MAUDY TH. SMITH1, j. p. VAN DER WALT 2 AND ELLA JOHANNSEN 2

Transcription

1 Antonie van Leeuwenhoek 42 (1976) The genus Stephanoascus gen. nov. (Ascoideaceae) MAUDY TH. SMITH1, j. p. VAN DER WALT 2 AND ELLA JOHANNSEN 2 Yeast Division of the Centraalbureau voor Schimmelcultures, Delft, The Netherlands, and 2Microbiology Research Group, Council for Scientific and Industrial Research. Pretoria, South Africa SMITH, M. TH., VAN DER WALT, J. P. and JOHANNSEN, E The genus Stephanoascus gen. nov. (Ascoideaceae). Antonie van Leeuwenhoek 42: The imperfect species currently cited as Candida ciferrii was found to constitute the haploid mating types of an undescribed, filamentous, heterothallic ascomycete. This perfect state has been transferred to the new genus Stephanoascus. The diagnosis of the genus and the description of the species Stephanoascus ciferrii are given. INTRODUCTION The yeast species currently cited as Candida ciferrii was described by Kregervan Rij (1965) on the basis of the study of four anascogenous yeast strains two of which were known to have derived from warm-blooded sources. The strains formed both true hyphae and budding yeast cells and in this respect resembled species of the genera Saccharomycopsis Schi0nning (1903) and Trichosporon Behrend (1890). As the strains lacked the ability to form fission cells and failed to yield an a scigerous state in either single or mixed cultures, the species was assigned to the genus Candida. A new isolate of this species was recently obtained from uncultivated soil from a locality near Pretoria. This isolate, CBS 6699, was found to mate with strains of this species held by the Yeast Division of the Centraalbureau voor Schimmelcultures, and to yield the perfect state of an undescribed, filamentous heterothallic ascomycete. MATERIALS, METHODS AND RESULTS Cultures. The following six cultures were studied: CBS 4856; recovered from the neck of a cow (Klokke and Kamp, 1962). CBS 5165; recovered from a wooden pole in a cow shed (Klokke and Kamp, 1962).

2 120 M. TH. SMITH, J. P. VAN DER WALT AND E. JOHANNSEN CBS 5166; received from Dr. H. Seeliger and presumed to be of human origin. CBS 5295; recovered from a human throat. CBS 5646; recovered from a bovine placenta. CBS 6699; recovered from soil near Pretoria. Mating types. Fresh transfers of the six cultures were mated in all fifteen possible combinations on V-8 agar and incubated at 28 C. In fertile crosses a mating reaction and the emergence of the perfect state was observable within 3-7 days. On the basis of these results CBS 6699 and CBS 5646 were found to be representative of one mating type, and CBS 5295, CBS 5166, CBS 5165 and CBS 4856 of the other. The most fertile crosses were obtained by mating CBS 6699 and CBS Crosses involving CBS 5166 led to the formation of aberrant ascospores. The perfect state obtained by mating of CBS 6699 and CBS 5295 on V-8 agar. The mating reaction and ascus formation are generally initiated by the convergence and fusion upon contact and dissolution of the cell walls of two hyphal extremities or by the anastomoses of lateral branchlets of two hyphae (Figs. 1 and 2). Upon further development an evagination is formed at the site of hyphal fusion (Fig. 3). After enlargement of the evagination (Fig. 4), vacuolization and the deposition of lipid bodies, two septa are formed in succession. The first is delimited at the base of the enlargement (Fig. 5) and the second at the distal extremity leading to the abscission of a small, persisting, apical cell which may occasionally form a bud (Fig. 6). The subapical cell upon further enlargement (Fig. 7) and formation of a second inner wall-layer is then transformed into the ascus, in which 1-4 (usually 2 or 3) ascospores develop. In young states hemispherical bodies can be observed (Fig. 8) around each of which a membrane is laid down (Fig. 9). The latter then contract to form somewhat irregular, hatshaped ascospores (Fig. 10). Ascospores of one-spored asci occasionally lack a brim and are oblate or nearly spherical. The mature ascus is crowned by the persisting apical cell. The mature asci measure #m, the mature ascospores, including the brims, x pm and the central ellipsoidal bodies /2m. The conidia formed on denticles (Fig. 11) are usually ovate or pyriform and measure x /~m. The fusion of yeast cells was not observed and efforts to obtain the perfect state as a single phase heterozygous for the mating type gene(s), were unsuccessful. Electron microscopy of the asci and ascospores. Asci containing mature ascospores deriving from a 7-day-old culture on V-8 agar were fixed for 6 h at 4 C in 5 (v/v)~ glutaraldehyde in 0.1 M phosphate buffer (ph 7.3) and post-fixed for 2 h in 1 ~ osmium tetroxide in the same buffer at 4 C. Ultra-thin sections of the material imbedded in an Araldite-Epon mixture were stained by consecutive treatments with saturated aqueous uranyl acetate and Reynold's lead citrate solution. Examination of ultra-thin sections of the mature ascospores established that

3 TI-IE GENUS STEPHANOASCUS GEN. NOV. 121 Figs Stephanoascus cijerrii on corn meal agar ~. Figs. 1 and 2. Convergence and fusion upon contact and dissolution of the cel! walls. Fig. 3. Development of an evagination. Fig. 4. Enlargement of the evagination.

4 122 M. TH. SMITH, J. P. VAN DER WALT AND E. JOHANNSEN Fig. 5. Delimitation of the first septum at the base of the enlargement. Fig. 6. Delimitation of the second septum at the distal extremity of the enlargement. Fig. 7. Enlargement of the subapical cell. Fig. 8. Hemispherical body in a young state of the ascus.

5 THE GENUS STEPHANOASCUS GEN. NOV. 123 Fig. 9. Membrane is laid down around a hemispherical body. Fig. 10. Ascus with two hat-shaped ascospores. Fig. 11. Hyphe with denticles and pyriform conidium. the latter were irregularly hat-shaped with characteristic thickening of the outer wall on the ventral and dorsal sides of the spores (Fig. 12). In some ascospores bifurcate brims were detected. The central ellipsoidal body was surrounded by a distinct membrane (Figs. 12, 13). Sections through mature asci confirmed that these have a double wall of which the outer layer is continuous with the original wall of the evagination (Fig. 12). This layer ruptures with age, while the inner wall is persistent. DISCUSSION The formation of hat-shaped ascospores and the copious development of true mycelium assigns the perfect state to the Ascoideaceae as defined by von Arx (1972). On the basis of the presence of both budding cells (blastoconidia) and hyphae with straight septa, this state shows some similarity to the genus Sac'-

6 124 M. TH. SMITH, J. P. VAN DER WALT AND E. JOHANNSEN Figs. 12 and 13. Stephanoascus cijerrii CBS 6699 x CBS 5295 on V-8 agar. Fig. 12. Section through mature ascus: ascospores with thickenings of the outer wall on ventral and dorsal side x. Fig. 13. Ascus with immature ascospore: central ellipsoidal body surrounded by a distinct membrane

7 THE GENUS STEPHANOASCUS GEN. NOV. 125 charomycopsis. However, its characteristic mode of ascus formation within a small subapical, ascogenous cell and the distinctive, crowned, mature asci not only differentiates this species from all representatives of the genus Saccharomycopsis but in fact distinguishes it from all the genera of the Ascoideaceae. In typical species of the genus Saccharomycopsis the asci develop in great numbers usually laterally on the diploid hyphae or, more rarely, intercalarily, in which case they are usually catenulate; the conidia are also elongated, often clavate, have a rather broad base and develop from scars. In Candida ciferrii the blastoconidia are pyriform with an attenuated base and are borne on small denticles of the conidiogenous cells. Such denticles are unknown in Saccharomycopsis. The authors therefore propose assigning this species to the new genus Stephanoascus Smith, van der Walt et Johannsen, gen. nov. Cellulae zymoticae gemmiferae, pseudohyphae et hyphae septatae ramosae adsunt. Conidia continua, ovata, hyalina, e cellulis conidiogenis denticulatis oriuntur. Asci singuli e cellula subapicali evaginationis brevis formantur quae e duabus hyphis copulantibus oritur, globosi, crassitunicati, persistentes, uni- ad quadrispori, cellula apicali parva vesieulosa coronati. Ascosporae semiglobosae petasiformes, margine basilari circumdatae. Species typica Stephanoascus ciferii Smith et al. Budding cells, pseudohyphae and septate branching hyphae present. Conidia unicellular, ovate, hyaline, formed on denticulate conidiogenous cells.asci developing singly in the subapical cell of a short evagination arising from two fusing hyphae, spherical, thick-walled, persistent, one- to four-spored, crowned by a small, blister-shaped apical cell. Ascospores hemispherical, hat-shaped, with a basal brim. Stephanoascus ciferrii Smith, van der Walt et Johannsen, spec. nov. Species heterothallica, statusascigerus Candidaeciferrii Kreger-vanRij. Coloniae in agaro maltoso post 6 dies 28 C 5 6 mm diametro, elevatae, restrictae, tenaces, rugosae, vel crispatulae, pubescentes vel floccosae, eburneae, marginibus fimbriatis. Cellulae zymoticae gemmiferae, ellipsoideae vel cylindricae, 3.(~11.0 x #m, singulae, binae vel in catenis brevibus connexae et pseudohyphae adsunt. Hyphae septatae dominantes, ramosae, septatae, pm latae, hyalinae. Cellulae conidiogenae intercalates vel terminales, saepe e hyphis erectis oriuntur, denticulis vulgo intercalares vel terminales acervati. Conidia ovata vel pyriforma, attenuata ad basim, #m. Asci singuli e conjugatione duarum hypharum orti in evaginatione inflata biseptata, globosi vel subglobosi, pariete crasso persistente objecti, uni- ad quadrispori (plerumque bi- vel trispori), 7-14 #m diametro, cellula rotundata vel cylindrica 2 6 x 2 3/~m coronati. Ascosporae semiglobosae vel asymmetrice oblatae, plerumque petasiformes, marine basilari circumdatae, 5-8 x 3 5#m. Fermentatio abest. Glucosum, galactosum, L-sorbosum, sucrosum, maltosum, cellobiosum, trehalosum, melibiosum, raffinosum, D-xylosum, L-arabinosum, D-arabinosum, D-ribosum, L-rhamnosum, ethanolum, glycerolum, i-erythritolum, ribitolum, galactitolum, D-mannitolum, o- glucitolum, ct-methyl-d-glucosidum (lente), salicinum, acidum ol-lacticum, acidum succinicure, acidum citricum et i-inositolum assimilantur at non lactosum, melezitosum, inulinum nec nitras. Assimilatio amyli solubilis variat. Ad crescentiam vitaminae externae non necessariae sunt. Crescere potest in 37C. Holotypus cultura conjugata CBS 6699 CBS 5295, exsiccata, in collectione zymotica Centraalbureau voor Schimmelcultures, Baarn praeservatur.

8 126 M. TH. SMITH, J. P. VAN DER WALT AND E. JOHANNSEN Heterothallic; colonies on malt agar after 6 days at 28 C reaching a diameter of 5-6 mm, raised, restricted, tough, rugose to crispate, pubescent to floccose, pale cream, with fimbriate margin. Yeast cells ellipsoidal, ovate or cylindrical, /~m, often catenulate and forming pseudohyphae. Septate hyphae predominant, branched, septate, #m in diameter, hyaline. Conidiogenous cells intercalary or terminal, often on erect hyphae, with denticles generally in intercalary or terminal clusters. Conidia ovate or pyriform, attenuated at the base, #m. Asci developing singly after fusion of two conjugating hyphae in the central swollen cell of a lateral, biseptate evagination, spherical or nearly so, with a thick, persistent wall, one- to four-(usually 2-3) spored, 7-14 ~m in diameter, crowned with a roundish or cylindrical cell, ~m in size. Ascospores hemispherical or asymmetrically oblate, usually hat-shaped, at the base surrounded by a brim, #m. No fermentation. Assimilation of glucose, galactose, L-sorbose, sucrose, maltose, cellobiose, trehalose, melibiose, raffinose, D-xylose, e-arabinose, n-arabinose, D-ribose, L-rhamnose, ethanol, glycerol, i-erythritol, ribitol, galactitol, o-mannitol, D-glucitol, ~-methyl-d-glucoside (slow), salicin, De-lactic acid, succinic acid, citric acid and i- inositol; no assimilation of lactose, melezitose, inulin and nitrate. Assimilation of soluble starch variable. No vitamins required for growth. Growth occurs at 37C. S. ~ciferrii is heterothallic and has thus far been recovered from nature only in the conidial form. The sexual state has been obtained under laboratory conditions. As efforts to obtain the species as a single phase, heterozygous for the mating type gene(s), were unsuccessful and as the International Code for Botanical Nomenclature does not deal adequately with the typification of haploid heterothallic yeasts (Wickerham, 1965), the holotype for the species is constituted by a dried specimen of a mixed culture of the two mating types CBS 6699 and CBS 5295, the isotypes for the species. By its formation of apically clustered eonidia borne on denticles formed by the hyphae, the conidial state of S. ciferrii shows some agreement with the fungal genus Sporothrix Hektoen et Perkins (de Hoog, 1974). Although the majority of cultures of the conidial state of S. ciferrii have been recovered from warm-blooded sources, this restricted occurrence might be due to isolation techniques which are inadequate for detecting the wider natural distribution of the species. The authors thank Dr. J. A. von Arx and Dr. W. Gams for examining the sporulating cultures, helpful suggestions and criticism of the manuscript, Mr. N. V. D. W. Liebenberg for his services at the electron microscope and Mr. J. A. Schuur for photographic assistance. Received 2 February 1976

9 THE GENUS STEPHANOASCUS GEN, NOV. 127 REFERENCES YON ARX, J. A. 1972, On Endomyces, Endomycopsis and related yeast-like fungi. -- Antonie van Leeuwenhoek 38: BEHRENO, G Ueber Trichomycosis nodosa (Juhel-R6noy); Piedra (Osorio) -- Berl. Klin. Wochenschr. 21 : DE HOOG, G. S. 1974, The genera Blastobotrys, Sporothrix, Calcarisporum and Calcarisporella gen. nov. -- Stud. Mycol. 7: KLOKKE, A. H. en KAMP, H Een systematisch onderzoek naar animale mycosen bij vee en mens.- Ned. Tijdschr. Geneesk. 106: KREGER-VAN RlJ, N. J. W Candida ciferrii, a new yeast species. -- Mycopathol. Mycol. Appl. 26: SCHI/~NNING, H Nouveau genre de la famille des Saccharomyc~tes.--C. R, Trav. Lab. Carlsberg 6: WICKERHAM, L. J Opposite sexes as type specimen for heterothallic haploid yeasts. -- Taxon 14: