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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83. (Fig. MEA.um. 2 months. CBS 514. For transmission electron microscopy (TEM) cultures were grown on YPGA at 25~ After the cells were washed with water. cylindricae vel obclavatae. Smith. ChA. washed again with water and centrifuged in Beem capsules. subinde olivascens. Th. TAXONOMY Hyphozyma roseonigra de Hoog & M. S. Cellulae gemmantes marginales hyalinae. young hyphae. cherry decoction agar (ChA) or malt extract agar (MEA) at room temperature. The pellets were dehydrated in an ethanol series. Several months-old colonies on agar slants were also studied. budding cells. nov. A. Th. Assimilation and fermentation abilities were tested three times by the methods standardized by Van der Walt (1970). mucidae. dilute aurantiae. C. Fig. Smith (YPGA). 1. 10 and 30 days.10. ChA. stained with 1. Mycelium submersum ex muco extendens. anastomosing cells. Plate 1) Coloniae in agaro YPGA dicto ad 8 mm diam post 10 dies.5% uranyl acetate for 2 h at the 50% ethanol step and finally embedded in Spurr'resin. . de Hoog & M. Hyphozyma roseonigra. 1 week. sp. Water-mounted preparations were examined after 3. circa .5% aqueous KMnO4 for 30 min at room temperature.40 G. B. thery were fixed with 1. 1. Ultrathin sections were cut with a Reichert model OM-04 ultra-microtome. I week. poststained with Reynolds lead citrate for 10 sec and examined with a Philips model EM 201 electron microscope at 60 kV.

all about . cellulas secundarias in successione basipetali producentes. fuscescentes ad 2. after 30 days the colony is dense.5-3. deinde crassitunicatas.CBS514. Colonies on ChA attain a diam of 4 mm in 10 days. somewhat lobed margin. dirty-white central patches which later develop dense fascicles of aerial mycelium. isolatus e solo.8-2. flat.5 lain. 6-9 • 4--6 lam.0 lam. Kornerup & Wanscher 1978) with a sharp. In parte vetustiore coloniae cellulae late ellipsoideae.I.2 ~tm inflatae.83. with light brown (6D6) to dark brown (5F7) reverse. USA.Key to the species of Hyphozyma 41 Plate 1. onemonth old colonies remain pink and slimy. slimy.83. Cellulae submersae saepe hyphas hyalinas tenuitunicatas. Protuberantiae ad 2 lam longae in una vel parte. After 10 days a submerged mycelium extends from the mucous colony. olive brown (4F4). deinde ad 10--13 x 2. 6-8 x 1. Marginal budding cells hyaline.2 ~tm latas proferentes. Farbood. with local thin. after 3 weeks becoming centrally olivaceous. pale orange (6A3. Colonies on YPGA attaining a diam of 8 mm in 10 days. Hyphozymaroseonigra. raro utrinque formata. Temperatura maxima 27~ Typus vivus et exsiccatus CBS 514.TEM micrographs. a slimy zone remains around the centra which shows fine pink/olivaceous-black radial striation. a M.Barsrepresent0. New Jersey.

At a young stage cells may show vague annellations which may disintegrate into mucous. formed at one end of the cell (rarely at both ends). USA. Type strain (living and dried) CBS 514. 1.42 G. by M. local hyphae begin to develop. daughter cells are produced in basipetal order. With age the (sub)hyaline cells are dark brown. After two months some of the cells develop thick walls. and have granular contents. 1). a dark brown colour.0-1. Single cells often aggregate in peculiarly shaped strings or clusters (Fig. fermentative ability absent. Cardinal temperatures: minimum 6 ~ optimum 15-24~ maximum 27 ~ Growth responses on various carbon compounds is given in Table 1. After two weeks on several media. de Hoog & M. Th. and strongly reminiscent of ordinary red yeasts. thin-walled. isolated from soil. DISCUSSION Young cultures of Hyphozyma roseonigra are yeast-like.2 gm wide. Young daughter cells are approximately 6-8 x 1.2 gm. and form a compact mycelium or dense fascicles. From a short butt of up to 2 gm in length. and may be up to 2.0/am and soon inflate to 10--13 x 2.5-3.8-2. slightly roughened. Dark hyphae and discrete cells in 2-month old cultures show numerous anastomoses. irregularly branched. Smith Table 1. Farbood. 6-9 x 4-6 gm. central New Jersey. S.83. thick-walled. Cells in older parts of the colony are broadly eUipsoidal. and after two months patches of aerial mycelium and small .I.8 gm wide. 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. Submerged cells of 2-week-old colonies often form hyphae which are hyaline.

Similar pleomorphism is known in many yeast-like Hyphomycetes. Karyogamy and meiosis probably do occur and conjugation merely allows an asexual exchange of nuclei. However. variabilis repetitive conidiogenesis occurred on hyphae. 1. 1982). sanguinea (Ramirez) de Hoog & M. Hyphozyma is characterized by pink colonies and budding cells which are produced repetitively from the same scar. 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. In contrast to the situation in red yeasts and cultural states of Taphrinales.). In addition. it was initially suspected that the culture consisted of a mixture of a mould and a yeast. even when abundant. The frequent occurrence of anastomoses in all Hyphozyma species is striking (Fig. 1). wall relations between mother and daughter cells remained unclear. Hyphozyma thus is markedly different from Rhodotorula. W. clear annellated collars are absent from bud scars. Th. Th. comm. for example. Occasionally the budding cells were seen to reproduce repetitively from an apical scar. pers. roseonigra the hyphae. Kreger-van Rij. Cultural states of Taphrinales often also show repetitive conidiogenesis and have ascomycete-like cell walls (Von Arx et al. remained sterile. leading to an inconspicuous annellated zone which soon deteriorated and became granular (Fig. in H. which usually precedes the formation ofa teleomorph. and unrelated to the endomycetous or basidiomycetous yeasts. Due to phenotypic segregation (Van Oorschot & De Hoog 1981) a strain may propagate abundantly in both forms. J. arrowheads). Some capsular material was seen on the wall of young cells. The cell walls were of the ascomycete type. which showed minute annellated zones. and Hormonema Lagerberg & Melin. similar to Hyphomycete genera such as Lecythophora Nannf. undifferentiated cells with a single nucleus. In H. A similar dimorphism has. In contrast to the formation of true hyphae in the red yeasts. the scars remaining invisible with light microscopy (De Hoog & Smith 1981). With TEM. comm. pers. which has a basidiomycete affinity. Smith this was mainly seen on discrete budding cells. Smith. where some culture no longer produced budding cells. particularly concentrated around the bud neck (Plate 1). been observed in Hyphozyma variabilis de Hoog & M. Since both types could be isolated separately. Bauer. The ascomycete-like character of H. The genus is considered to be ascomycetous. variabilis was supported by the occurrence of Woronin-bodies close to the septal pores (N. on repeated transfer each growth type was observed to revert to the original form.Key to the species of Hyphozyma 43 hyphal sectors are formed. none of the Hyphozyma isolates stained with Diazonium Blue B. In H.). but the colonies are usually purplish and lack true hyphae. The nuclei of both the yeast cells and the hyphae are haploid (R. . hyphae ofHyphozyma roseonigra are often produced from single.

. . . roseonigra lb. . growth at 37 ~ . V. . for information on ultrastructure of related Hyphozyma species. & De Hoog. . myo-inositol. New York. & Liebenberg. . . . . . . . . . . variabilis var. . . . . Methuen. . . N. . . J. . . . sucrose. hyphae. thick-walled. . Lehrstuhl fiir spezielle Botanik. . . . (1970) Criteria and methods used in classification. Mycologia 74:285-196 Received and accepted 13 September 1985 . . . Amsterdam/London Van Oorschot. . . M. . roseonigra. 3 3a. . . . . . H. . . . . . . North-Holland Publ. .. . A. . . . . . 2 2a. . . . & Smith. Growth with D-glucitol . . J. a new genus of yeast-like Hyphomycetes. . . .A taxonomic study. . . . 2 1b. W. The Netherlands. . Antonie van Leeuwenhoek 47:353-366 Von Arx. 3rd ed. Lodder (ed). . Batenburg-Van der Vegte. In: J. variabilis Key based on physiology: la. . M. . . . The Netherlands is acknowledged for making TEM micrographs. . . . J. . . . No growth with the above compounds nor at 37 ~ . . . . . . University of Technology. . No growth with D-glucitol . P. 3 2a. . . A. . methyl alpha-D-glucopyranoside. . REFERENCES De Hoog. . variabilis var. . Cultures on ChA after three weeks with abundant dark brown. S. Co. . sterile hyphae . . Antonie van Leeuwenhoek 47:339-352 Kornerup. Delft. No growth with the above compounds . N. melezitose . . . . . H . (1982) The classification of Taphrina and other fungi with yeast-like cultural states. . . de H o o g & M . variabilis Acknowledgements. . variabilis var. Cultures remaining pink on all media. . . G. J. . . H. . pp 34-113. P. . H . S. London Van der Walt. FRG. Growth with D-arabinose. H . sanguinea 2b. Growth with L-rhamnose. University of Tiibingen. H. Laboratory for Medical Microbiology. . . .44 G. when present. . . . .. . (1981) Hyphozyma. . . o d o r a 3b. . and to Dr R. . . Th. Initial growth with coherent strings ofglobose cells . variabilis var. & Wanscher. . hyaline. . C. The Yeasts . . . .. D. (1978) Methuen handbook of colour. thinwalled . . USA for their cooperation in the publication of this article. H .. Laboratory for Microbiology. . . . . . . . . Mrs W. Th. . (1981) Dimorphic behaviour and taxonomy of Trichosporiella sporotrichoides. Bauer. . . roseonigra 3a. . H . . H. . for establishing ploidy in H. . . We thank Fritsche Dodge and Olcott Inc. University of Groningen. . . . . . Initial growth either with budding cells or with (pseudo-)mycelium . . S. o d o r a 3b. . J. . . . . . Kreger-van Rij. . . H. . . . . s a n g u i n e a 2b. G. 2nd ed. Van der Walt. S m i t h KEYS TO HYPHOZYMA TAXA Key based on cultural characteristics and morphology: la. Cultures without significant fragrance . . . . . Cultures with vinaceous fragrance . We are indebted to Dr N.