Journalof Medicaland VeterinaryMycology(1991),29, 387-393
Ultrastructure of budding process of
Malassezia pachydermatis K. N I S H I M U R A , Y. ASADA, 1 S. T A N A K A 2 AND S. W A T A N A B E 2
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1Department of Dermatology, Kansai Medical University, Fumizono-cho 1, Moriguchi, Osaka and 2Department of Dermatology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Japan (Accepted 2 September1991) The ultrastructure of Malasseziapachydermatisand its budding process was investigatedby scanning and transmission electron microscopy.The innermost layer of the cell wall showed the serrated structure characteristicof the genus Malassezia.In the daughter cell, this structure became more defined as the cell grew. The mode of conidium ontogeny was monopolar blastic development with a collarette. The appearance was similar to that reported previously for Malassezia furfur, with the following difference: the budding base was 0-9-1.1 /zm in diameter which was broader than that of M.furfur (0.54).7/tm).
Malassezia pachydermatis was first detected in 1925 in the scales of an Indian rhinoceros with severe exfoliative dermatitis. Since then, it has been isolated from both healthy and diseased skin of dogs, cats, humans, Indian elephants and black bears [6, 21], as well as from ulcerated conjunctiva of dogs, cats and pigs [12], and from the throats of scarlet macaws [4]. Although M. pachydermatis has been isolated from humans [16, 17, 21], the pathogenic role of this organism in humans has remained unclear. Recently, however, systemic infections due to M. pachydermatis have been reported in high-risk infants and immunocompromised patients [7, 13, 14]. Due to the difficulty of fixation, there have been few reports on the ultrastructure of this fungus [1, 18]. In the present study, the ultrastructure of M. pachydermatis and its budding process was investigated and compared with that of Malassezia furfur, which has been reported previously [2, 8-11, 18]. METHODS
Strain M. pachydermatis strain K H L 3 (Kobe Central Municipal Hospital Laboratory, Kobe, Japan) was inoculated onto modified Bacto Yeast Morphology Agar (Difco Laboratories, Detroit, MI) slants as described by Porro et al. [15], and incubated for 1 to 3 weeks at 30°C.
Electron microscopy Scanning electron microscopy (SEM). Yeasts grown as described above were harvested and fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7-4) for 2 h at 4°C. Correspondence address: K. Nishimura, Department of Dermatology, Kansai Medical University, Fumizono-cho1, Moriguchi, Osaka, 570, Japan. 387
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Med Mycol Downloaded from informahealthcare.com by Flinders University of South Australia on 01/15/15 For personal use only.
Post-fixation was performed with 1% osmium tetroxide in the same buffer for i h at 4°C. The cells were dehydrated in a graded series of ethanol, dried in a desiccator to a critical point, and platinum and palladium-enveloped with an ion coater. They were then examined with a Hitachi S-700 scanning electron microscope at 20 kV.
Transmission electron microscopy (TEM). Samples were fixed with 4% glutaraldehyde in 0.1 ~ cacodylate buffer (pH 7.2) for 2 h at 25°C. Post-fixation was performed with 2% osmium tetroxide in the same buffer for 2 h at 25°C. The cells were then dehydrated in an ethanol series and embedded in Spurr's resin. Ultrathin sections were stained with uranyl acetate and Reynolds' lead citrate, and examined with Hitachi H-300 and H-500 transmission electron microscopes at 75 kV.
RESULTS
Electron microscopic observations SEM. Cells were ovoid to cylindrical in shape. The surface topography of these cells was generally smooth and various stages of monopolar blastic reproduction were observed. Many cells were budding on a broad base and had a collarette at the base (Fig. 1). TEM. The cell wall was relatively thick (0.1-0-25/zm) and multilayered, consisting of fibrillar components. The electron density of the inner side of the cell wall was higher than that of the outer side. The outermost surface was slightly rough and reticular. The innermost surface of the cell wall showed a serrated structure, which is the characteristic feature of the genus Malassezia. On sagittal section, 14 to 16 ridges were observed (Fig. 2a). A plasma membrane was observed to follow the serrated curve of the innermost surface of the cell wall (Fig. 2b). The nucleus was approximately spherical in
FIG. 1. SEM of M. pachydermatis. Many cells budding on a broad base. Bar = 1/zm.
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a FIG. 2. Nucleus (N), cell wall (CW) which is serrated, plasma membrane (PM), glycogen-like granules (G) and vacuoles (V) can be observed. A part of the cytoplasm near the bud base is bulging, while the cell wall of this portion has become thin (asterisks). Bar = 0.5 pin. (a) Separation of an ear-like structure from the cell wall (arrowheads). (b) The ear-like structure growing outwards.
shape, with a diameter of about 0-8 #m. The nucleoplasm had the same or lower electron density compared to the cytoplasm and was partly granular (Fig. 2a). Vacuoles of various sizes and shapes were present, some containing osmiophilic dense material (Figs. 2 and 3). Glycogen-like granules were widely distributed in the cytoplasm, however, no mitochondria were observed.
Budding process A part of the cell wall was first observed to separate from that of the mother cell and protrude outwards forming an ear-like shape. At the same time, a part of the cytoplasm of the mother cell near the bud base was bulging, and the cell wall of this portion became thin and dimpled. The cell wall of the bud tip was thin and rough compared with that of the mother cell (Fig. 2a). The ear-like projection continued growing outwards, separating from the mother cell, and the cell wall of the bud followed by the plasma membrane and the cytoplasm continued growing forwards (Fig. 2b). This earlike projection appeared to correspond to a collarette. The cell wall of the bud tip was thinner and less dense than that of the mother cell. No serrated structure could be detected in the bud at this stage. The bud base was now about 0-9 pm in diameter. No apical vesicles were observed. The cytoplasm protruded partially and the cell wall of this portion was dimpled near the bud base, as observed earlier. The cell wall of the daughter cell became slightly ridged at this stage, and the bud base was now 1.0/.tm wide (Fig. 3a). As the daughter cell grew, its inner cell wall became ridged but to a lesser degree than that of the mother cell. In the daughter cell as well as the mother cell, partial protrusion of the cytoplasm and dimpling of the cell wall was
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FIG. 3. (a) The cell wall of the daughter cell becomes slightly ridged. (b) A partial protrusion of the cytoplasm and dimpling of the cell wall are observed near the bud base in the daughter cell as well as in the mother cell (arrows). (c) The cell wall of the daughter cell becomes clearly serrated as it grows. Bar = 0.5/lm.
observed near the bud base (Fig. 3b). As the daughter cell grew, its cell wall became thicker and layered, and developed a serrated structure (Fig. 3c). Eventually, the daughter cell reached approximately the same length as the mother cell with a total length of about 7-0/.tm (Fig. 4a). The cytoplasm and cell wall of the daughter cell had almost the same structure as the mother cell, and 14 to 16 ridges were observed on each
FIG. 4. (a) The daughter cell becomes as long as the mother cell. (b) Formation of a cross wall by a protruding sickle-like structure. (c) The cross wall is continuous with the cell wall and has a narrow lumen. (d) Fission takes place at the lumen. Bar - 0-5/~m.
391
BUDDING PROCESS OF M. PACHYDERMATIS
side. At this stage, a cross wall started to form by the protrusion of a sickle-like structure from the cell wall at the bud base (Fig. 4b). The cross wall was continuous with the cell wall and had a narrow lumen (Fig. 4c). No pores, such as dolipores, were observed. After completion of cross wall formation, fission took place at the lumen of the cross wall and budding was complete. The ear-like projection of the mother cell remained as a bud scar (Fig. 4d).
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DISCUSSION The cell wall of M. pachydermatis was thick (0-1-0-25/.tin) and multilayered, but the three layers described previously [1] could not be differentiated. The innermost layer of the cell wall had a serrated structure which is characteristic of members of the genus Malassezia [18]. In the bud, this structure became apparent as it grew. About 14 to 16 ridges were observed on sagittal section. The plasma membrane ran adjacent to the serrated curve of the cell wall as a pair of unit membranes. Breathnach et al. [3] and Takeo & Nakai [19] reported that such serrated structures appeared to be spirals when observed by freeze-fracture techniques. The size of the nucleus was approximately the same as that of M. furfur. Vacuoles varied in shape and size, and some of them contained electron-dense material Wikler et al. [20] reported that this electron-dense material decreased or disappeared under ultraviolet light radiation and thought it could be used in cell metabolism. The bud base measured 0-9-1.1 /lm in diameter and was broader than that of Pityrosporum orbiculare and Pityrosporum ovale (0.5-0.7 #m) [11], whose names are
a
d
b
l
e
f
C
FIG. 5. Diagram of budding process of M. pachydermatis. (a) A n ear-like structure separating from the cell wall. (b) An ear-like structure growing outwards. (c) The cell wall of the daughter cell becomes serrated as it grows. (d) The daughter cell becomes as long as the mother cell and cross-wall formation begins as a protruding sickle-like structure. (e) The cross-wall has a narrow lumen. (f) Fission occurs at the cross-wall and the ear-like projection persists as a bud scar.
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synonyms of M. furfur. The ring swelling of the plasma membrane described by Takeo & Nakai [19] was not observed in the present study. The budding process is summarized in Fig. 5. First, an ear-like projection separated from the cell wall as described by Abou-Gabal & Fagerland [1] and developed in an outward direction (Fig. 5a). During the early stage, partial protrusion of the cytoplasm and dimpling of the cell wall of this portion were observed near the bud base, not only in the mother cell but also in the daughter cell (Fig. 5b). Such structures were thought to correspond to the circumvallate bulgings observed between ridges near the base by Breathnach et al. [3] and Takeo & Nakai [19]. As the bud grew, its cell wall became thick, layered and ridged (Fig. 5c). When the daughter cell was as long as the mother cell, a cross-wall was formed (Fig. 5d). The cross-wall had a narrow lumen where fission took place (Fig. 5e). The ear-like projection persisted as a bud scar after budding was complete (Fig. 5f). This mode of conidium ontogeny is monopolar blastic development [5]. The budding process of the genus Malassezia has been reported previously [1, 8, 11]. Compared with these reports, the budding process of M. pachydermatis appears to be similar to that of M. furfur. The present study shows that the broader base is a characteristic feature of M. pachydermatis, but we do not consider this difference to be of taxonomic value. ACKNOWLEDGEMENTS The authors thank Dr Matsuda for kindly supplying the strain of M. pachydermatis and Dr Saito for helpful advice.
REFERENCES 1. ABOU-GABAL, M. & FAGERLAND,J. A. 1979. Electron microscopy of Pityrosporum canis "pachydermatis". Mykosen, 22, 85-90. 2. BARFATANI,M., MUNN, R. J. & SCHJEIDE, O. A. 1964. An ultrastructure study of Pityrosporum orbiculare. Journal of Investigative Dermatology, 43, 231-233. 3. BREATHNACH,A. S., GROSS, M. & MARTIN, B. 1976. Freeze-fracture replication of cultured Pityrosporum orbiculare. Sabouraudia, 14, 105-113. 4. BREUER-STROSBERG,R., HOCHLEITHNER, M. 8z KUTTIN, E. S. 1990. Malassezia pachydermatis isolation from a scarlet macaw. Mycoses, 33, 247-250. 5. COLE, G. T. 1981. Conidiogenesis and conidiomatal ontogeny. In: G. T. COLE & B. KENDRICK (Eds) Biology of Conidial fungi. Vol. 2, pp. 271-327. Academic Press, New York. 6. GORDON, M. A. 1979. Malassezia (Pityrosporum) pachydermatis (Weidman) Dodge 1935. Sabouraudia, 17, 305-309. 7. GUEHO, E., SIMMONS, R. B., PRUITr, W. R., MEYER, S. A. & AHEARN, D. G. 1987. Association of Malassezia pachydermatis with systemic infections of humans. Journal of Clinical Microbiology, 25, 1789-1790. 8. IKEYA, T. 1974. Electron microscopic study on Malassezia furfur in horny layer. Japanese Journal of Medical Mycology, 15, 39-51. 9. KEDDIE, F. M. 1966. Electron microscopy of Malassezia furfur in tinea versicolor. Sabouraudia, 5, 134-137. t0. KEDDIE, F. M. ~,z BARAJAS,L. 1969. Three-dimensional reconstruction of Pityrosporum yeast cells based on serial section electron microscopy. Journal of Ultrastructure Research, 29, 260-275. 11. KEDDIE, F. M. & BARAJAS,L. 1972. Quantitative ultrastructural variations between Pityrosporum ovate and P orbiculare based on serial section electron microscopy. International Journal of Dermatology, 11, 40-48. 12. KOCKOVA-KRATOCHViLOVA,A., LADZIANSKA,K. & BUCKO, S. 1987. Malassezia pachydermatis in small animals. Mykosen, 30, 541-543.
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13. LAROCCO,M., DORENBAUM,A., ROBINSON,A. & PICKERING,L. K. 1988. Recovery of Malasseziapachydermatis from eight infants in a neonatal intensive care nursery: clinical and laboratory features. Pediatric Infectious Diseases Journal, 7, 398-401. 14. MICKELSEN, P. A., VIANO-PAULSON, M. C , STEVENS, D. A. & DIAZ, P. S. 1988. Clinical and microbiological features of infection with Malassezia pachydermatis in high-risk infants. Journal of Infectious Diseases, 157, 1163-1168. 15. PORRO, M. N., PASSI, S., CAPRILLI, F. & MERCANTINI, R. 1977. Induction of hyphae in cultures of Pityrosporum by cholesterol and cholesterol esters. Journal oflnvestigative Dermatology, 69, 531-534. 16. ROMANO. A., SEGAL,E. & BLUMENTHAL,M. 1978. Canaliculitis with isolation of Pityrosporum pachydermatis. British Journal of Ophthalmology, 62, 732-734. 17. SOMERVILLE,D. A. 1972. Yeasts in a hospital for patients with skin diseases. Journal of Hygiene, 70, 667-675. 18. SWIFT.J. A. & DUNBAR,S. F. 1965. Ultrastructure of Pityrosporum ovale and Pityrosporum canis. Nature, 206, 1174-1175 19. TAKEO, K. & NAKAI, E. 1986. Mode of cell growth of Malassezia (Pityrosporum) as revealed by using plasma membrane configurations as natural markers. Canadian Journal of Microbiology, 32, 389-394. 20. WIKLER, J. R., JANSSEN, N., BRUYNZEEL, D. P. & NIEBOER, C. 1990. The effect of UV-light on Pityrosporum yeasts: ultrastructural changes and inhibition of growth. Acta Dermato-Venereologica, 70, 69-71. 21. YARROW,D. & AHEARN, D. G. 1984. Malassezia Baillon. In: N. J. W. KREGER-VANRJJ (Ed.) The yeasts. A taxonomic study, pp. 882-885. Elsevier Science Publishers B. V., Amsterdam.
Ultrastructure of budding process of
Malassezia pachydermatis K. N I S H I M U R A , Y. ASADA, 1 S. T A N A K A 2 AND S. W A T A N A B E 2
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1Department of Dermatology, Kansai Medical University, Fumizono-cho 1, Moriguchi, Osaka and 2Department of Dermatology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Japan (Accepted 2 September1991) The ultrastructure of Malasseziapachydermatisand its budding process was investigatedby scanning and transmission electron microscopy.The innermost layer of the cell wall showed the serrated structure characteristicof the genus Malassezia.In the daughter cell, this structure became more defined as the cell grew. The mode of conidium ontogeny was monopolar blastic development with a collarette. The appearance was similar to that reported previously for Malassezia furfur, with the following difference: the budding base was 0-9-1.1 /zm in diameter which was broader than that of M.furfur (0.54).7/tm).
Malassezia pachydermatis was first detected in 1925 in the scales of an Indian rhinoceros with severe exfoliative dermatitis. Since then, it has been isolated from both healthy and diseased skin of dogs, cats, humans, Indian elephants and black bears [6, 21], as well as from ulcerated conjunctiva of dogs, cats and pigs [12], and from the throats of scarlet macaws [4]. Although M. pachydermatis has been isolated from humans [16, 17, 21], the pathogenic role of this organism in humans has remained unclear. Recently, however, systemic infections due to M. pachydermatis have been reported in high-risk infants and immunocompromised patients [7, 13, 14]. Due to the difficulty of fixation, there have been few reports on the ultrastructure of this fungus [1, 18]. In the present study, the ultrastructure of M. pachydermatis and its budding process was investigated and compared with that of Malassezia furfur, which has been reported previously [2, 8-11, 18]. METHODS
Strain M. pachydermatis strain K H L 3 (Kobe Central Municipal Hospital Laboratory, Kobe, Japan) was inoculated onto modified Bacto Yeast Morphology Agar (Difco Laboratories, Detroit, MI) slants as described by Porro et al. [15], and incubated for 1 to 3 weeks at 30°C.
Electron microscopy Scanning electron microscopy (SEM). Yeasts grown as described above were harvested and fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7-4) for 2 h at 4°C. Correspondence address: K. Nishimura, Department of Dermatology, Kansai Medical University, Fumizono-cho1, Moriguchi, Osaka, 570, Japan. 387
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NISHIMURA ET AL.
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Post-fixation was performed with 1% osmium tetroxide in the same buffer for i h at 4°C. The cells were dehydrated in a graded series of ethanol, dried in a desiccator to a critical point, and platinum and palladium-enveloped with an ion coater. They were then examined with a Hitachi S-700 scanning electron microscope at 20 kV.
Transmission electron microscopy (TEM). Samples were fixed with 4% glutaraldehyde in 0.1 ~ cacodylate buffer (pH 7.2) for 2 h at 25°C. Post-fixation was performed with 2% osmium tetroxide in the same buffer for 2 h at 25°C. The cells were then dehydrated in an ethanol series and embedded in Spurr's resin. Ultrathin sections were stained with uranyl acetate and Reynolds' lead citrate, and examined with Hitachi H-300 and H-500 transmission electron microscopes at 75 kV.
RESULTS
Electron microscopic observations SEM. Cells were ovoid to cylindrical in shape. The surface topography of these cells was generally smooth and various stages of monopolar blastic reproduction were observed. Many cells were budding on a broad base and had a collarette at the base (Fig. 1). TEM. The cell wall was relatively thick (0.1-0-25/zm) and multilayered, consisting of fibrillar components. The electron density of the inner side of the cell wall was higher than that of the outer side. The outermost surface was slightly rough and reticular. The innermost surface of the cell wall showed a serrated structure, which is the characteristic feature of the genus Malassezia. On sagittal section, 14 to 16 ridges were observed (Fig. 2a). A plasma membrane was observed to follow the serrated curve of the innermost surface of the cell wall (Fig. 2b). The nucleus was approximately spherical in
FIG. 1. SEM of M. pachydermatis. Many cells budding on a broad base. Bar = 1/zm.
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BUDDING PROCESS OF M. P A C H Y D E R M A T 1 S
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a FIG. 2. Nucleus (N), cell wall (CW) which is serrated, plasma membrane (PM), glycogen-like granules (G) and vacuoles (V) can be observed. A part of the cytoplasm near the bud base is bulging, while the cell wall of this portion has become thin (asterisks). Bar = 0.5 pin. (a) Separation of an ear-like structure from the cell wall (arrowheads). (b) The ear-like structure growing outwards.
shape, with a diameter of about 0-8 #m. The nucleoplasm had the same or lower electron density compared to the cytoplasm and was partly granular (Fig. 2a). Vacuoles of various sizes and shapes were present, some containing osmiophilic dense material (Figs. 2 and 3). Glycogen-like granules were widely distributed in the cytoplasm, however, no mitochondria were observed.
Budding process A part of the cell wall was first observed to separate from that of the mother cell and protrude outwards forming an ear-like shape. At the same time, a part of the cytoplasm of the mother cell near the bud base was bulging, and the cell wall of this portion became thin and dimpled. The cell wall of the bud tip was thin and rough compared with that of the mother cell (Fig. 2a). The ear-like projection continued growing outwards, separating from the mother cell, and the cell wall of the bud followed by the plasma membrane and the cytoplasm continued growing forwards (Fig. 2b). This earlike projection appeared to correspond to a collarette. The cell wall of the bud tip was thinner and less dense than that of the mother cell. No serrated structure could be detected in the bud at this stage. The bud base was now about 0-9 pm in diameter. No apical vesicles were observed. The cytoplasm protruded partially and the cell wall of this portion was dimpled near the bud base, as observed earlier. The cell wall of the daughter cell became slightly ridged at this stage, and the bud base was now 1.0/.tm wide (Fig. 3a). As the daughter cell grew, its inner cell wall became ridged but to a lesser degree than that of the mother cell. In the daughter cell as well as the mother cell, partial protrusion of the cytoplasm and dimpling of the cell wall was
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FIG. 3. (a) The cell wall of the daughter cell becomes slightly ridged. (b) A partial protrusion of the cytoplasm and dimpling of the cell wall are observed near the bud base in the daughter cell as well as in the mother cell (arrows). (c) The cell wall of the daughter cell becomes clearly serrated as it grows. Bar = 0.5/lm.
observed near the bud base (Fig. 3b). As the daughter cell grew, its cell wall became thicker and layered, and developed a serrated structure (Fig. 3c). Eventually, the daughter cell reached approximately the same length as the mother cell with a total length of about 7-0/.tm (Fig. 4a). The cytoplasm and cell wall of the daughter cell had almost the same structure as the mother cell, and 14 to 16 ridges were observed on each
FIG. 4. (a) The daughter cell becomes as long as the mother cell. (b) Formation of a cross wall by a protruding sickle-like structure. (c) The cross wall is continuous with the cell wall and has a narrow lumen. (d) Fission takes place at the lumen. Bar - 0-5/~m.
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BUDDING PROCESS OF M. PACHYDERMATIS
side. At this stage, a cross wall started to form by the protrusion of a sickle-like structure from the cell wall at the bud base (Fig. 4b). The cross wall was continuous with the cell wall and had a narrow lumen (Fig. 4c). No pores, such as dolipores, were observed. After completion of cross wall formation, fission took place at the lumen of the cross wall and budding was complete. The ear-like projection of the mother cell remained as a bud scar (Fig. 4d).
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DISCUSSION The cell wall of M. pachydermatis was thick (0-1-0-25/.tin) and multilayered, but the three layers described previously [1] could not be differentiated. The innermost layer of the cell wall had a serrated structure which is characteristic of members of the genus Malassezia [18]. In the bud, this structure became apparent as it grew. About 14 to 16 ridges were observed on sagittal section. The plasma membrane ran adjacent to the serrated curve of the cell wall as a pair of unit membranes. Breathnach et al. [3] and Takeo & Nakai [19] reported that such serrated structures appeared to be spirals when observed by freeze-fracture techniques. The size of the nucleus was approximately the same as that of M. furfur. Vacuoles varied in shape and size, and some of them contained electron-dense material Wikler et al. [20] reported that this electron-dense material decreased or disappeared under ultraviolet light radiation and thought it could be used in cell metabolism. The bud base measured 0-9-1.1 /lm in diameter and was broader than that of Pityrosporum orbiculare and Pityrosporum ovale (0.5-0.7 #m) [11], whose names are
a
d
b
l
e
f
C
FIG. 5. Diagram of budding process of M. pachydermatis. (a) A n ear-like structure separating from the cell wall. (b) An ear-like structure growing outwards. (c) The cell wall of the daughter cell becomes serrated as it grows. (d) The daughter cell becomes as long as the mother cell and cross-wall formation begins as a protruding sickle-like structure. (e) The cross-wall has a narrow lumen. (f) Fission occurs at the cross-wall and the ear-like projection persists as a bud scar.
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synonyms of M. furfur. The ring swelling of the plasma membrane described by Takeo & Nakai [19] was not observed in the present study. The budding process is summarized in Fig. 5. First, an ear-like projection separated from the cell wall as described by Abou-Gabal & Fagerland [1] and developed in an outward direction (Fig. 5a). During the early stage, partial protrusion of the cytoplasm and dimpling of the cell wall of this portion were observed near the bud base, not only in the mother cell but also in the daughter cell (Fig. 5b). Such structures were thought to correspond to the circumvallate bulgings observed between ridges near the base by Breathnach et al. [3] and Takeo & Nakai [19]. As the bud grew, its cell wall became thick, layered and ridged (Fig. 5c). When the daughter cell was as long as the mother cell, a cross-wall was formed (Fig. 5d). The cross-wall had a narrow lumen where fission took place (Fig. 5e). The ear-like projection persisted as a bud scar after budding was complete (Fig. 5f). This mode of conidium ontogeny is monopolar blastic development [5]. The budding process of the genus Malassezia has been reported previously [1, 8, 11]. Compared with these reports, the budding process of M. pachydermatis appears to be similar to that of M. furfur. The present study shows that the broader base is a characteristic feature of M. pachydermatis, but we do not consider this difference to be of taxonomic value. ACKNOWLEDGEMENTS The authors thank Dr Matsuda for kindly supplying the strain of M. pachydermatis and Dr Saito for helpful advice.
REFERENCES 1. ABOU-GABAL, M. & FAGERLAND,J. A. 1979. Electron microscopy of Pityrosporum canis "pachydermatis". Mykosen, 22, 85-90. 2. BARFATANI,M., MUNN, R. J. & SCHJEIDE, O. A. 1964. An ultrastructure study of Pityrosporum orbiculare. Journal of Investigative Dermatology, 43, 231-233. 3. BREATHNACH,A. S., GROSS, M. & MARTIN, B. 1976. Freeze-fracture replication of cultured Pityrosporum orbiculare. Sabouraudia, 14, 105-113. 4. BREUER-STROSBERG,R., HOCHLEITHNER, M. 8z KUTTIN, E. S. 1990. Malassezia pachydermatis isolation from a scarlet macaw. Mycoses, 33, 247-250. 5. COLE, G. T. 1981. Conidiogenesis and conidiomatal ontogeny. In: G. T. COLE & B. KENDRICK (Eds) Biology of Conidial fungi. Vol. 2, pp. 271-327. Academic Press, New York. 6. GORDON, M. A. 1979. Malassezia (Pityrosporum) pachydermatis (Weidman) Dodge 1935. Sabouraudia, 17, 305-309. 7. GUEHO, E., SIMMONS, R. B., PRUITr, W. R., MEYER, S. A. & AHEARN, D. G. 1987. Association of Malassezia pachydermatis with systemic infections of humans. Journal of Clinical Microbiology, 25, 1789-1790. 8. IKEYA, T. 1974. Electron microscopic study on Malassezia furfur in horny layer. Japanese Journal of Medical Mycology, 15, 39-51. 9. KEDDIE, F. M. 1966. Electron microscopy of Malassezia furfur in tinea versicolor. Sabouraudia, 5, 134-137. t0. KEDDIE, F. M. ~,z BARAJAS,L. 1969. Three-dimensional reconstruction of Pityrosporum yeast cells based on serial section electron microscopy. Journal of Ultrastructure Research, 29, 260-275. 11. KEDDIE, F. M. & BARAJAS,L. 1972. Quantitative ultrastructural variations between Pityrosporum ovate and P orbiculare based on serial section electron microscopy. International Journal of Dermatology, 11, 40-48. 12. KOCKOVA-KRATOCHViLOVA,A., LADZIANSKA,K. & BUCKO, S. 1987. Malassezia pachydermatis in small animals. Mykosen, 30, 541-543.
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