an electron microscope study of exogenously dormant spores, spore germination, hyphae and...

An Electron Microscope Study of Exogenously Dormant Spores, Spore Germination, Hyphaeand Conidiophores of Alternaria brassicicolaAuthor(s): Richard CampbellSource: New Phytologist, Vol. 69, No. 2 (Apr., 1970), pp. 287-293Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2429939 .

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New Phytol. (I970) 69, 287-293.

AN ELECTRON MICROSCOPE STUDY OF EXOGENOUSLY DORMANT SPORES, SPORE

GERMINATION, HYPHAE AND CONIDIOPHORES OF ALTERNARIA BRASSICICOLA

BY RICHARD CAMPBELL

Department of Botany, University of Bristol

(Received 22 August I969)

SUMMARY

Freeze-etching and chemical fixation techniques were used to study exogenously dormant spores, spore germination, hyphae and conidiophores of Alternaria brassicicola (Schw.) Wiltshire. Dormant spores have very thick, heavily pigmented, melanized walls with plugged septal pores. The small amounts of endoplasmic reticulum and the fewmitochondria lie near the plasmalemma. The germ-tube walls arise from the inner layers of the spore walls; lomasomes and endoplasmic reticulum vesicles are probably concerned with this wall formation. During germination mitochondria and ribosomes increase in numbers, first in the germinating cell and then in the germ tube. As the hyphae age they produce lipid droplets and vacuoles; the latter finally fill most of the cell as the cytoplasm degenerates. Conidiophores have a similar structure to mature hyphae except that they have, after spore production, a pore in the tip and an annulus.

INTRODUCTION

The spores of Alternaria brassicicola (Schw.) Wiltshire are produced in acropetal chains and at maturity they have melanized walls and are usually multicellular. When mature and in a suitable environment they are capable of germination. If environmental conditions prevent germination, mature spores may enter a quiescent period and they can remain viable for many weeks. Spores which have been quiescent for some time are referred to as 'exogenously dormant'.

The development, maturation and ultrastructure of the spores of A. brassicicola have been described already (Campbell, I968, I969), but no reports on the ultrastructure of conidia after a period of exogenous dormancy are known. Germination has been studied in a number of species and was reviewed by Hawker (I966) who concluded that the walls of the germ tube generally arise from the inner layers of the conidium wall. Remsen, Hess and Sassen (I967), using both chemically fixed and freeze-etched material, described the germination of Penicillium spores and support this generalization. The ultrastructure of hyphae has been reviewed by Hawker (I965) and Bracker (I967). Two important papers have since been published (Brenner and Carroll, I968; Hess, I968) which extend earlier findings; hyphae contain the usual organelles and their walls are generally thin, though they may be two-layered when pigmented (Werner and Lindberg, I966).

MATERIALS AND METHODS

Alternaria brassicicola was grown on potato dextrose agar (Oxoid CMI39) at 250 C. Spores were collected by washing the colony surface and centrifuging the resulting spore

287



288 RICHARD CAMPBELL

suspension. Exogenously dormant spores were obtained by collecting spores from suspensions on Millipore filters and storing these in the laboratory for periods of 4-17 weeks. To study germination fresh spore suspensions were transferred aseptically to a glucose- asparagine liquid medium agitated and aerated simultaneously with a stream of sterile air. Germ tubes were produced after about 3 hours at 250 C and germinated spores were fixed after 6-I2 hours. Hyphae were removed from colonies with a small piece of the agar on which they were growing. The following methods of preparation for electron microscopy were used:

(i) Potassium permanganate: 2% (w/v) unbuffered solution was used at 40 C for 2 hours with dormant spores and for i hour with germinating spores and hyphae. The material was washed in distilled water, stained in saturated aqueous uranyl acetate for 2 hours, dehydrated in an ethanol: water series, soaked in propylene oxide and embedded in epon. Sections were cut on an LKB ultramicrotome and viewed with an AEI EM6B electron microscope after staining with lead citrate (Reynolds, i963).

(2) Glutaraldehyde-acrolein: a i: i mixture of 3%0 (w/v) purified and neutralized glutaraldehyde and 300 (v/v) purified acrolein in O.I M sodium cacodylate buffer at pH 7.0 was used for I5 minutes at o-40 C (Campbell, i969). Material was washed in buffer and post-fixed in i% (w/v) buffered osmium tetroxide for 5 hours at 0-40 C, stained, dehydrated, sectioned and viewed as above.

(3) Freeze-etching: Balzer's freeze-etching equipment and the method of Moor and Muhlethaler (i963) were used. Germinating spores and hyphae were soaked in 2000 glycerol for 6 hours prior to freezing in liquid 'Freon' at - I850 C. After preparing the fracture face the frozen drop was warmed to - I00? C and etched for 40 seconds, shadowed with platinum-carbon and a carbon replica prepared. The replica was warmed to room temperature and cleaned by washing successively in sodium hypochlorite, water, concentrated sulphuric acid and water and mounted on 'Formvar' coated grids.

(4) Heavy metal shadowing: hyphae were suspended in saturated potassium hy- droxide and autoclaved at I5 lb/in.2 (io5N/m2) for three periods of i hour each, changing the solution each time. The material was washed in 2% sulphuric acid and then in water. Drops of aqueous suspension were placed on 'Formvar' coated grids and dried at room temperature.

RESULTS

Ultrastructure of exogenously dormant spores The ultrastructure of the exogenously dormant spores is dominated by the very thick,

heavily melanized wall (Plate i, No. i). The pores in the walls and septa are plugged (Plate i, Nos. 2 and 3). This effectively isolates the spore cells from each other and from the environment. After several weeks of dormancy the food reserves of the spore are largely used (Plate i, No. i; the spore has only a few lipid droplets in the lower cell). Mitochondria become very sparse and have few cristae. They, and the endoplasmic reticulum, lie near the periphery of the cells (Plate i, No. i). The nuclear envelopes have few, if any, pores.

Ultrastructure of germinating spores and hyphae Germination of spores taken direct from the colony to a suitable environment will

be described. Spores germinating after exogenous dormancy have a similar appearance to



Electron microscopy of Alternaria 289

freshly collected spores but have reduced food reserves, thicker walls and plugged pores in the walls.

The wall of the germ tube is an extension of the inner layer of the spore wall (the secondary wall; see Campbell, I968), and grows through the outer primary wall by mechanically breaking it (Plate 2, Nos. 4 and 6). The wall of the germ tube is thin, 0.2-0.5 pm, electron-transparent and frequently surrounded by a mass of electron-dense particles (Plate 3, No. 7). The walls after chemical cleaning have a fibrous structure (Plate 3, No. 8), indistinguishable from that of the spores. Cytochemical tests show that the wall is mainly chitin. Shortly after emergence, although not at any particular stage in germ tube elongation, a cross wall forms at the junction of the germ tube and the mother cell (Plate 2, Nos. 4 and 5). The plasmalemma invaginates and wall material is laid down on the side of the membrane away from the cytoplasm (Plate 2, No. 5). The mature septum has a three-layered structure and has a pore at its centre (Plate 3, No. 7).

Throughout germination the cytoplasmic structure is often distorted. Its appearance suggests a rapid streaming of cytoplasm into the germ tube (Plate 2, No. 4). Although vacuoles develop within the germinating cell swelling cannot be detected (Plate 2, No. 4). It is therefore possible that imbibition of water into the inelastic spore could produce the pressure which causes the cytoplasmic streaming. The most prominent organelles in the germinating cell and the germ tube are the mitochondria (Plate 3, Nos. 7 and 9), which are usually quite small though occasionally a larger invaginated one occurs (Plate 3, No. 9).

Vesicles presumably produced by the endoplasmic reticulum (Plate 3, No. 9) appear to pass through the plasmalemma of the germ tube (Plate 3, No. 9) or spore-cell near to the point of germ tube emergence (Plate 4, No. io). These vesicles give rise to lomasomes (Plate 4, No. io) associated with the rapid wall formation. This is particularly evident in the germ tube near the tip of which it is difficult to identify any single bounding membrane of the cytoplasm (Plate 3, No. 9). Lomasomes also occur in young hyphae where they appear as irregular raised areas on the inside surface of the plasmalemma (Plate 4, Nos. i i and I2) and reveal the vesicles lying within them when fractured (Plate 4, Nos. i i and I2). They are very similar to lomasomes found in chemically fixed material (Plate 4, No. I3). The endoplasmic reticulum sometimes seems to be nearly continuous between the nucleus and the plasmalemma (Plate 5, No. I4). I suggest that this indicates that wall-building activity may be under the direct control of the nucleus.

The plasmalemma of the germ tubes and hyphae has a smooth surface (Plate 4, No. i i) without the rectangular invaginations that occur in spores of this and other species.

Ribosomes are more frequent in the germinating cell than in the germ tube itself (Plate 5, No. I 5). This is presumably the result of the increase in the metabolic activity prior to and during germination; later the germ tube also becomes densely packed with ribosomes (Plate 5, No. i6).

Young hyphae have the same structure as the germ tubes. As they become older the first change is the formation of vacuoles (Plate 6, No. I7; Plate 7, No. I9). The cytoplasmic organelles are still densely packed and the vacuoles are often distorted. Storage products such as lipids are also present (Plate 7, No. I9). The wall is beginning to develop two ill- defined layers denoted by differences in texture in Plate 7, No. i9. In chemically fixed and sectioned material this layering of the wall is more distinct (Plate 6, No. I7). A very thin electron-transparent layer is formed next to the cytoplasm, and the outer wall and the septa are melanized, though not so much as in the spore walls. The walls are covered with a loose membranous layer (Plate 6, No. I7). The septa have Woronin bodies near



290 RICHARD CAMPBELL

them which plug the pores when the hyphae are broken (Plate 6, No. I7). The endoplasmic reticulum and ribosomes are less prominent than in the young hypha or germ tube (compare Plate 6, No. I7 with Plate 5, No. i6).

Cytoplasm degenerates as a hypha ages and finally most of the cell is filled with one or more vacuoles and the thin lining of cytoplasm contains little but a few mitochondria and disorganized membranous material (Plate 6, No. I8).

Conidiophore structure The ultrastructure of the conidiophores is indistinguishable from that of the

mature hyphae except that the terminal cell has a pore in its tip, and, after spore production, an annulus. Spores are produced in the same way as already described (Campbell, I968), by the extension of the inner wall layer of the conidiophore through the pore at its apex. The apical cell, which was the mother cell of the first spore of the chain, becomes sealed off from the rest of the conidiophore by a plug in the septal pore and its cytoplasm degenerates at maturity (Plate 8, No. 20).

DISCUSSION

This study has utilized several different preparatory techniques for electron microscopy; the methods were complimentary. Freeze-etching is a comparatively new technique but some excellent reviews are available (Moor, I966; Kochler, I968; Staehelin, I968).

The ultrastructure of exogenously dormant spores is compatible with the cells being in a quiescent state. The peripheral arrangement of most of the organelles in the cell would allow any change in the environment favouring germination, to be rapidly detected.

The conidiophores were very similar to the secondary conidiophores which develop from spores, as previously described (Campbell, I968).

The generalization that conidia germinate by the growth of the inner wall layer is confirmed by this study. Growth of this wall is thought to be brought about, in part at least, by the discharge of endoplasmic reticulum vesicles through the plasmalemma to form lomasomes. Lomasomes have often been reported (see Marchant and Robards, I968 for a review) but they have not previously been described in freeze-etched material. Bracker (I967) considered it possible that they were artifacts of chemical fixation. Their demonstration by freeze-etching makes it highly probable that they are real structures, but none of the very complex forms, usually referred to as plasmalemmasomes (Marchant and Robards, I968) have been seen, though they occur in some material of Alternaria brassicicola that has been chemically fixed (Campbell, I968). I suggest that the large membranous, tubular or vesicular invaginations of the plasmalemma may be fixation artifacts but that the simple vesicles between the plasmalemma and the cell wall are real structures concerned with wall formation.

Though lomasomes and endoplasmic reticulum vesicles contribute to wall formation other structures have also been assigned this role. Moor and Muhlethaler (I963) and Sassen, Remsen and Hess (I967) suggested that some of the particles on the plasmalemma surfaces were enzyme complexes concerned with the synthesis of fibrous wall polymers (see Northcote, I969 for review). No evidence for this has been obtained here, apart from the presence of some plasmalemma particles, but such a mechanism of wall formation is not precluded.

The main methods of wall formation are therefore centred on the plasmalemma. All major growth in the walls is from the secondary, inner wall layer in contact with the



Electron microscopy of Alternaria 29I

plasmalemma. The primary wall shows no appreciable growth, only melanization. As the walls thicken only the region next to the plasmalemma retains the capacity for further growth and all the new growth points therefore arise in this region. Thus the walls of spores and secondary conidiophores are produced from the secondary wall (Campbell, I968) as are the germ tubes described in this study. This results in an alternation of the wall layers, the secondary wall of the conidiophore or spore growing out and becoming the primary of the next spore or germ tube.

The lack of rectangular grooves on the outer surface of the plasmalemma of germ tubes and hyphae is interesting since they occur in the spores of Alternaria brassicicola (Campbell, I969), in Penicillium conidia (Sassen et al., I967) and in yeast (Moor and Muhlethaler, I963). Remsen et al. (I967) noted that they were absent from the germ tubes of the Penicillium and Hess (I968) reported without photographs that the grooves were uncommon in hypae of Pyrenochaeta. The abundance of the grooves apparently varies with the stage in the life cycle; they may therefore be concerned with the production of some part of the wall that is characteristic of conidia rather than of vegetative cells of filamentous fungi. This would not, however, account for their presence in yeast, where it has been pointed out that they increase the plasmalemma surface area (Moor and Muhlethaler, I963).

There are many more mitochondria in the germinating spore than in either the dormant or the mature one. These new mitochondria are thought to arise by the division of the pre-existing ones and constricted mitochondria, which could be interpreted as being in the process of division, have been seen.

ACKNOWLEDGMENTS

I wish to express my thanks to the following: Mr A. B. Britton for instruction in the use of the freeze-etching apparatus and for much helpful advice during this study, and to Professor L. E. Hawker, Dr M. F. Madelin and Dr A. Beckett for advice and discussion in the preparation of this paper. The work here reported formed part of a Ph.D. thesis presented at Bristol University.

REFERENCES

BRACKER, C. E. (I967). Ultrastructure of fungi. A. Rev. Phytopath., 5, 343. BRENNER, D. M. & CARROLL, G. C. (I968). Fine-structural correlates of growth in hyphae of Ascodesmis

sphaerospora. J7. Bact., 95, 658. CAMPBELL, R. (I968). An electron microscope study of spore structure and development in Alternaria

brassicicola. Jt. gen. Microbiol., 54, 38 I . CAMPBELL, R. (I969). Further electron microscope studies of spore structure in Alternaria brassicicola.

Arch. Mikrobiol., 69, 6o. HAWKER, L. E. (I965). Fine structure of fungi as revealed by electron microscopy. Biol. Rev., 40, 52. HAWKER, L. E. (I966). Germination: morphological and anatomical changes. In: The Fungus Spore (Ed.

by M. F. Madelin), p. I5x. London. HESS, W. M. (I968). Ultrastructural comparisons of fungus hyphal cells using frozen-etched replicas and

thin sections of the fungus Pyrenochaeta terrestris. Can. 3t. Microbiol., 14, 205. KOEHLER, J. K. (I968). The technique and application of freeze-etching in ultrastructural research.

Adv. biol. med. Phys., 12, I. MARCHANT, R. & ROBARDS, A. W. (I968). Membrane systems associated with the plasmalemma of plant

cells. Ann. Bot., 32, 457. MOOR, H. (I966). Use of freeze-etching in the study of biological ultrastructure. Int. Rev. expl. Path., 5,

179. MOOR, H. & MUHLETHALER, K. (I963). Fine structure in frozen-etched yeast cells. 5'. Cell Biol., 17, 609. NORTHCOTE, D. H. (I969). Fine structure of cytoplasm in relation to synthesis and secretion in plant cells.

Proc. R. Soc. B, 173, 2I. REMSEN, C. C., HESS, W. M. & SASSEN, M. M. A. (I967). Fine structure of germinating Penicillium mega-

sporum conidia. Protoplasma, 64, 439.



292 RICHARD CAMPBELL REYNOLDS, E. S. (I963). The use of lead citrate at high pH as an electron-opaque stain in electron micro-

scopy. J. Cell Biol., 17, 208. SASSEN, M. M. A., REMSEN, C. C. & HESS, W. M. (I967). Fine structure of Penicillium megasporum conidio-

spores. Protoplasma, 64, 75. STAEHELIN, L. A. (I968). The interpretation of freeze-etched artificial and biological membranes. Y'.

Ultrastruct. Res., 22, 326. WERNER, H. J. & LINDBERG, G. D. (I966). Electron microscope observations of Helminthosporium victoriae.

Y'. gen. Microbiol., 45, I23.

EXPLANATION OF PLATES i-8 Key to lettering: A, annulus; ER, endoplasmic reticulum; GC, germinating cell of spore; GT, germ tube; L, lipid droplet; Lo, lomasome; M, mitochondrion; Me, membrane covering wall; N, nucleus; NP, nuclear pores; P, plasmalemma; PW, primary wall of spore; R, ribosome; SW, secondary wall of spore; V, vacuole; Ve, vesicle. The broad arrow at the bottom left of photographs of freeze-etched replicas indicates the direction of the platinum- carbon shadow.

PLATE I

No. i. A spore that has been kept exogenously dormant for I7 weeks. The primary wall is thick and very heavily melanized. The endoplasmic reticulum and mitochondria lie around the periphery of the cells and the nuclei have no pores in their envelopes. Note the scarcity of storage products, only a little lipid remains in the lower cell. KMnO4; x 7300. No. 2. The plugged basal pore in the main spore wall. Glutaraldehyde-acrolein; x 32,000. No. 3. The plugged septal pore of a spore that has only just entered dormancy. The abundant endoplasmic reticulum suggests that the walls are still being thickened. KMnO4; X 25,000.

PLATE 2

No. 4. A germinating cell in a mature spore. Note how the wall of the germ tube arises from the secondary wall of the spore and a septum forms across the base of the germ tube. There are vacuoles in the germinating cell and the germ tube is surrounded by electron-dense particles. KMnO4; x 6500. No. 5. Detail of the septum formation at the base of the germ tube. The wall material is laid down within the invaginated plasmalemma. KMnO4; x 45,000. No. 6. Light micrograph of a germinating spore showing the mechanical splitting of the melanized primary wall. X I200.

PLATE 3 No. 7. A completed septum with a central pore across the base of a germ tube. Note the large numbers of mitochondria. KMnO4; x I4,500. Inset: The septum is composed of three ill- defined layers, two granular ones on either side of an electron-transparent one. KMnO4; x 37,500. No. 8. The wall of the germ tube, after chemical cleaning and heavy metal shadowing, showing the fibres which are probably chitin. x 67,500. No. 9. A young germ tube with much endoplasmic reticulum and many mitochondria. The endoplasmic reticulum is producing vesicles which appear to pass through the plasmalemma (at arrow) and probably contribute to wall formation. KMnO4; X 29,000.

PLATE 4 No. io. Part of the wall of a spore near to the point of germ tube emergence. Vesicles lie near the plasmalemma and appear to pass through it (at arrow) to form lomasomes. Freeze-etched; x 32,000.

No. i I. The inner surface of the plasmalemma of a young hypha with raised areas caused by the underlying lomasomes, vesicles of which can be seen in cross-fracture (at arrow). Freeze-etched; X 26,250.

No. I2. A fracture across a lomasome containing a single vesicle. Note, just under the plasmalemma, other vesicles which may later form lomasomes. Freeze-etched; x 6o,ooo. No. I3. A lomasome in sectioned material. KMnO4; x 76,ooo.



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No. I4. A part of a germinating cell with the endoplasmic reticulum producing vesicles. The endoplasmic reticulum is associated with both the nucleus and the plasmalemma. KMnO4; X I7,500.

No. I 5. The junction of a germinating cell and the germ tube; the former has many more ribosomes than the latter. Note the Woronin body near the pore in the septum. Glutaraldehyde- acrolein; x 48,ooo. No. I6. The cytoplasm of a young hypha with more ribosomes than the germ tube of No. I 5. Glutaraldehyde-acrolein; x 48,ooo.

PLATE 6 No. I7. A mature hypha; the walls are melanized, have two indistinct layers and are covered with a loosely attached membrane. The septal pore is plugged, probably by a Woronin body, where the hypha has been broken. The cytoplasm contains vacuoles, mitochondria and a nucleus. There are some ribosomes but not as many as in the young hypha of No. i6. Glutaraldehyde-acrolein; x I2,750. No. i8. An old hypha with a large central vacuole and degenerating cytoplasm containing only a few mitochondria with sparse cristae. Glutaraldehyde-acrolein; x I5,000.

PLATE 7 No. i9. A mature hypha showing a similar structure to that of No. I7. In addition lipid droplets, pores in the nuclear envelope and lomasomes are present. The layers of the wall are not as distinct as in sectioned material but are denoted by a difference in texture (see on the right of the photograph). Freeze-etched; x 28,ooo.

PLATE 8 No. 20. The tip of a conidiophore after spore production. The terminal cell has been sealed off by a plug in the septal pore (at arrow) and its cytoplasm has degenerated. Note the annulus around the pore from which the spore was produced. The cytoplasm of the penulti- mate cell is very similar to that of the mature hypha (compare with No. I7). Glutaraldehyde- acrolein; x 27,000.



an electron microscope study of exogenously dormant spores, spore germination, hyphae and...

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