J. Cell Sci. 18, 123-132 (1975) 123

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ADHESION OF PHYTOPHTHORA PALMIVORA

ZOOSPORES: ELECTRON MICROSCOPY OF

CELL ATTACHMENT AND CYST WALL

FIBRIL FORMATION

V. O. SING AND S. BARTNICKI-GARCIADepartment of Plant Pathology, University of California,Riverside, California, 92502, U.S.A.

SUMMARY

Zoospores of Phytophthora palmivora adhered to a plastic film surface were examined byelectron microscopy. Three stages of adhesion were compared: (1) non-adhesive, unencystedzoospores, (2) adhered incipient cysts, and (3) adhered mature cysts. Thin sections of incipientcysts revealed cells attached to the film surface through the partially discharged contents of theso-called peripheral vesicles; this seems to be the first step in cell adhesion. In mature cysts, theadhesive appeared to have been compacted into an electron-dense deposit binding the cystwall to the plastic surface. The adhesion zone was also examined in face view after lysingattached incipient cysts with sodium dodecyl sulphate. Cyst wall microfibrils were seen togetherwith an amorphous substance (presumably the adhesive material). The microfibrils were invarious stages of formation. Seemingly, adhesion and microfibril formation take place con-currently. The possibility was considered that the material contained in the peripheral vesiclesserves in both cell adhesion and microfibril elaboration.

INTRODUCTION

After a period of active swimming, zoospores of Phytophthora palmivora becomesluggish, settle down and encyst. Zoospores have a tendency to become firmlyattached to any substratum on to which they settle. The adhesive phase is brief; ittakes place during the early stages of encystment, before a recognizable (alkali-resistant) cyst wall is formed (Sing & Bartnicki-Garcia, 1972). Thus, if a zoosporeencysts before reaching a solid surface, it will probably not adhere to it.

This communication is part of a series dealing with cytological, physiological, andbiochemical aspects of adhesion of zoospores of P. palmivora to living or inanimatesurfaces. Presently, we describe ultrastructural details of the adhesion phenomenonand the genesis of the cyst wall. Both events appear to be intimately related.

MATERIALS AND METHODS

Zoospores of Phytophthora palmivora, isolate Pi 13, were obtained as previously described(Tokunaga & Bartnicki-Garcia, 1971a).

Electron microscopy of spores attached to plastic surfaces. Samples (5 ml) of a swimmingzoospore suspension, at room temperature, were transferred to 2 precooled 30-ml beakers heldat 1 °C. Several thin plastic disks of Epon 812 had been placed at the bottom of the beakers.

124 V• O- ^"W and S. Bartnicki-Garcia

After 20 min of incubation, one sample was fixed immediately with 5 ml of 2 % glutaraldehydein o-i M sodium cacodylate buffer, pH 7-2; under these conditions, most zoospores were ableto initiate the processes of encystment and adhesion but did not develop further (incipientcysts). The other sample was incubated at room temperature for an additional 30 min - to allowzoospores to complete their encystment and adhesion - and then fixed with glutaraldehyde for1 h (mature cysts). The plastic disks with the attached cells were washed with cacodylate buffer,postfixed with 1 % osmium tetroxide in cacodylate buffer for 1 h, washed, dehydrated with anethanol series and embedded in Epon 812. Thin sections were prepared and stained withuranyl acetate and lead citrate. The specimens were viewed in a Hitachi HU-12 electronmicroscope.

Samples of unencysted, unattached zoospores were fixed with glutaraldehyde, centrifugedand prepared for electron microscopy by a similar procedure.

Electron microscopy of fibrillar cyst wall formation. Zoospores were allowed to adhere toelectron-microscope grids (coated with Formvar film) placed at the bottom of a precooledbeaker as described above. After 20 min incubation, the attached incipient cysts were lysedwith 1 % sodium dodecyl sulphate (SDS) and washed extensively with a jet of distilled water.The grids were dried and shadow cast with palladium at a 190 angle. Mature cyst walls wereisolated by sonication (Tokunaga & Bartnicki-Garcia, 19716) and shadow cast as above.

RESULTS

Thin sections of zoospores and attached cysts

Fig. 1 shows the typical appearance of an unencysted zoospore of Phytophthorapalmivora. This appearance is closely similar to that described previously for thisorganism (Hemmes & Hohl, 1971), other species of Phytophthora (Ho, Zachariah &Hickman, 1968; Reichle, 1969a) and Pythium (Grove, 1971). The plasmalemma is theoutermost boundary of the cell. Conspicuous under the plasmalemma are numerousspheroidal vesicles with a homogeneous finely granular content. These vesicles havebeen called 'peripheral' vesicles (Grove, 1971), vesicles with 'granular to fibrous' con-tent (Reichle, 1969a), or 'fibrillar' vacuoles (Hemmes & Hohl, 1971). In addition,there are also many flattened vesicles, with mostly electron-transparent contents, inclose proximity to the plasmalemma.

Fig. 2 shows an incipient cyst beginning to adhere to an inanimate surface. The cellhas lost most of its peripheral and flattened vesicles, but has not yet rounded off. Theprominent groove of the zoospore is still conspicuous. The incipient cyst lacks a welldefined cell wall and shows only a fine fuzz (perhaps nascent wall) covering theplasmalemma surface (Fig. 3). In these fortuitous sections, we can see that the cell isanchored to the plastic surface through the partially secreted contents of a peripheralvesicle (Figs. 2, 3).

Fig. 4 shows a mature cyst attached to a solid surface. The cell has completelyrounded off and a thin, electron-transparent wall is evident. Essentially all the peri-pheral and flattened vesicles have disappeared by this time. Electron-dense cementingmaterial can be seen in the area between the cyst wall and the plastic surface. Thezone of attachment is rather small; sections which failed to cut through this zone werecommonly found. They showed a cell 'suspended' above the plastic film with emptyspace in between.

Zoospore adhesion and wall formation 125

Face view of the adhesion zone

Zoospores were allowed to attach themselves to electron-microscope grids, treatedwith ionic detergent to lyse the cells, mainly incipient cysts, washed extensively toeliminate loose material and shadow cast. Figs. 5-7 show the materials that remainedattached to the grids. They consist mainly of microfibrils with some amorphousmaterial. From the overall appearance, it was concluded that in most instances eachdeposit corresponded to the remnants of an individual cyst. The appearance of theseremnants varied considerably, reflecting different stages of cyst wall formation(Figs. 5-7); some contained only short microfibrils while others were meshworks oflong microfibrils, interwoven into loose or tight fabrics. For comparison, the appear-ance of a broken fragment of a mature cyst wall isolated by sonication (Tokunaga &Bartnicki-Garcia, 1971 A) is shown in Fig. 8.

DISCUSSION

By a mild cold shock, zoospore populations were induced to adhere to plastic disksplaced at the bottom of a small beaker. This cold treatment also triggered the onset ofencystment. Both processes, encystment and adhesion, are intimately related (seebelow). Seemingly, the gradual drop in temperature caused the zoospores to (a)settle down on to the plastic disks, (b) become adhesive and (c) begin wall formation.

In samples kept at low temperature, the cells remained attached to the plastic disksbut did not complete their encystment (incipient cysts). Presumably the enzymesresponsible for wall synthesis were slowed down and wall formation ceased pre-maturely. In such samples, we found cyst walls in widely different stages of develop-ment (Figs. 5-7). Most of the cells that were brought back to room temperature afterthe cold shock resumed and completed the synthesis of a cyst wall and remainedfirmly adhered to the plastic surface (mature cysts).

Our present results on the fine structure of incipient and mature cysts of P. palmi-vora attached to plastic films agree with the previous conclusion that zoosporesadhere to surfaces early in the encystment process (Sing & Bartnicki-Garcia, 1972),i.e. before an alkali-resistant cyst wall is formed. They also show that adhesion isprobably established through the newly discharged contents of the peripheral vesicles.These vesicles occur abundantly next to the cell surface and disappear early duringencystment by fusing with the plasmalemma and discharging their contents to theoutside. This secretory process was described earlier by Hemmes & Hohl (1971) forP.palmivora and by Grove (1971) for Pythium aphanidermatum. The secreted materialforms an irregular amorphous coat on the cyst surface (Sing & Bartnicki-Garcia,1975 a). Apparently, if the zoospore is in contact with a solid surface as it initiatesencystment, the peripheral vesicle secretion at the point of contact would cement thecell to the solid surface (Figs. 2, 3).

After the cyst wall is fully developed, the cell remains firmly attached by anelectron-dense substance located between the wall and the plastic surface. The higherelectron density of the adhesive in the mature cyst, as compared with the incipient

126 V. O. Sing and S. Bartnicki-Garcia

cyst (Figs. 2, 3 vs 4), could be largely due to compaction of the adhesive materialoriginally discharged by the peripheral vesicles.

To obtain face views of the adhesion zone we took advantage of two distinct actionsof SDS on encysting zoospores. First, this detergent causes complete cell disintegra-tion of incipient cysts of P. palmivora (mature cysts are also lysed by SDS but thewall prevents complete dispersal of cell particles) (S. Bartnicki-Garcia, unpublished).Secondly, SDS cannot detach adhered cysts of P. palmivora, probably because thecement is unaffected by the detergent (Sing & Bartnicki-Garcia, 19756). It followsthen that SDS treatment of incipient cysts adhered to a film surface would remove allcell components, leaving behind only the cementing material attached to the filmsurface together with any other cell structures associated with the adhesive. Thelatter proved to be microfibrils in various stages of formation.

The zoospore of P. palmivora lacks preformed microfibrils; these must be assembledat the cell surface during encystment (Bartnicki-Garcia, 1973). It has been proposedthat peripheral vesicles (fibrillar vacuoles) play a role in cyst wall elaboration (Hemmes& Hohl, 1971; Grove, 1971). Conceivably these vesicles and/or the flattened vesiclesmay deliver enzymes and/or precursors for microfibril synthesis. We have evidencethat the peripheral vesicles contain a glycoprotein with receptor sites for concana-valin A; the former substance is probably involved in adhesion (Sing & Bartnicki-Garcia, 1975 a). We have therefore conjectured that the materials discharged by thesevesicles may serve for both microfibril synthesis and cell adhesion. Conceivably, themain role of the adhesive glycoprotein would be that of a matrix for wall neogenesis,binding nascent microfibrils to the cell surface and to one another. Additionally, theadhesive would serve to anchor the cell to any external surface with which the zoosporemight be in contact at the time of encystment.

Our present findings together with other recent electron-microscopic studies(Hemmes & Hohl, 1971; Grove, 1971; Tokunaga & Bartnicki-Garcia, 19716) permita deeper understanding of the process of encystment of an oomycetous zoospore. Theyindicate the crucial role played by preformed vesicles in wall formation and develop-ment of adhesiveness - two key events in encystment. The classical description ofevents in zoospore encystment (Waterhouse, 1962; Hickman, 1970) may thus be up-dated to include~the following: (1) cessation of motility; (2) loss of flagella either byretraction or shedding (see Reichle, 19696); (3) discharge of peripheral and flattenedvesicles and formation of an amorphous cyst wall coat; (4) acquisition of adhesiveness;(5) microfibril synthesis and development of an alkali-resistant cyst wall; and (6)change from pyriform to spherical shape (see Desjardins, Wang & Bartnicki-Garcia,1973)-

This investigation was supported in part by a research grant from the National ScienceFoundation and by a predoctoral fellowship from the University of California to V. O. Sing.We are much indebted to D. E. Hemmes and W. Van Der Woude for their valuable advice.

Zoospore adhesion and wall formation 127

REFERENCES

BARTNICKI-GARCIA, S. (1973). Cell wall genesis in a natural protoplast: the zoospore of Phytoph-tliora palmivora. Jn Yeast, Mould and Plant Protoplasts (ed. J. R. Villanueva, I. Garcia-Acha,S. Gascon & F. Uruburu), pp. 77-91. London: Academic Press.

DESJARDINS, P. R., WANG, M. C. & BARTNICKI-GARCIA, S. (1973). Electron microscopy ofzoospores and cysts of Phytophtliora palmivora: morphology and surface texture. Arch.Mikrobiol. 88, 61-70.

GROVE, S. N. (1971). Protoplasmic Correlates of Hyfilial Tip Initiation and Development in Fungi.Ph.D. Dissertation, Purdue University.

HEMMES, D. E. & HOHL, H. R. (1971). Ultrastructural aspects of encystation and cyst germina-tion in Phytophtliora parasitica. J. Cell Sci. 9, 175-191.

HICKMAN, C. J. (1970). Biology of Phytophtliora zoospores. Phytopathology 60, 1128-1135.Ho, H. H., ZACHARIAH, K. & HICKMAN, C. J. (1968). The ultrastructure of zoospores of

Phytophtliora megasperma var. sojae. Can.J. Bot. 46, 37-41.REICHLE, R. E. (1969a). Fine structure of Phytophtliora parasitica zoospores. Mycologia 61,

3O-5 '•REICHLE, R. E. (19696). Retraction of nagella of Phytophtliora parasitica var. nicotiana zoospores.

Arch. Mikrobiol. 66, 340-347.SING, V. O. & BARTNICKI-GARCIA, S. (1972). Adhesion of zoospores of Phytophthora palmivora

to solid surfaces. Phytopathology 62, 790.SING, V. O. & BARTNICKI-GARCIA, S. (19750). Adhesion of zoospores of Phytophthora palmivora.

Detection and ultrastructural visualization of concanavalin A receptor sites appearing duringencystment. J. Cell Sci. (in press.)

SlNC, V. O. & BARTNICKI-GARCIA, S. (:975ft). Adhesion of Phytophthora palmivora zoospores:detachment by enzymic and chemical treatments. J. gen. Microbiol. (Submitted for publica-tion.)

TOKUNACA, J. & BARTNICKI-GARCIA, S. (1971a). Cyst wall formation and endogenous carbo-hydrate utilization during synchronous encystment of Phytophthora palmivora zoospores.Arch. Mikrobiol. 79, 283-292.

TOKUNACA, J. & BARTNICKI-GARCIA, S. (19716). Structure and differentiation of the cell wall ofPhytophthora palmivora: cysts, hyphae and sporangia. Arch. Mikrobiol. 79, 293-310.

WATERHOUSE, G. M. (1962). The zoospore. Trans. Br. mycol. Soc. 45, 1-20.

(Received 29 November 1974)

128 V. O. Sing and S. Bartnicki-Garcia

pm

Fig. i. Cross-section of an. unencysted zoospore of Phytophtiiora palmivora. fp,finger-print vacuoles; fv, flattened vesicles; /, lipid-like body; m, mitochondria;n, nucleus; pm, plasma membrane; pv, peripheral vesicles, x 18500.

Zoospore adhesion and wall formation 129

Figs. 2, 3. Cross-sections of an encysting zoospore (incipient cyst) as it initiatesadhesion to a plastic surface.

Fig. 2. Overall view of the zoospore anchored to a plastic surface through the newlydischarged contents of a peripheral vesicle (pv). The encysting zoospore has not yetrounded off, and a prominent groove (g) is still present, x, microbody-like structureswith electron-dense cortex and core. Other symbols as in Fig. 1. x 16000.

Fig. 3. Details of the adhesion area in a different serial section of the same zoospore.x 37000.

Q C E L I S

130 V. 0. Sing and S. Bartnicki-Garcia

pm

cm

Fig. 4. Cross-section of a mature cyst adhered to a plastic surface. A thin, electron-transparent cyst wall (ca. 60 nm thick) covers the cell but it is not visible on thisreproduction. Electron-dense adhesive material (cm) cements the cyst to the plasticsurface. Other symbols as in Fig. 1. x 16000.

Zoospore adhesion and wall formation

Figs, s, 6. Face views of the adhesion zone showing early stages in cyst wall assembly.From a sample of incipient cysts of P. palmivora lysed in situ with i % SDS and shadowcast, x 32000.

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V. O. Sing and S. Bartnicki-Garcia

Fig. 7. A more advanced stage in cyst wall .assembly"(cf. Figs. 5, 6). x 32000.Fig. 8. Fragment of a fully assembled cyst wall of P. palmivora prepared by soni-cation. x 32000.