Antonie van Leeuwenhoek 52 (1986) 39-44 9 Martinus NijhoffPublishers, Dordrecht - Printed in the Netherlands

K e y to the species o f Hyphozyma (yeast-like H y p h o m y c e t e s ) a n d d e s c r i p t i o n o f H. roseonigra sp. n o v .

G. S. DE HOOG 1 & M. TH. SMITH 2

t Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 AG Baarn, The Netherlands Yeast Division, Centraalbureau voor Schimmelcultures, Laboratory of Microbiology, Delft University of Technology, Julianalaan 67a, 2628 BC Delft, The Netherlands

Abstract. The new species, Hyphozyma roseonigra, characterized by pink colonies, budding cells with minute annellated zones and brown, septate hyphae, is described. It is non-fermentative, shows no colouration with Diazonium Blue B, and has an ascomycete-type cell wall ultrastructure. A key to the accepted species and varieties of Hyphozyma is given.

INTRODUCTION

In 1981 a pink, yeast-like fungus was submitted by M. I. Farbood for identification. Subcultures eventually turned olivaceous-black and the thin- or thickwalled, true hyphae showed numerous anastomoses. The strain could not be identified either with any of the known hyphal 'black yeasts', or with any of the red yeasts. The form-genus Hyphozyma de Hoog & M. Th. Smith (1981), recently introduced for yeast-like Hyphomycetes that are superficially similar to both Rhodotorula Harrison and PhialophoraMedlar and allied genera, seems appropriate to accommodate this fungus. The genus was not intended to reflect any natural relationships but merely described to classify those pink anamorphic fungi, which are capable of abundant budding, and also form narrow, septate hyphae without chlamydospores. The fungus under consideration also shows these key-features, but is considered sufficiently different from the known species to warrant its description as a new one.

MATERIALS AND METHODS

Cultures were grown in petri dishes containing 15 ml yeast-pepton-glucose agar

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G. S. de Hoog & M. Th. Smith

(YPGA), cherry decoction agar (ChA) or malt extract agar (MEA) at room temperature. Water-mounted preparations were examined after 3, 10 and 30 days. Several months-old colonies on agar slants were also studied. Assimilation and fermentation abilities were tested three times by the methods standardized by Van der Walt (1970). For transmission electron microscopy (TEM) cultures were grown on YPGA at 25~ After the cells were washed with water, thery were fixed with 1.5% aqueous KMnO4 for 30 min at room temperature, washed again with water and centrifuged in Beem capsules. The pellets were dehydrated in an ethanol series, stained with 1.5% uranyl acetate for 2 h at the 50% ethanol step and finally embedded in Spurr'resin. Ultrathin sections were cut with a Reichert model OM-04 ultra-microtome, poststained with Reynolds lead citrate for 10 sec and examined with a Philips model EM 201 electron microscope at 60 kV.

TAXONOMY

Hyphozyma roseonigra de Hoog & M. Th. Smith, sp. nov. (Fig. 1, Plate 1)
Coloniae in agaro YPGA dicto ad 8 mm diam post 10 dies, mucidae, dilute aurantiae. Mycelium submersum ex muco extendens, subinde olivascens. Cellulae gemmantes marginales hyalinae, cylindricae vel obclavatae, circa

,10.um,
Fig. 1. Hyphozyma roseonigra, CBS 514.83. A. budding cells, ChA, I week; B. young hyphae, ChA,
1 week; C. anastomosing cells, MEA, 2 months.

Key to the species of Hyphozyma

41

Plate 1. Hyphozymaroseonigra,CBS514.83,TEM micrographs.Barsrepresent0.5 lain. 6-8 x 1.8-2.0 lam, deinde ad 10--13 x 2.5-3.2 ~tm inflatae. Protuberantiae ad 2 lam longae in una vel parte, raro utrinque formata, cellulas secundarias in successione basipetali producentes. In parte vetustiore coloniae cellulae late ellipsoideae, 6-9 4--6 lam. Cellulae submersae saepe hyphas hyalinas tenuitunicatas, deinde crassitunicatas, fuscescentes ad 2.2 ~tm latas proferentes. Temperatura maxima 27~ Typus vivus et exsiccatus CBS 514.83, isolatus e solo, New Jersey, USA, a M.I. Farbood. Colonies on YPGA attaining a diam of 8 mm in 10 days, flat, slimy, pale orange (6A3; Kornerup & Wanscher 1978) with a sharp, somewhat lobed margin; onemonth old colonies remain pink and slimy. Colonies on ChA attain a diam of 4 mm in 10 days, after 3 weeks becoming centrally olivaceous, with light brown (6D6) to dark brown (5F7) reverse. After 10 days a submerged mycelium extends from the mucous colony; after 30 days the colony is dense, olive brown (4F4), with local thin, dirty-white central patches which later develop dense fascicles of aerial mycelium; a slimy zone remains around the centra which shows fine pink/olivaceous-black radial striation. Marginal budding cells hyaline, all about

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G. S. de Hoog & M. Th. Smith

Table 1. Growth responses of Hyphozyrna roseonigra

D-glucose D-galactose L-sorbose D-ribose D-xylose L-arabinose D-arabinose L-rhamnose Sucrose Maltose Trehalose Methyl ec-D-glucopyranoside Cellobiose Salicin Arbutin Melibiose Lactose Raffinose + + + + + + + + + + + + + + + v + Melezitose Inulin Soluble starch Glycerol Meso-erythritol Ribitol D-glucitol D-mannitol Galactitol Myo-inositol DL-lactate Suceinate Citrate KNO 3 NaNO2 Ethylamine Without vitamins DBB + + + + + + + v + + + + + + + -

equal in size and cylindrical to obclavate in shape. Young daughter cells are approximately 6-8 x 1.8-2.0/am and soon inflate to 10--13 x 2.5-3.2 gm. From a short butt of up to 2 gm in length, formed at one end of the cell (rarely at both ends), daughter cells are produced in basipetal order. At a young stage cells may show vague annellations which may disintegrate into mucous. Cells in older parts of the colony are broadly eUipsoidal, 6-9 x 4-6 gm. With age the (sub)hyaline cells are dark brown, thick-walled, slightly roughened, and have granular contents. Submerged cells of 2-week-old colonies often form hyphae which are hyaline, thin-walled, irregularly branched, 1.0-1.8 gm wide, and form a compact mycelium or dense fascicles. After two months some of the cells develop thick walls, a dark brown colour, and may be up to 2.2 gm wide. Dark hyphae and discrete cells in 2-month old cultures show numerous anastomoses. Single cells often aggregate in peculiarly shaped strings or clusters (Fig. 1). Cardinal temperatures: minimum 6 ~ optimum 15-24~ maximum 27 ~ Growth responses on various carbon compounds is given in Table 1; fermentative ability absent. Type strain (living and dried) CBS 514.83, isolated from soil, central New Jersey, USA, by M.I. Farbood.

DISCUSSION

Young cultures of Hyphozyma roseonigra are yeast-like, and strongly reminiscent of ordinary red yeasts. After two weeks on several media, local hyphae begin to develop, and after two months patches of aerial mycelium and small

Key to the species of Hyphozyma

43

hyphal sectors are formed. Since both types could be isolated separately, it was initially suspected that the culture consisted of a mixture of a mould and a yeast. However, on repeated transfer each growth type was observed to revert to the original form. Similar pleomorphism is known in many yeast-like Hyphomycetes. Due to phenotypic segregation (Van Oorschot & De Hoog 1981) a strain may propagate abundantly in both forms. Reversion to the original condition may sometimes be difficult, but in initial stages this seems to be determined by environmental factors rather than by changes in genotype. A similar dimorphism has, for example, been observed in Hyphozyma variabilis de Hoog & M. Th. Smith, where some culture no longer produced budding cells. Hyphozyma is characterized by pink colonies and budding cells which are produced repetitively from the same scar. In H. sanguinea (Ramirez) de Hoog & M. Th. Smith this was mainly seen on discrete budding cells, which showed minute annellated zones; in H. variabilis repetitive conidiogenesis occurred on hyphae, the scars remaining invisible with light microscopy (De Hoog & Smith 1981). In H. roseonigra the hyphae, even when abundant, remained sterile. Occasionally the budding cells were seen to reproduce repetitively from an apical scar, leading to an inconspicuous annellated zone which soon deteriorated and became granular (Fig. 1, arrowheads). With TEM, wall relations between mother and daughter cells remained unclear. In contrast to the situation in red yeasts and cultural states of Taphrinales, clear annellated collars are absent from bud scars. Some capsular material was seen on the wall of young cells, particularly concentrated around the bud neck (Plate 1). The cell walls were of the ascomycete type. The ascomycete-like character of H. variabilis was supported by the occurrence of Woronin-bodies close to the septal pores (N. J. W. Kreger-van Rij, pers. comm.). In addition, none of the Hyphozyma isolates stained with Diazonium Blue B. Hyphozyma thus is markedly different from Rhodotorula, which has a basidiomycete affinity. The genus is considered to be ascomycetous, similar to Hyphomycete genera such as Lecythophora Nannf. and Hormonema Lagerberg & Melin, and unrelated to the endomycetous or basidiomycetous yeasts. Cultural states of Taphrinales often also show repetitive conidiogenesis and have ascomycete-like cell walls (Von Arx et al. 1982), but the colonies are usually purplish and lack true hyphae. The frequent occurrence of anastomoses in all Hyphozyma species is striking (Fig. 1). In contrast to the formation of true hyphae in the red yeasts, which usually precedes the formation ofa teleomorph, hyphae ofHyphozyma roseonigra are often produced from single, undifferentiated cells with a single nucleus. The nuclei of both the yeast cells and the hyphae are haploid (R. Bauer, pers. comm.). Karyogamy and meiosis probably do occur and conjugation merely allows an asexual exchange of nuclei.

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G. S. de H o o g & M . Th. S m i t h

KEYS TO HYPHOZYMA TAXA

Key based on cultural characteristics and morphology: la. Cultures on ChA after three weeks with abundant dark brown, thick-walled, sterile hyphae . . . . . . . . . . . . . . . . . . . . . . . . . . H. roseonigra lb. Cultures remaining pink on all media; hyphae, when present, hyaline, thinwalled . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2a. Initial growth with coherent strings ofglobose cells . . . . . H. sanguinea 2b. Initial growth either with budding cells or with (pseudo-)mycelium . . . 3 3a. Cultures with vinaceous fragrance . . . . . . . . . H . variabilis var. o d o r a 3b. Cultures without significant fragrance . . . . . H . variabilis var. variabilis Key based on physiology: la. Growth with L-rhamnose, sucrose, methyl alpha-D-glucopyranoside, melezitose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1b. No growth with the above compounds . . . . . . . . . . . . . . . . . . 3 2a. Growth with D-arabinose, myo-inositol; growth at 37 ~ . . H . s a n g u i n e a 2b. No growth with the above compounds nor at 37 ~ . . . . . . H. roseonigra 3a. Growth with D-glucitol . . . . . . . . . . . . . . H . variabilis var. o d o r a 3b. No growth with D-glucitol . . . . . . . . . . . . H . variabilis var. variabilis
Acknowledgements. We thank Fritsche Dodge and Olcott Inc., New York, USA for their cooperation in the publication of this article. Mrs W. Batenburg-Van der Vegte, Laboratory for Microbiology, University of Technology, Delft, The Netherlands is acknowledged for making TEM micrographs. We are indebted to Dr N. J. W. Kreger-van Rij, Laboratory for Medical Microbiology, University of Groningen, The Netherlands, for information on ultrastructure of related Hyphozyma species, and to Dr R. Bauer, Lehrstuhl fiir spezielle Botanik, University of Tiibingen, FRG, for establishing ploidy in H. roseonigra.

REFERENCES De Hoog, G. S. & Smith, M. Th. (1981) Hyphozyma, a new genus of yeast-like Hyphomycetes. Antonie van Leeuwenhoek 47:339-352 Kornerup, H, & Wanscher, J. H. (1978) Methuen handbook of colour, 3rd ed. Methuen, London Van der Walt, J. P. (1970) Criteria and methods used in classification. In: J, Lodder (ed), The Yeasts - A taxonomic study, 2nd ed., pp 34-113. North-Holland Publ. Co., Amsterdam/London Van Oorschot, C. A. N. & De Hoog, G. S. (1981) Dimorphic behaviour and taxonomy of Trichosporiella sporotrichoides. Antonie van Leeuwenhoek 47:353-366 Von Arx, J. A., Van der Walt, J. P. & Liebenberg, N. V. D. M. (1982) The classification of Taphrina and other fungi with yeast-like cultural states. Mycologia 74:285-196 Received and accepted 13 September 1985