Ultrastructural aspects of encystation and cyst-germination in Phytophthora parasitica

Zoospores released from sporangia of Phytophthora parasitica rapidly encyst in an aqueous medium. The cysts, like those of several other species of Phytophthora (Drechsler, 1930; Ho & Hickman, 1967), may germinate by the production of germ tubes or secondary zoospores analagous to direct and indirect germination in the sporangium (Hohl & Hamamoto, 1967; Hemmes & Hohl, 1969). There have been several recent studies on the ultrastructure of fungal zoospores (Ho, Zachariah & Hickman, 1968; Reichle, 1969) and on hyphal morphogenesis (Girbardt, 1969; Grove, Bracker & Morré, 1970), but none of these deal specifically with the formation and germination of cysts. As it is possible to obtain uniform cultures of encysting and directly or indirectly germinating cysts in P. parasitica, this organism appears to be particularly suited for a study of the aspects of encystation and cyst germination. The results of such a study are presented in this report, with emphasis on the role of dictyosomes and lomasomes in wall formation.

¹ Present address: Cytological laboratory, University of Zurich, Birchstrasse 95, 8050 Zurich, Switzerland.

PhytophthoraparasiticaDost. P 113, used throughout this study, was provided by DrR. B.Hine of the Department of Plant Pathology, University of Hawaii. It represents a single spore isolate and has the advantage that sporangium formation can be induced by exposing the dark-grown mycelium to light (Aragaki & Hine, 1963).

The fungus was grown in darkness at 31 °C on a vegetable-juice agar (10% Campbell Soup Corp. V-8 juice, 0·1 % CaCO₃, 1·5 % agar) in 60 × 15 mm Petri dishes. After 3–4 days the cultures were transferred to room temperature under fluorescent light of approximately 1715 lx (500 ft-c) for 24 h (temperature at level of plates approximately 28 °C).

Petri dishes heavily covered with sporangia were flooded with 5 ml of the desired incubation medium. Submerged sporangia were then brushed loose from the hyphae with a transferring loop and transferred to 60 × 15 mm plastic Petri dishes. The suspensions were incubated for 20 min and then decanted twice in order to separate the zoospores from sporangial cases and ungerminated sporangia. Environmental parameters, such as temperature, pH, age of cultures, and density of germinating sporangia, were considered to obtain the following procedure for cyst germination.

To induce direct cyst germination zoospores were incubated in 5 % unbuffered, aqueous sucrose solution at 28 °C that was aerated by stirring with a magnetic stirring bar. Under these conditions over 90 % of the zoospores encyst and produce germ tubes within 90 min.

To induce indirect cyst germination zoospores were incubated in 10 mM Sorenson’s phos phate buffer solution at pH 7·5. Here too the solution was aerated by stirring with a magnetic stirrer. After encystation germ tubes are not extended until 4·5 h of incubation and average only 6 μm in length. On average, 70–80 % of the cysts germinate indirectly under these conditions and approximately 30 % of the germinating cysts release secondary zoospores over a period of approximately 2h.

Photomicrographs were taken with a Zeiss phase-contrast microscope using Kodak Plus-X Pan sheet film.

Suspensions of encysting zoospores were fixed with an equal volume of 2 % glutaraldehyde in 0·5 M sodium cacodylate buffer at pH 7·2. The fixative was added 0·5 h after zoospores were released from the sporangia, when approximately 50% of the cells had encysted. The suspensions were fixed for 1 h, centrifuged at low speeds, embedded in 1-5% agar, and washed in cacodylate buffer at pH 7·2 for 0·5 h. Postfixation was carried out in 1 % Palade’s osmium tetroxide for 1 h after washing in phosphate buffer. The agar blocks were then stained with °’5 % veronal-acetate buffered uranyl acetate for 1 h.

Suspensions of germinating cysts were fixed in the incubation Petri dish with an equal volume of ice-cold solution containing as final concentration 6·25 % glutaraldehyde, 2 % osmium tetroxide, and 1 % phosphotungstic acid at pH 7·0 (GOP), a fixation-procedure described by Schafer-Danneel (1967). The fixative was added 15 min after induction of excystment in the case of directly germinating cysts and after 4h in the case of indirectly germinating cysts, at the time when germ-tube formation had been initiated. The suspensions were fixed for 05 h, centrifuged at low speeds, embedded in 1·5 % agar, and resuspended in fresh fixative for 0·5 h. Agar blocks were then washed in Sorenson’s phosphate buffer, pH 7·4, for 0·5 h and stained with 0·5 % veronal-acetate buffered uranyl acetate for 1 h.

Dehydration of all specimens was carried out in ethanol and followed by embedding in Epon 812. Thin sections were post-stained in lead citrate (Reynolds, 1963) and viewed in a Hitachi HU-11A electron microscope.

Cultures of sporangia were flooded with 5 % aqueous sucrose or 10 mM Sorenson’s phosphate buffer solution, each containing 10^-3 g/ml of cycloheximide (Upjohn Company), and transferred to 60 × 15 mm plastic Petri dishes. Samples were fixed after 1, 3, and 5 h with an equal volume of GOP fixative. The suspensions were fixed for 1 h and embedded in Epon as described above.

The ultrastructure of Phytophthora parasitica P 113 zoospores is similar to that described for other members of Phytophthora (Ho et al. 1968; Reichle, 1969). Median sections through the cell demonstrate an ordered, consistent arrangement of organelles (see Reichle, 1969, figs. 1 and 3). The pyriform nucleus and associated flagellar apparatus occupy the central portion of the cell and are flanked by several elongated dictyosomes and a water-expulsion vesicle (contractile vacuole). The bulk of the cyto plasm contains membrane-free ribosomes, lipid droplets, and vacuoles containing lamellar inclusions (‘crystalline vacuoles’, Reichle, 1969; liposomes, Williams & Webster, 1970) which remain randomly distributed throughout encystation and germination. At the cell periphery ribosome-free flattened vesicles varying from 0·1 to 0·3 μm in thickness line the plasmalemma and are interspersed with mito chondria, fibrillar vacuoles 0·5–0·8 μm in diameter, and microbody-like organelles (Fig. 1). The microbody-like structures are bullet-shaped, enclosed by a unit mem brane, and consist of a central core of fibres surrounded by one or two cisternae (Fig. 5). These organelles are observed throughout encystation and germination and are again found at the cell periphery in the mature secondary zoospore (Fig. 18).

In the rounded zoospore the flagella are shed with no evidence of retraction of the flagellar axonemes into or around the cyst. The basal bodies, mastigonemes, and associated microtubules remain intact near the nucleus and are interspersed with densely staining vacuoles containing small vesicles and membranous elements in myelin-like configurations. The major ultrastructural change appears to be a decrease in the number of flattened vesicles lining the plasmalemma. In their place is a fibrillar zone of ectoplasm devoid of ribosomes (Fig. 2). At this stage the periphery of the cell is irregular and no wall is detectable.

Once a wall matrix is observed, the cell periphery appears smoother (Fig. 3). Double staining with lead citrate and uranyl acetate reveals a barely stainable wall matrix 0·1–0·15 μm thick which, however, can be clearly stained with 0·5 % barium permanganate. At this stage fibrillar vacuoles and flattened vesicles have disappeared, but fibrillar areas may still be seen near the plasmalemma. The dictyosomes are morpho logically similar to those seen in the zoospore (Fig. 11), i.e. the cisternae are not dis tended and are associated with large numbers of small vesicles averaging 75 nm in diameter. We have not observed a water expulsion vesicle once the cyst wall has formed.

Maturation of the cyst wall coincides with the initiation of germ-tube formation. At this time dictyosomes are no longer arranged in a Golgi complex near the nucleus as in the zoospore and early cyst, but appear individually, often near the cyst wall (Fig. 6). The cisternae of the dictyosomes are distended and enlarge into irregularly shaped vesicles 150–350 nm in diameter (Fig. 12). The vesicles are also found free in the surrounding cytoplasm and contain small amounts of condensed amorphous material.

Where vesicles occur in close proximity to the cyst periphery, the plasmalemma and vesicular membrane often appear disrupted, thus providing continuity between the vesicular contents and the cyst wall. In these areas aggregations of membranous material in the form of short segments of membrane, small vesicles, or tubules are observed (Figs. 4, 14). At this time the cyst wall has become readily stainable with lead citrate alone.

The first indication of germ-tube formation is an evagination of the cyst wall and plasmalemma proximal to a large accumulation of vesicles morphologically identical to those surrounding the dictyosomes (Fig. 13). In germ tubes of increasing length, the vesicles always occur at the tip and occupy approximately the first 2–4 μm of the germ tube cytoplasm (Fig. 15). As in the cyst, when vesicles occur in close proximity to the germ-tube periphery the plasmalemma and vesicular membranes often appear dis rupted, proving continuity between the vesicular contents and the germ-tube wall (Fig. 14). These pockets of membranous material remain lining the germ tube as it extends (Fig. 15).

Indirect cyst germination begins identical to direct cyst germination, with the production of a germ tube. However, the germ tubes extended average only 6 μm in length and are terminated by a cap (Fig. 16) resembling the papillar cap seen in the sporangium (see Hemmes & Hohl, 1969, fig. 20). The sporangial papillar cap differs, however, in that it stains densely and can be distinguished from the sporangial walls which enclose it. The cap at the hyphal tip is similar to the hyphal walls in texture and density of staining. In sections taken during the development of the cap, the vesicles at the germ-tube tip decrease in number as the cap becomes larger and are absent in older stages where the caps average 2–3 μm in thickness. As in direct germination the plasmalemma and vesicle membranes appear disrupted in the developing cap region, thus establishing continuity between the vesicular contents and cap material.

Flagella are first seen in an aggregation of vacuoles near the nucleus (Fig. 17). Dictyosomes surrounded by numerous vesicles are closely associated with the vacuo lated areas, suggesting that at least some of these vacuoles are derived from dictyo-somes. In different sections flagella are seen in varying stages of development and are eventually located outside the cell between the plasmalemma and cyst wall (Fig. 18). After release, the secondary zoospore once again assumes the ultrastructural organi-zation and morphology characteristic of the primary zoospore. We have no ultra-structural indication as to the fate of the cap, which appears to dissolve upon zoospore release when viewed with light microscopy.

Sporangia incubated in cycloheximide do release their zoospores in a normal fashion, but the developing cysts do not mature beyond a stage comparable to the early cyst in normal encystation. A cyst wall is formed, but is only barely stainable with uranyl acetate and lead citrate. The cisternae of the dictyosomes are not distended as they would be in germinating cysts of comparable age. Instead an aggregation of individual flattened cisternae and vesicles are found surrounding the dictyosomes near the nucleus (Fig. 19). The cisternae appear to bend on themselves and thus enclose and isolate segments of cytoplasm (arrows, Fig. 19). The aggregation of vesicles does not move to the periphery of the cyst, but remains in close proximity to the nucleus even after 5 h of incubation. Lomasomes are conspicuously absent in these inhibited cysts.

Encystation in Phytophthora may be conveniently divided into 3 stages. The first, preparative stage begins at the time of sporangial cleavage, when the forming zoospores line their periphery with flattened vesicles and fibrillar vacuoles (see Hohl & Hamamoto, 1967, figs. 9 and 11). After release from the sporangium and a brief period of motility, the zoospores then shed their flagella and round up just prior to cyst wall formation. In the second stage the initial wall layer is deposited – a process most likely involving the flattened vesicles and the fibrillar vacuoles, which are no longer found after the initial wall has been deposited. Although no cytochemical tests have been performed, it is assumed that the fibrillar material from the vacuoles serves as wall material at this stage (Grove, 1970). The third stage involves the formation of the final cyst wall by addition of materials from dictyosome-derived vesicles. These vesicles are produced in large numbers during this stage and accumulate at the cell periphery, thereby emptying their contents to the outside. Distinct wall layers cannot be distinguished, but the wall increases noticeably in density

The first morphological indication of cyst germination is in the form of an aggregate of dictyosome-derived vesicles. The cyst wall proximal to this peripheral accumulation bulges outward and eventually forms the tip of the germ tube. The involvement of an aggregation of vesicles in initiating germ-tube formation and in the extending hyphal tip has been shown to be typical for growing hyphae in general (Girbardt, 1969; Grove et al. 1970).

When no exogenous energy is supplied during germination by incubating the cysts in distilled water or buffer solution, germ-tube extension terminates after a short period of time. Interestingly enough, the accumulation of vesicles at the germ-tube tip and the incorporation of the contents of the vesicles into the apical wall continue. This leads to the formation of the cap. From this it appears that germ-tube extension on one side, and production, transportation, and deposition of dictyosome-derived materials at the other, are not strictly correlated.

A further consequence of the lack of exogenous energy supply, or perhaps of the low osmotic value of the medium, is the formation of the secondary zoospore after the cap has been formed. Thus the cyst has become a miniature sporangium with the rudimentary germ tube as an exit pore. From this it appears that zoospore production can be induced by preventing germ-tube extension, a corollary to the observation in the sporangia of this species (Hemmes & Hohl, 1969), where the suppression of zoospore formation leads to the production of germ tubes.

The following generalizations can be made concerning the role of cellular organelles in germination and wall formation in Phytophthora: as is the case in other systems, the Golgi apparatus performs various tasks, i.e. during cyst formation and direct cyst germination it acts mainly in wall construction, whereas in indirect germination it provides membrane material for the vacuole into which the flagellum grows.

The results furthermore suggest that lomasomes are not cell organelles with a specific function such as wall synthesis, but rather consist of aggregations of excess mem branous material that have formed as a result of the discharge of vacuoles and vesicles at the cell periphery during wall formation. The following observations would be consistent with this interpretation. (1) Lomasomes are not observed in early stages of encystment, or in inhibited cysts, showing that lomasomes are not prerequisite for wall formation. (2) Lomasomes occur only as vesicles begin to arrive at the hyphal tip during germ-tube formation or in the later stages of cyst wall formation. (3) At hyphal tips there are fewer lomasomes than in the subapical portion of the hypha, indicating the incorporation of the membranous material into the rapidly expanding plasma membrane. (4) When inhibited by cycloheximide, the formation of normal vesicles from the dictyosomes is impaired, and concomitantly lomasomes are not found.

This paper is part of a thesis submitted by D.E.H. in partial fulfilment of the requirements for the Ph.D. degree, University of Hawaii.