Pleoanamorphic life cycle of Exophiala (Wangiella) dermatitidis

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1 Antonie van Leeuwenhoek 65 : , (g) 1994 Kluwer Academic Publishers. Printed in the Netherlands. Pleoanamorphic life cycle of Exophiala (Wangiella) dermatitidis G.S. de Hoog 1'3, K. Takeo 1, S. Yoshida 1, E. G6ttlich 2, K. Nishimura 1 & M. Miyaji 1 1Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Inohana, Chuo-ku, Chiba 260, Japan; 2Institut fiir Siedlungswasserbau, Universitdt Stuttgart, Bandtiile 1, Stuttgart, Germany (3present address: Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740AG Baarn, The Netherlands.) Accepted 9 December 1993 Key words: black yeasts, Capronia, conidiogenesis, Exophiala dermatitidis, life cycle, pleomorphism, Wangiella dermatitidis Abstract The anamorph life cycle of the black yeast Exophiala (Wangiella) dermatitidis is described. The fungus is dimorphic, yeast cells being the prevalent form of propagation. The fungus is strongly hydrophilic, probably completing its anamorph life cycle in submersion. Adaptation to dry conditions is slow. Types of conidiogenesis comprise annellidic, phialidic and sympodial reproduction, in addition to isotropic development. Phialoconidia fail to germinate under the conditions tested, and thus may have a function other than dispersal. Sterile, multicellular bodies resembling a Capronia teleomorph are described. Introduction Recent findings on the relatively common pulmoneous occurrence of Exophiala dermatitidis (Kano) de Hoog [= WangielIa dermatitidis (Kano) McGinnis & Padhye] in patients with cystic fibrosis (Haase et al. 1991; Kusenbach et al. 1992) and its neurotropic pathology in otherwise healthy patients (Hiruma et al. 1993) have stimulated interest in its vectors of dispersal and route of infection. The prevalent types of propagation as routinely seen in the laboratory, viz. by budding and by percurrent conidiogenesis from hyphae, as well as isotropic development, have extensively been described by RJ. Szaniszlo and coworkers. However, the complete anamorph life cycle also involves sympodial reproduction, phialoconidia and sclerotial bodies. The present study aims at placing the divergent morphologies of E. dermatitidis in an ecological perspective. Due to the rarity of some of the synanamorphs we have not used a single model strain, but selected the most pronounced representatives from collections of strains present at IFM (Chiba University, Chiba, Japan) and CBS (Baarn, The Netherlands). Material and methods Strains and culture conditions Strains studied are listed in Table 1. Identities were verified by morphology, absence of sodium nitrite assimilation (De Hoog & Haase 1993) and growth at 40 C. Stock cultures were maintained on PDA slants. For morphological observation, the following media were used: Sabouraud's glucose agar (SGA), Czapek Dox agar (CzA) and Synthetischer nahrstoffarmer Agar (SNA), the latter medium containing glucose as the only C-source. The N-source in CzA is KNO3 while E. dermatitidis is nitrate-negative; the medium is thus N-limiting. SNA is C- and N-liiniting. Transfers on solid media were made (1) by bringing a loopful of cells from 3-day-old cultures into sterile water and inoculating 0.1 ml suspension onto CzA, SGA or SNA by means of a Drigalski spathula; (2) by transferring yeast cells from 1-month-old cultures directly onto solid substrates using a wire loop; (3) by transferring fragments of hyphal, sporulating thalli onto solid substrates using a needle. Suspensions in liquid SN medium with sterilized tomato stems, basically following the procedure of Untereiner (1994) were shaken for 10 d and subsequently incubated on moist filter paper

2 144 Table 1. List of strains studied IFM 4826 = MM-7, ex humidifier, Japan, K. Nishimura (Nishimura & Miyaji 1982). IFM 4827 = CBS = ATCC 28869, with mutants IFM 4828 and IFM , ex human skin, Japan, K. Kano (Kano 1937). IFM 4829, ex human oropharynx, Japan, K. Yamashita (Mizoue et al. 1959). IFM ATCC 46435, ex chromoblastomycosis, Japan, S. Watanabe (Hironaga et al. 1981). IFM 4835, ex neurotropic infection, Japan, Y. Shimazono (Shimazono et al. 1963). IFM 4837, ex disseminated infection, Japan, M. Sugawara (Sugawara et al. 1964). IFM 4840, ex nenrotropic infection, Japan, A. Hariyama (Hariyama et al. 1971). IFM 4842, ex systemic infection, Japan, T. Kusunoki (Harada et al. 1976). IFM 4846 = NCMH 253, M.R, McGinnis. IFM 4848 = INPA 109, ex bat Sturnira lilium, Brazil, W.Y. Mok (Reiss & Mok 1979). IFM 4850 = INPA 122, ex bat Sturnira lilium, Brazil, W.Y. Mok (Reiss & Mok 1979). IFM 4958, ex public bath, Japan, K. Nishimura (Nishimura et al. 1987). IFM 4964, ex house bath, Japan, K. Nishimura (Nishimura et al. 1987). IFM 5383, Japan, K. Iwata. IFM 40903, source unknown. IFM 41480, ex humidifier, Japan, K. Nishimura. IFM 41489, ex human skin, Japan, T. Takase. IFM 41689, ex neurotropic infection, China, D. Wang. IFM 41822, ex rotten wood, Venezuela, K. Nishimura. IFM 41823, ex cactus, Venezuela, K. Nishimura. IFM 41825, ex rotten wood, Venezuela, K. Nishimura. IFM 41826, ex rotten wood, Venezuela, K. Nishimura. IFM 41827, ex soil, Brazil, K. Nishimura. IFM 45986, ex tap water, Japan, K. Takatori. CBS = ATCC , ex skin, Japan. CBS , ex sputum of CF patient, Norway, P. Sandven. CBS , ex sputum of CF patient, Germany, G. Haase. CBS , ex sputum of CF patient, Germany, B. H6vener. CBS , ex sputum of patient with pneumonia, The Netherlands, W. Pauw. CBS , ex onychomycosis, Mauritius. df19814, ex blood, Germany. dh 9816, ex sputum, Germany, K. Tintelnot. in Petri-dishes. Critical cultures on stems were verified by re-isolation on SGA plates. Strains were also inoculated into liquid glucose- ethylamine medium (EA, ph 5.0) in erect test tubes shaken at 160 rpm. Yeast cells were grown in shaken liquid culture and observed in exponential phase (3 d) and in maximum (8 d) and late (15-25 d) stationary phase, determined on the basis of growth curves. Germination ability of phialoconidia was tested by vortexing phialidic colonies on stems in sterile water and bringing 0.1 ml suspension onto a 2 mm CzA layer on a sterilized slide; annelloconidia from the same substrate were observed as positive control. Occurrence of phialides was verified in Petridishes with varying sugar concentrations (CzA and SNA containing %, 0.05%, 0.4% and 3.75% glucose or saccharose, respectively) and on SNA at ph 9.2. Effect of drying was evaluated by using PDA in Petri-dishes which had been tilted during solidification. Mating experiments were performed by mixing exponential phase cells in suspension with stems as described above. Staining Viability of cells was tested with Rhodamin 123 (Sigma) by observation with a Nikon fluorescence microscope using filter system B 1. Scanning electron microscopy (SEM) For observation of phialides, hyphal thalli were carefully peeled off from tomato stems, washed twice in

3 t45 Table 2. Presence of anastomoses (A) and phialides (P) using exponentialphaseinocula on complete medium (SGA), nitrogen depleted medium (CzA), nitrogen- and carbon-depleted medium (SNA) and on moistened tomato stems; formation of multicel- lular structures on stems, either sclerotial bodies (S) or teleo- morph-initials (T). Strain SGA CzA SNA Stem Multicellular 4826 A P A P A P A P S T t dh M phosphate buffer (ph 7) and centrifuged 3 rain at 3000 rpm. Specimens were fixed in freshly prepared 2.5% glutaraldehyde / 0.1% phosphate buffer for a few days at 4 C, followed by washing in phosphate buffer. Samples were critical-point-dried, sputtered with platinum for 1 rain, and observed in a Hitachi S-800 instrument operated at 10 KV. Results Yeast cells Initial growth of suspended yeast cells in exponential growth phase at room temperature and near-neutral ph was almost invariably with budding. Budding was also promoted by vigorous shaking (300 rpm) followed by repeated transfer. Exponential phase budding was predominantly percurrent from one or several scars at the distal apex; flat annellated zones were thus developed. Daughter cells measured about 4 x 2 #m, but soon inflated and rounded off to about 7 x 5 #m. Maturation of cells continued by increasing pigmentation and wall thickness. At transfer onto fresh media, the older, swollen cells germinated with hyphae. Under conditions of oxygen limitation (submerged in agar or in liquid EA medium with poor aeration) budding was predominantly multilateral from restricted areas at opposite poles, leading to asteriskshaped microcolonies. When mother cells became older, daughter cells were still produced, but the process of their delimitation and secession slowed down. Consequently, daughter cells might still be attached while a subtending cell was already produced. Increasing coherence between daughter cells was also noted with adjacent scars, resulting in a shift towards multilateral rather than percurrent budding. Under conditions of environmental stress, i.e. at ph 9.0 and at 42 C, yeast cells were the most prevalent form of sporulation (Table 3). In a small number of strains a granular, meristematic colony type was obtained at this temperature (Table 3). Hyphal thallus Torulose hyphae When late stationary phase cells were seeded out onto solid substrata, torulose hyphae of 1-5 cells were produced, which changed over into hyphae of equal width throughout. Conidia were produced from flat or slightly extending annellated zones close to the distal septurn. Homogeneous layers of young budding cells developed differently on either side of slant Petri-dishes. At the thin side, where drying started within about a week, the crust of yeast cells was finally noted dead. At the thicker agar layer aerial hyphae developed in about 30 d. Colonies derived from old aerial cells almost invariably developed a hydrophobic mycelial

4 146 Table 3. Stress morphology. Reactions to prolongedincubation at initial ph 5.0, ph 9.2, 42 C, and after drying. Stages I and II represent mono- and multi-celled isotropic growth, respectively; DI and DII represent growth of young inoculum on slant dish, thin and thick side of the agar layer, respectively; S is growth of old inoculum, y = yeast; h = hyphae; g = granular. Strain ph 5.0 ph C Drying I II DI DII S 4826 y y y h h 4827 y y h h y y y y y/h 4830 y y h h 4835 y y y y h 4837 y y y y/h y y y h 4842 y y h h 4846 y y h h y y y y h y y h h 4958 y 4964 y y y y h 5383 y h g h h h y y h h y y y h y y h h y y y y h y y h y/h y y y y h h y y y y/h y y h y/h y y h h y y y y y y y h dh y y y y/h mat (Table 3) with abundant annellidic conidiation. Additional conidia strongly adhering in chains were occasionally present. True hyphae Slimy, relatively hydrophilic hyphae arose from torulose hyphae. On SGA this thallus consisted of melanized hyphae of 2-3 #m width. The thallus finally acquired a somewhat rubbery texture. Additional, hyaline, submerged hyphae, #m in width were sometimes present. Anastomoses were absent on rich media but often present under nutritionally limiting conditions (Table 2). The hyphal thallus was predominantly composed of dry, non-wettable hyphae when hyphal fragments from 30-day-old colonies were used as inoculum. A transition from percurrent to sympodial conidiogenesis was observed. Forms of propagation Annellidic Conidiogenesis on agar media (SGA, CzA, SNA) was predominantly percurrent, particularly when exponential-phase inocula were used. 1-5 Annellated zones were located at the distal end of the cell. Conidiogenous cells were either discrete budding cells, or they were intercalary or terminal hyphal cells or formed on short lateral branches composed of flask-shaped cells, which in some strains were aggregated in dense brushes. Annelloconidia adhered in slimy heads. After 2 wk growth they were embedded in pale yellowish-brown, extracellular material. On stems the annellidic morph often extended above the thallus, and could be recognized by irregular growth and light yellowish brown clumps of conidia (Figs la,c). Phialidic Phialides remained absent on SGA and were occasionally present on nitrogen-limiting media (CzA, SNA). The availability of carbon in CzA proved insignificant (Table 2). This was confirmed in a test series using varying glucose or saccharose concentrations in CzA and SNA, respectively: carbon limitation (0.0125%) did not stimulate phialide formation (results not shown). Phialides were commonly produced in abundance in colonies on tomato stems which had been soaked in a yeast suspension in SN medium (Table 2; Fig. 2). Often they were the main anamorph in colonies between 1 and 4 wk old. On the stems small, white slime droplets were observed which extended slightly above the thallus (Figs lb,d). These droplets were seen in strains which on stems almost exclusively produced phialides and remained absent in strains without phialides; it was therefore supposed that they were phialoconidial aggregates. Often compact phialidic sporodochia were formed (Figs 3a,b,d), which firmly adhered to hard substrata such as filter paper fibres (Fig. 3a). Phialoconidia proved unable to germinate on CzA, while adjacent annelloconidia produced yeast-like microcolonies.

5 147 b Fig. 1. Mass-growth on tomato stems, a,c. irregular, yellowish brown slimy clumps of annellidic anamorph; b,d, regular, white drops of phialidic anamorph. Bar represents 200 Izm. Sympodial In late stationary phase, reproduction in the hyphal thallus tended to become predominantly sympodial, comparable to the shift towards multilateral reproduction in budding cells. Acropetal chains A dry, hyphal thallus, often containing somewhat hydrophobic, firmly coherent, acropetal chains of cells, was developed after 3 weeks when late stationary phase cells from predominantly hyphal thalli were used (Table 3). The catenate morph consisted of coherent, lateral and terminal chains of ellipsoidal to barrelshaped cells, arising at acute angles from ascending or erect, cylindrical hyphae. Liberation of single cells was rarely observed. Isotropic development and endoconidia Chlamydospore-like cells were frequently produced in acidic EA medium. They were spherical, up to 15 #m diam, and were produced on hyphae or were discrete. Cell walls were comparatively thin and rather delicate, subhyaline. In a later stage they often shed off an outer wall layer, liberating one or several endoconidium-like cells. Frequently the inner cell appeared deteriorated; such cells were found dead using Rhodamin staining. Alternatively, they developed several transverse and oblique septa before attaining maximum size. Cell clumps remained subhyaline and did not seem to develop any further. In a number of strains, local series of intercalary hyphal cells developed muriform septation. These cells became olivaceous black and thick-walled and developed into sclerotial bodies. The bodies often had an irregular outline, the wall being composed of

6 148 Fig. 2. Phialides in 3-5 wk old cultures on tomato stems, a. first initial of phialide breaking half-way to form collarette; b. lateral phialide; c. intercalary phialide; d. phialide (arrow-head indicating collarette) developing with phialidic branches; e. complex phialidic branching leading to sporodochium; f. SEM of compacted phialide openings; g. collarette on young sclerotial body. Bar represents 10/~m.

7 149 C e :?;... Fig. 3. Sporodochiaand teleomorph initials, a. phialidic sporodochiumadhering to filter paper fibre; b,d. compacted phialides on sporodochium; c,e,f, teleomorph initials; g. detail of teleomorph initial wall showing plate-like parenchymatous cell complexes. Bars represent 25 #m in a,c,e,f, 10 #m in b,d,g. inflated cells of variable size. In six strains sclerotial bodies developed into bodies resembling Capronia ascomata, bearing blackish brown, thick-walled setae (Figs 3c,e-g) and measuring up to 55/~m diam. The contents of the bodies were hyaline, with texture clearly different from the surrounding, membranaceous body wall, but no development of asci was observed. Mating experiments thus far proved unsuccessful.

8 150 Discussion Yeast cells of Exophiala dermatitidis are known to be delimited from the mother cell by non-perforated septa (Grove et al. 1973). After liberation they inflate, accumulate polysaccharides as storage compounds (Oujezdsky et al. 1973), and soon reproduce with budding cells. The wall of the bud is continuous with the innermost layer of the mother cell wall (Grove et al. 1973). Several buds are produced percurrently through the same scar (Oujezdsky & Szaniszlo 1974). Multiple conidiogenous loci are found near the poles of the cells, leading to short and flat annellated zones (Nishimura & Miyaji 1985). Daughter cells produce subsequent buds first at their distal, later also at their proximal ends. Polarity in liberated cells is controlled by the same mechanism as found in hyphae, in both cases suggested to be cell wall proteins which function as tubulin attachment sites (Jacobs & Szaniszlo 1982). Lateral buds are extremely rare. Young annelloconidia, liberated from the hyphal thallus, swell and enter the yeast cell cycle. They are comparable to yeast cells in having a similar, nonperforate delimiting septum (Grove et al. 1973). The preponderance of budding cells over hyphae is promoted by the use of exponential-phase inocula and by vigorous shaking. In nature, the yeast anamorph thus may serve an efficient occupation of dynamic, watery systems under favourable nutritional conditions. When the medium approaches exhaustion, the cells develop thicker walls, become melanized and accumulate lipids (Oujezdsky et al. 1973). They utilize less nutritional sources from their environment by showing endogenous respiration of fatty acids (Calderone 1976). When such cells are inoculated onto fresh substrates, they germinate with torulose hyphae, which soon change into true, cylindrical hyphae. Butterfield & Jong (1976) supposed that the abundance of yeast cells compared to hyphae was dependent on the carbon source used, but their results are probably explained by different growth kinetics of carbon sources. The formation of aerial thallus via lipid accumulating cells is rather slow, as is clearly shown in the reactions to drought in slant Petri-dishes. Exponential phase colonies show a considerable lag in adaptation to dry environmental conditions (Table 3). It is therefore likely that the anamorph life cycle is principally completed in a moist habitat. The prevalent process of conidiogenesis on hyphae is annellidic (Hironaga et al. 1981). The yeast cycle and its interchange with the hyphal, annellidic thal- lus is the dimorphism routinely seen in the laboratory (Geis & Jacobs 1985), comprising the prevalent growth forms under favourable culture conditions. The yeastlike and hyphal forms of growth were referred to by De Hoog (1985) as micro- and macrocycles, respectively. Late stationary-phase hyphal thalli reproduce less vigorously. Daughter cells are not liberated by subsequent conidia but rather pushed aside and remain attached for a long time. The number of buds produced per conidiogenous locus becomes lower, but each locus remains productive, so that the process of bud formation gradually becomes multilateral rather than percurrent. This results in a morphology similar to that of Exophiala negronii (Pereira) de Hoog et al. (1994). The asterisk-shaped, submerged microcolonies have very narrow isthmi between cells. Possibly they are comparable to pseudomycelium reported by Geis & Jacobs (1985) which lacks septal pores with Woronin bodies. Conjugation between flocculated yeast cells, as reported by Oujezdsky & Szaniszlo (1973), was frequently seen on tomato stems but rarely on other substrata. Szaniszlo et al. (1976) found that cellular extension becomes predominantly isotropic within 5 d at a ph below 3.5. We found this morphology in the majority of the strains kept over prolonged periods in EA medium (ph 5.0). Butterfield & Jong (1976) reported its frequent occurrence in numerous assimilation tests in liquid media, probably due to acidification of medium during growth. The availability of Ca 2+ ions may be the crucial factor in phenotypic switching towards isotropic growth (Mendoza et al. 1993). This would explain the occurrence of muriform cells in human tissue in cases of chromoblastomycosis (Szaniszlo et al. 1993). The occurrence of this kind of switching in a limited number of species would suggest a taxonomic relationship between the etiologic agents of chromoblastomycosis. Cellular division in isotropically enlarging cells (Stage I of Cooper & Szaniszlo 1993) is often found to be slower than mitosis, so that large cells (up to 15 #m diam) sometimes are found to be multinucleate (Roberts et al. 1979). These cells may develop an internal envelope, the outer, galactomannan-rich wall layer frequently being shed off (Geis & Jacobs 1985). One or several cells (endoconidia), which Matsumoto et al. (1990) reported to bud or to germinate, may be liberated. They often die without further development, as was noted by Cooper & Szaniszlo (1993) in a mutant strain. Irregularly muriform septation of swollen cells (Stage II of Cooper & Szaniszlo 1993) leads to subhyaline cell

9 yeast cell J 151 intracel ular 0 ~ 0 polysaccharidc annclloconidium cndoconidium 0 [' annellidic lipid ~ hypha hyl)ha multilateral phialidic sympodial isotropic Stage l: cmamydospore phialidic sporodochium isotropic sclerotial body O Stage U: m ulticellular body teleomorph initial MICROCYCLE MACROCYCLE Fig. 4. Diagram of possible developmentai routes and types of conidiogenesis. clumps resembling Botryomyces caespitosus de Hoog & Rubio (1982). They are nearly exclusively found as discrete cells rather than on hyphae, the clumps swelling meristematically and fragmenting into smaller parts when they have attained a particular size. Melanized multicellular bodies, which also develop by isotropic growth, are borne on hyphae in a limited number of strains (Table 2) after prolonged incubation on plant stems. These probably are primordia of a Capronia teleomorph (compare Janex-Favre 1988). Some of them (Table 2) develop large spines (Figs 3c,e,f), as reported recently in related Herpotrichiellaceae by Untereiner (1994). The bodies develop very slowly and may serve hibernation. In a final stage of development they had a hyaline content, clearly different from the body wall, but no asci were observed. The occurrence of phialides with collarettes was first observed by Jotisankasa et al. (1970). De Hoog (1977, 1985), Matsumoto & Matsuda (1984) and Muotoe-Okafor & Gugnani (1993) stressed that this was a type of conidiogenesis additional to the annel- lidic anamorph, the latter being referred to as 'phialides without collarettes' by McGinnis (1977) and many subsequent workers. Phialides are produced in relative abundance on tomato stems. In later stages of development they are often aggregated in dense clusters resembling sporodochia (Figs 3a, b,d), which show a remarkable adhesion to the substrate. In Cladosporium carrionii phialides were found to be produced particularly on nutritionally deficient media (Honbo et al. 1984). Our experiments with C- and N-limited media have shown that in E. dermatitidis other factors are concerned. The abundance of phialides on tomato stems, prior to the emergence of teleomorph-initials, suggests that phialides might play a role in the saprophytic, possibly sexual phase of the life cycle. Phialoconidia lack the yellowish brown, slimy capsules which are present on annelloconidia. Their production in tiny slime droplets slightly raised above the thallus (Figs lb,c) would favour dispersal by small arthropods such as mites, but their apparent inability to germinate suggests a possible sexual function.

10 152 This would imply that the phialides produced in supposed anamorph members of Herpotrichiellaceae are not always comparable: in the monomorphic species Phialophora verrucosa Medlar they serve propagation. The smaller, nearly spherical additional phialides known to occur in low abundance in addition to the main form on conidiation in Cladosporium carrionii, Exophiala spinifera, Fonsecaea compacta and E pedrosoi are morphologically similar to those of Exophiala dermatitidis; their function and taxonomic significance require further study. Acknowledgements The authors are indebted to W.A. Untereiner for protocols of cultivation on natural substrates and to RJ. Szaniszlo for critically reading the manuscript. Financial support for this research was provided by the Japanese Ministry of Education to the senior author. References Butterfield W & Jong SC (1976) Effect of carbon source on conidiogenesis in Fonsecaea dermatitidis, agent of chromomycosis. 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