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Electron microscopy of genesis, maturation and wall structure of conidia of Aspergillus terreus
[ 27 ] Trans. Br. mycol, Soc. 66 (1) 27-34 (1976)
Printed in Great Britain
ELECTRON MICROSCOPY OF GENESIS, MATURATION AND WALL STRUCTURE OF CONIDIA OF ASPERGILLUS TERREUS By J. FLETCHER Department of Plant Biology, The University, Newcastle upon Tyne* First conidium initials of Aspergillus terreus Thorn were each formed blastically from the tip of a phialide. The wall surrounding a well-swollen, first conidium initial appeared continuous only with an inner wall layer of the phialide neck. Septa delimiting conidium initials were two-layered and perforate. The upper layer of each septum was continuous with the wall of the conidium initial which the septum delimited. The lower layer of each septum was continuous with a layer of wall material lining the phialide neck to form a thimble-shaped structure within the phialide neck. Budding of second and subsequent conidium initials was by distension of the upper, periclinal part of the thimble-shaped structure associated with the last-formed septum. Each developing spore chain and its subtending phialide was covered by a thin, electron-opaque surface-coating. Subsequent to delimitation of each conidium initial, a new, inner wall layer developed around the conidium protoplast. The material of the septa which initially separated consecutive conidium-initials of a chain was broken down leaving mature conidia mutually joined by the surface-coating, Trinci, Peat & Banbury (1968) described partially formed, first conidium initials of Aspergillus giganteus Wehmer. Tsukahara (1970) described the formation of condium initials, including the first, and some features of wall development subsequent to delimitation of conidium initials in Aspergillus niger van Tieghem. Both A. niger and A. giganteus are species producing phialides and conidia at the upper end of the size range found in the genus Aspergillus (Raper & Fennell, 1965). Oliver (1972) and Ghiorse & Edwards (1973) working with Aspergillus nidulans van Tieghem and Aspergillus fumigatus Fresenius, respectively (both species having phialides and conidia at the lower end of the size range found in the genus) did not report on the formation of first conidium initials. Though both Oliver and Ghiorse & Edwards thought that conidium maturation in the species they examined might involve changes in wallstructure similar to those observed in three Penicillium spp. by Fletcher (1971), they were unable to obtain clear evidence of this. This paper presents results of an investigation of the formation of first and subsequent conidium initials and of the changes in wall structure during maturation of conidia in Aspergillus terreus which has sporing heads, phialides and conidia of a size comparable to those of A. nidulans and A.jumigatus (Raper & Fennell, 1965).
* Present address: Department of Botany, University College Dublin, Belfield, Dublin, Ireland.
MATERIALS AND METHODS
To obtain part formed conidium initials and partially mature conidia, Aspergillus terreus was grown in slide culture on Oxoid potato dextrose agar (PDA). Each slide culture consisted of a flamed, microscope slide bearing a 1-2 mm thick square of PDA which was inoculated along its edges, covered with a flamed coverslip and placed in a Petri dish moist chamber. Slide cultures were fixed entire by immersion in fixative after 5-7 days incubation at 20-22 "C. Fixation procedures used were 1, treatment for 1'5 h at room temperature with potassium permanganate (1' 5 %) in veronal buffer (pH 7'2) and 2, treatment for 2 h with cooled (4°), mixed formaldehyde (4 %) and glutaraldehyde (5 %) in cacodylate buffer (pH 7'2) followed, after washing with 6-8 changes of cacodylate buffer and transfer to veronal buffer, by treatment with potassium permanganate solution as in procedure 1. Teepol (0'2 %) was added to the potassium permanganate solution used in procedure 1 and to the aldehyde solution used in procedure 2 to ensure wetting of sporing heads, and the first 10 min of fixation was carried out under vacuum to remove trapped air. Both fixation procedures were followed by washing with veronal buffer. Small pieces of PDA bearing sporing conidiophores were cut from the edges of fixed cultures and embedded in agar (0' 5 %) to protect sporulating
Conidiogenesis in Aspergillus
28
Figs.
1-10.
For description see opposite.
J. Fletcher regions from mechanical damage during subsequent handling. To obtain fully-mature conidia, clumps of mature conidial chains from 6 week old Petri dish cultures on PDA were embedded in agar (0 · 5 %) and fixed by procedure 1. Fixed, washed material from cultures of all ages was dehydrated in an ethanol series, transferred to acetone and embedded in Araldite. Sections were stained for 30 min with lead citrate (Reynolds, 1963) . For surface examination, mature conidia from 6 week old Petri dish cultures were dusted onto formvarfilmed grids and carbon replicas prepared, with carbon deposited either on one side only or on both sides of each grid, using th e procedures described by Bradley (1965). Replicas were examined unshadowed. Electron micrographs of sections were printed to show wall details at maximum contrast. RESULTS
First conidium initials were each formed blastically (Kendrick, 1971) from the apex of a phialide (Figs. 1-3). Prior to budding of the first con idium initial, the wall at the tip of a phialide was differentiated into an electron-opaque, apical zone and an electron-transparent, su bapical zone (Fig. 4). During formation of the first initial the electron-transparent part of the wall at the phialide neck thickened (Figs . 4-6) but distension during swelling of the initial appeared to be restricted to the electron-opaque, apical zone (Figs. 4-6). The wall surrounding an initial in an early stage of swelling appeared continuous with the main wall of the phialide (F ig. 5). The wall surrounding a well-swollen initial appeared continuous only with an inner wall layer of the phialide neck, the outer, electron-transparent wall layer of the phialide
neck appearing to terminate at the base of the bud (Fig. 6). The protoplast of the first conidium initial was delimited from the phialide protoplast by a two-layered septum (Fig. 7). A second conidium initial was formed percurrently below the septum (F igs. 7, 8). The protoplast of a second conidium initial was delimited from the phialide protoplast by a two-layered septum below which budding occurred to form a third conidium initial (Fig. 9). Septa formed at phialide necks, whether delimiting first or subsequent conidium initials, were initially thin but showed some indication of being two-layered (Fig. 11). Later, septa thickened and the layering became more apparent (Fig. 12). A narrow perforation traversed both layers of a septum (Fig. 12) . In each septern, the layer distal to the phialide appeared continuous with the basal part of the wall of the conidium initial which the septum delimited (Fig. 12); the layer proximal to the phialide was continuous with a periclinal layer ofwall material within the neck ofthe phialide, forming a thimble-shaped structure (Fig. 12). Formation of a second or subsequent conidium initial was by distension of the apical, periclinal part of the thimble-shaped structure associated with the last-formed septum (Figs. 7-9). During the swelling and delimitation of a conidium initial, the septum immediately distal to the initial became considerably thickened and sometimes biconvex (Figs. 7-9). Following fixation with permanganate alone, the neck of a phialide bearing one or more delimited conidium initials contained a layer of electronopaque material lying between the outer wall layer of the phialide neck and the inner wall layer continuous with the wall surrounding the currentlyforming conidium initial (Figs. 8, 10). Following
A spergillus terreus. Abbrevia tions: DS, upper (distal) layer of delimiting septum ; IW, inner wall layer at phialide neck; M, metulus ; OW, outer wall layer at phialide neck; P, perforation of septum ; PL, plasmalemma; PS, lower (proximal) layer of delimiting septum ; SC, surface-coating; VW, wall of conidiophore vesicle. All magnifications are approximate.
Fig.
1.
29
Section of a phialide apex prior to formation of the first conidium initial. Permanganate fixation,
x 15000.
Figs. 2, 3. Sections of phialide apices showing consecutive stages in budding of the first conidium initial. Permanganate fixation, x 15000. Fig. 4. Enlargement of the marked rectangle in Fig. 1, x 45000. Fig. 5. Enlargement of the marked rectangle in Fig. 2, x 45000. Fig. 6. EnlarIJement of the marked rectangle in Fig. 3, x 45000. Fig. 7, 8. Sections of phialides each bearing a delimited, first conidium initial showing consecutive stages in budding of the second conidium initial. The arrow in Fig. 7 indicates the centre line of the twolayered septum delimiting the first conidium initial. Permanganate fixation, x 15000. Fig. 9. Section of ~ phialide apex bearing two, delimited conidium initials. The arrow indicates the centre line ofthe two-layered septum delimiting the second condium initial. Perrnanganate fixation, x 15000. Fig. 10. Enlargement of the marked rectangle in Fig. 8, x 45000.
3°
Conidiogenesis in Aspergillus
Fig. 11. A non-median section of a recently formed septum at the base of a conidium initial. The arrows indicate the centre line of the septum profile. Permanganate fixation, x 45000. Fig. 12. Section of a thickened septum at the base of a conidium initial. Permanganate fixation, x 45000. Fig. 13. Part of the neck of a phialide bearing three, delimited conidium initials and a part-swollen fourth initial. Aldehyde and permanganate fixation, x 45000. Figs. 14-17. Sections of conidium initials and of partially mature conidia showing possible consecutive stages in development of a new, inner wall layer (arrowed) around conidium protoplasts. Series constructed from several spore chains. Aldehyde and permanganate fixation, x 22500. Fig. 18. Part of a chain of conidium initials and partially mature conidia showing the change in appearance of the septa (arrowed) separating conidium initials with increasing distance from the parent phialide. The conidium protoplast shown in part at the bottom of the figure is that of a partswollen, undelimited initial at the phialide apex. Aldehyde and permanganate fixation, x 22500.
J. Fletcher fixation with aldehyde and permanganate, this layer of material sometimes appeared thickened and of an electron-opacity similar to that of the inner wall layer continuous with the wall surrounding the currently-forming initial (Fig. 13). No such layer was present in the neck of a phialide bearing only an undelimited, first conidium initial (Fig. 6). During maturation of conidium initials, an inner, electron-transparent wall layer became visible around each conidium protoplast (Figs. 14-17). In parts of developing conidial chains distal to the parent phialide, the material of the thickened septa separating adjacent initials had an appearance suggesting breakdown of this material, the extent of breakdown increasing with increasing distance from the phialide (Fig. 18). A thin, electronopaque surface coating was present on walls of maturing conidia (Figs. 16-18), phialides and partformed conidium initials (Figs. 4-6), metulae (Fig. 26) and the conidiophore vesicle (Fig. 26). This coating was often separated from the wall (Figs. 5, 13) and was sometimes continuous around profiles of spaces formed by narrowed bases of closely-packed metulae (Fig. 26). Mature conidial chains from 6 week old cultures were each clothed externally by an electron-opaque surface-coating similar to that seen on phialides, metulae and maturing conidium-initials (Figs. 20, 21). In longitudinal sections of conidia, the surface profile appeared smooth (Figs. 19, 20, 24). In transverse sections of conidia, the surface-coating appeared undulate (Fig. 21) due to irregular, longitudinal corrugation of the coating which was seen best in surface replicas (Fig. 23). In surface view, surfaces of conidia showed parallel striations with a periodicity of approximately 10 nm . These striations were sometimes seen on replicas but were seen best in what appeared to be fragments of spore-surface material which occasionally adhered to the carbon film during preparation of replicas (Fig. 27). The protoplast of a mature conidium was surrounded by a thick, electrontransparent wall layer (Figs. 20, 21, 24) apparently homologous with the electron-transparent wall layer immediately surrounding the protoplast of a partially mature conidium (Fig. 17). Internal to the electron-transparent layer was a thin layer of granular material which filled invaginations of the spore plasmalemma (Fig. 24). In fully mature conidia, a layer of granular, electron-opaque material was present immediately beneath the surface-coating (Figs. 20, 21), occupying the same position as the outer wall layer of partially mature conidia (Figs. 14-17). Variation of the thickness of this electron-opaque material due to the corrugation of the surface-coating caused the outer layers of a conidium to appear longitudinally and
irregularly barred when seen in surface view in grazing sections of parts of spore chains (Fig. 22). Oblique sections of partly formed conidium initials showed the outer surface of the original walls of initials to have longitudinal corrugations similar to the corrugations seen on mature spore chains (Fig. 25). Consecutive conidia of a mature spore chain were held together by a narrow connective (Fig. 19). The connective appeared to consist of the electronopaque surface-coating together with an irregular deposit of electron-opaque material similar to that lying between the surface-coating and the electrontransparent wall layer of a conidium (Fig . 20) . DISCUSSION
The appearance of wall layers at necks of phialides of A. terreus bearing part-formed second or third conidium initials suggests formation of these and later formed initials by distension of the upper, periclinal part of a thimble-shaped wall layer with a new thimble-shaped layer being laid down within the phialide neck prior to formation of each initial. Material lying between the outer wall layer and the innermost layer at necks of phialides bearing one or more delimited initials may represent partially crushed remains of the basal parts of thimble-shaped layers associated with previously formed conidium-initials. The absence of such material from necks of phialides bearing well swollen first initials only is consistent with this . This interpretation of formation of second and subsequent conidium initials in A. terreus is substantially consistent with the interrelationships of wall layers at necks of phialides hypothesised by Morgan-Jones, Nag Raj & Kendrick (1972), though whether the transverse, apical part of the thimble-shaped layer is, ontogenetically, the lower layer of a two-layered septum or, as suggested by Morgan-Jones et al, (1972), a separate layer laid down below a singlelayered septum is not clear. The two transverse layers delimiting conidium initials in A . terreus, together with the periclinal part of the thimbleshaped structure, appear to be homologous with the structure in phialides of Penicillium spp, which Fletcher (1971 ) called the apical plug. The appearance of the wall at necks of phialides of A. terreus bearing first conidium initials at an early stage of swelling suggests holoblastic formation (Kendrick, 1971) of the first initial although the appearance at a late stage of swelling suggests enteroblastic formation (Kendrick, 1971) of the first initial. It is possible that prior to budding of the first initial the wall at the phialide apex consists of two layers which the present study has failed to
32
Conidiogenesis in Aspergillus
•
24
Figs. 19-27. For description see opposite.
J. Fletcher reveal, the outer layer being stretched and broken during swelling of the first initial. Figures of partformed conidium-initials of A.giganteusandA. niger presented by Trinci et al. (1968) and Tsukahara (1970), respectively, are consistent with enteroblastic formation of the first conidium initial in these species. Vuiicic & Muntaniola-Cvetkovic (1973) reported that emergence of conidia (conidium initials) in Aspergillus aereolatus followed rupture of the wall at the phialide apex. Changes occurring during maturation of conidia of A. terreus in relation to the development of a new, inner wall-layer around conidium protoplasts and breakdown of the material of the septa separating conidium-initials, are similar to the changes which occur during maturation of conidia of the three Penicillium species examined by Fletcher (1971). Secession of mature conidia of A. terreus would appear to be by eventual mechanical breakage of the narrow connective which joins them and which appears to consist primarily of the surfacecoating. The appearance of the surface-coating profile, particularly around spaces between bases of metulae, suggests that the coating may form at the air interface of material exuded from the vesicle, mutulae, phialides and developing conidia. This is consistent with the appearance of apparently homologous surface layers in developing conidial heads of A. giganteus (Trinci et al. 1968) and of A. niduians (Oliver, 1972). The striations seen on surfaces of mature spore chains of A. terreus show a spacing similar to and possibly represent structures homologous with the rod lets seen on
33
surfaces of mature conidia of several species of Aspergillus (Hess & Stocks, 1969) and of Penicillium (Hess, Sassen & Remsen, 1968) by freeze-etching and in sections of mature conidia of A. niduians (Florance et al, 1972). The layer of electron-opaque material immediately internal to the surface-coating in mature conidia of A. terreus appears to be homologous with the layer which Oliver (1972) identified as the pigment containing layer in conidia of A. nidulans. REFERENCES BRADLEY, D. E. (1965). Replica and shadowing techniques. Techniques for electron microscopy (ed. D. Kay), ch. 5, pp. 96-152. Oxford and Edinburgh: Blackwell Scientific Publications. FLETCHER, J. (1971). Conidium ontogeny in Penicillium. Journal of General Microbiology 67, 207-214. FLORANCE, E. R, DENISON, W. C. & ALLEN, T. C., JR. (1972). Ultrastructure of dormant and germinating conidia of Aspergillus nidulans. Mycologia 64, 115-123. GHIORSE, W. C. & EDWARDS, M. R. (1973). Ultrastructure of Aspergillus fumigatus conidia. Development and maturation. Protoplasma 76, 49-59. HESS, W. M., SASSEN, M. M. A. & REMSEN, C. C. (1968). Surface characteristics of Penicillium conidia. Mycologia 60, 290-303. HESS, W. M. & STOCKS, D. L. (1969). Surface characteristics of Aspergillus conidia. Mycologia 61, 560571. KENDRICK, B. (1971). Conclusions and recommendations. Taxonomy of Fungi Imperfecti (ed. B. Kendrick), ch. 16, pp. 253-262. Toronto: University of Toronto Press.
Fig. 19. Parts of two, mature spore chains from 6 week old culture. Permanganate fixation, x 9000. Fig. 20. Enlargement of the marked rectangle in Fig. 19, x 35000. Fig. 21. Part of the wall of a mature conidium from 6 week old culture. Section cut transversely to the long axis of the spore chain. Permanganate fixation, x 90000. Fig. 22. Grazing section of the surface of a conidium from a mature spore chain from 6 week old culture. The arrow indicates the direction of the long axis of the spore chain. Permanganate fixation, x 22500. Fig. 23. Surface replica of parts of two, adjacent conidia of a mature spore chain from 6 week-old culture, x 20000. Fig. 24. Part of the wall of a mature conidium from 6 week old culture. Section cut parallel to the long axis of the spore chain. Permanganate fixation, x 90000. Fig. 25. Section of a conidium initial with a partially formed delimiting septum showing obliquely sectioned, longitudinal corrugations (arrowed) of the wall of the initial. Aldehyde and permanganate fixation, x 22500. Fig. 26. Parts of walls of a conidiophore vesicle and of two metulae showing the arrangement of the surface-coating. Aldehyde and permanganate fixation, x 45000. Fig. 27. Surface material (surface-coating?) from a mature spore chain from 6 week old culture, x 55000. 2
MYC
61i
34
Conidiogenesis in Aspergillus
MORGAN-JONES, G., NAG RAJ, T . R. & KENDRICK, B. (1972). Conidium ontogeny in coelornycetes . IV. Percurrent proliferating phialides. Canadian Jo urnal of Botany SO, 2009-2 0 14 . OLIVER, P . T . P . (1972). Con idioph ore and spore development in A spergillus nidulans. J ournal of General M icrobiology 73, 45-45· RAPER, K. B. & FENNELL, D.!. (1965). The genus Aspergillus. Baltimore: Williams and Wilkins. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron -opaque stain in electron microscopy. Journa l of Cell B iology 17, 208-212.
TRINCI, A. P. J., PEAT, A. & BANBURY, G. H. (1968). Fine structure of ph ialide and conidium development in A spergillus giganteus Wehmer. Annals of Botany 32, 241-249. TSUKAHARA, T. (1970). Electron microscopy of conidiospore formation in A spergillus niger. Sabouraudia 8, 93-97· VUJICIC, R. & MUNTANJOLA-CVETKOVIC, M. (1973). A comparative ultrastructural study on conidium differen tiation in the Cladosarum-like mutant 22B of A spergillus aereolatus. Journal of General Microbiology 79, 45-51.
(Accepted f or publication
11
April 1975)
Printed in Great Britain
ELECTRON MICROSCOPY OF GENESIS, MATURATION AND WALL STRUCTURE OF CONIDIA OF ASPERGILLUS TERREUS By J. FLETCHER Department of Plant Biology, The University, Newcastle upon Tyne* First conidium initials of Aspergillus terreus Thorn were each formed blastically from the tip of a phialide. The wall surrounding a well-swollen, first conidium initial appeared continuous only with an inner wall layer of the phialide neck. Septa delimiting conidium initials were two-layered and perforate. The upper layer of each septum was continuous with the wall of the conidium initial which the septum delimited. The lower layer of each septum was continuous with a layer of wall material lining the phialide neck to form a thimble-shaped structure within the phialide neck. Budding of second and subsequent conidium initials was by distension of the upper, periclinal part of the thimble-shaped structure associated with the last-formed septum. Each developing spore chain and its subtending phialide was covered by a thin, electron-opaque surface-coating. Subsequent to delimitation of each conidium initial, a new, inner wall layer developed around the conidium protoplast. The material of the septa which initially separated consecutive conidium-initials of a chain was broken down leaving mature conidia mutually joined by the surface-coating, Trinci, Peat & Banbury (1968) described partially formed, first conidium initials of Aspergillus giganteus Wehmer. Tsukahara (1970) described the formation of condium initials, including the first, and some features of wall development subsequent to delimitation of conidium initials in Aspergillus niger van Tieghem. Both A. niger and A. giganteus are species producing phialides and conidia at the upper end of the size range found in the genus Aspergillus (Raper & Fennell, 1965). Oliver (1972) and Ghiorse & Edwards (1973) working with Aspergillus nidulans van Tieghem and Aspergillus fumigatus Fresenius, respectively (both species having phialides and conidia at the lower end of the size range found in the genus) did not report on the formation of first conidium initials. Though both Oliver and Ghiorse & Edwards thought that conidium maturation in the species they examined might involve changes in wallstructure similar to those observed in three Penicillium spp. by Fletcher (1971), they were unable to obtain clear evidence of this. This paper presents results of an investigation of the formation of first and subsequent conidium initials and of the changes in wall structure during maturation of conidia in Aspergillus terreus which has sporing heads, phialides and conidia of a size comparable to those of A. nidulans and A.jumigatus (Raper & Fennell, 1965).
* Present address: Department of Botany, University College Dublin, Belfield, Dublin, Ireland.
MATERIALS AND METHODS
To obtain part formed conidium initials and partially mature conidia, Aspergillus terreus was grown in slide culture on Oxoid potato dextrose agar (PDA). Each slide culture consisted of a flamed, microscope slide bearing a 1-2 mm thick square of PDA which was inoculated along its edges, covered with a flamed coverslip and placed in a Petri dish moist chamber. Slide cultures were fixed entire by immersion in fixative after 5-7 days incubation at 20-22 "C. Fixation procedures used were 1, treatment for 1'5 h at room temperature with potassium permanganate (1' 5 %) in veronal buffer (pH 7'2) and 2, treatment for 2 h with cooled (4°), mixed formaldehyde (4 %) and glutaraldehyde (5 %) in cacodylate buffer (pH 7'2) followed, after washing with 6-8 changes of cacodylate buffer and transfer to veronal buffer, by treatment with potassium permanganate solution as in procedure 1. Teepol (0'2 %) was added to the potassium permanganate solution used in procedure 1 and to the aldehyde solution used in procedure 2 to ensure wetting of sporing heads, and the first 10 min of fixation was carried out under vacuum to remove trapped air. Both fixation procedures were followed by washing with veronal buffer. Small pieces of PDA bearing sporing conidiophores were cut from the edges of fixed cultures and embedded in agar (0' 5 %) to protect sporulating
Conidiogenesis in Aspergillus
28
Figs.
1-10.
For description see opposite.
J. Fletcher regions from mechanical damage during subsequent handling. To obtain fully-mature conidia, clumps of mature conidial chains from 6 week old Petri dish cultures on PDA were embedded in agar (0 · 5 %) and fixed by procedure 1. Fixed, washed material from cultures of all ages was dehydrated in an ethanol series, transferred to acetone and embedded in Araldite. Sections were stained for 30 min with lead citrate (Reynolds, 1963) . For surface examination, mature conidia from 6 week old Petri dish cultures were dusted onto formvarfilmed grids and carbon replicas prepared, with carbon deposited either on one side only or on both sides of each grid, using th e procedures described by Bradley (1965). Replicas were examined unshadowed. Electron micrographs of sections were printed to show wall details at maximum contrast. RESULTS
First conidium initials were each formed blastically (Kendrick, 1971) from the apex of a phialide (Figs. 1-3). Prior to budding of the first con idium initial, the wall at the tip of a phialide was differentiated into an electron-opaque, apical zone and an electron-transparent, su bapical zone (Fig. 4). During formation of the first initial the electron-transparent part of the wall at the phialide neck thickened (Figs . 4-6) but distension during swelling of the initial appeared to be restricted to the electron-opaque, apical zone (Figs. 4-6). The wall surrounding an initial in an early stage of swelling appeared continuous with the main wall of the phialide (F ig. 5). The wall surrounding a well-swollen initial appeared continuous only with an inner wall layer of the phialide neck, the outer, electron-transparent wall layer of the phialide
neck appearing to terminate at the base of the bud (Fig. 6). The protoplast of the first conidium initial was delimited from the phialide protoplast by a two-layered septum (Fig. 7). A second conidium initial was formed percurrently below the septum (F igs. 7, 8). The protoplast of a second conidium initial was delimited from the phialide protoplast by a two-layered septum below which budding occurred to form a third conidium initial (Fig. 9). Septa formed at phialide necks, whether delimiting first or subsequent conidium initials, were initially thin but showed some indication of being two-layered (Fig. 11). Later, septa thickened and the layering became more apparent (Fig. 12). A narrow perforation traversed both layers of a septum (Fig. 12) . In each septern, the layer distal to the phialide appeared continuous with the basal part of the wall of the conidium initial which the septum delimited (Fig. 12); the layer proximal to the phialide was continuous with a periclinal layer ofwall material within the neck ofthe phialide, forming a thimble-shaped structure (Fig. 12). Formation of a second or subsequent conidium initial was by distension of the apical, periclinal part of the thimble-shaped structure associated with the last-formed septum (Figs. 7-9). During the swelling and delimitation of a conidium initial, the septum immediately distal to the initial became considerably thickened and sometimes biconvex (Figs. 7-9). Following fixation with permanganate alone, the neck of a phialide bearing one or more delimited conidium initials contained a layer of electronopaque material lying between the outer wall layer of the phialide neck and the inner wall layer continuous with the wall surrounding the currentlyforming conidium initial (Figs. 8, 10). Following
A spergillus terreus. Abbrevia tions: DS, upper (distal) layer of delimiting septum ; IW, inner wall layer at phialide neck; M, metulus ; OW, outer wall layer at phialide neck; P, perforation of septum ; PL, plasmalemma; PS, lower (proximal) layer of delimiting septum ; SC, surface-coating; VW, wall of conidiophore vesicle. All magnifications are approximate.
Fig.
1.
29
Section of a phialide apex prior to formation of the first conidium initial. Permanganate fixation,
x 15000.
Figs. 2, 3. Sections of phialide apices showing consecutive stages in budding of the first conidium initial. Permanganate fixation, x 15000. Fig. 4. Enlargement of the marked rectangle in Fig. 1, x 45000. Fig. 5. Enlargement of the marked rectangle in Fig. 2, x 45000. Fig. 6. EnlarIJement of the marked rectangle in Fig. 3, x 45000. Fig. 7, 8. Sections of phialides each bearing a delimited, first conidium initial showing consecutive stages in budding of the second conidium initial. The arrow in Fig. 7 indicates the centre line of the twolayered septum delimiting the first conidium initial. Permanganate fixation, x 15000. Fig. 9. Section of ~ phialide apex bearing two, delimited conidium initials. The arrow indicates the centre line ofthe two-layered septum delimiting the second condium initial. Perrnanganate fixation, x 15000. Fig. 10. Enlargement of the marked rectangle in Fig. 8, x 45000.
3°
Conidiogenesis in Aspergillus
Fig. 11. A non-median section of a recently formed septum at the base of a conidium initial. The arrows indicate the centre line of the septum profile. Permanganate fixation, x 45000. Fig. 12. Section of a thickened septum at the base of a conidium initial. Permanganate fixation, x 45000. Fig. 13. Part of the neck of a phialide bearing three, delimited conidium initials and a part-swollen fourth initial. Aldehyde and permanganate fixation, x 45000. Figs. 14-17. Sections of conidium initials and of partially mature conidia showing possible consecutive stages in development of a new, inner wall layer (arrowed) around conidium protoplasts. Series constructed from several spore chains. Aldehyde and permanganate fixation, x 22500. Fig. 18. Part of a chain of conidium initials and partially mature conidia showing the change in appearance of the septa (arrowed) separating conidium initials with increasing distance from the parent phialide. The conidium protoplast shown in part at the bottom of the figure is that of a partswollen, undelimited initial at the phialide apex. Aldehyde and permanganate fixation, x 22500.
J. Fletcher fixation with aldehyde and permanganate, this layer of material sometimes appeared thickened and of an electron-opacity similar to that of the inner wall layer continuous with the wall surrounding the currently-forming initial (Fig. 13). No such layer was present in the neck of a phialide bearing only an undelimited, first conidium initial (Fig. 6). During maturation of conidium initials, an inner, electron-transparent wall layer became visible around each conidium protoplast (Figs. 14-17). In parts of developing conidial chains distal to the parent phialide, the material of the thickened septa separating adjacent initials had an appearance suggesting breakdown of this material, the extent of breakdown increasing with increasing distance from the phialide (Fig. 18). A thin, electronopaque surface coating was present on walls of maturing conidia (Figs. 16-18), phialides and partformed conidium initials (Figs. 4-6), metulae (Fig. 26) and the conidiophore vesicle (Fig. 26). This coating was often separated from the wall (Figs. 5, 13) and was sometimes continuous around profiles of spaces formed by narrowed bases of closely-packed metulae (Fig. 26). Mature conidial chains from 6 week old cultures were each clothed externally by an electron-opaque surface-coating similar to that seen on phialides, metulae and maturing conidium-initials (Figs. 20, 21). In longitudinal sections of conidia, the surface profile appeared smooth (Figs. 19, 20, 24). In transverse sections of conidia, the surface-coating appeared undulate (Fig. 21) due to irregular, longitudinal corrugation of the coating which was seen best in surface replicas (Fig. 23). In surface view, surfaces of conidia showed parallel striations with a periodicity of approximately 10 nm . These striations were sometimes seen on replicas but were seen best in what appeared to be fragments of spore-surface material which occasionally adhered to the carbon film during preparation of replicas (Fig. 27). The protoplast of a mature conidium was surrounded by a thick, electrontransparent wall layer (Figs. 20, 21, 24) apparently homologous with the electron-transparent wall layer immediately surrounding the protoplast of a partially mature conidium (Fig. 17). Internal to the electron-transparent layer was a thin layer of granular material which filled invaginations of the spore plasmalemma (Fig. 24). In fully mature conidia, a layer of granular, electron-opaque material was present immediately beneath the surface-coating (Figs. 20, 21), occupying the same position as the outer wall layer of partially mature conidia (Figs. 14-17). Variation of the thickness of this electron-opaque material due to the corrugation of the surface-coating caused the outer layers of a conidium to appear longitudinally and
irregularly barred when seen in surface view in grazing sections of parts of spore chains (Fig. 22). Oblique sections of partly formed conidium initials showed the outer surface of the original walls of initials to have longitudinal corrugations similar to the corrugations seen on mature spore chains (Fig. 25). Consecutive conidia of a mature spore chain were held together by a narrow connective (Fig. 19). The connective appeared to consist of the electronopaque surface-coating together with an irregular deposit of electron-opaque material similar to that lying between the surface-coating and the electrontransparent wall layer of a conidium (Fig . 20) . DISCUSSION
The appearance of wall layers at necks of phialides of A. terreus bearing part-formed second or third conidium initials suggests formation of these and later formed initials by distension of the upper, periclinal part of a thimble-shaped wall layer with a new thimble-shaped layer being laid down within the phialide neck prior to formation of each initial. Material lying between the outer wall layer and the innermost layer at necks of phialides bearing one or more delimited initials may represent partially crushed remains of the basal parts of thimble-shaped layers associated with previously formed conidium-initials. The absence of such material from necks of phialides bearing well swollen first initials only is consistent with this . This interpretation of formation of second and subsequent conidium initials in A. terreus is substantially consistent with the interrelationships of wall layers at necks of phialides hypothesised by Morgan-Jones, Nag Raj & Kendrick (1972), though whether the transverse, apical part of the thimble-shaped layer is, ontogenetically, the lower layer of a two-layered septum or, as suggested by Morgan-Jones et al, (1972), a separate layer laid down below a singlelayered septum is not clear. The two transverse layers delimiting conidium initials in A . terreus, together with the periclinal part of the thimbleshaped structure, appear to be homologous with the structure in phialides of Penicillium spp, which Fletcher (1971 ) called the apical plug. The appearance of the wall at necks of phialides of A. terreus bearing first conidium initials at an early stage of swelling suggests holoblastic formation (Kendrick, 1971) of the first initial although the appearance at a late stage of swelling suggests enteroblastic formation (Kendrick, 1971) of the first initial. It is possible that prior to budding of the first initial the wall at the phialide apex consists of two layers which the present study has failed to
32
Conidiogenesis in Aspergillus
•
24
Figs. 19-27. For description see opposite.
J. Fletcher reveal, the outer layer being stretched and broken during swelling of the first initial. Figures of partformed conidium-initials of A.giganteusandA. niger presented by Trinci et al. (1968) and Tsukahara (1970), respectively, are consistent with enteroblastic formation of the first conidium initial in these species. Vuiicic & Muntaniola-Cvetkovic (1973) reported that emergence of conidia (conidium initials) in Aspergillus aereolatus followed rupture of the wall at the phialide apex. Changes occurring during maturation of conidia of A. terreus in relation to the development of a new, inner wall-layer around conidium protoplasts and breakdown of the material of the septa separating conidium-initials, are similar to the changes which occur during maturation of conidia of the three Penicillium species examined by Fletcher (1971). Secession of mature conidia of A. terreus would appear to be by eventual mechanical breakage of the narrow connective which joins them and which appears to consist primarily of the surfacecoating. The appearance of the surface-coating profile, particularly around spaces between bases of metulae, suggests that the coating may form at the air interface of material exuded from the vesicle, mutulae, phialides and developing conidia. This is consistent with the appearance of apparently homologous surface layers in developing conidial heads of A. giganteus (Trinci et al. 1968) and of A. niduians (Oliver, 1972). The striations seen on surfaces of mature spore chains of A. terreus show a spacing similar to and possibly represent structures homologous with the rod lets seen on
33
surfaces of mature conidia of several species of Aspergillus (Hess & Stocks, 1969) and of Penicillium (Hess, Sassen & Remsen, 1968) by freeze-etching and in sections of mature conidia of A. niduians (Florance et al, 1972). The layer of electron-opaque material immediately internal to the surface-coating in mature conidia of A. terreus appears to be homologous with the layer which Oliver (1972) identified as the pigment containing layer in conidia of A. nidulans. REFERENCES BRADLEY, D. E. (1965). Replica and shadowing techniques. Techniques for electron microscopy (ed. D. Kay), ch. 5, pp. 96-152. Oxford and Edinburgh: Blackwell Scientific Publications. FLETCHER, J. (1971). Conidium ontogeny in Penicillium. Journal of General Microbiology 67, 207-214. FLORANCE, E. R, DENISON, W. C. & ALLEN, T. C., JR. (1972). Ultrastructure of dormant and germinating conidia of Aspergillus nidulans. Mycologia 64, 115-123. GHIORSE, W. C. & EDWARDS, M. R. (1973). Ultrastructure of Aspergillus fumigatus conidia. Development and maturation. Protoplasma 76, 49-59. HESS, W. M., SASSEN, M. M. A. & REMSEN, C. C. (1968). Surface characteristics of Penicillium conidia. Mycologia 60, 290-303. HESS, W. M. & STOCKS, D. L. (1969). Surface characteristics of Aspergillus conidia. Mycologia 61, 560571. KENDRICK, B. (1971). Conclusions and recommendations. Taxonomy of Fungi Imperfecti (ed. B. Kendrick), ch. 16, pp. 253-262. Toronto: University of Toronto Press.
Fig. 19. Parts of two, mature spore chains from 6 week old culture. Permanganate fixation, x 9000. Fig. 20. Enlargement of the marked rectangle in Fig. 19, x 35000. Fig. 21. Part of the wall of a mature conidium from 6 week old culture. Section cut transversely to the long axis of the spore chain. Permanganate fixation, x 90000. Fig. 22. Grazing section of the surface of a conidium from a mature spore chain from 6 week old culture. The arrow indicates the direction of the long axis of the spore chain. Permanganate fixation, x 22500. Fig. 23. Surface replica of parts of two, adjacent conidia of a mature spore chain from 6 week-old culture, x 20000. Fig. 24. Part of the wall of a mature conidium from 6 week old culture. Section cut parallel to the long axis of the spore chain. Permanganate fixation, x 90000. Fig. 25. Section of a conidium initial with a partially formed delimiting septum showing obliquely sectioned, longitudinal corrugations (arrowed) of the wall of the initial. Aldehyde and permanganate fixation, x 22500. Fig. 26. Parts of walls of a conidiophore vesicle and of two metulae showing the arrangement of the surface-coating. Aldehyde and permanganate fixation, x 45000. Fig. 27. Surface material (surface-coating?) from a mature spore chain from 6 week old culture, x 55000. 2
MYC
61i
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Conidiogenesis in Aspergillus
MORGAN-JONES, G., NAG RAJ, T . R. & KENDRICK, B. (1972). Conidium ontogeny in coelornycetes . IV. Percurrent proliferating phialides. Canadian Jo urnal of Botany SO, 2009-2 0 14 . OLIVER, P . T . P . (1972). Con idioph ore and spore development in A spergillus nidulans. J ournal of General M icrobiology 73, 45-45· RAPER, K. B. & FENNELL, D.!. (1965). The genus Aspergillus. Baltimore: Williams and Wilkins. REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron -opaque stain in electron microscopy. Journa l of Cell B iology 17, 208-212.
TRINCI, A. P. J., PEAT, A. & BANBURY, G. H. (1968). Fine structure of ph ialide and conidium development in A spergillus giganteus Wehmer. Annals of Botany 32, 241-249. TSUKAHARA, T. (1970). Electron microscopy of conidiospore formation in A spergillus niger. Sabouraudia 8, 93-97· VUJICIC, R. & MUNTANJOLA-CVETKOVIC, M. (1973). A comparative ultrastructural study on conidium differen tiation in the Cladosarum-like mutant 22B of A spergillus aereolatus. Journal of General Microbiology 79, 45-51.
(Accepted f or publication
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April 1975)