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Conidium ultrastructure and wall architecture in the Chalara state of Ceratocystis adiposa
Vol. 72, Part
April 1979
2
Printed in Great Britain
Trans. Br, mycol, Soc. 72 (2) 177-187 (1979)
CONIDIUM ULTRASTRUCTURE AND WALL ARCHITECTURE IN THE CHALARA STATE OF CERATOCYSTIS ADIPOSA By C. R. HAWES Department of Botany, The University of Bristol, Bristol, BS8
1 UG,
U.K.
When cultured on different media the Chalara state of Ceratocystis adiposa (Butl.) C. Moreau forms conidia of varying morphology. The major variation is in the thickness, elaboration and pigmentation of the wall of the coniduim. The appearance of conidial walls after chemical and enzymic treatments, and their binding properties with plant lectins is described. A model is proposed to show the distribution of polymers throughout the three wall layers. Little is known of the chemical composition and structure of conidium walls (Marchant, 1966;'Akai, Fukutomi, Kunoh& Shiraishi, 1976), although the cytoplasmic fine structure of conidia has been the subject of various studies (Campbell, 1969; Richmond & Pring, 1971; Hammill, 1972; Griffiths, 1973; Ellis & Griffiths, 1975a, b). Biochemical analyses of the walls of higher fungi such as Aspergillus nidulans (Eidam) Wint. (Bull, 1970a; Zonneveld, 1971) have produced no information as to the distribution of polymers. The structure of vegetative hyphal walls of various species has been elucidated by means of chemical and enzymic digestion (Mahadevan & Tatum, 1967; Hunsley & Burnett, 1970; Michalenco, Hohl & Rast, 1976), but only a few comparable studies have been carried out on spore walls (Rast & Hollenstein, 1977). The Chalara state of Ceratocystis adiposa (Butl.) C. Moreau when cultured on malt agar (MA) has been shown to produce conidia which are variable in size, shape, degree of pigmentation and ornamentation (Hawes & Beckett, 1977a, b). Conidia from cultures grown on different media also exhibit variation in the above features (Hawes, 1977) and the cytoplasmic and wall structure of these conidia is reported in this paper. MATERIALS AND METHODS
The culture of C. adiposa used (Hawes & Beckett, 1977 a) was maintained on Czapek-Dox (CzA), glucose asparagine (GAA) and malt agar (MA) Petri plates. Histochemical staining Cultures were obtained on cellophane discs (Hawes & Beckett, 1977a). Conidia were stained for lipids using Sudan Black Band benzpyrenecaffeine techniques (Jensen, 1962). Polysaccharides with a-D-manopyranosyl and «-n-glucanopy-
ranosyl residues were stained with fluoresceinisothiocyanate (FITC) conjugated Concanavalin A (Con. A) (Miles Yeda Ltd). This consisted of a stock solution of 8 mg of the protein per ml of 0'1 M-phosphate buffer at pH 7"4. 10,tt1 of the stock solution were diluted to 100 ,ttl with 1-0 MNaCl and used in 10,tt1 drops for staining. Chitin was stained with FITC wheat germ aglutinin (WGA) (Miles Yeda Ltd) which consisted of a solution of 1'85 mg of the protein per rnl of phosphate buffered saline at pH 7'4. The staining procedure was as with the FITC Con. A. Micrographs were recorded using Ilford Pan F 35 mm film on a Zeiss Photomicroscope II (Figs. 1,2,4,6) and Ilford HP4 35 rom film on a Zeiss Photomicroscope III with epifluorescence condenser III/RS, dark ground illumination and ultra-violet light (Figs. 3, 5, 33-37). Electron microscopy Material for transmission and scanning electron microscopy was fixed and prepared as described in Hawes & Beckett (1977 b). For freeze-fracturing, conidia were harvested in distilled water from an agar plate culture, centrifuged into a pellet and embedded in 2 % agar. The pellet was then split into 1 rom cubes, frozen on a precooled lead block, immersed in liquid nitrogen and fractured with a razor blade. The whole block was put under vacuum in an Edwards high vacuum coating unit and after sublimation of the nitrogen and water the dried material was mounted on stubs, coated with gold and observed with a Cambridge Scientific Instrument Company S4 scanning electron microscope (SEM). Carbon replicas of conidial surfaces were obtained by drying conidia suspended in drops of distilled water onto formvar-coated grids. These were coated from above with carbon and shadowed at 20°C with carbon/platinum. Replicas were cleaned by dipping the grids in chloroform
0007-1536/79/2828-4780 $01.00 © 1979 The British Mycological Society Vol. 72, Part 7
1,
was issued 20 February 1979 MYC 72
Ultrastructure of Ceratocystis adiposa followed by immersion in a mixture of potassium permanganate and potassium dichromate in concentrated sulphuric acid overnight. After washing in distilled water the replicas were treated with a 1 :1 (vIv) mixture of glacial acetic acid and 30 % hydrogen peroxide, picked up on new grids, washed and observed with an AE1 EM 6G transmission electron microscope (TEM). Chemical digestion of conidium walls Conidia from cultures on CzA and GAA were collected from suspensions in distilled water on millipore filter papers and treated as follows: (a) autoclaved in 4'5 % (w/v) KOH at 103'5 RN/m 2 for 15 min and washed in distilled water; (b) treated with a 1 : 1 (vIv) mixture of 30 % hydrogen peroxide and concentrated glacial acetic acid at 100 0 for 0'5-7 h and washed in distilled water. After each treatment conidia were (i) observed with the light microscope, (ii) dried onto forrnvarcoated grids, shadowed with carbon/platinum and observed with the TEM, and (iii) dehydrated in a graded water/ethanol series, embedded in Spurr's resin (Spurr, 1969), sectioned and observed with the TEM. Enzymic wall digestion Chitinase (fungal) and pronase (ex-Streptomyces griseusy were obtained from Koch-Light Laboratories Ltd and a laminarinase (ex mollusca) from Calbiochem. For each experiment conidia were incubated in flasks for 48 h at 37 with the appropriate enzyme in a sodium phosphate-citric acid buffer. With pronase, a potassium phosphate buffer was used. Laminarinase was incubated at pH 5'0, chitinase at pH 5"5 and pronase at pH 7'5 (Hunsley & Burnett, 1970). 2 ml of a suspension containing 0'5 mg enzyme per ml were added to 18 ml of
buffer, to this was added 2 ml of a conidial suspension in the appropriate buffer. The control for each treatment contained boiled enzyme. A drop of toluene was added to all treatments as a bacteriostat. After incubation, conidia were collected by centrifugation, washed four times in distilled water, dried onto formvar-coated grids, shadowed with carbon platinum and observed with the TEM. Conidia from CzA cultures were treated with all the enzymes. Chemically digested conidia from GAA cultures were treated with combinations of chitinase and pronase, and fresh conidia from these cultures were treated with combinations of laminarinase and pronase or autoclaved in 4'0% (w/v) potassium hydroxide prior to enzymic treatment. RESULTS
Conidia produced by cultures on CzA are large ( > 15 pm diam), globose, deeply pigmented and show little variation in shape (Figs. 2, 6, 7, 11). Easily fragmented chains of small, less-pigmented conidia are occasionally found (Fig. 1). Cultures on GAA form long conidial chains composed of hyaline slightly pigmented, small, round to doliiform occasionally pyriform spores « 12 pm diam) (Figs. 4, 9, 15). Conidia produced by cultures on MA (Fig. 10) are variable in size, shape, degree of pigmentation and wall ornamentation.
0
Cytoplasmic ultrastructure Lipid is the prominent storage product in conidial cytoplasm. Large globose conidia from CzA and MA cultures have a central aggregation of lipid (Figs. 2, 3), seen in section as numerous electron opaque bodies (Figs. 6, 7). In many of the chains
Fig. 1. Bright field micrograph of thin-walled conidia stained with Sudan Black B showing polar aggregations of lipid (CzA culture). x 1500. Fig. 2. Bright field micrograph of large globose conidia stained with Sudan Black B (CzA culture). x 1500. Fig. 3. Fluorescence micrograph of conidia stained using benzpyrene-caffeine (CzA culture). x 1300. Fig. 4. Bright field micrograph of thin-walled conidia stained with Sudan Black B (GAA culture). x 1200. Fig. 5. Fluorescence micrograph of conidia stained using benzpyrene-caffeine (GAA culture). x 1900. Fig. 6. Phase contrast micrograph of a large globose conidium (CzA culture). L = lipid. x 3800. Fig. 7. TEM large globose conidium with a 3-layered wall (CzA culture). L = lipid. x 6600. Fig, 8. TEM part of a conidium showing the association of vesiculate endoplasmic reticulum (VER) with the wall (CzA culture). x 20400. Fig. 9. TEM ovoid conidium with flocculose material in the cytoplasm and 3 wall layers (GAA culture). x 11400. Fig. 10. A cylindrical conidium with a 3-layered wall and polar aggregations of lipid (MA culture). x 5700.
C. R. Hawes
179
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,
.
·f ·
•
._ -
W2 W3
7-2
180
17
17:
Ultrastructure of Ceratocystis adiposa
C. R. Hawes of ovoid, less pigmented conidia from cultures on these media, the lipid is in polar clusters (Figs. 1, 10). Conidia from cultures on GAA stain less with Sudan Black Band benzpyrene-caffein and the lipid is scattered (Figs. 4, 5). Conidial cytoplasm contains smooth endoplasmic reticulum, varying numbers of mitochondria, vacuoles which may contain electron dense material, ribosomes, and from one to six nuclei (Figs. 7-10). The ribosome content of conidia from GAA cultures is considerably greater than that in other conidia (Fig. 9). Flocculose material, probably polysaccharide, is present in some conidia (Fig. 9). In the thick-walled conidia, areas of vesicular, smooth membrane are associated with the plasmalemma (Fig. 8). Surface structure of the conidium The conidial wall is verrucose/spiny to smooth, and all intermediates between these extremes in surface topography are seen (Figs. 11-15). The verrucose/spiny nature of the large globose conidia from CzA and MA cultures is often masked by the sheath (Fig. 13) but may be revealed by part removal of the sheath by freeze-fracturing (Fig. 12). Ornamentation of the wall of the conidium does not extend over the basal septal plate (Fig. 12, arrows). The intermediate forms of surface ornamentation may be reticulate or slightly verrucose as shown by conidia from MA cultures (Fig. 14). Conidia from easily fragmented chains are always smooth-walled as are conidia from GAA cultures where tearing of the sheath reveals no wall ornamentation (Fig. 15, arrows). Conidium wall in section With the light microscope the wall of a mature, globose, pigmented conidium from a CzA culture appears three-layered (Fig. 6). In thin sections the walls of mature conidia from cultures on all three media exhibit the same basic three-layered
181
structure (Figs. 7, 9, 10). An outer layer (W1) consisting of spines of pigment embedded in electron transparent material and surrounded by the sheath, overlies a middle pigmented layer (W2) to the inside of which is an electron transparent layer (W 3) (Fig. 39). The major variation between the different conidium types is in the thickness, elaboration and degree of pigmentation of the outer two layers. The two extremes of wall development are represented by the deeply pigmented, large, globose, and spiny-walled conidia from CzA cultures where the wall may be up to 2'6 pm thick (Fig. 7) and the slightly pigmented, globose to doliiform, smooth-walled conidia from GAA cultures where the wall may be as thin as 0'2 psi». (Fig. 9). In the latter conidia the outer two layers are poorly differentiated with little pigment deposition (Fig. 9). Chemical digestion of conidia The effect of prolonged chemical digestion on conidia from CzA cultures is shown in Figs. 16-22. Autoclaving in potassium hydroxide removes the material from the outer wall layer (W 1) in which the spines of pigment are embedded and the sheath collapses around them (Fig. 16). Prolonged boiling in a glacial acetic acid/hydrogen peroxide mixture slowly digests the conidial wall (Figs. 17-19) until, after 5-7 h, only a layer of closely intermeshed microfibrils remains (Fig. 22). After digestion for 0'5 h thin sections reveal some bleaching or removal of the pigment but the sheath remains intact (Fig. 20 arrows). After 1 h individual layers cannot be distinguished (Fig. 21). In fully digested conidia a circular arrangement of microfibrils around the septal pore at the base ofthe conidium is often seen (Fig. 22 arrows). Chemical digestion of the thin-walled conidia from GAA cultures proceeds more rapidly than in those described above, and the microfibrillar layer is exposed after digestion for 0'5-1 h (Fig. 23).
Fig. 11. SEM large globose conidium (CzA culture). x 3700. Fig. 12. SEM large globose conidium with the sheath partly removed revealing spines of pigmented material (CzA culture, freeze-fractured material). x 3700. Fig. 13. TEM replica of a large globose conidium (CzA culture). x 2700. Fig. 14. SEM conidium with reticulate ornamentation of the wall (MA culture). x 4100. Fig. 15. SEM ovoid smooth-walled conidium (GAA culture). x 8000. Fig. 16. SEM large globose conidium after autoclaving in potassium hydroxide. Much of the outer wall layer has been removed (CzA culture). x 4°°00. Fig. 17. LM large globose conidium (CzA culture). x 2(';00. Fig. 18. LM large globose conidium after digestion for 0-5 h in hydrogen peroxide/acetic acid (CzA culture). x 1900. Fig. 19. LM large globose conidium after chemical digestion for 7 h (CzA culture). x 2200.
182
Ultrastructure of Ceratocystis adiposa
..
~
23 Fig. 20. TEM section of the wall of a large globose comuium atter digestion for 0'5 h in hydrogen peroxide/acetic acid. The sheath (arrows) remains intact around the outer wall layer (CzA culture). x 18000. Fig. 21. TEM section of the wall of a large globose conidium after chemical digestion for 1 h (CzA culture). x 13300. Fig. 22. TEM large globose conidium after digestion for 7 h showing the circular arrangement of microfibrils around the septal pore (arrows) (CzA culture). x 9000. Fig. 23. TEM part of a thin-walled, ovoid conidium after digestion for 1 h (GAA culture). x 18000.
C. R. Hawes Enzymic digestion of conidia
Treatment of conidia from cultures on CzA with laminarinase, pronase and chitinase individually, has no effect on the wall structure. After incubation of chemically digested conidia from GAA cultures in chitinase the microfibrils become more clearly defined (Figs. 24, 25). Treatment of these digested conidia with pronase prior to chitinase results in some disruption of the microfibrils (Fig. 26). Shadowed preparations of fresh conidia from GAA cultures are electron opaque (Fig. 27), but digestion with laminarinase renders the conidium less opaque. The surface of the conidium is amorphous to granulate and an underlying microfibrillar structure can be seen (Fig. 28). Autoclaving in potassium hydroxide before laminarinase treatment results in a less granulate wall texture (Fig . 29). Preparations of conidia treated sequentially with laminarinase and pronase show an amorphous surface structure and microfibrils are not discernible (Fig. 30). The microfibrillar layer as revealed by chemical digestion is exposed by laminarinase and pronase treatment only after prior autoclaving in potassium hydroxide (F igs. 31, 32). Fluorescent staining Thin-walled conidia from GAA cultures fluoresce brightly with ultra-violet light when stained with FITC Con. A (Fig. 33), but onl y show a dim fluorescence with FITC WGA (Fig. 34). Chemically digested conidia from both GAA and CzA cultures fluoresce brightly with FITC WGA (Figs. 35,36), but only the central portion, that marks the remains of the protoplast, (Fig. 38) fluoresces when stained with FITC Con. A and then onl y faintly (Fig. 37). This area, however, also shows weak autofluorescence with ultra-violet light . DISCUSSION
, Cytoplasmic ultrastructure With the exception of the numerous lipid bodies, the organelle complement of mature conidia is similar to that of the conidiophores and developing conidia (Hawes & Beckett, 1977b). The areas of vesicular smooth endoplasmic reticulum found in thick-walled conidia are probably involved in the centripetal deposition of wall material around the protoplast as conidia in the chain continue to mature. The association of endoplasmic reticulum with the plasmalemma has also been seen in the conidia of Botrytis fabae Sardina (Richmond & Pring, 1971) and in the exogenously dormant conidia of Pleiochaeta setosa (Kirchn.) Hughes (Harvey, 1975). The large ribosome content of
conidia from GAA cultures is similar to that found in the conidiophores and young conidia from these cultures (Hawes, 1977). Conidium wall structure Several tentative conclusions can be drawn as to the structure of the wall and the distribution of polymers. It is the degree of development of the outer layer (W 1) that governs the surface ornamentation. Thus in the large globose conidia from GAA cultures, W 1 is thick with well-developed spines of pigment making the surface verrucose/ spiny. In contrast in the thin-walled conidia from GAA cultures, this layer is thin without much pigment and the surface is correspondingly smooth. The electron-transparent material in which the spines of pigment are embedded is soluble in potassium hydroxide and when removed leaves the pigment surrounded by the alkali resistant sheath. This electron-transparent material could be an alkali soluble a - 1,3-glucan similar to that which makes up the amorphous outer wall layer of the hyphae of Sch izophyllum commune Fr. (Hunsley & Burnett, 1970; van der Valk & Wessels, 1976). Prolonged chemical digestion of conidia from both CzA and GAA cultures removes all the wall material with the exception of a microfibrillar layer. Treatment of this layer with chitinase makes the fibrils more distinct, but only after pronase followed by chitinase treatment is there any disruption of the microfibrils. This suggests that the layer is composed of chitin microfibrils bound with protein. A similar structure has been proposed for layer II of the Agaricus bisporus spore wall (Rast & Hollenstein, 1977). Fluorescence under ultra-violet light of chemically digested conidia stained with FITC WGA, which binds to N-acetylglucosamine (Lotan & Sharon, 1973), verifies the presence of chitin in this layer . Melanin in the hyphal walls of Aspergillus nidulans has been shown to inhibit the activity of a ;J-1,3-g1ucanase and a chitinase (Bull, 1970b). It is possibly melanin that prevents enzymic digestion of the large pigmented conidia of C. adiposa. Enzyme work was therefore restricted to the thinwalled, less-pigmented conidia from GAA cultures. Sequential treatment of these conidia with laminarinase (a ;J- 1,3-g1ucanase) and pronase, removes material from the wall. However, only after the complete removal of the outer layer (W 1) with pota ssium hydroxide prior to enzyme treatment, is the microfibrillar layer clearly revealed. The results from light microscopy of conidia treated with FITC Con. A and FITC WGA confirm some of the ultrastructural observations. Large pigmented conidia do not fluoresce with these stains
Ultrastructure of Ceratocystis adiposa
C. R. Hawes presumably because the lectin and/or the ultraviolet light cannot penetrate the layer of pigment in the wall. Conidia from GAA cultures fluoresce brightly with FITC Con. A but only dimly with FITC WGA. This supports the suggestion that the ch itinous layer is the innermost layer of the wall as the fluorescence of this layer is bright when all other material is removed prior to staining. FITC Con. A would bind to the alkali soluble a- 1,3glucans in the outer layer (W 1) of the wall and to any other polysaccharides with a-n-mannopyranosyl and a-n-glucanopyranosyl residues at the terminal end of the chain (Liener, 1976). This explains the lack of fluorescence with digested conidia as Con. A has no binding sites with fJ-N-acetylglucosamine. If it is assumed that the distribution of polymers throughout the wall is similar in those conidia produced by cultures on the different media, then a tentative model of the wall structure can be constructed. Fig. 39 shows a reconstruction of the wall based on the layers visible in a thin section of a large globose conidium from a CzA culture. A basal layer of chitin microfibrils bound with protein underlies a layer consisting of fJ-1,3-glu cans and protein which together make up W3 . Towards the outside of W 3 is a pigmented layer which is assumed to be melanized (W 2). Arising from this layer of pigment are spines of melanin embedded in an amorphous layer of a -1,3-glucans (W 1). These spines are only found in the larger pigmented conidia from CzA and MA cultures. The entire wall is surrounded by an alkali insoluble sh eath. I wish to thank Dr A. Beckett for valuable advice during the study and preparation of the manuscript, Mr R . Porter for technical assistance and Dr A. J. Abbott, Long Ashton Research Station, University of Bristol, for use of the fluore scence
microscope. Thanks are also due to the Science Research Council for a Research Studentship to the author. REFERENCES
AKAI, S., FUKUTOMI, M., KUNOH, H. & SHIRAISHI, M. (1976). Fine structure of the spore wall and germ tube change during germination. In Th e Fungal Spore (ed. D. J. Weber and W. M. Hess), ch, 9, pp. 355-411. New York, London, Sydney, Toronto : J. Wiley. BULL, A. T. (1970a). Chemical composition of wild type and mutant Aspergillus nidulans cell walls. The nature of the polysaccharide and melanin constituents. Journal of General Microbiology 63, 75--94. BULL, A. T. (1970b ). Inhibition of polysaccharases by melanin. Enzyme inhibition in relation to mycolysis. Archives of Biochemistry and Biophysics 137,345-356. CAMPBELL, R. (1969). An electron microscope study of spore structure and development in Alternaria brassicicola.Journal of General Microbiology 54, 381392. ELLIS, D. H. & GRIFFITHS, D. A. (1975 a). The fine structure of conidial development in the genus Torula. I. T. herbarum (Pers.) Link ex S. F. Gray and T. herbarum f. quaternella Sacco Canadian Journal of Microbiology %1, 1661-1675. ELLIS, D. H. & GRIFFITHS, D. A. (1975 b). The fine structure of conidial development in the genus Torula. II. T. caligans (Batista & Upadhyay) M. B. Ellis and T . terrestris Misra. Canadian Journal of M icrobiology %1, 1921-1929. GRIFFITHS, D. A. (1973). The fine structure of conidial development in Epicoccum nigrum.Journal de Microscopie 17, 55-64. HAMMILL, T. M. (1972). Electron microscopy of phialoconidiogenesis in Metarrhizium anisopliae. American Journal of Botany 59, 317-326. HARVEY, I. C. (1975). Studies on the growth and development of the hyphomycete, Pleiochaeta setosa, a pathogen of lupins. Ph.D. Thesis, University of Bristol.
Fig. 24. TEM chemically digested conidium (CzA culture). x 13700. Fig. 25. TEM chemically digested conidium incubated with chitinase (CzA culture). x 23400. Fig. 26. TEM chemicallydigested conidium incubated with pronase followed by chitinase (CzA culture). x 23300. Fig. 27. TEM untreated conidium (GAA culture). x 5900. Fig. 28. TEM conidium incubated with laminarinase. Some microfibrils are evident in the wall (GAA culture). x 5500. Fig. 29. TEM conidium autoc1aved in potassium hydroxide and incubated with laminarinase (GAA culture). x 6300. Fig. 30. TEM wall of a conidium after incubation with laminarinase followedby pronase (GAA culture), x 19700. Fig. 31. TEM wall of a conidium autoc1aved in potassium hydroxide and incubated with laminarinase followed by pronase (GAA culture). x 18000. Fig. 32. TEM wall of a conidium treated as in Fig. 31 (GAA culture). x 35500.
186
Ultrastructure of Ceratocystis adiposa
o
o
G G)
W3
39
W2
WI
C. R. Hawes HAWES, C. R. (1977). Light and electron microscope studies on the Chalara state of Ceratocystis adiposa (Butl.) C. Moreau. Ph.D. Thesis, University of Bristol. HAWES, C. R. & BECKETT, A. (1977a). Conidium ontogeny in the Chalara state of Ceratocystis adiposa. 1. Light microscopy. Transactions of the British Mycological Society 68, 259-265. HAWES, C. R. & BECKETT, A. (1977b). Conidium ontogeny in the Chalara state of Ceratocystis adiposa, II. Electron microscopy. Transactions of the British Mycological Society 68, 267-276. HUNSLEY, D. & BURNETT, J. H. (1970). The ultrastructural architecture of the walls of some hyphal fungi. Journal of General Microbiology 62, 2°3-218. JENSEN, W. A. (1962). Botanical Histochemistry. San Francisco and London: W. H. Freeman. LIENER, 1. E. (1976). Phytohemagglutinins (phytolectins). Annual Review of Plant Physiology 27, 291-319. LOTAN, R. & SHARON, N. (1973). The fluorescence of wheat germ agglutinin and of its complexes with saccharides. Biochemical and Biophysical Research Communications 55, 134°-1346. MAHADEVAN, P. R. & TATUM, E. L. (1967). Localisation of structural polymers in the cell wall of Neurospora crassa.Journal of Cell Biology 35, 295-302.
MARCHANT, R. (1966). Wall structure and spore germination in Fusarium culmorum. Annals of Botany 30, 821-83°. MICHALENCO, G. 0., HOHL, H. R. & RAST, D. (1976). Chemistry and architecture of the mycelial wall of Agaricus bisporus.tfournal of General Microbiology 92, 25 1-262. RAST, D. & HOLLENSTEIN, G. O. (1977). Architecture of the Agaricus bisporus spore wall. Canadian Journal of Botany 55, 2251-2262. RICHMOND, D. V. & PRING, R. J. (1971). Fine structure of Botrytis [abae Sardina conidia. Annals of Botany 35, 175-1 82. SPURR, A. R. (1969). A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 26, 31-43. VALK, P. VAN DER & WESSELS, J. G. H. (1976). Ultrastructure and localisation of wall polymers during regeneration of protoplasts of Schizophyllum commune. Protoplasma 90, 65-87. ZONNEVELD, B. J. M. (1971). Biochemical analysis of the cell wall of Aspergillus nidulans. Biochimica er Biophysica Acta 249, 506-514.
(Accepted for publication 10 May 1978)
Fig. 33. Fluorescence micrograph of thin-walled conidia stained with FITC Con. A showing strong fluorescence of the walls (GAA culture). x 1200. Fig. 34. Fluorescence micrograph of thin-walled conidia stained with FITC WGA (GAA culture).
x iooo. Fig. 35. Fluorescence micrograph of chemically digested conidia stained with FITC WGA showing strong fluorescence of the remaining wall material (GAA culture). x 1150. Fig. 36. Fluorescence micrograph of chemically digested large globose conidia stained with FITC WGA (CzA culture). x 990. Fig. 37-38. Fluorescence and bright field micrographs respectively of chemically digested conidia stained with FITC Con. A. The central portion of the conidium which represents the remains of the protoplast shows dim fluorescence (CzA culture). x 1300. Fig. 39. Diagrammatic interpretation of the wall structure of a large globose conidium. PM = plasma membrane; S = sheath; possible components of wall layers: W 1; «-glucans, melanin. W2; 8-glucans, melanin. W 3; Chitin microfibrils, fJ-glucans, protein.
April 1979
2
Printed in Great Britain
Trans. Br, mycol, Soc. 72 (2) 177-187 (1979)
CONIDIUM ULTRASTRUCTURE AND WALL ARCHITECTURE IN THE CHALARA STATE OF CERATOCYSTIS ADIPOSA By C. R. HAWES Department of Botany, The University of Bristol, Bristol, BS8
1 UG,
U.K.
When cultured on different media the Chalara state of Ceratocystis adiposa (Butl.) C. Moreau forms conidia of varying morphology. The major variation is in the thickness, elaboration and pigmentation of the wall of the coniduim. The appearance of conidial walls after chemical and enzymic treatments, and their binding properties with plant lectins is described. A model is proposed to show the distribution of polymers throughout the three wall layers. Little is known of the chemical composition and structure of conidium walls (Marchant, 1966;'Akai, Fukutomi, Kunoh& Shiraishi, 1976), although the cytoplasmic fine structure of conidia has been the subject of various studies (Campbell, 1969; Richmond & Pring, 1971; Hammill, 1972; Griffiths, 1973; Ellis & Griffiths, 1975a, b). Biochemical analyses of the walls of higher fungi such as Aspergillus nidulans (Eidam) Wint. (Bull, 1970a; Zonneveld, 1971) have produced no information as to the distribution of polymers. The structure of vegetative hyphal walls of various species has been elucidated by means of chemical and enzymic digestion (Mahadevan & Tatum, 1967; Hunsley & Burnett, 1970; Michalenco, Hohl & Rast, 1976), but only a few comparable studies have been carried out on spore walls (Rast & Hollenstein, 1977). The Chalara state of Ceratocystis adiposa (Butl.) C. Moreau when cultured on malt agar (MA) has been shown to produce conidia which are variable in size, shape, degree of pigmentation and ornamentation (Hawes & Beckett, 1977a, b). Conidia from cultures grown on different media also exhibit variation in the above features (Hawes, 1977) and the cytoplasmic and wall structure of these conidia is reported in this paper. MATERIALS AND METHODS
The culture of C. adiposa used (Hawes & Beckett, 1977 a) was maintained on Czapek-Dox (CzA), glucose asparagine (GAA) and malt agar (MA) Petri plates. Histochemical staining Cultures were obtained on cellophane discs (Hawes & Beckett, 1977a). Conidia were stained for lipids using Sudan Black Band benzpyrenecaffeine techniques (Jensen, 1962). Polysaccharides with a-D-manopyranosyl and «-n-glucanopy-
ranosyl residues were stained with fluoresceinisothiocyanate (FITC) conjugated Concanavalin A (Con. A) (Miles Yeda Ltd). This consisted of a stock solution of 8 mg of the protein per ml of 0'1 M-phosphate buffer at pH 7"4. 10,tt1 of the stock solution were diluted to 100 ,ttl with 1-0 MNaCl and used in 10,tt1 drops for staining. Chitin was stained with FITC wheat germ aglutinin (WGA) (Miles Yeda Ltd) which consisted of a solution of 1'85 mg of the protein per rnl of phosphate buffered saline at pH 7'4. The staining procedure was as with the FITC Con. A. Micrographs were recorded using Ilford Pan F 35 mm film on a Zeiss Photomicroscope II (Figs. 1,2,4,6) and Ilford HP4 35 rom film on a Zeiss Photomicroscope III with epifluorescence condenser III/RS, dark ground illumination and ultra-violet light (Figs. 3, 5, 33-37). Electron microscopy Material for transmission and scanning electron microscopy was fixed and prepared as described in Hawes & Beckett (1977 b). For freeze-fracturing, conidia were harvested in distilled water from an agar plate culture, centrifuged into a pellet and embedded in 2 % agar. The pellet was then split into 1 rom cubes, frozen on a precooled lead block, immersed in liquid nitrogen and fractured with a razor blade. The whole block was put under vacuum in an Edwards high vacuum coating unit and after sublimation of the nitrogen and water the dried material was mounted on stubs, coated with gold and observed with a Cambridge Scientific Instrument Company S4 scanning electron microscope (SEM). Carbon replicas of conidial surfaces were obtained by drying conidia suspended in drops of distilled water onto formvar-coated grids. These were coated from above with carbon and shadowed at 20°C with carbon/platinum. Replicas were cleaned by dipping the grids in chloroform
0007-1536/79/2828-4780 $01.00 © 1979 The British Mycological Society Vol. 72, Part 7
1,
was issued 20 February 1979 MYC 72
Ultrastructure of Ceratocystis adiposa followed by immersion in a mixture of potassium permanganate and potassium dichromate in concentrated sulphuric acid overnight. After washing in distilled water the replicas were treated with a 1 :1 (vIv) mixture of glacial acetic acid and 30 % hydrogen peroxide, picked up on new grids, washed and observed with an AE1 EM 6G transmission electron microscope (TEM). Chemical digestion of conidium walls Conidia from cultures on CzA and GAA were collected from suspensions in distilled water on millipore filter papers and treated as follows: (a) autoclaved in 4'5 % (w/v) KOH at 103'5 RN/m 2 for 15 min and washed in distilled water; (b) treated with a 1 : 1 (vIv) mixture of 30 % hydrogen peroxide and concentrated glacial acetic acid at 100 0 for 0'5-7 h and washed in distilled water. After each treatment conidia were (i) observed with the light microscope, (ii) dried onto forrnvarcoated grids, shadowed with carbon/platinum and observed with the TEM, and (iii) dehydrated in a graded water/ethanol series, embedded in Spurr's resin (Spurr, 1969), sectioned and observed with the TEM. Enzymic wall digestion Chitinase (fungal) and pronase (ex-Streptomyces griseusy were obtained from Koch-Light Laboratories Ltd and a laminarinase (ex mollusca) from Calbiochem. For each experiment conidia were incubated in flasks for 48 h at 37 with the appropriate enzyme in a sodium phosphate-citric acid buffer. With pronase, a potassium phosphate buffer was used. Laminarinase was incubated at pH 5'0, chitinase at pH 5"5 and pronase at pH 7'5 (Hunsley & Burnett, 1970). 2 ml of a suspension containing 0'5 mg enzyme per ml were added to 18 ml of
buffer, to this was added 2 ml of a conidial suspension in the appropriate buffer. The control for each treatment contained boiled enzyme. A drop of toluene was added to all treatments as a bacteriostat. After incubation, conidia were collected by centrifugation, washed four times in distilled water, dried onto formvar-coated grids, shadowed with carbon platinum and observed with the TEM. Conidia from CzA cultures were treated with all the enzymes. Chemically digested conidia from GAA cultures were treated with combinations of chitinase and pronase, and fresh conidia from these cultures were treated with combinations of laminarinase and pronase or autoclaved in 4'0% (w/v) potassium hydroxide prior to enzymic treatment. RESULTS
Conidia produced by cultures on CzA are large ( > 15 pm diam), globose, deeply pigmented and show little variation in shape (Figs. 2, 6, 7, 11). Easily fragmented chains of small, less-pigmented conidia are occasionally found (Fig. 1). Cultures on GAA form long conidial chains composed of hyaline slightly pigmented, small, round to doliiform occasionally pyriform spores « 12 pm diam) (Figs. 4, 9, 15). Conidia produced by cultures on MA (Fig. 10) are variable in size, shape, degree of pigmentation and wall ornamentation.
0
Cytoplasmic ultrastructure Lipid is the prominent storage product in conidial cytoplasm. Large globose conidia from CzA and MA cultures have a central aggregation of lipid (Figs. 2, 3), seen in section as numerous electron opaque bodies (Figs. 6, 7). In many of the chains
Fig. 1. Bright field micrograph of thin-walled conidia stained with Sudan Black B showing polar aggregations of lipid (CzA culture). x 1500. Fig. 2. Bright field micrograph of large globose conidia stained with Sudan Black B (CzA culture). x 1500. Fig. 3. Fluorescence micrograph of conidia stained using benzpyrene-caffeine (CzA culture). x 1300. Fig. 4. Bright field micrograph of thin-walled conidia stained with Sudan Black B (GAA culture). x 1200. Fig. 5. Fluorescence micrograph of conidia stained using benzpyrene-caffeine (GAA culture). x 1900. Fig. 6. Phase contrast micrograph of a large globose conidium (CzA culture). L = lipid. x 3800. Fig. 7. TEM large globose conidium with a 3-layered wall (CzA culture). L = lipid. x 6600. Fig, 8. TEM part of a conidium showing the association of vesiculate endoplasmic reticulum (VER) with the wall (CzA culture). x 20400. Fig. 9. TEM ovoid conidium with flocculose material in the cytoplasm and 3 wall layers (GAA culture). x 11400. Fig. 10. A cylindrical conidium with a 3-layered wall and polar aggregations of lipid (MA culture). x 5700.
C. R. Hawes
179
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.
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17
17:
Ultrastructure of Ceratocystis adiposa
C. R. Hawes of ovoid, less pigmented conidia from cultures on these media, the lipid is in polar clusters (Figs. 1, 10). Conidia from cultures on GAA stain less with Sudan Black Band benzpyrene-caffein and the lipid is scattered (Figs. 4, 5). Conidial cytoplasm contains smooth endoplasmic reticulum, varying numbers of mitochondria, vacuoles which may contain electron dense material, ribosomes, and from one to six nuclei (Figs. 7-10). The ribosome content of conidia from GAA cultures is considerably greater than that in other conidia (Fig. 9). Flocculose material, probably polysaccharide, is present in some conidia (Fig. 9). In the thick-walled conidia, areas of vesicular, smooth membrane are associated with the plasmalemma (Fig. 8). Surface structure of the conidium The conidial wall is verrucose/spiny to smooth, and all intermediates between these extremes in surface topography are seen (Figs. 11-15). The verrucose/spiny nature of the large globose conidia from CzA and MA cultures is often masked by the sheath (Fig. 13) but may be revealed by part removal of the sheath by freeze-fracturing (Fig. 12). Ornamentation of the wall of the conidium does not extend over the basal septal plate (Fig. 12, arrows). The intermediate forms of surface ornamentation may be reticulate or slightly verrucose as shown by conidia from MA cultures (Fig. 14). Conidia from easily fragmented chains are always smooth-walled as are conidia from GAA cultures where tearing of the sheath reveals no wall ornamentation (Fig. 15, arrows). Conidium wall in section With the light microscope the wall of a mature, globose, pigmented conidium from a CzA culture appears three-layered (Fig. 6). In thin sections the walls of mature conidia from cultures on all three media exhibit the same basic three-layered
181
structure (Figs. 7, 9, 10). An outer layer (W1) consisting of spines of pigment embedded in electron transparent material and surrounded by the sheath, overlies a middle pigmented layer (W2) to the inside of which is an electron transparent layer (W 3) (Fig. 39). The major variation between the different conidium types is in the thickness, elaboration and degree of pigmentation of the outer two layers. The two extremes of wall development are represented by the deeply pigmented, large, globose, and spiny-walled conidia from CzA cultures where the wall may be up to 2'6 pm thick (Fig. 7) and the slightly pigmented, globose to doliiform, smooth-walled conidia from GAA cultures where the wall may be as thin as 0'2 psi». (Fig. 9). In the latter conidia the outer two layers are poorly differentiated with little pigment deposition (Fig. 9). Chemical digestion of conidia The effect of prolonged chemical digestion on conidia from CzA cultures is shown in Figs. 16-22. Autoclaving in potassium hydroxide removes the material from the outer wall layer (W 1) in which the spines of pigment are embedded and the sheath collapses around them (Fig. 16). Prolonged boiling in a glacial acetic acid/hydrogen peroxide mixture slowly digests the conidial wall (Figs. 17-19) until, after 5-7 h, only a layer of closely intermeshed microfibrils remains (Fig. 22). After digestion for 0'5 h thin sections reveal some bleaching or removal of the pigment but the sheath remains intact (Fig. 20 arrows). After 1 h individual layers cannot be distinguished (Fig. 21). In fully digested conidia a circular arrangement of microfibrils around the septal pore at the base ofthe conidium is often seen (Fig. 22 arrows). Chemical digestion of the thin-walled conidia from GAA cultures proceeds more rapidly than in those described above, and the microfibrillar layer is exposed after digestion for 0'5-1 h (Fig. 23).
Fig. 11. SEM large globose conidium (CzA culture). x 3700. Fig. 12. SEM large globose conidium with the sheath partly removed revealing spines of pigmented material (CzA culture, freeze-fractured material). x 3700. Fig. 13. TEM replica of a large globose conidium (CzA culture). x 2700. Fig. 14. SEM conidium with reticulate ornamentation of the wall (MA culture). x 4100. Fig. 15. SEM ovoid smooth-walled conidium (GAA culture). x 8000. Fig. 16. SEM large globose conidium after autoclaving in potassium hydroxide. Much of the outer wall layer has been removed (CzA culture). x 4°°00. Fig. 17. LM large globose conidium (CzA culture). x 2(';00. Fig. 18. LM large globose conidium after digestion for 0-5 h in hydrogen peroxide/acetic acid (CzA culture). x 1900. Fig. 19. LM large globose conidium after chemical digestion for 7 h (CzA culture). x 2200.
182
Ultrastructure of Ceratocystis adiposa
..
~
23 Fig. 20. TEM section of the wall of a large globose comuium atter digestion for 0'5 h in hydrogen peroxide/acetic acid. The sheath (arrows) remains intact around the outer wall layer (CzA culture). x 18000. Fig. 21. TEM section of the wall of a large globose conidium after chemical digestion for 1 h (CzA culture). x 13300. Fig. 22. TEM large globose conidium after digestion for 7 h showing the circular arrangement of microfibrils around the septal pore (arrows) (CzA culture). x 9000. Fig. 23. TEM part of a thin-walled, ovoid conidium after digestion for 1 h (GAA culture). x 18000.
C. R. Hawes Enzymic digestion of conidia
Treatment of conidia from cultures on CzA with laminarinase, pronase and chitinase individually, has no effect on the wall structure. After incubation of chemically digested conidia from GAA cultures in chitinase the microfibrils become more clearly defined (Figs. 24, 25). Treatment of these digested conidia with pronase prior to chitinase results in some disruption of the microfibrils (Fig. 26). Shadowed preparations of fresh conidia from GAA cultures are electron opaque (Fig. 27), but digestion with laminarinase renders the conidium less opaque. The surface of the conidium is amorphous to granulate and an underlying microfibrillar structure can be seen (Fig. 28). Autoclaving in potassium hydroxide before laminarinase treatment results in a less granulate wall texture (Fig . 29). Preparations of conidia treated sequentially with laminarinase and pronase show an amorphous surface structure and microfibrils are not discernible (Fig. 30). The microfibrillar layer as revealed by chemical digestion is exposed by laminarinase and pronase treatment only after prior autoclaving in potassium hydroxide (F igs. 31, 32). Fluorescent staining Thin-walled conidia from GAA cultures fluoresce brightly with ultra-violet light when stained with FITC Con. A (Fig. 33), but onl y show a dim fluorescence with FITC WGA (Fig. 34). Chemically digested conidia from both GAA and CzA cultures fluoresce brightly with FITC WGA (Figs. 35,36), but only the central portion, that marks the remains of the protoplast, (Fig. 38) fluoresces when stained with FITC Con. A and then onl y faintly (Fig. 37). This area, however, also shows weak autofluorescence with ultra-violet light . DISCUSSION
, Cytoplasmic ultrastructure With the exception of the numerous lipid bodies, the organelle complement of mature conidia is similar to that of the conidiophores and developing conidia (Hawes & Beckett, 1977b). The areas of vesicular smooth endoplasmic reticulum found in thick-walled conidia are probably involved in the centripetal deposition of wall material around the protoplast as conidia in the chain continue to mature. The association of endoplasmic reticulum with the plasmalemma has also been seen in the conidia of Botrytis fabae Sardina (Richmond & Pring, 1971) and in the exogenously dormant conidia of Pleiochaeta setosa (Kirchn.) Hughes (Harvey, 1975). The large ribosome content of
conidia from GAA cultures is similar to that found in the conidiophores and young conidia from these cultures (Hawes, 1977). Conidium wall structure Several tentative conclusions can be drawn as to the structure of the wall and the distribution of polymers. It is the degree of development of the outer layer (W 1) that governs the surface ornamentation. Thus in the large globose conidia from GAA cultures, W 1 is thick with well-developed spines of pigment making the surface verrucose/ spiny. In contrast in the thin-walled conidia from GAA cultures, this layer is thin without much pigment and the surface is correspondingly smooth. The electron-transparent material in which the spines of pigment are embedded is soluble in potassium hydroxide and when removed leaves the pigment surrounded by the alkali resistant sheath. This electron-transparent material could be an alkali soluble a - 1,3-glucan similar to that which makes up the amorphous outer wall layer of the hyphae of Sch izophyllum commune Fr. (Hunsley & Burnett, 1970; van der Valk & Wessels, 1976). Prolonged chemical digestion of conidia from both CzA and GAA cultures removes all the wall material with the exception of a microfibrillar layer. Treatment of this layer with chitinase makes the fibrils more distinct, but only after pronase followed by chitinase treatment is there any disruption of the microfibrils. This suggests that the layer is composed of chitin microfibrils bound with protein. A similar structure has been proposed for layer II of the Agaricus bisporus spore wall (Rast & Hollenstein, 1977). Fluorescence under ultra-violet light of chemically digested conidia stained with FITC WGA, which binds to N-acetylglucosamine (Lotan & Sharon, 1973), verifies the presence of chitin in this layer . Melanin in the hyphal walls of Aspergillus nidulans has been shown to inhibit the activity of a ;J-1,3-g1ucanase and a chitinase (Bull, 1970b). It is possibly melanin that prevents enzymic digestion of the large pigmented conidia of C. adiposa. Enzyme work was therefore restricted to the thinwalled, less-pigmented conidia from GAA cultures. Sequential treatment of these conidia with laminarinase (a ;J- 1,3-g1ucanase) and pronase, removes material from the wall. However, only after the complete removal of the outer layer (W 1) with pota ssium hydroxide prior to enzyme treatment, is the microfibrillar layer clearly revealed. The results from light microscopy of conidia treated with FITC Con. A and FITC WGA confirm some of the ultrastructural observations. Large pigmented conidia do not fluoresce with these stains
Ultrastructure of Ceratocystis adiposa
C. R. Hawes presumably because the lectin and/or the ultraviolet light cannot penetrate the layer of pigment in the wall. Conidia from GAA cultures fluoresce brightly with FITC Con. A but only dimly with FITC WGA. This supports the suggestion that the ch itinous layer is the innermost layer of the wall as the fluorescence of this layer is bright when all other material is removed prior to staining. FITC Con. A would bind to the alkali soluble a- 1,3glucans in the outer layer (W 1) of the wall and to any other polysaccharides with a-n-mannopyranosyl and a-n-glucanopyranosyl residues at the terminal end of the chain (Liener, 1976). This explains the lack of fluorescence with digested conidia as Con. A has no binding sites with fJ-N-acetylglucosamine. If it is assumed that the distribution of polymers throughout the wall is similar in those conidia produced by cultures on the different media, then a tentative model of the wall structure can be constructed. Fig. 39 shows a reconstruction of the wall based on the layers visible in a thin section of a large globose conidium from a CzA culture. A basal layer of chitin microfibrils bound with protein underlies a layer consisting of fJ-1,3-glu cans and protein which together make up W3 . Towards the outside of W 3 is a pigmented layer which is assumed to be melanized (W 2). Arising from this layer of pigment are spines of melanin embedded in an amorphous layer of a -1,3-glucans (W 1). These spines are only found in the larger pigmented conidia from CzA and MA cultures. The entire wall is surrounded by an alkali insoluble sh eath. I wish to thank Dr A. Beckett for valuable advice during the study and preparation of the manuscript, Mr R . Porter for technical assistance and Dr A. J. Abbott, Long Ashton Research Station, University of Bristol, for use of the fluore scence
microscope. Thanks are also due to the Science Research Council for a Research Studentship to the author. REFERENCES
AKAI, S., FUKUTOMI, M., KUNOH, H. & SHIRAISHI, M. (1976). Fine structure of the spore wall and germ tube change during germination. In Th e Fungal Spore (ed. D. J. Weber and W. M. Hess), ch, 9, pp. 355-411. New York, London, Sydney, Toronto : J. Wiley. BULL, A. T. (1970a). Chemical composition of wild type and mutant Aspergillus nidulans cell walls. The nature of the polysaccharide and melanin constituents. Journal of General Microbiology 63, 75--94. BULL, A. T. (1970b ). Inhibition of polysaccharases by melanin. Enzyme inhibition in relation to mycolysis. Archives of Biochemistry and Biophysics 137,345-356. CAMPBELL, R. (1969). An electron microscope study of spore structure and development in Alternaria brassicicola.Journal of General Microbiology 54, 381392. ELLIS, D. H. & GRIFFITHS, D. A. (1975 a). The fine structure of conidial development in the genus Torula. I. T. herbarum (Pers.) Link ex S. F. Gray and T. herbarum f. quaternella Sacco Canadian Journal of Microbiology %1, 1661-1675. ELLIS, D. H. & GRIFFITHS, D. A. (1975 b). The fine structure of conidial development in the genus Torula. II. T. caligans (Batista & Upadhyay) M. B. Ellis and T . terrestris Misra. Canadian Journal of M icrobiology %1, 1921-1929. GRIFFITHS, D. A. (1973). The fine structure of conidial development in Epicoccum nigrum.Journal de Microscopie 17, 55-64. HAMMILL, T. M. (1972). Electron microscopy of phialoconidiogenesis in Metarrhizium anisopliae. American Journal of Botany 59, 317-326. HARVEY, I. C. (1975). Studies on the growth and development of the hyphomycete, Pleiochaeta setosa, a pathogen of lupins. Ph.D. Thesis, University of Bristol.
Fig. 24. TEM chemically digested conidium (CzA culture). x 13700. Fig. 25. TEM chemically digested conidium incubated with chitinase (CzA culture). x 23400. Fig. 26. TEM chemicallydigested conidium incubated with pronase followed by chitinase (CzA culture). x 23300. Fig. 27. TEM untreated conidium (GAA culture). x 5900. Fig. 28. TEM conidium incubated with laminarinase. Some microfibrils are evident in the wall (GAA culture). x 5500. Fig. 29. TEM conidium autoc1aved in potassium hydroxide and incubated with laminarinase (GAA culture). x 6300. Fig. 30. TEM wall of a conidium after incubation with laminarinase followedby pronase (GAA culture), x 19700. Fig. 31. TEM wall of a conidium autoc1aved in potassium hydroxide and incubated with laminarinase followed by pronase (GAA culture). x 18000. Fig. 32. TEM wall of a conidium treated as in Fig. 31 (GAA culture). x 35500.
186
Ultrastructure of Ceratocystis adiposa
o
o
G G)
W3
39
W2
WI
C. R. Hawes HAWES, C. R. (1977). Light and electron microscope studies on the Chalara state of Ceratocystis adiposa (Butl.) C. Moreau. Ph.D. Thesis, University of Bristol. HAWES, C. R. & BECKETT, A. (1977a). Conidium ontogeny in the Chalara state of Ceratocystis adiposa. 1. Light microscopy. Transactions of the British Mycological Society 68, 259-265. HAWES, C. R. & BECKETT, A. (1977b). Conidium ontogeny in the Chalara state of Ceratocystis adiposa, II. Electron microscopy. Transactions of the British Mycological Society 68, 267-276. HUNSLEY, D. & BURNETT, J. H. (1970). The ultrastructural architecture of the walls of some hyphal fungi. Journal of General Microbiology 62, 2°3-218. JENSEN, W. A. (1962). Botanical Histochemistry. San Francisco and London: W. H. Freeman. LIENER, 1. E. (1976). Phytohemagglutinins (phytolectins). Annual Review of Plant Physiology 27, 291-319. LOTAN, R. & SHARON, N. (1973). The fluorescence of wheat germ agglutinin and of its complexes with saccharides. Biochemical and Biophysical Research Communications 55, 134°-1346. MAHADEVAN, P. R. & TATUM, E. L. (1967). Localisation of structural polymers in the cell wall of Neurospora crassa.Journal of Cell Biology 35, 295-302.
MARCHANT, R. (1966). Wall structure and spore germination in Fusarium culmorum. Annals of Botany 30, 821-83°. MICHALENCO, G. 0., HOHL, H. R. & RAST, D. (1976). Chemistry and architecture of the mycelial wall of Agaricus bisporus.tfournal of General Microbiology 92, 25 1-262. RAST, D. & HOLLENSTEIN, G. O. (1977). Architecture of the Agaricus bisporus spore wall. Canadian Journal of Botany 55, 2251-2262. RICHMOND, D. V. & PRING, R. J. (1971). Fine structure of Botrytis [abae Sardina conidia. Annals of Botany 35, 175-1 82. SPURR, A. R. (1969). A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 26, 31-43. VALK, P. VAN DER & WESSELS, J. G. H. (1976). Ultrastructure and localisation of wall polymers during regeneration of protoplasts of Schizophyllum commune. Protoplasma 90, 65-87. ZONNEVELD, B. J. M. (1971). Biochemical analysis of the cell wall of Aspergillus nidulans. Biochimica er Biophysica Acta 249, 506-514.
(Accepted for publication 10 May 1978)
Fig. 33. Fluorescence micrograph of thin-walled conidia stained with FITC Con. A showing strong fluorescence of the walls (GAA culture). x 1200. Fig. 34. Fluorescence micrograph of thin-walled conidia stained with FITC WGA (GAA culture).
x iooo. Fig. 35. Fluorescence micrograph of chemically digested conidia stained with FITC WGA showing strong fluorescence of the remaining wall material (GAA culture). x 1150. Fig. 36. Fluorescence micrograph of chemically digested large globose conidia stained with FITC WGA (CzA culture). x 990. Fig. 37-38. Fluorescence and bright field micrographs respectively of chemically digested conidia stained with FITC Con. A. The central portion of the conidium which represents the remains of the protoplast shows dim fluorescence (CzA culture). x 1300. Fig. 39. Diagrammatic interpretation of the wall structure of a large globose conidium. PM = plasma membrane; S = sheath; possible components of wall layers: W 1; «-glucans, melanin. W2; 8-glucans, melanin. W 3; Chitin microfibrils, fJ-glucans, protein.