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Conidium ontogeny in the Chalara state of Ceratocystis adiposa
Trans. Br, mycol. Soc. 68 (2) 267-276 (1977)
Printed in Great Britain
CONIDIUM ONTOGENY IN THE CHALARA STATE OF CERATOCYSTIS ADIPOSA II. ELECTRON MICROSCOPY By C. R. HAWES AND A. BECKETT Department of Botany, The University of Bristol The conidiogenous locus in the Chalara state of Ceratocystis adiposa (Butl.) C. Moreau is a meristem. A mechanism is proposed to account for the formation of conidia of different shapes within a single chain. The results are discussed in relation to the concept of a typical phialide and to the recommendations of the Kananaskis conference on the Taxonomy of Fungi Imperfecti. Ontogenetic modes in the hyphomycetes are currently distinguished on the basis of variations in the relationship between wall layers of conidia and conidiogenous cells. These variations usually occur at the apex of the conidiogenous cell and are frequently beyond the limits of resolution of the light microscope. Recent ultrastructural studies on conidiogenesis in phialidic deuteromycetes have helped to clarify the relationship between the walls of the newly formed conidium (Buckley, Wyllie & DeVay, 1969; Campbell, 1972, 1975; Hammill, 1972, 1974; Carroll & Carroll, 1974; Olah & Reisinger , 1974). These studies dealt only with conidial production by 'blowout' at the tip of a phialide as defined in subsection IVA of Tubaki's (1958) revision of Hughes's (1953) scheme. The only ultrastructural study of an example of Tubaki's subsection IVB in which conidia are formed in basipetal succession within a conidiogenous cell (endogenously) is that by Delvecchio, Corbaz & Turian (1969) on Thielaviopsis basicola (Berk. & Br.) Ferraris. They showed that the conidial wall was distinct from the conidiogenous cell wall, but they did not show the conidiogenous locus and therefore the origin of the conidial wall. Subramanian (1971, 1972) suggested that conidia of Thielauiopsis were formed by cleavage of the cytoplasm in the conidiogenous cell and were subsequently enclosed by a wall, which was formed de novo. Hawes & Beckett (1977a) showed that Ceratocystis adiposa (Butl.) C. Moreau produced conidia both from the tip and within the conidiogenous cell. The work reponed here concerns the ultrastructural details of the ontogenetic process involved.
MA TERIALS AND METHODS
The culture of Ceratocystis adiposa used was as described in Hawes & Beckett (1977a).
Transmission electron microscopy Sterile cellophane disks on the surface of 3 % (w jv) malt agar plates were inoculated with a few drops of a concentrated conidial suspension of C. adiposa. This was spread evenly over the surface of the cellophane and incubated at 25 °C for 2 days. Cultures were fixed by flooding the plates with a mixture of 1 % (v jv) glutaraldehyde and 0'5 % (w jv) formaldehyde in 0'1 M sodium cacodylate buffer at pn 7'2 for 45 min ..The cellophane disks were then stripped off the agar surface, washed in buffer and post-fixed in 1 % (w jv) osmium tetroxide for 2 h. To ensure adequate wetting of the conidiophores the surfactant' Brij 35' was used as a 0'1 % (w jv) solution in the fixative. After washing in buffer and distilled water the disks were soaked overnight in 0'5 % aqueous uranyl acetate at 4 °C. Following dehydration in a graded ethanol /water series the disks were embedded in Spurr's resin (Spurr, 1969), sectioned with a diamond knife on an LKB Ultrotome III, stained with lead citrate and examined with an AEI EM 6 G electron microscope. Scanning electron microscopy Cultures were prepared on cellophane disks as described above and fixed for 1 h in 4 % (v jv) glutaraldehyde in 0'1 M sodium cacodylate buffer at pn 7'2. Pieces of the disks were washed and dehydrated in graded ethanoljwater and ethanol j amyl acetate series culminating in pure amyl acetate. The specimens were then dried in a Polaron E 3000 critical point drying apparatus, mounted on stubs, coated with gold in a modified
268
Conidium ontogeny in Ceratocystis. II
... " .
Fig . 1. First conidium initial. Note newly formed inner wall layer (small arrows) and break in conidiogenous cell wall (large arrows). x 28000. Fig. 2. Later stage of first conidium formation showing a break in the conidiogenous cell wall (large arrows), and the proximal extension of the new wall layer to the point marked with double arrow. x 13700.
Polaron sputter coating unit and examined with a Cambridge Scientific Instrument Company s4 Scanning electron microscope. RESULTS
The first conidium is formed as a 'blowout' at the tip of the conidiogenous cell and is enclosed by a new wall formed on the inner side of the conidiogenous cell wall (Fig. 1, small arrows). The outer wall subsequently breaks (Fig. 2, large arrows) and in so doing marks the tip of the conidiogenous cell. At this stage the conidium wall tapers off at a point some 3 pm below the conidiogenous cell tip (Fig. 2 double arrow). Continued growth of this layer results in subsequent conidium formation (see below). Smooth endoplasmic reticulum occurs at
the periphery of the conidiogenous cell (Figs. 1-3 a). At the apex of the developing conidium the endoplasmic reticulum is vesicular (Fig. 1). As the conidiogenous cell matures the outer wall becomes electron-opaque owing to deposition of a pigment, presumably melanin (Figs. 3-8). The inner wall is seen as an electron-transparent cylinder of material within the neck of the conidiogenous cell and is tapered at the base (Figs. 3) 5). The conidiogenous cell contains a single nucleus) endoplasmic reticulum, numerous mitochondria and ribosomes (Figs. 2-5). Young conidia often contain lipid droplets (Figs. 4, 6) 8). After the first conidium has formed, the pigmented wall of the conidiogenous cell becomes flared at the tip (Figs. 3, 14, 18). Subsequent conidium production occurs by growth and septation
C. R. Hawes and A. Beckett
.'. .'
Fig. Fig. Fig. Fig.
3. Mature conidiogenous cell and meristem prior to formation of a new conidium. x 11000. 3 Q • Portion of meristem lined with smooth endoplasmic reticulum. x 23500. 4. Blowout of a globose conidium at the tip of a conidiogenous cell. x 6400. 5. Area where new wall tapers off at the base of the meristem. x 30000.
27°
Conidium ontogeny in Ceratocystis. II
t
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c. R. Hawes and A. Beckett of the inner wall. This produces a variety of conidial sizes and shapes ranging between globose (Figs. 4, 13, 17, 19, 20), when septation is at the apex of the conidiogenous cell and the conidium expands (Fig. 12 A1); pyriform (Figs. 6-8, 12 A2, 12B), when septation is within the neck of the conidiogenous cell, and the conidium expands at the apex; and cylindrical (Figs. 12 A3, 16), when septation takes place nearer the base of the conidiogenous cell so that the conidium is formed entirely within it and does not subsequently expand. The septum is single layered (Fig. 7) and perforate when formed, but becomes multilayered as the conidium wall matures (Fig. 6). As the next conidium starts to develop the septum partially splits along the septal plate (Fig. 8, arrows; Fig. 11, arrows), leaving the cytoplasm of the conidiogenous cell capped with wall material (Fig. 3). If these conidia occur in permanent chains (Hawes & Beckett, 1977a) the septal pores are often plugged by Woronin bodies or other material (Figs. 10, 12C, 19, 20) thus maintaining structural continuity between walls of adjacent conidia. At maturity conidial walls are three layered (Fig. 6). The basipetal chains of conidia (Fig. 13) are enclosed within a membranous sheath which extends from the neck of the conidiogenous cell to the apical conidium of the chain. This is clearly seen in disrupted chains (Figs. 19,20, arrows). In thin sections the sheath is seen as a thin electronopaque skin which is closely appressed to the outer layer of the conidium and which extends down into the neck of the conidiogenous cell between the outer wall of the conidium and the wall of the conidiogenous cell (Fig. 6, arrows; Fig. 12B). The basal cell of the conidiophore is delimited from its subtending hypha by a layered, perforate septum (Fig. 9). The wall of the conidiophore is continuous with the wall of the hypha (Figs. 9, 15). Electron-opaque material at the base of the conidiophore is probably mucilage from the surface of the hypha (Fig. 9, arrows). DISCUSSION
Conidium ontogeny 'Blowout' of the first conidium in a chain of C. adiposa involves rupture of the conidiogenous cell
271
wall before the conidium has completely formed. Subsequently the conidium enlarges by expansion of a newly formed inner wall. Since the conidium is not formed from material from the wall of the conidiogenous cell, the process is enteroblastic. Synthesis of this new wall layer within the conidiogenous cell continues proximally, forming a cylinder of wall material enclosing the protoplast in the upper half of the cell (e.g. Fig. 3). Subsequent conidial production involves the distal extension of this new wall, material being incorporated by intussusception, and an apical extension of the protoplast. Other ultrastructural studies of typical phialides have shown a thickening and lamellation of wall material at the apex. This thickening forms as a result of the sequential layering of wall material, each layer corresponding to the production of one conidium. Repeated conidium production ultimately leads to a plugging of the neck of the phialide (Campbell, 1972, 1975; Hammill, 1972; Beckett, Heath & McLaughlin, 1974; Olah & Reisinger, 1974). This apposition of new wall material as in a typical phialide does not occur in C. adiposa. Its absence is an important feature of distinction between the ontogenetic mode of this fungus and that of other phialidic hyphomycetes so far critically studied at the ultrastructural level. Associated with the area of wall synthesis in C. adiposa is the presence at the periphery of the protoplast of sheets of smooth endoplasmic reticulum. The latter is possibly involved in the mediation of wall synthesis. A similar association of endoplasmic reticulum with specific areas of wall synthesis occurs in developing ascospores of Saccharomyces cerevisiae Hansen (Beckett et al. 1974). This zone of wall synthesis in the conidiogenous cell corresponds with the area of fluorescence reported by Hawes & Beckett (1977 a). Both of these features support the suggestion that this region is a meristem. Conidia are delimited from the conidiogenous cell by septation at a variable point along this meristem. Thus a cylindrical conidium formed in the neck of the conidiogenous cell (Fig. 12 A3), has the same mode of ontogeny as a globose conidium 'blown out' from the tip (12 Al), except that in the former, the wall of the conidiogenous cell prevents any expansion of the conidium before the conidium wall becomes inextensible. This mode of
Fig. 6. Pyriform conidium in neck of a conidiogenous cell. x 9500. Fig. 7. Early stage in formation of pyriform conidium. x 9300. Fig. 8. Pyriform conidium. Note the split (arrows) along the septal plate. x 8900. Fig. 9. Basal cell of conidiophore. Note deposit (mucilage or pigment?) at point of emergence from repent hypha (arrows). x 14300.
Conidium ontogeny in Ceratocystis. II
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C. R. Hawes and A. Beckett ontogeny has also been found in Thielaoiopsis basicola (Hawes & Beckett, 1977b). However in T. basicola the conidia are delimited by septation at a fixed point within the conidiogenous cell. What controls the location of the septum within the meristem of C. adiposa? Several factors, either individually or in combination, may regulate septation within the meristem on both a spacial and temporal basis. Structural or pressure changes within the cytoplasm, the point at which the nucleus divides within the conidiogenous cell, the relative rates of synthesis and /or ageing of the wall at the proximal and distal ends of the meristem are examples of such factors. If septation was a regular cyclic process at the temporal level, then an imbalance between the above factors could lead to irregularities in the spacial control of the septum. Once the septum has formed, the wall distal to it might change chemically and rigidify. This would presumably prevent further alteration in shape and size of the conidium. Relatively little is known of this process of wall rigidification but it has been suggested that it may involve the deposition of non-extensible secondary wall material onto the extensible primary wall and /or the formation of cross-linkages between existing primary wall polymers. Robertson (1968) suggested the former process in rigidification of hyphal tips but recent work with Neurospora crassa Shear & Dodge and Geotrichum candidum Link ex Persoon (Trinci & Collinge, 1975), has shown that rigidification at vegetative hyphal tips is more likely to involve either cross-linkage between primary wall polymers or the cessation of fusions between apical wall vesicles and the plasma membrane/cell wall. In C. adiposa no observable change in thickness of the conidium wall occurs until septation is complete. Rigidification, as in N. crassa, is not therefore likely to involve a deposition of secondary wall material. A model for conidium formation in C. adipose must assume a balance between wall synthesis along the meristem (Fig. 12B, small arrows) and the apical extension growth of the wall (Fig. 12B, large arrow), since there is no recurrent thickening of the wall along the meristem,
273
Conidial chains The sheath surrounding permanent conidial chains is shown to originate from inside the neck of the conidiogenous cell (see also Hawes & Beckett, 1977 a). This sheath is equivalent to the 'inner wall' of the conidiogenous cell which Hutchinson (1939) described as surrounding the conidia of C. major. Although the ontogeny of the easily fragmented chains (Hawes & Beckett, 1977a) is not shown here, all conidiogenous cells so far observed in thin sections have shown the same mode of conidium formation and this probably includes the easily fragmented chain type. In median sections a pore can be seen in the septum between the newly formed conidium and the conidiogenous cell protoplast (Figs. 10, 12C). Contrary to the observation on Phialocephala of Carroll & Carroll (1974), in C. adiposa this pore can persist while the conidia within a chain mature. For the chain to fragment this septum has to split (Figs. 8, 11). If the septum happened to form without a pore or if the pore was plugged soon after formation, the septum could split while the conidium was still at the base of a chain, thu s an easily fragmented chain, possibly held together only by a sheath, could arise. As in Stachybotrys and Memnoniella (Campbell, 1972, 1975) the chain type formed is dependent on the strength of the septum between adjacent conidia. In T. basicola (Hawes & Beckett, 1977 b), no sheath is present and persistent chains do not form. Concept of the phialide The Kananaskis conference (Kendrick, 1971) made a series of recommendations on the terminology to be used when describing conidiation in Deuteromycotina. Although the results reported here show conidiogenesis in C. adiposa to be of a mode hitherto undescribed, we will as far as possible adhere to the terminology recommended. The inner wall in the neck of the conidiogenous cell is a meristem since along this region growth occurs. The whole of this meristem is the conidiogenous locus which is defined as 'a point, area, or zone of a conidiogenous cell at which a conidium arises'. The conidiogenous cell of C. adiposa therefore complies with the definition of a phialide
Fig. 10. Septum and pore between conidium and conidiogenous cell. x 18500. Fig. 11. Septum betweentwo conidia in a chain. x 13500. Fig. 1~ . (A) Diag~nlIn.atic representation of the v~ation possible in conidium shape; 1, globose; 2., pyriform; 3, cylindrical. m = menstem. (B) DIagram of the wall layers involved in conidium formation showing the originof the sheath (s). Seediscussion for explanation of arrows. c.w. = conidium wall; .c.c.w. = conidiogenous cell wall; c.t, = collarette; m = meristem; p = plasma membrane. (C) DIagram of the septum between conidium (c) and conidiogenous cell (c.c). 10
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274
Conidium ontogeny in Ceratocystis. II
Fig. 13. Permanent chains. x 1600. Fig. 14. High power of Fig. 13. Note flared wall of conidiogenous cell (arrow). x 8400. Fig. 15. Base of conidiophore. x 6900. Fig. 16. Cylindrical conidium in neck of conidiogenous cell. x 7100.
C. R. Hawes and A. Beckett
275
Fig. 17. Globose conidium. x 7000. Fig. 18. Conidium in flared neck of conidiogenous cell. x 9200. Figs. 19, 20. The sheath around conidial chains (arrows). Note also the septal scars at the tip of each conidium. x 7500. 10-2
276
Conidium ontogeny in Ceratocystis. II
which is given as 'a conidiogenous cell which produces from a fixed conidiogenous locus, a basipetal succession of enteroblastic conidia whose walls arise de novo'. We do not consider the meristem to be a wall layer of the conidiogenous cell but to be a new wall which lines that of the conidiogenous cell. It was also recommended (Kendrick, 1971) that' any phialide wall distal to the conidiogenous locus ...' be termed the collarette. We suggest that in C. adiposa the portion of the wall of the conidiogenous cell which overlies the meristem be termed the collarette irrespective of where along its length septation and thus conidiation occurs. As a result of this and previous ultrastructural studies on phialides it can be seen that the subsections IVA and IVB of Tubaki's (1958) scheme are very clearly separated when the role of wall layers in conidiation are considered. Subsection IVA includes phialides where the conidia are formed at the apex by apposition of new wall material which 'blows out' to form a conidium, e.g, Stachybotrys and Memnoniella (Campbell, 1972, 1975). Subsection IVB includes phialides where conidia are produced at the apex or in the neck by septation of a meristem bounded by a collarette, e.g , the Chalara state of C. adiposa and T. basicola (Hawes & Beckett, 1977b). We are grateful to Drs R. Campbell and M. F. Madelin for helpful discussion during preparation of the manuscript and to Mr R. Porter for skilful technical assistance and for building the sputter coating unit. Thanks are also due to the Science Research Council for a Research Studentship (to C.R.H.) and for a Research Grant (BjSRj90718 to A.B.). REFERENCES
BECKETT, A., HEATH, I. B. & McLAUGHLIN, D. J. (1974). An atlas of fungal ultrastructure. London: Longman. BUCKLEY, P. M., WYLLIE, T. D. & DEVAY, J. E. (1969). Fine structure of conidia and conidium formation in Verticillium albo-atrum and V. nigrescens, Mycologia 61, 240-250. CAMPBELL, R. (1972). Ultrastructure of conidium ontogeny in the deuteromycete fungus Stachybotrys atra Corda. New Phytologis: 71, 1143-1149.
CAMPBELL, R. (1975)· The ultrastructure of the formation of chains of conidia in Memnoniella echinata. Mycologic 67, 760-769· CARROLL, G. C. & CARROLL, F. E. (1974)· The fine structure of conidium development in Phialocephala dimorphospora. Canadian Journal of Botany 52, 2119-2128. D ELVECCHIO,. V G ., CORBAZ,. R & T URIAN,. G (196) 9. An ultrastructural study of the hyphae, endoconidia and chlamydospores of Thielaoiopsis basicola, Journal of General Microbiology 58, 23-27. HAMMILL, T. M. (1972). Electron microscopy ofphialo conidiogenesis in Metarhizium anisopliae. American Journal of Botany 59, 317-326. HAMMILL, T. M. (1974)· Electron microscopy of phialides and conidiogenesis in Trichoderma saturnisporium. American Journal of Botany 61, 15-2 4. HAWES, C. R. & BECKETT, A. (1977a) . Conidium ontogeny in the Chalara state of Ceratocystis adiposa. I. Light microscopy. Transactions of the British Mycological Society 68, 259-265. HAWES, C. R. & BECKETT, A. (1977 b). Conidium ontogeny in Thielaviopsis basicola. Transactions of the British Mycological Society 68,304-3°7. HUGHES, S. J. (1953). Conidiophores, conidia and classification. Canadian Journal of Botany 31, 5776 59· HUTCHINSON, S. A. (1939). Macroconidial formation in Ophiostoma majus (van Beyma) Goidanich . Annals of Botany 3, 795-802. KENDRICK, W. B. (1971). Taxonomy of the Fungi Imperfecti, Toronto: University of Toronto Press. OLAH, G. M. & REISINGER, O. (1974). Etude ultrastructurale et cytochirnique de l'appareil sporifere chez Phialophora richardsiae. Canadian Journal of Botany 52, 2473-2480. ROBERTSON, N. F. (1968). The growth process in fungi. Annual Review of Phytopathology 6, 115-136. SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research 26, 31-43. SUBRAMANIAN, C. V. (1971). The phialide. In Taxonomy of Fungi Imperfecti (ed. W. B. Kendrick), pp. 92-119. Toronto: University of Toronto Press. SUBRAMANIAN, C. V. (1972). Conidial chains, their nature and significance in the taxonomy of hyphomycetes. Current Science 41, 43-49. TRINCI, A. P. J. & COLLINGE, A. J. (1975). Hyphal wall growth in Neurospora crassa and Geotrichum candidum. Journal of General Microbiology 91, 355361. TUBAKI, K. (1958). Studies on the Japanese hyphomycetes: V. Journal of the Hattori Botanical Laboratory 20, 142-244.
(Accepted for publication 29 September 1976)
Printed in Great Britain
CONIDIUM ONTOGENY IN THE CHALARA STATE OF CERATOCYSTIS ADIPOSA II. ELECTRON MICROSCOPY By C. R. HAWES AND A. BECKETT Department of Botany, The University of Bristol The conidiogenous locus in the Chalara state of Ceratocystis adiposa (Butl.) C. Moreau is a meristem. A mechanism is proposed to account for the formation of conidia of different shapes within a single chain. The results are discussed in relation to the concept of a typical phialide and to the recommendations of the Kananaskis conference on the Taxonomy of Fungi Imperfecti. Ontogenetic modes in the hyphomycetes are currently distinguished on the basis of variations in the relationship between wall layers of conidia and conidiogenous cells. These variations usually occur at the apex of the conidiogenous cell and are frequently beyond the limits of resolution of the light microscope. Recent ultrastructural studies on conidiogenesis in phialidic deuteromycetes have helped to clarify the relationship between the walls of the newly formed conidium (Buckley, Wyllie & DeVay, 1969; Campbell, 1972, 1975; Hammill, 1972, 1974; Carroll & Carroll, 1974; Olah & Reisinger , 1974). These studies dealt only with conidial production by 'blowout' at the tip of a phialide as defined in subsection IVA of Tubaki's (1958) revision of Hughes's (1953) scheme. The only ultrastructural study of an example of Tubaki's subsection IVB in which conidia are formed in basipetal succession within a conidiogenous cell (endogenously) is that by Delvecchio, Corbaz & Turian (1969) on Thielaviopsis basicola (Berk. & Br.) Ferraris. They showed that the conidial wall was distinct from the conidiogenous cell wall, but they did not show the conidiogenous locus and therefore the origin of the conidial wall. Subramanian (1971, 1972) suggested that conidia of Thielauiopsis were formed by cleavage of the cytoplasm in the conidiogenous cell and were subsequently enclosed by a wall, which was formed de novo. Hawes & Beckett (1977a) showed that Ceratocystis adiposa (Butl.) C. Moreau produced conidia both from the tip and within the conidiogenous cell. The work reponed here concerns the ultrastructural details of the ontogenetic process involved.
MA TERIALS AND METHODS
The culture of Ceratocystis adiposa used was as described in Hawes & Beckett (1977a).
Transmission electron microscopy Sterile cellophane disks on the surface of 3 % (w jv) malt agar plates were inoculated with a few drops of a concentrated conidial suspension of C. adiposa. This was spread evenly over the surface of the cellophane and incubated at 25 °C for 2 days. Cultures were fixed by flooding the plates with a mixture of 1 % (v jv) glutaraldehyde and 0'5 % (w jv) formaldehyde in 0'1 M sodium cacodylate buffer at pn 7'2 for 45 min ..The cellophane disks were then stripped off the agar surface, washed in buffer and post-fixed in 1 % (w jv) osmium tetroxide for 2 h. To ensure adequate wetting of the conidiophores the surfactant' Brij 35' was used as a 0'1 % (w jv) solution in the fixative. After washing in buffer and distilled water the disks were soaked overnight in 0'5 % aqueous uranyl acetate at 4 °C. Following dehydration in a graded ethanol /water series the disks were embedded in Spurr's resin (Spurr, 1969), sectioned with a diamond knife on an LKB Ultrotome III, stained with lead citrate and examined with an AEI EM 6 G electron microscope. Scanning electron microscopy Cultures were prepared on cellophane disks as described above and fixed for 1 h in 4 % (v jv) glutaraldehyde in 0'1 M sodium cacodylate buffer at pn 7'2. Pieces of the disks were washed and dehydrated in graded ethanoljwater and ethanol j amyl acetate series culminating in pure amyl acetate. The specimens were then dried in a Polaron E 3000 critical point drying apparatus, mounted on stubs, coated with gold in a modified
268
Conidium ontogeny in Ceratocystis. II
... " .
Fig . 1. First conidium initial. Note newly formed inner wall layer (small arrows) and break in conidiogenous cell wall (large arrows). x 28000. Fig. 2. Later stage of first conidium formation showing a break in the conidiogenous cell wall (large arrows), and the proximal extension of the new wall layer to the point marked with double arrow. x 13700.
Polaron sputter coating unit and examined with a Cambridge Scientific Instrument Company s4 Scanning electron microscope. RESULTS
The first conidium is formed as a 'blowout' at the tip of the conidiogenous cell and is enclosed by a new wall formed on the inner side of the conidiogenous cell wall (Fig. 1, small arrows). The outer wall subsequently breaks (Fig. 2, large arrows) and in so doing marks the tip of the conidiogenous cell. At this stage the conidium wall tapers off at a point some 3 pm below the conidiogenous cell tip (Fig. 2 double arrow). Continued growth of this layer results in subsequent conidium formation (see below). Smooth endoplasmic reticulum occurs at
the periphery of the conidiogenous cell (Figs. 1-3 a). At the apex of the developing conidium the endoplasmic reticulum is vesicular (Fig. 1). As the conidiogenous cell matures the outer wall becomes electron-opaque owing to deposition of a pigment, presumably melanin (Figs. 3-8). The inner wall is seen as an electron-transparent cylinder of material within the neck of the conidiogenous cell and is tapered at the base (Figs. 3) 5). The conidiogenous cell contains a single nucleus) endoplasmic reticulum, numerous mitochondria and ribosomes (Figs. 2-5). Young conidia often contain lipid droplets (Figs. 4, 6) 8). After the first conidium has formed, the pigmented wall of the conidiogenous cell becomes flared at the tip (Figs. 3, 14, 18). Subsequent conidium production occurs by growth and septation
C. R. Hawes and A. Beckett
.'. .'
Fig. Fig. Fig. Fig.
3. Mature conidiogenous cell and meristem prior to formation of a new conidium. x 11000. 3 Q • Portion of meristem lined with smooth endoplasmic reticulum. x 23500. 4. Blowout of a globose conidium at the tip of a conidiogenous cell. x 6400. 5. Area where new wall tapers off at the base of the meristem. x 30000.
27°
Conidium ontogeny in Ceratocystis. II
t
.
,
t
I J
9
•
J
I
c. R. Hawes and A. Beckett of the inner wall. This produces a variety of conidial sizes and shapes ranging between globose (Figs. 4, 13, 17, 19, 20), when septation is at the apex of the conidiogenous cell and the conidium expands (Fig. 12 A1); pyriform (Figs. 6-8, 12 A2, 12B), when septation is within the neck of the conidiogenous cell, and the conidium expands at the apex; and cylindrical (Figs. 12 A3, 16), when septation takes place nearer the base of the conidiogenous cell so that the conidium is formed entirely within it and does not subsequently expand. The septum is single layered (Fig. 7) and perforate when formed, but becomes multilayered as the conidium wall matures (Fig. 6). As the next conidium starts to develop the septum partially splits along the septal plate (Fig. 8, arrows; Fig. 11, arrows), leaving the cytoplasm of the conidiogenous cell capped with wall material (Fig. 3). If these conidia occur in permanent chains (Hawes & Beckett, 1977a) the septal pores are often plugged by Woronin bodies or other material (Figs. 10, 12C, 19, 20) thus maintaining structural continuity between walls of adjacent conidia. At maturity conidial walls are three layered (Fig. 6). The basipetal chains of conidia (Fig. 13) are enclosed within a membranous sheath which extends from the neck of the conidiogenous cell to the apical conidium of the chain. This is clearly seen in disrupted chains (Figs. 19,20, arrows). In thin sections the sheath is seen as a thin electronopaque skin which is closely appressed to the outer layer of the conidium and which extends down into the neck of the conidiogenous cell between the outer wall of the conidium and the wall of the conidiogenous cell (Fig. 6, arrows; Fig. 12B). The basal cell of the conidiophore is delimited from its subtending hypha by a layered, perforate septum (Fig. 9). The wall of the conidiophore is continuous with the wall of the hypha (Figs. 9, 15). Electron-opaque material at the base of the conidiophore is probably mucilage from the surface of the hypha (Fig. 9, arrows). DISCUSSION
Conidium ontogeny 'Blowout' of the first conidium in a chain of C. adiposa involves rupture of the conidiogenous cell
271
wall before the conidium has completely formed. Subsequently the conidium enlarges by expansion of a newly formed inner wall. Since the conidium is not formed from material from the wall of the conidiogenous cell, the process is enteroblastic. Synthesis of this new wall layer within the conidiogenous cell continues proximally, forming a cylinder of wall material enclosing the protoplast in the upper half of the cell (e.g. Fig. 3). Subsequent conidial production involves the distal extension of this new wall, material being incorporated by intussusception, and an apical extension of the protoplast. Other ultrastructural studies of typical phialides have shown a thickening and lamellation of wall material at the apex. This thickening forms as a result of the sequential layering of wall material, each layer corresponding to the production of one conidium. Repeated conidium production ultimately leads to a plugging of the neck of the phialide (Campbell, 1972, 1975; Hammill, 1972; Beckett, Heath & McLaughlin, 1974; Olah & Reisinger, 1974). This apposition of new wall material as in a typical phialide does not occur in C. adiposa. Its absence is an important feature of distinction between the ontogenetic mode of this fungus and that of other phialidic hyphomycetes so far critically studied at the ultrastructural level. Associated with the area of wall synthesis in C. adiposa is the presence at the periphery of the protoplast of sheets of smooth endoplasmic reticulum. The latter is possibly involved in the mediation of wall synthesis. A similar association of endoplasmic reticulum with specific areas of wall synthesis occurs in developing ascospores of Saccharomyces cerevisiae Hansen (Beckett et al. 1974). This zone of wall synthesis in the conidiogenous cell corresponds with the area of fluorescence reported by Hawes & Beckett (1977 a). Both of these features support the suggestion that this region is a meristem. Conidia are delimited from the conidiogenous cell by septation at a variable point along this meristem. Thus a cylindrical conidium formed in the neck of the conidiogenous cell (Fig. 12 A3), has the same mode of ontogeny as a globose conidium 'blown out' from the tip (12 Al), except that in the former, the wall of the conidiogenous cell prevents any expansion of the conidium before the conidium wall becomes inextensible. This mode of
Fig. 6. Pyriform conidium in neck of a conidiogenous cell. x 9500. Fig. 7. Early stage in formation of pyriform conidium. x 9300. Fig. 8. Pyriform conidium. Note the split (arrows) along the septal plate. x 8900. Fig. 9. Basal cell of conidiophore. Note deposit (mucilage or pigment?) at point of emergence from repent hypha (arrows). x 14300.
Conidium ontogeny in Ceratocystis. II
272
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m
m
m
p
c.w.
~
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.... _ . .. -
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2
3
c.c.w, (C.I. )
m
_
c B
12
-
-
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C. R. Hawes and A. Beckett ontogeny has also been found in Thielaoiopsis basicola (Hawes & Beckett, 1977b). However in T. basicola the conidia are delimited by septation at a fixed point within the conidiogenous cell. What controls the location of the septum within the meristem of C. adiposa? Several factors, either individually or in combination, may regulate septation within the meristem on both a spacial and temporal basis. Structural or pressure changes within the cytoplasm, the point at which the nucleus divides within the conidiogenous cell, the relative rates of synthesis and /or ageing of the wall at the proximal and distal ends of the meristem are examples of such factors. If septation was a regular cyclic process at the temporal level, then an imbalance between the above factors could lead to irregularities in the spacial control of the septum. Once the septum has formed, the wall distal to it might change chemically and rigidify. This would presumably prevent further alteration in shape and size of the conidium. Relatively little is known of this process of wall rigidification but it has been suggested that it may involve the deposition of non-extensible secondary wall material onto the extensible primary wall and /or the formation of cross-linkages between existing primary wall polymers. Robertson (1968) suggested the former process in rigidification of hyphal tips but recent work with Neurospora crassa Shear & Dodge and Geotrichum candidum Link ex Persoon (Trinci & Collinge, 1975), has shown that rigidification at vegetative hyphal tips is more likely to involve either cross-linkage between primary wall polymers or the cessation of fusions between apical wall vesicles and the plasma membrane/cell wall. In C. adiposa no observable change in thickness of the conidium wall occurs until septation is complete. Rigidification, as in N. crassa, is not therefore likely to involve a deposition of secondary wall material. A model for conidium formation in C. adipose must assume a balance between wall synthesis along the meristem (Fig. 12B, small arrows) and the apical extension growth of the wall (Fig. 12B, large arrow), since there is no recurrent thickening of the wall along the meristem,
273
Conidial chains The sheath surrounding permanent conidial chains is shown to originate from inside the neck of the conidiogenous cell (see also Hawes & Beckett, 1977 a). This sheath is equivalent to the 'inner wall' of the conidiogenous cell which Hutchinson (1939) described as surrounding the conidia of C. major. Although the ontogeny of the easily fragmented chains (Hawes & Beckett, 1977a) is not shown here, all conidiogenous cells so far observed in thin sections have shown the same mode of conidium formation and this probably includes the easily fragmented chain type. In median sections a pore can be seen in the septum between the newly formed conidium and the conidiogenous cell protoplast (Figs. 10, 12C). Contrary to the observation on Phialocephala of Carroll & Carroll (1974), in C. adiposa this pore can persist while the conidia within a chain mature. For the chain to fragment this septum has to split (Figs. 8, 11). If the septum happened to form without a pore or if the pore was plugged soon after formation, the septum could split while the conidium was still at the base of a chain, thu s an easily fragmented chain, possibly held together only by a sheath, could arise. As in Stachybotrys and Memnoniella (Campbell, 1972, 1975) the chain type formed is dependent on the strength of the septum between adjacent conidia. In T. basicola (Hawes & Beckett, 1977 b), no sheath is present and persistent chains do not form. Concept of the phialide The Kananaskis conference (Kendrick, 1971) made a series of recommendations on the terminology to be used when describing conidiation in Deuteromycotina. Although the results reported here show conidiogenesis in C. adiposa to be of a mode hitherto undescribed, we will as far as possible adhere to the terminology recommended. The inner wall in the neck of the conidiogenous cell is a meristem since along this region growth occurs. The whole of this meristem is the conidiogenous locus which is defined as 'a point, area, or zone of a conidiogenous cell at which a conidium arises'. The conidiogenous cell of C. adiposa therefore complies with the definition of a phialide
Fig. 10. Septum and pore between conidium and conidiogenous cell. x 18500. Fig. 11. Septum betweentwo conidia in a chain. x 13500. Fig. 1~ . (A) Diag~nlIn.atic representation of the v~ation possible in conidium shape; 1, globose; 2., pyriform; 3, cylindrical. m = menstem. (B) DIagram of the wall layers involved in conidium formation showing the originof the sheath (s). Seediscussion for explanation of arrows. c.w. = conidium wall; .c.c.w. = conidiogenous cell wall; c.t, = collarette; m = meristem; p = plasma membrane. (C) DIagram of the septum between conidium (c) and conidiogenous cell (c.c). 10
M
v c fiR
274
Conidium ontogeny in Ceratocystis. II
Fig. 13. Permanent chains. x 1600. Fig. 14. High power of Fig. 13. Note flared wall of conidiogenous cell (arrow). x 8400. Fig. 15. Base of conidiophore. x 6900. Fig. 16. Cylindrical conidium in neck of conidiogenous cell. x 7100.
C. R. Hawes and A. Beckett
275
Fig. 17. Globose conidium. x 7000. Fig. 18. Conidium in flared neck of conidiogenous cell. x 9200. Figs. 19, 20. The sheath around conidial chains (arrows). Note also the septal scars at the tip of each conidium. x 7500. 10-2
276
Conidium ontogeny in Ceratocystis. II
which is given as 'a conidiogenous cell which produces from a fixed conidiogenous locus, a basipetal succession of enteroblastic conidia whose walls arise de novo'. We do not consider the meristem to be a wall layer of the conidiogenous cell but to be a new wall which lines that of the conidiogenous cell. It was also recommended (Kendrick, 1971) that' any phialide wall distal to the conidiogenous locus ...' be termed the collarette. We suggest that in C. adiposa the portion of the wall of the conidiogenous cell which overlies the meristem be termed the collarette irrespective of where along its length septation and thus conidiation occurs. As a result of this and previous ultrastructural studies on phialides it can be seen that the subsections IVA and IVB of Tubaki's (1958) scheme are very clearly separated when the role of wall layers in conidiation are considered. Subsection IVA includes phialides where the conidia are formed at the apex by apposition of new wall material which 'blows out' to form a conidium, e.g, Stachybotrys and Memnoniella (Campbell, 1972, 1975). Subsection IVB includes phialides where conidia are produced at the apex or in the neck by septation of a meristem bounded by a collarette, e.g , the Chalara state of C. adiposa and T. basicola (Hawes & Beckett, 1977b). We are grateful to Drs R. Campbell and M. F. Madelin for helpful discussion during preparation of the manuscript and to Mr R. Porter for skilful technical assistance and for building the sputter coating unit. Thanks are also due to the Science Research Council for a Research Studentship (to C.R.H.) and for a Research Grant (BjSRj90718 to A.B.). REFERENCES
BECKETT, A., HEATH, I. B. & McLAUGHLIN, D. J. (1974). An atlas of fungal ultrastructure. London: Longman. BUCKLEY, P. M., WYLLIE, T. D. & DEVAY, J. E. (1969). Fine structure of conidia and conidium formation in Verticillium albo-atrum and V. nigrescens, Mycologia 61, 240-250. CAMPBELL, R. (1972). Ultrastructure of conidium ontogeny in the deuteromycete fungus Stachybotrys atra Corda. New Phytologis: 71, 1143-1149.
CAMPBELL, R. (1975)· The ultrastructure of the formation of chains of conidia in Memnoniella echinata. Mycologic 67, 760-769· CARROLL, G. C. & CARROLL, F. E. (1974)· The fine structure of conidium development in Phialocephala dimorphospora. Canadian Journal of Botany 52, 2119-2128. D ELVECCHIO,. V G ., CORBAZ,. R & T URIAN,. G (196) 9. An ultrastructural study of the hyphae, endoconidia and chlamydospores of Thielaoiopsis basicola, Journal of General Microbiology 58, 23-27. HAMMILL, T. M. (1972). Electron microscopy ofphialo conidiogenesis in Metarhizium anisopliae. American Journal of Botany 59, 317-326. HAMMILL, T. M. (1974)· Electron microscopy of phialides and conidiogenesis in Trichoderma saturnisporium. American Journal of Botany 61, 15-2 4. HAWES, C. R. & BECKETT, A. (1977a) . Conidium ontogeny in the Chalara state of Ceratocystis adiposa. I. Light microscopy. Transactions of the British Mycological Society 68, 259-265. HAWES, C. R. & BECKETT, A. (1977 b). Conidium ontogeny in Thielaviopsis basicola. Transactions of the British Mycological Society 68,304-3°7. HUGHES, S. J. (1953). Conidiophores, conidia and classification. Canadian Journal of Botany 31, 5776 59· HUTCHINSON, S. A. (1939). Macroconidial formation in Ophiostoma majus (van Beyma) Goidanich . Annals of Botany 3, 795-802. KENDRICK, W. B. (1971). Taxonomy of the Fungi Imperfecti, Toronto: University of Toronto Press. OLAH, G. M. & REISINGER, O. (1974). Etude ultrastructurale et cytochirnique de l'appareil sporifere chez Phialophora richardsiae. Canadian Journal of Botany 52, 2473-2480. ROBERTSON, N. F. (1968). The growth process in fungi. Annual Review of Phytopathology 6, 115-136. SPURR, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research 26, 31-43. SUBRAMANIAN, C. V. (1971). The phialide. In Taxonomy of Fungi Imperfecti (ed. W. B. Kendrick), pp. 92-119. Toronto: University of Toronto Press. SUBRAMANIAN, C. V. (1972). Conidial chains, their nature and significance in the taxonomy of hyphomycetes. Current Science 41, 43-49. TRINCI, A. P. J. & COLLINGE, A. J. (1975). Hyphal wall growth in Neurospora crassa and Geotrichum candidum. Journal of General Microbiology 91, 355361. TUBAKI, K. (1958). Studies on the Japanese hyphomycetes: V. Journal of the Hattori Botanical Laboratory 20, 142-244.
(Accepted for publication 29 September 1976)