Light and electron microscopy of oospore maturation in Saprolegnia furcata

[ 25 ] Trans. Br, mycol. Soc. 71 (1) 25-35 (1978)

Primed in Great Britain

LIGHT AND ELECTRON MICROSCOPY OF OOSPORE MATURATION IN SAPROLEGNIA FURCATA 2.

WALL DEVELOPMENT

By G . W. BEAKES

AND

J. L.

GAY

Department of Plant Biology, The University, N ewcastle upon Tyne, NE1 7RU, U.K., and Department of Botany, Imperial College, London, SW7 2BB, U.K.

Wall formation in maturing oospores of Saprolegnia fu rcata Maurizio has been studied by light and electron microscopy. Following oosphere delimitation an outer (epispore) wall is secreted by Golgi-derived vesicles. Outer wall formation continues after fertilization but ceases prior to inner (endospore) wall initiation. The inner wall of the oospore is accreted centripetally until it reaches a thickness of about r-5 pm. Wall vesicles do not appear to be involved and only cisternae of endoplasmic reticulum lie close to the growing wall. Inner and outer walls remain separated by an electron-dense tripartite, membrane-like, layer. The appearance of the wall of the mature oospore varies with the fixative employed. Whereas the inner wall appears uniformly electron transparent in glutaraldehyde-osmic fixed oospores, both osmic acid and permanganate fixation reveals electron-dense inner and outer zones within this layer. The inner zone, also discernible in living spor es, is bounded on one side by the cytoplasmic membrane and has a distinct convolute outer margin. The outer zone is bounded by the tripartite layer and has an indistinct inner margin. Permanganate fixation also reveals the fibrillar sub structure of the inner wall and staining with periodic acid-silver methenamine indicates that it is rich in polysaccharides. In add ition, the unusual wall structure of abnormally swollen but well-fixed oospores is described. Oospore wall development in S. furcata is compared with that in other oomycetes and the origin and nature of cell wall components is discussed. While several studies have been made on the differentiation of oogonia and oospores in the Saprolegniales (Howard & Moore, 1970; Gay, Greenwood & Heath, 1971) there are no accounts of the development of the complex wall of the mature oospore. This study of oospore wall development in Saprolegnia [urcata Maurizio, complements a preceding paper in which the corresponding cytoplasmic changes which accompany oospore maturation are described (Beakes & Gay, 1978). As with cytoplasmic change s, oospore wall development in Saprolegnia is compared with that in members of the Peronosporales (Hemmes & Bartnicki-Garcia, 1975; McKeen, 1975; Hegnauer, 1977). MATERIALS AND METHODS

Culture techniques

The strain of Saprolegnia fu rcata used in this study was obtained from the Aquatic Ph ycomycete Culture Collection (no. 205a ) at the University of Reading, England. Oospores were obtained as described by Beakes & Gay (1978).

Microscopy The techniques for both electron and light microscopy were as described by Beakes & Gay (1978). In addition to uranyl acetate-lead citrate staining, the following cytochemical tests were carried out on ultrathin sections. (1) 1 % phosphotungstic acid-10 % chromic acid solution (PT CA, Roland, Lembi & Moore, 1972) was used as a selective plasma membrane stain. (2) Cell wall polysaccharide localisation was determined by the periodic acid-silver methenamine staining procedure (PASM, Dhir & Boatman, 1972). For controls, periodic acid treatment was omitted from the procedure. In thes e tests, sections were transferred from one solution to another using a small platinum loop. RESUL TS

Oospore wall development as seen with the light microscope is summarized in Figs. 1-5. Oospore maturation is divided into the developmental

26

6

Oospore maturation in Saprolegnia furcara. II

9

G. W. Beakes and]. L. Gay stages (I-IV) defined by Beakes & Gay (1978). Inner and outer oospore wall layers are distinguished, a situation which parallels that in PhytophthoracapsiciLeonin(Hemmes&BartnickiGarcia, 1975). In Saprolegnia these are equivalent to the epispore and endospore of de Bary (1887). Outer wall formation stages I-II The outer wall (W1) is secreted during the 2030 min period following oosphere differentiation during which the oosphere has an irregular and changing surface (Fig. 1). Shrinkage in 0·8 M sucrose fails to reveal a cell wall. The peripheral cytoplasm of Stage I oospheres contains abundant vesicles close to the plasmalemma (Fig. 6) similar in appearance to, but larger (200- 400 nm) than the peripheral wall vesicles (90-150 nm ) in Stage II oospheres (Fig. 7). Many of these larger vesicles appear to fuse with the plasma membrane, possibly resulting in the irregular spore outline (Fig. 6). They presumably discharge precursors and enzymes involved in the synthesis of the outer wall and are equivalent in function to the apical vesicles of hyphae (Heath, Gay & Greenwood, 1971). Some loose fibrillar material is present around these oospheres. Owing to the rapid development, more advanced Stage I oospheres were not found and subsequent stages in outer wall formation could not be followed . When an oosphere has acquired a stable, smooth outline (Fig. 2) it can be plasmolysed in 0·8 M sucrose to reveal the newly formed wall. Sections of such Stage II oospheres show an electron-

27

transparent W1 wall, 100-150 om in thickness (Fig. 7). W1 is similar in appearance to walls surrounding developing gametangia (Gay et al., 1971). In the region of fertilization tube contact W1 is locally thickened and contains lomasomes (Beakes & Gay, 1977b). The peripheral cytoplasm of early Stage II oospheres contains abundant wall vesicles and their proximity to the Golgi vesicles suggests that active production of vesicles is still occurring (Fig. 7). W 1 formation probably continues slightly beyond oosphere fertilization as discharged penetration pegs become coated in a thin layer of wall material (Beakes & Gay, 1977 b). However, in late Stage II oospheres the absence of peripheral wall vesicles and the quiescent Golgi apparatus (Fig. 8) indicate cessation of W 1 secretion. In sections of Stage II oospheres stained with PTCA, the plasma membrane stains densely (Fig. 9) together with a slight increase in cell wall staining as noted by Roland et at. (1972) in higher plant cells. Inner scall formation Stage III Centripetal accretion of the inner wall (W2) takes approximately 10 h and reaches about l ' 5 pm in thickness (Figs. 3-5). The inner wall appears uniformly refractile before ooplast development (Fig. 3) but soon acquires an optically distinct inner zone (Figs. 3-5). Once W2 has formed the cell can no longer be plasmolysed in 0·8 M sucrose, indicating that the wall is nc longer permeable to water. Ultrastructural study of W2 formation is

Saprolegnia [urcata

ER, endoplasmic reticulum ; GV, golgi vesicle; L1, inner zone of inner wall; L2, outer zone of inner wall; M, mitochondrion; T, tripartite layer separating inner and outer oospore walls; W1, outer (epispore) wall; W2, inner (endospore) wall; WV, wall vesicles. Unle ss otherwise stated oospores were induced in sterile distilled water and electron-microscope prep arations stained in uranyl acetate-lead citrate. Figs. 1-5 . Light micrographs showing stages of oospore wall development. Fig. 1. Stage I oosphere (naked) x 2300. Fig. 2. Stage II oosphere (walled) x 2200. Figs. 3-4. Stage III oospores showing the developing inner wall, The differentiating inner layer is arrowed, x 2200. Fig. 5. Mature Stage IV oospore . Note the optically dense inner layer (arrowed), x 2500. Fig. 6. Peripheral cytoplasm of naked Stage I oospheres showing large per ipherall y distributed wall vesicles (WV). GaOs x 25000. Fig. 7. Peripheral cytoplasm of an early Stage II oosphere showing the newly formed outer wall (W 1) and small peripheral wall vesicles (WV). Note the abundance of Golgi-derived vesicles (GV) in per ipheral cytoplasm. Os x 37500 . Fig. 8. Peripheral cytoplasm of a late Stage I oosphere, Wall-vesicles are absent. Note the quiescent dictyosorne (arr owed). GaOs x 39000. Fig. 9. Peripheral cytoplasm of a PTCA stained Stage II oosphere showing an electron-dense tripartite plasma membrane. KMnO. (20 min) x 56000.

28

Oospore maturation in Saprolegnia furcata. II

WI

WI

W2

WI

-10

11

12

WI

W2

13

G. W. Beakes and]. L. Gay hampered by fixation difficulties (cf. Os-fixed cytoplasm in Figs . 7 and 10), which coincide with initiation of this layer. Fortunately, KMn04 preserves the cytoplasm well enough to demonstrate the absence of peripheral wall vesicles and Golgi vesicles during the period of W2 accretion (Fig. 11). Cisternae of endoplasmic reticulum aligned close to the growing wall are, however, present (Fig. 11) although plasma membrane is difficult to detect even in potassium permanganate-fixed spores (Figs. 10-12). In both Os and GaOs W2 appears largely structureless (Figs. 10, 12) although KMn0 4 fixation shows that it has a more finely granular texture than Wl (Fig. 11). The latter remains unaltered throughout Stage III (Figs. 7, 10-13). Layers Wl and W2 are separated by a thin (10 nm) tripartite, membrane-like (Fig. 13) electron-dense layer (Figs. 10-12). During the early stages of inner wall accretion this layer cannot be seen in all regions (Figs. 10, 11), possibly through being cut obliquely in parts. During Stage III, the inner wall in living oospores acquires a distinct, refractile, inner zone (Figs. 3, 4). Mature oospore wall structure Stage I V The outer wall (W1) of mature Stage III oospores appears similar in all fixatives (Figs. 14-16). It is more electron dense and fibrillar (Figs. 14, 15, 16), however, than at Stage II (F ig. 7) and resembles the outer 'fluffy' layer of the wall of the mature oogonium (Fig . 14), suggesting that W1 alters somewhat during oospore maturation. In contrast, the appearance of the inner wall (W2) in mature oospores is extremely variable, depending upon the fixative used (Figs. 12-15). In GaOs-fixed spores W2 appears almost uniformly electron transparent with little discernible substructure (Fig. 12). The uneven thickness of W2 and the fact that it is significantly thinner than in other fixatives (1'0 pm, cf. l '5 Ilm) suggests that W2 is only partially preserved in GaOs. In both KMn04 and Os W2 is of even thi ckness (Figs. 14,

29

15) as observed in living spores (Fig. 5) and a more complex substructure is revealed. There are two electron-dense zones within W2, although with KMn0 4 the innermost zone is only revealed with fixation times of 20 min or more. The outer electron-dense zone (Li ) ranges from 0'2 to 0'4 pm diam and grades into the W2 matrix along its inner margin (Figs. 14, 15, 16). It is bordered by the tripartite layer (T) and comprises fine, possibly fibrillar, material (Fig. 16). The more coarsely particulate inner zone (L2) varies in thickness from 0'2 to 0'5 pm (Figs . 14, 15, 17) and corresponds to the optically dense inner layer often observed in living oospores (Fig. 5). The L2 zone has an irregular, convolute, outer margin form ing a labyrinth of electron-dense material within the more electron-transparent W2 matrix. The L2 zone is bounded by a more electron-dense inner margin (40-50 nm) separated by a narrow (10-15 nm) electron-transparent layer from the cytoplasmic membrane (Fig. 17). Most of the W2 matrix has a finely granular substructure (Figs. 14, 15) which in KMn0 4-fixed spores allows the frequently undulating, wall fibrils to be resolved (Fig. 14). PASM-staining results in a dense deposit of silver grains over both W1 and, more particularly, W2 showing that both are rich in polysaccharides (Fig. 18). The L1 and L2 zones, however, stain less densely (Fig. 18) suggesting either that the electron-dense material is not composed of polysaccharides or that it screens polysaccharides from the stain. These observations suggest that W2 is composed of a skeleton of microfibrillar polysaccharide in which other materials are incorporated form ing the Li and L2 zones. A continuous tr ipartite layer separates W 1 from W2 (Figs. 13-16). In GaOs and Os-fixed spores this layer has a regular membrane-like appearance, approximately 15-20 nm diam (Figs . 13, 15). However, in KMn0 4 it appears less membrane-like, with a much thicker outer (20 nm ) than inner layer (5 nm) (Fig. 16). The tripartite

Fig. 10. Peripheral cytoplasm of an early Stage III, oospore, with a newly initiated inner wall (Wz). Note the poor cytoplasmic preservation and the tripartite layer (closed arrow) between the outer (Wl) and inner walls (Wz) and possible inner plasma membrane (open arrow). Os x 85000. Fig. 11. Peripheral cytoplasmof a Stage III oospore showing the developing inner wall (Wz). Note the tripartite layer (arrowed). Endoplasmic reticulum (ER) lies close to wall. KMn04 (ao min) x 60000. Fig. 12. A Stage III oospore with a developing inner (Wz) wall and a clearly defined tripartite layer (arrowed). GaOs x 45000. Fig. 13. Detail of the tripartite wall layer shown in Fig. 13. GaOs x 71500. Figs. 14-20. A series of mature (Stage IV) oospore walls. Fig. 14. A mature oospore showing fibrillar sub-structure of the inner wall (Wz) and electron-dense outer (L i) and inner (Lz) zones. Note the increased density of the outer wall (W1) in comparison with the much thicker oogoniumwall (OW). KMn0 4 (25 min) x 40000.

Oospore maturation in Saprolegnia furcata. II

W2 <,

.

.'

WI T LI

W2

18

WI

, • 22

W2

G. W. Beakes and]. L. Gay layer stains densely with PTCA (Fig. 19) whereas the inner membrane bounding the cytoplasm stains barely at all (Fig. 20). In addition PTCA stains both W 1 and the inner and outer parts « 100 run) of W2 (Figs. 19, 20). This suggests that the tripartite layer may have originated from the plasma membrane. The highest density of deposited silver grains with PASM staining, however, also coincides with the tripartite layer (Fig. 18) indicating a polysaccharide component. On the basis of these observations the normal pattern of oospore wall formation in Saprolegnia furcata is summarized diagrammatically in Fig. 24. Abnormal oospore wall structure Stage IV A number of oospores with unusual W2 walls were observed. Significantly, the cytoplasm of most of these oospores fixed well in Os (Beakes & Gay, 1978) which indicates the critical role of W2 in governing the permeability of oospores to fixatives. While some of these abnormal oospores were induced in sterile distilled water, most were encountered from tap water induced material (Figs. 21-23). In both cases, abnormal oospores were frequently larger (Beakes & Gay, 1978, Fig. 37) and W2 thinner (0'5-1'0 Ifm) than in normal oospores (Fig. 21), although some were of normal size and wall thickness (Fig. 22). W 1 and W2 walls are separated by the usual membranelike layer (Figs. 21, 22) although the tripartite structure could not always be resolved (Fig. 21). W2 is bounded by a clearly tripartite plasma membrane (Fig. 23) even in those walls with an uneven inner margin.

The texture of the inner wall is variable (Figs. 21,22). Usually W2 contains granular and electrontransparent zones and small (30-50 run) compressed electron-dense globules (Figs. 21, 22). These are either distributed uniformly throughout W2 (Fig. 22) or may be concentrated into localized bands (Fig. 21). No substructure can be distinguished in these walls in living oospores. DISCUSSION

As in most other fungal resting spores (Beckett et al., 1974) oospores acquire a thick multilayered wall. Such walls afford protection against microbiological and physical degradation and act as a food reservoir. In oospores the thick inner wall is almost certainly the major carbohydrate reserve (Bartnicki-Garcia & Hernmes, 1976), which must be mobilized prior to germ-tube formation (Blackwell, 1943a; Clausz, 1968; Zentmeyer & Erwin, 1970; Beakes & Gay, 1977a). Spore wall development is often accompanied by cytoplasmic dehydration enabling spores to withstand extremes of temperature and water stress (Sussman & Halvorson, 1966). However, it is difficult to ascertain the extent to which this occurs in Saprolegnia since oospores readily lyse at temperatures above 30°C and do not recover following dehydration (Clausz, 1968). Oospore wall development is also of interest since Blackwell (1943b) suggested that the structure of oospore walls probably governs spore dormancy. Certainly an understanding of the mechanism of constitutive dormancy is desirable,

Fig. 15. A mature oospore wall showing an electron-dense outer (L1) and on inner (L2) zone of the inner wall (W2). The unusually thick outer wall (W1) is the result of streptomycin treatment (100 mg/l), Os x23000. Fig. 16. Detail of the outer region of an oospore wall showing the outer wall (W1), the tripartite layer (T) and the outer electron-dense zone (L1) of W2. KMnO. (25 min) x 110000. Fig. 17. Detail of the inner region of the inner oospore wall showing the inner electron-dense zone (L2) and plasma membrane (arrowed). KMnO. (25 min). x 105000. Fig. 18. Oospore wall as shown in Fig. 17, but stained by PASM to reveal localizationof polysaccharides. Os x 23000. Fig. 19. Detail of a PTCA-stained oospore wall showing the densely stained outer wall (W1), tripartite layer (T) and outer part of the L1 zone. KMnO. (25 min x 75000. Fig. 20. Detail of a PTCA-stained oospore wall showing the densely stained margin of the W2 zone and the unstained plasma membrane (arrowed). KMnO. (25 min) x 75000. Figs. 21-23. Abnormal walls in oospores induced in distilled water (21) and tap water (22). Fig. 21. The wall of an abnormally well-fixedoospore. Note variation in density of inner wall (W2) and bands of small osmiophilic globules. KMnO. (25 min) x 25000. Fig. 22. An abnormal oospore wall with an inner layer of normal thickness but containing electron-dense globules. Os x 29000. Fig. 23. Detail of Fig. 22, showing the plasma membrane between inner wall (W2) and cytoplasm. Os x 90000.

Oospore maturation in Saprolegnia furcata . II

32 2

WI

3

5

4

T

T

6

W2

WIT LI

L2

Fig. z4. A diagramsummarizing oosporewall development in Saprolegnia. The cytoplasm is to the right of each Figure (stippled). (1) Stage I oosphere (naked) with underlying wall vesicles and dictyosomes. (z) An early Stage II oosphere with the newly formed outer wall (W1) and wall vesicles fusing with plasma membrane, contributing to (W1). (3) A late Stage II oosphere, after completion of outer wall secretion. (4- 5) Stage III oospores with the developing inner wall (Wz). A tripartite layer (T) separates outer and inner walls. (6) A mature, Stage IV, oospore wall with electron-dense inner (L 1) and outer (Lz) zones formed in the inner wall.

since the erratic germination of oospores has been walls in this fungus (Heath et al., 1971). It corresan intractable problem in genetic studies in these ponds to an epispore wall, defined by de Bary fungi. Cytological study of oospores has always (1887) as the first-formed wall of what was then been difficult (Trow, 1895; Blackwell, 1943 a). thought to be a zygote. In contrast, in PeronoThe improved fixation of cytoplasm in oospores sporales (Hemmes & Bartnicki-Garcia, 1975; formed in the presence of agents which result in McKeen, 1975), the outer wall is a thin electronabnormal wall development suggests that the inner dense layer derived from the peripheral periplasm, layer (Wz) is the main barrier to the penetration and therefore corresponds to an exospore wall as of dyes and fixatives. It seems likely, therefore, defined by de Bary (1887). that Wz also precludes diffusion of prerequisites Outer wall secretion by naked oospheres of for germination and thus may be a major factor Saprolegnia may be compared with cyst wall contributing to dormancy. A more detailed study secretion by naked zoospores (Heath & Greenwood, of the much more permeable oospores with 1970). As Bartnicki-Garcia (1973) pointed out, abnormal inner walls may, therefore, afford cyst wall formation may be taken as an example valuable information concerning the nature of this of wall regeneration in a 'natural protoplast'. However, cyst wall formation in Saprolegnia barrier. The present account and the recent studies on (Heath & Greenwood, 1970) and Phytophthora oospore development in Phytophthora capsici (Bartnicki-Garcia, 1973) involves an immediate Leonin (Hemmes & Bartnicki-Garcia, 1975) and discharge of an outer amorphous adhesive layer a Pythium sp. (McKeen, 1975) togeth er with before the microfibrillar glucan wall is secreted a largely unpublished freeze etch, and thin section beneath it. In oospheres, only a layer correspondstudy of oospore wall structure in Phytophthora . ing to the glucan wall is formed and thus they caetorum (Lebert & Cohn) Schroeter (Hegnauer, closely resemble such artificially prepared fungal 1977) allow comparison of oospore wall structure protoplasts as those described in Aspergillus to be made between the Saprolegniales and (Gibson & Peberdy, 1972). In Saprolegnia Peronosporales. A major difference in oospore oospheres, however, there is only a delay of 5 min wall development lies in the formation of the or so before wall secretion commences and a disouter wall. In Saprolegn ia the outer wall is tinct wall forms within 30 min compared with the secreted by dictyosome-derived vesicles and is Z4-48 h required for protoplast wall regeneration. similar in appearance to the other vesicle secreted Saprolegnia would clearly provide an excellent

G. W. Beakes and]. L. Gay system for the comparison of natural and artificial wall regeneration in fungi. In contrast the inner (endospore) wall (de Bary, 1887), appears remarkably similar in both Saprolegnia and the Peronosporales. While, in general, GaOs reveals little substructure to the inner wall (W2) both the use of KMn0 4 in Saprolegnia and freeze etching in Phytophthora (Hegnauer, 1977) demonstrate a microfibrillar component and a well-defined amorphous inner zone. However, in Phytophthora the inner zone does not have a convolute margin and there is no evidence of an outer electron-dense layer (Hegnauer, 1977). Furthermore, freeze-etch profiles of the electrondense outer wall of Phytophthora oospores resolves two layers, the outer of which probably corresponds to the periplasm-derived exospore and the inner to the tripartite layer observed in Saprolegnia. Biochemical analysis of combined oogoniumoospore walls of Phytophthora megasperma var, sojae Hildebrand has shown them to compri se 80 % carbohydrate and approximately 10 % lipid and 10 % protein (Lippman, Erwin & BartnickiGarcia, 1974). Amorphous fJ-1,3-linked glucans account for the majority of carbohydrate with microfibrillar cellulose contributing only 7 %. It, therefore, seems likely that, as in hyphal walls (Hunsley & Burnett, 1970), the inner oospore wall has a microfibrillar cellulose skeleton impregnated with amorphous fJ-l,3 glucans, The electron-dense zones within the inner wall may be the location of most of the protein and lipid fractions. The latter are probably derived from breakdown of the main cell organelles which occurs during Stage III (Beakes & Gay, 1978). Stereological analysis of maturing oospores shows that the inner wall in mature oospores accounts for approximately 20 % of the total volume fraction and that its development occurs mainly at the expense of the ' cytoplasmic phase' and neutral lipid fractions (Beakes & Gay, 1978). Although the inner wall of the oospore in Saprolegnia is entirely cytoplasmically derived its accretion does not appear to depend on the usual Golgi-derived wall vesicles. Hemmes & BartnickiGarcia (1975) also reported the apparent absence of wall vesicles during inner wall secretion, but as in this study, poor cytoplasmic preservation makes it difficult to be certain of this. However, the decline in the number of Goigi bodies and activity in late Stage II oospheres strongly suggests that the usual wall vesicles are not involved in inner wall secretion. Further evidence supporting a fundamentally different mode of inner wall accretion is provided by studies with the streptomycin, which normally stimulates wall vesicle

33

production in sporulating colonies (Beakes, 1976). While this antibiotic leads to increase in the thickness of the outer wall it has no effect on the extent of inner wall development (compare Figs. 18,17)· The possible origin of the electron-dense tripartite layer separating the outer and inner oospore walls merits further consideration. This layer is of approximately the same dimensions as the plasmalemma and stains densely with PTCA, reported to be diagnostic for the plasma membrane (Roland et al., 1972), suggesting that it may be derived from the plasma membrane. Further support for this idea is provided by the absence of a clearly defined membrane between the developing inner wall and cytoplasm (although this may be due to poor cytoplasmic fixation), the inability to plasmolyse spores in 0·8 M sucrose, and the report of an indistinct inner margin of the developing endospore wall in Phytophthora oospores (Murphy, 1918). The inner endospore wall may thus be internal to the plasma membrane and be regarded as a phase separation of carbohydrate and other reserves in the peripheral cytoplasm. This would also explain the absence of the usual wall vesicles in its secretion. There are, however, a number of objections to this hypothesis. The formation of a wall or any similar structure immediately inside the plasma membrane is extraordinary, although Robards et al. (1972) have shown a plasma membrane-like structure trapped betw een two wall layers in the primary Casparian strip of barley roots. More importantly, mature oospores have a well-defined membrane between the cytoplasm and the inner wall and it is clear from a preliminary account of oospore germination in Saprolegnia f erax (Beakes & Gay, 1977a ) that the inner membrane becomes the functional plasma membrane. Thus if the original plasma membrane of the oospore becomes sequestered between the inner and outer walls, the problem of the origin of the inner membrane is raised . De novo membrane formation on a 'naked' wall has not previously been reported and it is believed that all membranes grow by the intussusception of material into a pre-existing membrane (Finean, Coleman & Mitchell, 1974). The possibility that the membrane arises from a layer of vesicles similar to that in the cell plate of a higher plant can be excluded, because the mechanism would result in two new membranes rather than the single one observed. It is conceivable that the abundant membrane fragments in the peripheral cytoplasm of the oospore (Beakes & Gay, 1978) may become aligned along the inner wall and coalesce to form a new membrane but we do not favour this hypothesis. MYC 7 1

34

Oospore maturation in Saprolegnia furcata. II

Alternatively, the tripartite layer between the inner and outer walls may be regarded as another special wall layer with the plasma membrane located at the cytoplasm-inner wall interface throughout development. Certainly the specificity of the PTCA test for all plant plasma membranes has been recently questioned (Hall & Flowers, 1976). Evidence of a high concentration of polysaccharides associated with the tripartite layer suggests that it may not be a predominantly phospholipid membrane. Furthermore, morphologically similar tripartite layers have been widely reported in algal cell walls. As in Saprolegnia such layers frequently separate inner and outer wall layers (e.g. sheath in Volvox, Burr & McCracken, 1973; and cyst wall of Polytomella agilis, Brown, Leppard & Massalski, 1976). The tripartite layer surrounding Chlorella walls is composed of sporopollenin (Atkinson, Gunning & John, 1972), a compound which has been recently demonstrated in the walls of fungal resting spores (e.g. zygospores of Mucor, Gooday et al., 1973; ascospores of Daldinia, Beckett, 1976). Further investigations, employing techniques such as freeze etching and the chemical and enzymic dissection of walls, will be needed to resolve the origin and nature of the tripartite layer in oospores of Saprolegnia. Thus, the wall of the mature oospore of Saprolegnia is structurally complex. The inner wall appears to govern permeability to dyes and fixatives and probably plays a crucial role in controlling oospore dormancy. Clearly oospore wall development in Saprolegnia merits detailed biochemical study to determine precisely how the structural features reported here are related to wall chemistry and function. We wish to thank Miss Marion Martin, Peter Webster and Bob Hewit for excellent technical assistance. Part of this study was carried out under the tenure by one of us (G. W.B.) of a Science Research Council Studentship and is included in a Ph.D. Thesis for the University of London. REFERENCES ATKINSON, A. W., GUNNING, B. E. S. & JOHN, P. C. L. (1972). Sporopollenin in the cell wall of Chlorella and other algae; ultrastructure, chemistry and incorporation of HC-acetate, studied in synchronous cultures. Planta (Berlin) 107, 1-32. BARTNICKI-GARCIA, S. (1973). Cell wall genesis in a natural protoplast: The zoospore of Phytophthora palmioora. In Yeast, Mould and Plant Protoplasts (eds. J. R. Villanueva, I. Garcia-Acha, S. Gascon and F. Umbur), PP.77-91. London: Academic Press.

BARTNICKI-GARCIA, S. & HEMMES, D. E. (1976). Some aspects of the form and function of oomycete spores. In The Fungal Spore: form and function (eds D. J. Weber and W. M. Hess), pp. 593-639. London, New York: John Wiley and Sons. BEAKES, G. W. (1976). The effects of streptomycin and divalent cations on the development of finestructure of Saprolegnia spp. Ph.D. Thesis, University of London. BEAKEs, G. W. & GAY, J. L. (1977a). Oospore maturation and germination in Saprolegnia. Proceedings of the znd International Mycological Congress, Tampa, Florida. BEAKES, G. W. & GAY, J. L. (1977b). Gametangial nuclear divisions and fertilization in Saprolegnia furcata as observed by light and electron microscopy. Transactions of the British Mycological Society 69, 459-471. BEAKES, G. W. & GAY, J. L. (1978). A light and electron microscopic study of oospore maturation in Saprolegnia furcata. I. Cytoplasmic changes. Transactions of the British Mycological Society 71, 11-24. BECKETT, A. (1976). Ultrastructural studies on exogenously dormant ascospores of Daldinia concentrica. Canadian Journal of Botany 54, 689-697. BECKETT, A., HEATH, I. B. & McLAUGHLIN, D. J. (1974). An Atlas of Fungal Ultrastructure. London: Longman. BLACKWELL, E. (1943a). The life history of Phytophthora cactorum (Leb & Cohen) Schroet. Transactions of the British Mycological Society 26, 71-89. BLACKWELL, E. (1943b). On germinating oospores of Phytophthora cactorum. Transactions of the British Mycological Society 26, 93-103. BROWN, D., LEPPARD, G. C. & MASSALSKI, A. (1976). Fine structure of encystment of the quadriflagellate alga, Polytomella agilis. Protoplasma 90, 139-154. BURR, F. A. & MCCRACKEN, M. D. (1973). Existence of a surface layer on the sheath of Volvox. Journal of Phycology 3, 345-346. CLAUSZ, J. C. (1968). Factors affecting oogenesis and oospore germination in Achlya hypogyna. Journal of the Elisha Mitchell Scientific Society 84, 199-206. DE BARY, A. (1887). Comparative Morphology and Biology of the Fungi, Mycetozoa and Bacteria. Oxford: Clarendon Press. DHlR, S. P. & BOATMAN, E. S. (1972). Localisation of polysaccharide on Chlamydia psittaci by silver methenamine staining and electron microscopy. Journal of Bacteriology 111, 267-271. FINEAN, J. B., COLEMAN, R. & MITCHELL, R. H. (1974). Membranes and their Cellular Functions. Oxford: Blackwell. GAY, J. L., GREENWOOD, A. D. & HEATH, I. B. (1971). The formation of vacuoles (vesicles) during oosphere development and zoospore germination in Saprolegnia. Journal of General Microbiology 65, 233-241. GIBSON, R. K. & PEBERDY, J. F. (1972). Fine structure of protoplasts of Aspergillus nidulans. Journal of General Microbiology 72, 529-538. GOODAY, G. W., FAWCETT, P., GREEN, D. & SHAW, G. (1973). The formation of fungal sporopollenin in the zygospore wall of Mucor mucedo: a role of the

G. W. Beakes and J. L. Gay sexual carotogenesis 10 the Mucorales. Journal of General Microbiology 74, 233-239. HALL, J . L. & FLOWERS, T . J . (1976) . Properties of membranes from the halophyte Suaeda maritima. 1. Cytochemical staining of membranes in relation to the validity of membrane markers. Journal of Experimental Botany 27, 658-671. HEATH, I. B., GAY, J. L. & GREENWOOD, A. D . (1971). Cell wall formation in the Saprolegniales: cytoplasmic vesicles underlying developing walls . Journal of General Microbiology 65, 225-232. HEATH, I. B. & GREENWOOD, A. D . (1970). Wall formation in the Saprolegniales. II. Formation of cysts of zoospores of Saprolegnia and Dictyuchus. Archives fur Mikrobiologie 75, 67-79. HEGNAUER, H . (1977). Bau, enuoicklung und chemische aspekte der Zellwandtypen von Phytophthora. Ph.D. Dissertation, University of Zurich. HEMMES, D. L. & BARTNICKI-GARCIA, S. (1975). Electron microscopy of gametangial interaction and oospore development in Phytophthora capsid. Archives for Microbiology 103,91-112. HOWARD, K. L. & MOORE, R. T. (1970). Ultrastructure of oogenesis in Saprolegnia terrestris. Botanical Gazette 131, 311-336. HUNSLEY, D . & BURNETT, J. H. (1970). The ultrastructural architecture of the walls of some hyphal fung LJournal of General Microbiology 6:z,203-218.

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LIPPMAN, E., ERWIN, D. C . & BARTNICKI-GARCIA, S. (1974) . Isolation and chemical composition of oospore-oogonium walls of Phytophthora megasperm a var. sojae. Journal of General Microbiology 80, 131-141. McKEEN, W. E. (1975). Electron microscopy of a developing Pythium oogonium. Canadian Journal of Botany 53,2354-2360. MURPHY, P. A. (1918). The morphology and cytology of the sexual organs of Phytophthora erythroseptica. Annals of Botany 32, 115-153. ROBARDS, A. W., JACKSON, M. S., CLARKSON, D. T. & SANDERSON, T . (1972) . The structure of barley roots in relation to transport of ions into the stele. Protoplasma 77, 291-3 11. ROLAND, J . C., LEMBI, C. A. & MORRE, D . D. J. (1972). Phosphotungstic acidchromic acid as a selective electron-dense stain for plasma membranes in plant cells. Stain Technology 47, 195-200. SUSSMAN, A. S . & HALVORSON, H. O. (1966). Spores: their Dormancy and Germination. New York, London: Harper and Row. TROW, A. H. (1895). The karyogamy of Saprolegnia. Annals of Botany 9, 609-652. ZENTMEYER, G. A. & ERWIN, D. C. (1970). Development and reproduction of Phytophthora. Phytopathology 60,1120-1127.

(Accepted for publication 17 January 1978)

ADDENDUM Since preparation of these manuscripts two additional accounts of Peronosporalean oospores have come to our attention tPythium acanthicum Drechsler, Haskins et al., 1976, and Albugo candida (Pers. ex Lev.) Ktze, Tewari & Skoropad, 1977). Oosphere wall development in P. acanthicum is particularly interesting since the secretion between the oosphere and periplasm of a vesicle mediated epispore wall is shown (Haskins et al., 1976, figs . 14, 1S). This suggests oospore wall development in this species is intermediate between that of Saprolegnia and the other members of the Peronosporales so far described. Mature oospores of Albugo (Tewari & Skoropad, 1977, figs. 9, 14) contain a small eccentricallyplaced ooplast, surrounded by a very extensive lipid-packed cytoplasm. The mature oospore wall of Albugo is extremely complex due to the pre-

sence of at least three periplasm derived layers. On the basis of our study it is suggested that the inner electron transparent zone (v) and the adjacent electron dense layer (w) are probably oospore derived and equivalent to the endospore (W2) and tripartite layers in Saprolegnia. The additional layers (x, y , z) are presumably secreted sequentially by the peripheral periplasmic cytoplasm. These studies clearly confirm the potential importance of mature oospore wall structures as a taxonomic character in oomycete fungi. HASKINS, R. H., BRUSHABER, J. A., CHILD, J. J. & HOLTBY, L. B. (1976). The ultrastructure of sexual reproduction in Pythium acanthicum, Canadian Journal of Botany 54, 21 9 3- 2 20 3 . TEWARI,J. P. & SKOROPAD, W. P. (1977). Ultrastructure of oospore development in Albugo candida on rape seed. Canadian Journal of Botany 55, 2348-2357.