infiltrated with Pseudomonas pisi. 4. Scan-ning electron microscope studies on fractured leaf tissue. Phy-topathol. Z. 106:1-15.

23. Thomson, S. V. 1986. The role of the stigma in fire blightinfections. Phytopathology 76:476-482.

24. Yamanaka, S., M. Ozaki, and S. Kato. 1980. Some observationson angular leaf spot of cucumber with a scanning electronmicroscope. Tohoku J. Agric. Res. 30:135-141.

25. Young, J. M. 1978. Survival of bacteria on Prunus leaves. PlantPathog. Bact. 4:779-786.

on May 24, 2018 by guest

http://aem.asm

.org/D

ownloaded from