hyphozyma - antonie van leeuwenhoek (1)

8/2/2019 HYPHOZYMA - Antonie Van Leeuwenhoek (1)

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An to n ie va n Leeu w en h o ek 5 2 ( 1 9 8 6 ) 3 9 - 4 49 M a r t in u s N i jh o f fPu b l i sh ers , Do rd rech t - Pr in ted in th e N e th er la n d s

K e y t o t h e s p e c i e s o f Hyphozyma ( y e a s t - l i k e H y p h o m y c e t e s ) a n dd e s c r i p t i o n o f H. roseonigra s p . n o v .

G. S . D E H O O G 1 & M . T H . S M I T H 2t Centraalbureau voor Schimm elcul tures , P .O. B ox 273 , 3740 AG Baarn, The Netherland sYe ast Div is ion , Centraalbureau voor Schim melcul tures , Lab orator y o f Micro bio logy, De l f t

Universi ty o f Technology, Ju l ianalaan 67a , 2628 B C D el f t , The Netherland s

Ab s t ra c t . The new species, Hyphozyma roseonigra , characterized by pink colonies, budding cellswith minute annellated zones and brown, septate hyphae, is described. It is non-fermentative, showsno colouration with Diazonium Blue B, and has an ascomycete-type cell wall ultrastructure. Akey to the accepted species and varieties o f H y p h oz y m a is given.

INTRODUCTION

In 1 9 8 1 a p in k , y eas t - l ik e fu n g u s w as s u b m i t t ed b y M . I . Fa rb o o d fo r id en t if i ca -t i o n . Su b cu l t u re s ev en t u a l l y t u rn ed o l i v aceo u s -b l ack an d t h e t h i n - o r t h i ck -w a l l e d , t r u e h y p h a e s h o w e d n u m e r o u s a n a s t o m o s e s . T h e s t r a i n c o u l d n o t b ei d en ti f ied e i th e r w i t h an y o f t h e k n o w n h y p h a l ' b l ack y eas t s ', o r w i t h an y o ft h e r ed y eas t s . Th e fo rm -g en u s Hyphozymad e Ho o g & M . Th . Sm i t h (1 9 8 1 ) ,r ecen t l y i n t ro d u ce d fo r y eas t - li k e Hy p h o m y ce t e s th a t a r e s u p e r f i c ia l ly s im i l a rt o b o t h RhodotorulaH a r r i s o n a n d PhialophoraM ed l a r an d a l l ied g en e ra , s eem sap p ro p r i a t e t o acc o m m o d a t e t h is fu n g u s. Th e g en u s was n o t i n t en d ed t o r e fl ec tan y n a t u ra l r e l a ti o n s h i p s b u t m ere l y d es c r ib ed t o c l a s si fy t h o s e p i n k an am o rp h i cfu n gi , wh i ch a re cap ab l e o f ab u n d an t b u d d i n g , an d a l s o fo rm n a r ro w , s ep t a t eh y p h a e w i t h o u t c h l a m y d o s p o r e s . T h e f u n g u s u n d e r c o n s i d e r a t i o n a l s o s h o w st h ese k ey - fea t u res , b u t is co n s i d e red s u f f ic i en tl y d i f f e ren t f ro m t h e k n o w n s p e -c ies to war ran t i t s desc r ip t ion as a new one .

MATERIALS AND METHODS

Cu l t u res w ere g ro w n i n p e tr i d i s h es co n t a i n i n g 1 5 m l y eas t -p ep t o n -g l u co s e ag a rNothing contained herein shall be construed to imply the non-existence or relevant patents norso constitute a permission, inducement, or recommendation to practice any convention coveredby any patent without authority from the owner of the patent.


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40 G. S. de Hoog & M. Th. Smith(YPGA), cherry decoction agar (ChA) or malt extract agar (MEA) at roomtemperature. Water-mounted preparations were examined after 3, 10 and 30days. Several months-old colonies on agar slants were also studied. Assimilationand fermentat ion abilities were tested three times by the methods standardizedby Van der Walt (1970).

For transmission electron microscopy (TEM) cultures were grown on YPGAat 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 waterand centrifuged in Beem capsules. The pellets were dehydrated in an ethanolseries, stained with 1.5% uranyl acetate for 2 h at the 50% ethanol step andfinally embedded in Spurr'resin. Ultrathin sections were cut with a Reichertmodel OM-04 ultra-microtome, poststained with Reynolds lead citrate for 10sec and examined with a Philips model EM 201 electron microscope at 60 kV.

TA X O N O MY

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, diluteaurantiae. Mycelium submersum ex muco extendens, subinde olivascens. Cel-lulae gemmantes marginales hyalinae, cylindricae vel obclavatae, circa

, 1 0 . u m ,Fi g . 1 . H yph oz y ma r oseon igr a , CB S 514.83 . A. budd ing ce ll s, ChA , I week; B. you ng hyphae, C hA ,1 week; C. anastom osing ce l ls , ME A, 2 mo nths .


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Key to the species of Hyphozyma 41

Plate 1. Hyphozyma roseonigra,CBS 514.83, TEM micrographs. Bars represent 0.5 lain.6-8 x 1.8-2.0 lam, deinde ad 10--13 x 2.5-3.2 ~tm inflatae. Protuberantiae ad2 lam longae in una vel parte, raro utrinque formata, cellulas secundarias insuccessione basipetali producentes. In parte vetustiore coloniae cellulae late el-lipsoideae, 6-9 4--6 lam. Cellulae submersae saepe hyphas hyalinas tenuituni-catas, deinde crassitunicatas, fuscescentes ad 2.2 ~tm latas proferentes. Tempera-tura maxima 27~ Typus vivus et exsiccatus CBS 514.83, isolatus e solo, NewJersey, 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; one-month old colonies remain pink and slimy. Colonies on ChA attain a diam of4 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 extendsfrom the mucous colony; after 30 days the colony is dense, olive brown (4F4),with local thin, dirty-white central patches which later develop dense fasciclesof aerial mycelium; a slimy zone remains around the centra which shows finepink/olivaceous-black radial striation. Marginal budding cells hyaline, all about


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42 G. S. de Hoog & M. Th. SmithTable 1. Growt h responses o f Hyphozyrna roseonigraD-glucose + Melezitose +D-galactose + Inulin -L-sor bose + Soluble starch +D-ribo se + Glycerol +D-xylose + Meso-erythr itol +L-ara binose + Ribitol +D-ar abin ose - D-glucitol +L-rhamn ose + D-mannitol +Sucrose + Galactitol vMaltose + Myo-inositol -Trehalose + DL-lacta te +Methyl ec-D-glucopyranoside + Suceinate +Cellobiose + Citrat e +Salicin + KN O 3 +Arbutin + NaNO2 +Melibiose + Ethylamin e +Lactose v Without vitamins +Raffinose + DBB -

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

DISCUSSION

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


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Key to the species o f Hyp hozym a 43hyphal sectors are formed. Since both types could be isolated separately, it wasinitially 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 theoriginal form.

Similar pleomorphism is known in many yeast-like Hyphomycetes. Due tophenotypic segregation (Van Oorschot & De Hoog 1981) a strain may propagateabundantly in both forms. Reversion to the original condition may sometimesbe difficult, but in initial stages this seems to be determined by environmentalfactors rather than by changes in genotype. A similar dimorphism has, for exam-ple, been observed in Hyphozyma variabil is de Hoog & M. Th. Smith, wheresome culture no longer produced budding cells.

H y p h o zy m a is characterized by pink colonies and budding cells which areproduced repetitively from the same scar. In H. sanguinea (Ramirez) de Hoog& M. Th. Smith this was mainly seen on discrete budding cells, which showedminute annellated zones; in H. variabilis repetitive conidiogenesis occurred onhyphae, the scars remaining invisible with light microscopy (De Hoog & Smith1981). In H. roseonigra the hyphae, even when abundant , remained sterile. Oc-casionally the budding cells were seen to reproduce repetitively from an apicalscar, leading to an inconspicuous annellated zone which soon deteriorated andbecame granular (Fig. 1, arrowheads). With TEM, wall relations betweenmother and daughter cells remained unclear. In contrast to the situation in redyeasts and cultural states of Taphrinales, clear annellated collars are absent frombud scars. Some capsular material was seen on the wall of young cells, particular-ly concentrated around the bud neck (Plate 1). The cell walls were of the ascomy-cete type.

The ascomycete-like character of H. variabilis was supported by the occur-rence of Woronin-bodies close to the septal pores (N. J. W. Kreger-van Rij,pers. comm.). In addit ion, none of the H y p h o zy m a isolates stained with Diazo-nium Blue B. H y p h o zy m a thus is markedly different from Rhodotorula, whichhas a basidiomycete affinity. The genus is considered to be ascomycetous, similarto Hyphomycete genera such as Lecythophora Nannf. and Hormonema Lager-berg & Melin, and unrelated to the endomycetous or basidiomycetous yeasts.Cultural states of Taphrinales often also show repetitive conidiogenesis and haveascomycete-like cell walls (Von Arx et al. 1982), but the colonies are usuallypurplish and lack true hyphae.

The frequent occurrence of anastomoses in all H y p h o zy m a species is striking(Fig. 1). In contrast to the formation of true hyphae in the red yeasts, whichusually precedes the formation of a teleomorph, hyphae o fH yph ozym a roseon i-gra are of ten produced from single, undifferentiated cells with a single nucleus.The nuclei o f both the yeast cells and the hyphae are haploid (R. Bauer, pers.comm.). Karyogamy and meiosis probably do occur and conjugation merelyallows an asexual exchange o f nuclei.


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44 G . S . d e H o o g & M . T h . S m i t hK E Y S T O H Y P H O Z Y M A T A X A

Key based on cultural characteristics and morphology:la. Cultures on ChA after three weeks with abundant dark brown, thick-walled,sterile hyphae . . . . . . . . . . . . . . . . . . . . . . . . . H . r o s e o n i g r a

lb. Cultures remaining pink on all media; hyphae, when present, hyaline, thin-walled . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2a. Initial growth with coherent strings ofglobose cells . . . . . H . s a n g u i n e a2b. Initial growth either with budding cells or with (pseudo-)mycelium . . . 33a. Cultures with vinaceous fragrance . . . . . . . . H . v a r i a b i l i s var. o d o r a3b. Cultures without significant fragrance . . . . . H . v a r i a b i l i s var. v a r i a b i l i sKey based on physiology:la. Growth with L-rhamnose, sucrose, methyl alpha-D-glucopyranoside, mele-

zitose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21b. No growth with the above compounds .. . . . . . . . . . . . . . . . . 32a. Growth with D-arabinose, myo-inositol; growth at 37 ~ . . H . s a n g u i n e a2b. No growth with the above compounds nor at 37 ~ . . . . . . H . r o s e o n i g r a3a. Growth with D-glucitol . . . . . . . . . . . . . . H . v a r i a b i l i s var. o d o r a3b. No growth with D-glucitol . . . . . . . . . . . H . v a r i a b i l i s var. v a r i a b i l i sAcknowledgements . W e t h a n k F r i ts c h e D o d g e a n d O l c o t t I n c. , N e w Y o r k , U S A f o r th e i r c o o p e r a -t i on in t he pub l i ca t ion o f t h is a r ti cl e. M rs W . Ba t enbu rg -V an de r V eg te, Labo ra to ry fo r M ic rob io -logy , U n ive rs i t y o f Techno logy , D e l f t, The N e the r l ands i s acknow ledged fo r mak ing T EM mic ro -g raphs . W e a re i ndeb ted t o D r N . J . W . K rege r -van R i j , Lab o ra to ry fo r Med ica l Mic rob io logy ,U n ive rs i t y o f G ron ingen , T he N e the r l ands , fo r i n fo rma t ion on u l t ra s truc tu re o f re l a ted Hyphozymaspecies , and to D r R . Bauer , L ehrs tuhl f i i r speziel le B otanik , Un ivers i ty of Ti ib ingen , FR G , fores tab l i sh ing p lo idy in H . roseonigra.

REFERENCESD e H oog , G . S . & Smi th , M. Th . (1981 ) Hyphozyma, a new genus o f yeas t- l ike H yphomy ce t e s .

A n t o n i e v a n L e e u w e n h o e k 4 7 : 3 3 9- 3 5 2K orne rup , H , & W ansche r , J . H . (1978 ) Me thuen h andbo ok o f co lou r , 3 rd ed . Me thuen , Lo ndonVan d er W al t , J . P. (1970) Cri ter ia and m ethod s used in c lassi fica tion . In : J , Lodd er (ed), The Y easts

- A t axonom ic s t udy , 2nd ed . , pp 34 -113. N or th -H o l l and Pub l . Co . , A m ste rdam /Lon donV an O orscho t , C . A . N . & D e H oo g , G . S . (1981 ) D im orph ic behav iou r and t axon om y o f Trichospo-riella sporotrichoides. A n t o n i e v a n L e e u w e n h o e k 4 7 : 3 53 - 3 6 6Vo n Arx , J . A. , Van der Wal t , J . P. & Liebenbe rg , N. V. D. M . (1982) The c lass i f ica t ion of Taphrinaand o the r fungi wi th yeast - l ike cu l tura l s ta tes . M ycolo gia 74:285 -196Received and accepted 13 Septem ber 1985

hyphozyma - antonie van leeuwenhoek (1)

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