Blossoms by Pseudomonas syringae pv. syringae

Size: px

Start display at page:

Download "Blossoms by Pseudomonas syringae pv. syringae"

Opal Floyd
6 years ago
Views:

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1989, p. 533-538 0099-2240/89/020533-06$02.00/0 Copyright ) 1989, American Society for Microbiology Vol. 55, No.

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1989, p /89/ $02.00/0 Copyright ) 1989, American Society for Microbiology Vol. 55, No. 2 Scanning Electron Microscopy of Invasion of Apple Leaves and Blossoms 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 through stomata and multiplied in the substomatal chambers. Strain L195 applied to blossoms colonized stigmas and also occurred in intercellular spaces of styles. Nonpathogenic strain L796 failed to colonize blossoms. This study suggests that inoculum of pathogenic P. syringae pv. syringae builds up on apple leaves and blossoms. Pseudomonas syringae pv. syringae van Hall causes bacterial blister bark of apple (Maluis domestica Borkh.) in South Africa (9). The pathogen frequently multiplies on apple leaves and blossoms during the growing season without causing lesions (13). However, it is uncertain where on these plant surfaces P. syringae pv. syringae multiplies and if underlying tissue is invaded. Scanning electron microscopy of other host-pathogen systems has shown that populations of bacteria on leaves colonize substomatal cavities (1, 2, 6, 14-16, 18, 19, 24, 25) or trichomes (3, 11, 21), whereas populations on blossoms occur on stigmas (4, 6, 12, 20, 23) and the hypanthium (6, 12, 20). This paper reports the ecological niche of P. syringae pv. syringae on apple leaves and 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 L795 are pathogenic on shoots of apple, cherry, pear, and plum, whereas L796 is nonpathogenic. Stock cultures were maintained at room temperature on slants of nutrient agar (Difco Laboratories, Detroit, Mich.) supplemented with 20% glycerol. Inoculum suspensions of the strains were prepared from cultures grown overnight on King medium B (7) at 26 C and suspended in sterile distilled water to 106 CFU/ml as previously described (11). Concentrations were confirmed by dilution plating. Potted Oregon Spur apple trees were used. Bacterial suspensions were applied to leaves or blossoms by gentle spraying with an atomizer until runoff. Controls were sprayed with sterile distilled water. Leaves on vigorous shoots (20 cm) on 1-year-old trees were treated with P. syringae pv. syringae L795. Individual shoots were covered with moist plastic bags for 24 h before leaves were sprayed. Trees were then left uncovered in a greenhouse kept at approximately 24 C. Blossom inoculations were performed on 3-year-old trees that were removed from cold storage and allowed to break dormancy in the greenhouse. Separate flower clusters in full bloom were sprayed with P. syringae pv. 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 were sampled after 1 and 2 days. Petals of blossoms were removed and discarded, and remaining floral parts were separated by cutting off stamens and filaments at the sites of attachment with a sterile blade (12). Sections were fixed in a 5% glutaraldehyde solution, dehydrated in an alcohol series, and * Corresponding author. 533 dried in a critical point drier under CO2 (11). Samples were mounted on stainless steel stubs. Some leaf samples were fractured through mesophyll tissue in the plane of the leaf surface as described by Sigee and Al-Issa (22). Segments of floral parts or longitudinal sections cut with a new razor blade were mounted. Specimens were gold-coated and examined with an ISI 10-nm scanning electron microscope (International Scientific Instruments, Santa Clara, Calif.) at 15 kv. Leaves. Forty-five minutes after application of pathogenic strain L795, bacterial cells were randomly dispersed over the leaf surface (Fig. 1A). After 3 days, small aggregates occurred near or over stomata. In fractured tissue, bacteria were also seen inside the substomatal chamber, where they had 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 within the substomatal cavity were embedded in a dense layer (Fig. 1D). Few bacteria were seen in the intercellular spaces of the spongy parenchyma of the mesophyll. Bacteria were otherwise unevenly distributed over the leaf surface and occurred sparsely at the base of trichomes (Fig. 1E). However, P. syringae pv. syringae L795 failed to invade trichomes on apple leaves, although the same strain is capable of colonizing trichomes on pear leaves extensively (11). We assume that substomatal chambers were directly colonized by P. syringae pv. syringae entering through stomata. The pathogen might also have reached some chambers after having moved from colonized pockets through the intercellular spaces of the spongy parenchyma. However, intercellular spread appears to have been fairly restricted, and bacteria were not seen beyond the outer cell layers of the spongy parenchyma. In contrast, P. syringae pv. morsprunorum entering stomata on cherry leaves spreads from the mesophyll, invades the xylem of minor veins, and then migrates to other regions in the leaf blade and petiole (17). Furthermore, P. syringae pv. syringae introduced into petioles 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 substomatal chamber were embedded in similar material (Fig. 2A). Aggregates of bacteria were sparsely dispersed in the intercellular spaces of the spongy parenchyma (Fig. 2B). Twelve days after spraying, only a few bacterial cells could be distinguished on the leaf surface or associated with fibrillar material present on stomata or in the substomatal chamber.

2 534 NOTES APPL. ENVIRON. MICROBIOL. FIG. 1. Scanning electron micrographs of apple leaves treated with pathogenic strain L795 of P. syringae pv. syringae. (A) Bacteria on abaxial 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), and single cells are present at the base of a trichome (E). (F) Fibrillar material on stoma after 9 days. Bars, 5 p.m.

VOL. 55, 1989 NOTES 535 FIG. 2. Scanning electron micrographs of apple leaves and blossoms treated, respectively, with pathogenic strains L795 and L195 of P. syringae

3 VOL. 55, 1989 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) Bacterial cells on stigmatic papillae and on surfaces of underlying cells (arrow). (D) Invaded papilla. Note the absence of bacteria from intercellular spaces. (E) Bacteria at penetration site of pollen tube (arrow). (F) Mass of bacterial cells on papillae 2 days after application. Bars, 5,um.

4 536 NOTES 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 applying nonpathogenic strain L796 (F) of P. syringae pv. syringae. (A) Bacteria (arrows) within collapsed regions of the cuticular layer of stigmatic cells beneath the protruding papillae. (B) Longitudinal section showing bacteria in the intercellular spaces of stigmatic tissue. (C to E) Bacteria associated with stylar tissue. Masses of bacteria in intercellular spaces (C) were confined to the outer cell layers of the style (D) but also occurred externally on the surface of the longitudinal groove of the conduplicate style (E). (F) Single bacterial cells on surface of papillae. Bars, 5 ~tm.

VOL. 55, 1989 Bacteria were not seen on the surfaces of control leaves sprayed with water or in cross sections or longitudinal sections of these leaves.

5 VOL. 55, 1989 Bacteria were not seen on the surfaces of control leaves sprayed with water or in cross sections or longitudinal sections of these leaves. No lesions developed on leaves sprayed with the bacterial suspension. P. syringae pv. syringae is not known to cause lesions on apple leaves. Nevertheless, the abundant presence of the pathogen in substomatal chambers and in intercellular spaces of the spongy parenchyma of the mesophyll has epidemiological implications. Apart from being sheltered in leaf tissue, P. syringae pv. syringae was also extruded through stomata after having multiplied extensively in substomatal chambers. Inoculum in orchards might be replenished in this way. Blossoms. One day after application of pathogenic strain L195, bacteria were noted on the surfaces of papillae and underlying cells of the stigma (Fig. 2C) but were not seen within tissues (Fig. 2D) or on other blossom parts. Bacteria were occasionally found where a pollen germ tube had entered 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 collapsed regions in the cuticular layer of underlying cells (Fig. 3A). Longitudinal sections revealed large numbers of bacteria in the intercellular spaces of stigmatic tissue (Fig. 3B) and stylar tissue (Fig. 3C). Bacteria within styles appeared to have been confined to the intercellular spaces of cells immediately below the epidermis (Fig. 3D). Bacteria were also present in the longitudinal groove of the conduplicate style (Fig. 3E). Failure of pathogenic P. syringae pv. syringae to cause necrosis of apple blossoms agrees with the results obtained with treated leaves of this host. Stigmatic papillae were preferentially colonized, and the pathogen spread intercellularly through the style. In contrast, P. syringae pv. syringae colonizing stigmas of pear blossoms causes blast (12). Erwinia amylovora brings about blossom necrosis of different ponp 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, discoloration and browning of stigmas associated with the natural degeneration that follows anthesis might have masked expression of disease symptoms. Likewise, the physical appearance of stigmas and styles in orchards is not correlated with the presence of E. amylovora, although degeneration 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. None were seen in the intercellular spaces of the style. Bacteria were not seen on any part of control blossoms sprayed with water. In view of the ability of saprophytic Erwinia herbicola to exploit this niche (4), we are unsure why nonpathogenic P. syringae pv. syringae L796 failed to multiply on the stigmatic surface. Klement and Goodman (8) suggested that avirulent bacteria elicit a hypersensitive reaction in apple tissue. However, strain L796 does not cause a hypersensitive reaction in tobacco leaf tissue (E. L. Mansvelt, unpublished 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 as it does on pear blossoms (12). The tight circular arrangement of the stamens and abundant stylar trichomes associated with apple blossoms might have prevented bacterial cells NOTES 537 from contacting the surface of this tissue. Under field conditions, E. amylovora is washed by rainwater from stigmas to hypanthia, where infection occurs (23), but we do not know if this applies to P. syringae pv. syringae. An early report (20) suggested that E. amylovora breaches the thin walls of papillae and spreads intercellularly to the receptacle. We found that intercellular spread of P. syringae pv. syringae from stigmas downward was limited to the periphery of the style. The core cells of the style are smaller and more compact, and the minute intercellular spaces apparently restrict bacterial migration in this region. The presence of bacteria on the surface of the groove of the conduplicate style indicates that the pathogen might have been extruded from heavily colonized underlying tissue. In conclusion, our investigation shows that large populations of pathogenic P. syringae pv. syringae can be associated with macroscopically symptomless apple leaves and blossoms. Pathogenic strains of P. syringae pv. syringae gain entry into Oregon Spur apple trees through stomata on leaves and stigmas on blossoms. The subsequent presence of large numbers of bacteria inside substomatal cavities and on stigmas suggests that these niches are preferred sites of colonization on apple trees. Occupation of protected sites might explain how bacterial populations survive dry, adverse conditions on leaf surfaces (5), why large proportions of resident populations of P. syringae pv. syringae on deciduous fruit trees are not killed by copper sprays (25), and why leaves carrying these populations cannot be surface disinfested (15). We thank C. R. Swart for assistance with scanning electron microscopy and P. S. Knox-Davies for reading the manuscript. LITERATURE CITED 1. Bashan, Y., E. Sharon, Y. Okon, and Y. Henis Scanning electron and light microscopy of infection and symptom development in tomato leaves infected with Pseudomonas tomato. Physiol. Plant Pathol. 19: Gitaitis, R. D., D. A. Samuelson, and J. 0. Strandberg Scanning electron microscopy of the ingress and establishment of Pseudomonas alboprecipitans in sweet corn leaves. Phytopathology 71: Haas, J. H., and J. Rotem Pseudomonas lachrymans adsorption, survival, and infectivity following precision inoculation of leaves. Phytopathology 66: Hattingh, M. J., S. V. Beer, and E. W. Lawson Scanning electron microscopy of apple blossoms colonized by Erwinia amylovora and E. herbicola. Phytopathology 76: Hirano, S. S., and C. D. Upper Ecology and epidemiology of foliar bacterial plant pathogens. Annu. Rev. Phytopathol. 21: Huang, J.-S Ultrastructure of bacterial penetration in plants. Annu. Rev. Phytopathol. 24: King, E. O., M. K. Ward, and D. E. Raney Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44: Klement, Z., and R. N. Goodman Hypersensitive reaction induced in apple shoots by an avirulent form of Erwinia amylovora. Acta Phytopathol. Acad. Sci. Hung. 1: Mansvelt, E. L., and M. J. Hattingh Bacterial blister bark and blight of fruit spurs of apple in South Africa caused by Pseudomonas syringae pv. syringae. Plant Dis. 70: Mansvelt, E. L., and M. J. Hattingh Pear blossom blast in South Africa caused by Pseudomonas syringae pv. syringae. Plant Pathol. 35: Mansvelt, E. L., and M. J. Hattingh Scanning electron microscopy of colonization of pear leaves by Pseudomonas syringae pv. syringae. Can. J. Bot. 65: Mansvelt, E. L., and M. J. Hattingh Scanning electron microscopy of pear blossom invasion by Pseudomonas syringae

538 NOTES APPL. ENVIRON. MICROBIOL. pv. syringae. Can. J. Bot. 65:2523-2529. 13. Mansvelt, E. L., and M. J. Hattingh. 1988. Resident populations of Pseudomonas syringae pv.

6 538 NOTES APPL. ENVIRON. MICROBIOL. pv. syringae. Can. J. Bot. 65: Mansvelt, E. L., and M. J. Hattingh Resident populations of Pseudomonas syringae pv. syringae on leaves, blossoms, and fruits of apple and pear trees. J. Phytopathol. 121: Mew, T. W., I. C. Mew, and J. S. Huang Scanning electron microscopy of virulent and avirulent strains of Xanthomonas campestris pv. oryzae on rice leaves. Phytopathology 74: Miles, W. G., R. H. Daines, and J. W. Rue Presymptomatic egress of Xanthomonas pruni from infected peach leaves. Phytopathology 67: Roos, I. M. M., and M. J. Hattingh Scanning electron microscopy of Pseudomonas syringae pv. morsprunorum on sweet cherry leaves. Phytopathol. Z. 108: Roos, I. M. M., and M. J. Hattingh Pathogenicity and numerical analysis of phenotypic features of Pseudomonas syringae strains isolated from deciduous fruit trees. Phytopathology 77: Roos, I. M. M., and M. J. Hattingh Systemic invasion of cherry leaves and petioles by Pseudomonas syringae pv. morsprunorum. Phytopathology 77: Roos, I. M. M., and M. J. Hattingh Systemic invasion of plum leaves and shoots by Pseudomonas syringae pv. syringae introduced into petioles. Phytopathology 77: Rosen, H. R Mode of penetration and of progressive invasion of fire-blight bacteria into apple and pear blossoms. Arkansas Agric. Exp. Stn. Bull Schneider, R. W., and R. G. Grogan Tomato leaf trichomes, a habitat for resident populations of Pseudomonas tomato. Phytopathology 67: Sigee, D. C., and A. N. Al-Issa. The hypersensitive reaction in tobacco leaf tissue infiltrated with Pseudomonas pisi. 4. Scanning electron microscope studies on fractured leaf tissue. Phytopathol. Z. 106: Thomson, S. V The role of the stigma in fire blight infections. Phytopathology 76: Yamanaka, S., M. Ozaki, and S. Kato Some observations on angular leaf spot of cucumber with a scanning electron microscope. Tohoku J. Agric. Res. 30: Young, J. M Survival of bacteria on Prunus leaves. Plant Pathog. Bact. 4: Downloaded from on June 14, 2018 by guest

Systemic Invasion of Plum Leaves and Shoots by Pseudomonas syringae pv. syringae Introduced into Petioles

Ecology and Epidemiology Systemic Invasion of Plum Leaves and Shoots by Pseudomonas syringae pv syringae Introduced into Petioles Isabel M M Roos and M J Hattingh Fruit and Fruit Technology Research Institute,