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Antonie van Leeuwenhoek 65: 41-54,1994. © 1994 KluwerAcademic Publishers. Printed in the Netherlands.
Conidiogenesis, nutritional physiology and taxonomy of Aureobasidium and Hormonema
G.S. de Hoog ~'2 & N.A. Yurlova 1'3 1 Centraalbureau voor Schimmelcultures, P.O. Box 273, 3740 A G Baarn, The Netherlands; 2 Institute for Molecular Cell Biology, BioCentrum Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands (3 Present address: Department of Microbiology, Chemico-Pharmaceutical Institute, 14, Prof. Popov Street, 197022 St. Petersburg, Russia)
Received 20 July 1993; accepted 17 September 1993
Key words: black yeasts, Aureobasidium, Hormonema, Kabatiella, Pringsheimia, Dothiora, Dothidea, Dothideaceae, conidiogenesis, physiology, taxonomy
Abstract
Diagnostic criteria for the distinction of the slightly osmophilic species, Aureobasidium pullulans, and the opportunistic pathogen on conifers, Hormonema dematioides, are provided. Reliable identification requires a combination of characters of conidiogenesis, expansion growth and assimilative abilities. Relationships with species of Kabatiella, and with the teleomorph genera Pringsheimia, Dothidea and Dothiora, all having Hor- monema-like cultural states, are discussed. An identification key is provided.
Introduction
Species of Aureobasidium and Hormonema have pinkish, rapidly expanding colonies which soon be- come slimy due to production of yeast cells. Colo- nies later often darken due to the development of chlamydospores. Endoconidia are common; extra- cellular polysaccharides are often produced. The species studied thus far have the same coenzyme Q system (Yamada et al. 1989) and show close simi- larities in thallus development and karyology (Takeo & De Hoog 1991).
The currently used criterion for distinction of the two genera is a difference in the mode of conidioge- nesis, viz. synchronous in Aureobasidium and per- current in Hormonema (Dennis & Buhagiar 1973; Hermanides-Nijhof 1977). Using this criterion, the latter author reclassified cultural states of Kabatiel-
la, a genus of plant pathogens forming sporodochia on the natural substrate, in Aureobasidium.
Synchronous versus percurrent conidiogenesis is, theoretically, a clear-cut generic marker for distinc- tion of Aureobasidium from Hormonema species, but has become difficult to apply since the conidio- genesis in an increasing number of strains cannot easily be attributed to either one of these types. The present article aims to re-evaluate this character. In addition, patterns of nutritional physiology are studied to see whether they provide useful key char- acters at generic or specific levels.
Materials and methods
Strains studied are listed in Table 1. Stock cultures were maintained on 2% malt/1% yeast extract agar (MYA) slants. Growth and fermentative ability
42
were tested at least twice after Van der Walt & Yar-
row (1984) in l iquid m e d i u m in test tubes at 25°C.
Final reading was done one week after the glucose
Concentra t ion in the posit ive control , measu red
with Glukotes t strips (Boehr inger) , was zero. Dia-
zon ium Blue B staining was pe r fo rmed according to
Hagler & A h e a r n (1981). Urease p roduc t ion was
tested in l iquid m e d i u m and on agar. Extracel lu lar
starch was visualized with 0.1% Lugol in l iquid
growth m e d i u m as well as microscopical ly using
Table 1. Strains examined.
Melzer ' s reagent . Acid p roduc t ion was tested on
2% MY A with calcium ca rbona te after Van der
Walt & Yarrow (1984). Cycloheximide to lerance
was tested bo th in l iquid m e d i u m at concen t ra t ions
of 0.01, 0.05 and 0.1% and on commercia l Mycosel
agar (0.04%; Bacto, Dickinson, USA) . Proteolysis
was tested in Petr i -dishes with t rypton/yeas t ex-
t ract /gelat in (Brocades) med ium, using HgC12 as in-
dicator. Growth at different t empera tu res was
scored in M E A slants after 7 d incubat ion . Dai ly
Group 1: pathogens on herbaceous plants CBS 242.64: as Kabatiella caulivora (Kirchner) Karakulin, ex anthracnose of Trifolium incarnatum (Leguminosae). CBS 767.71: as Kabatiella zeae Narita & Hiratsuka, ex leaf spots on Zea mays (Gramineae). CBS 125.21: T of Kabatiella lini (Lafferty) Karakulin, ex diseased Linum sp. (Linaceae). CBS 114.64: as Kabatiella microsticta Bub~k, ex leaf spots of Hemerocallis sp. (Liliaceae). CBS 873.71: AUT of Pringsheimia smilacis E. Mtiller, ex twig of Smilax aspera (Liliaceae). CBS 737.71: T of Dothiora cannabinae Froidevaux, ex twig of Daphne cannabina (Thymelaceaceae).
Group 2: pathogens on Pinaceae CBS 116.29: AUT of Hormonema dematioides Lagerb. & Melin, T of Pullulariafermentans Wynne & Gott var. melinii
Wynne & Gott, ex Pinus sp. CBS 215.50: as Dothichiza pithyophila (Corda) Petrak, ex dead twigs of Abies concolor. CBS 128.64: from ascospores of Sydowia polyspora (Bref. & Tavel) E. Mtiller, ex wood of Pinus sylvestris. CBS 750.71: from ascospores of Sydowia polyspora, ex twigs of Pinus strobus. CBS 719.76: as Hormonema dematioides, ex needle of Pinus sylvestris. CBS 765.84: as Hormonema prunorum (C. Dennis & Buhagiar) Hermanides-Nijhof, ex logs of Pinus radiata. CBS 906.85: T of Hormonema merioides Funk et al., ex needles of Pseudotsuga menziesii.
Group 3: pathogens on shrubs CBS 746.71: T of Pringsheimia chamaecyparidis Froidevaux, ex twig of Chamaecyparis lawsoniana (Cupressaceae). CBS 747.71: T of Pringsheimia euphorbiae Froidevaux, ex stem of Euphorbia rigida (Euphorbiaceae). CBS 748.71,749.71: from ascospores of Pringsheimia sepincola (Ft.) Hoehnel on twig of Rosa canina (Rosaceae). CBS 745.71: T of Dothiora rhamni-alpinae Froidevaux, ex twig of Rhamnus alpina (Rhamnaceae). CBS 739.71: T of Dothiora europaea Froidevaux, ex twig of Salix helvetica (Salicaceae). CBS 388.92: T of Selenophoma mahoniae Ramaley, ex leaf spots of Mahonia repens (Berberidaceae).
Group 4: non-pathogenic; on diverse, somewhat osmotic substrates CBS 933.72: T of Hormonerna prunorurn, ex fruit of Prunus domestica. CBS 105.22: T of Aureobasidium pullulans (De Bary) Arnaud var. melanogenum. CBS 626.85: ex peritoneal dialysis fluid. CBS 123.37: T of Pullularia fermentans var. schoenii (Roukhelman) Wynne & Gott, ex distilled glycerol. CBS 298.56: T of Pullularia fermentans var. fermentans, ex human lymph node. CBS 584.75: NT of Aureobasidium pullulans, ex fruit of Vitis vinifera. CBS 146.30: T of Dematoideum nigrescens Stautz, ex Quercus slime flux. CBS 701.76: T of Candida malicola Clark & Wallace, ex fruit of Malus sylvestris. CBS 247.92 = Helander 346F and CBS 244.82 = Helander 378F: ex phyllosphere of Betula pubescens. Yurlova-8: ex grass. Yurlova-ll: ex leaf of Fragaria vesca. Yurlova-16 and -18: ex metal parts of equipment. Yurlova-Flll6: ex deteriorated materials. Yurlova-Fl125: ex fruitbody of Inonotus obliquus. Yurlova-238: IFO 4466.
growth rates on 4% malt extract (MEA) and Sa-
bouraud 's glucose (SGA) agars were measured in
Petri-dishes incubated at 22 ° C. Conidiogenesis was
studied using Petri-dishes with potato carrot agar
(PCA).
R e s u l t s
The most pronounced types of conidiogenesis, i.e.
synchronous vs. annellidic, are found in Kabatiella and anamorphs of Dothideaceae, respectively (Ta-
ble 3). In Kabatiella conidiogenous cells are mostly terminal or lateral and produce 3-35 conidia next to each other. After secession, no further conidia are
produced f rom the same denticle. Hormonema anamorphs of Dothidea, Dothiora
and Pringsheimia mostly have only one conidioge- nous locus per cell; several conidia are produced percurrently through the same scar. More or less sympodial or spiral conidiogenesis on short butts may also occur, particularly on yeast-like cells in
shaken, liquid medium. Conidiogenesis in Hormonerna dematioides and
Aureobasidium pullulans is less easy to attribute to either one type. H. dematioides has 1-2 conidial loci
Table 2. Teleomorph-anamorph connections in Dothideaceae.
43
per cell, which produce rounded conidia percur-
rently in slimy heads. An extended annellated zone
with vague annellations is frequently observed. Co- nidiogenesis in Hormonema merioides and H. pru- norum is also annellidic.
Young, expanding hyphae of Aureobasidiurn pul- lulans show synchronous conidiation, 2-14 conidia being produced closely together f rom lateral and terminal as well as f rom intercalary cells. Conidial scars are slightly protuberant . Percurrent produc-
tion of conidia f rom single butts was generally ob-
served in a later stage of development. Dense clumps of conidia were seen alongside submerged
hyphae by direct observation of Petri-dishes with transparant, nutritionally deficient media.
The nutritional physiological pat tern of strains previously identified as Aureobasidium and Hor- monema by Hermanides-Nijhof (1977), supple- mented with the type strain of the later-described
H. merioides, is given in Tables 3.1 and 3.2. Diazonium Blue B staining mostly gave weak re-
actions which were difficult to interpret. Main dif-
ferences between the species are summarized in Ta- ble 5.
Notable intra-specific variations within A. pullu- lans are seen with growth responses to D-galactose,
Teleomorph: Anamorph:
Coelomycetous Acervular Hyphomycetous
- - m _
- Selenophoma mahoniae - - - Kabatiella microsticta - - Kabatiella caulivora - - Kabatiella zeae Discosphaerina fulvida - Kabatiella lini Discosphaerina miribelii Sarcophoma miribelii - Sydowia polyspora Dothichiza pithyophila -
Pringsheimia chamaecyparis unnamed Dothichiza Pringsheimia sepincola unnamed Dothichiza - Pringsheimia smilacis - Dothiora cannabinae - Dothiora europaea unnamed Dothichiza - Dothiora rhamni-alpinae unnamed Dothichiza -
Aureobasidium pullulans unnamed Aureobasidium Aureobasidium microstictum Aureobasidium caulivorum Aureobasidium zeae Aureobasidium lini unnamed Hormonema Hormonema dematioides Hormonema prunorum Hormonema merioides unnamed Hormonema unnamed Hormonema unnamed Uormonema unnamed Hormonema unnamed Hormonema unnamed Hormonema Hortaea werneckii
44
Table 3.1. G r o w t h r e a c t i o n s a n d o t h e r t e s t s o f Aureobasidium, Kabatiella a n d Selenophoma.
( ' , q . . .
D - g l u c o s e + + + + + + + + + + + +
D - g a l a c t o s e + + w w w w + - + + + +
L - s o r b o s e w w w - w w + - + + + +
D - g l u c o s a m i n e w w w w w w - + w w + w
D - r i b o s e + + w + + + + w w + + w
D - x y l o s e + + + + + + + + + + + +
L - a r a b i n o s e + + + + + + + + + + + +
D - a r a b i n o s e w w + w w w w w + + + w
L - r h a m n o s e + + + + + + + + + + + +
s u c r o s e + + + + + + + + + + + +
m a l t o s e + + + + + + + + + + + +
~ , c t t r e h a l o s e + + + + + + + + + + + +
m e t h y l c t - g l u c o s i d e + + + + + w w . . . . .
c e l l o b i o s e + + + + + + + + + + + +
s a l i c i n + + + + + + + + + + + +
a r b u t i n + + + + + + + + + w w +
m e l i b i o s e + + + + + + + + - - + +
l a c t o s e w + + + + + + w w w + +
r a f f i n o s e + + + + + + + + + + + +
m e l e z i t o s e + + + + + + + + + + + +
i n u l i n w w w w w + w w + + + -
s o l u b l e s t a r c h + + + + + + + + + + + +
g l y c e r o l + + + + + + + + + + + +
m e s o - e r y t h r i t o l + + + + + + + + + + - +
r i b i t o l w w + w + + + w + + + +
x y l i t o l + + + + + + + w + w + +
L - a r a b i n i t o l + + + + + + + + + + + +
D - g l u c i t o l + + + + + + + w + w + +
D - m a n n i t o l + + + + + - + + + + + +
g a l a c t i t o l + + + . . . . . . . + +
m y o - i n o s i t o l + + + + + + + + + + + +
g l u c o n o 8 - 1 a c t o n e w w w + + w + - + w - -
D - g l u c o n a t e + + + + + + w + - - + -
D - g l u c u r o n a t e + + + + + + w - w - + +
D - g a l a c t u r o n a t e + + + + + + + - + + + +
D L - l a c t a t e w w w w w w . . . . . .
s u c c i n a t e + + w + + + w - + w w -
c i t r a t e + + + + + + - - + + - +
m e t h a n o l . . . . . . . . . . . .
e t h a n o l + + + + + + w w + + + +
n i t r a t e + + + + + + + + + + + +
n i t r i t e + + + + + + + + + + + +
e t h y l a m i n e + + + + + + + + + + - +
L - l y s i n e + + + + + + + + + + + +
c a d a v e r i n e + + + + + + + + + + + +
Table 3.1. C o n t i n u e d ,
45
. ~ t-- eq. t--
c-,1
z z e _~ N "s =
~2 ~2
e~ t"q
t---
~2
¢¢3
O
~6
c r e a t i n e - - - w w w . . . . w -
c r e a t i n i n e - - - w w w . . . . . .
2 % MgC12 + + + + + + + w + + + +
5 % MgC12 + + + + + + w w + + + +
1 0 % MgC12 + + + + + + - w + w w +
2 % N a C 1 + + + + + + + w + + + +
5 % N a C 1 + + + + + + w w + w w +
1 0 % N a C l + + + + + + - w + - - w
0 . 0 1 % c y c l o h e x i m i d e . . . . . . . . . . . .
0 . 0 5 % c y c l o h e x i m i d e . . . . . . . . . . . .
m y c o s e l . . . . . . . . . . . .
a c i d p r o d u c t i o n w w w w w w + + - n d n d n d
u r e a s e t e s t + + + + + + + + + n d n d n d
s t a r c h p r o d u c t i o n ( l u g o l ) . . . . . . . . . . . .
s t a r c h p r o d . ( m e l z e r ) . . . . . . . . . . . .
f e r m e n t a t i o n . . . . . . . . . . . .
D B B . . . . . . . . . . . .
g e l a t i n w w - + + + + . . . . .
3 0 ° C + + + + - - - + - + + +
35 ° C + + . . . . . . . . . .
M E A 4 * 1.7 2 .7 3.5 2 .0 3,1 3.1 2.1 2 . 0 4 .3 3 .8 1.8 2 .2
S G A * 1.7 2 .7 3 .6 2.1 2 ,4 2 .6 1.9 1.4 3 .2 3 .6 1.3 1.8
a m y l a s e + + + + + + + + + + + +
c e l l u l a s e . . . . . . . . . . . .
p h o s p h o l i p a s e + + + + + + + + + + - +
c h i t i n a s e . . . . . . . . . . . .
D N A s e + + + + + + + + + + + +
* D a i l y g r o w t h r a t e a t r o o m t e m p e r a t u r e o n 4 % m a l t e x t r a c t a n d S a b o u r a u d ' s g l u c o s e a g a r s .
D-glucosamine, D-ribose, inulin, galactitol, DL-lac- tate, citrate and osmotolerance. No distinct infra- specific pattern can be observed. CBS 701.76 is ex- ceptional in not assimilating citrate and not being osmotolerant (Table 4). Notable intra-specific vari- ation within H. dematioides are with lactose, D- gluconate, D-galacturonate, succinate, 10% NaC1, production of extracellular starch and urease. The two strains of H. prunorum differ considerably in growth reactions to D-galactose, L-sorbose, L-ara- binose, glycerol, meso-erythritol, D-glucuronate, D-galacturonate and ethanol. CBS 765.84 was iso- lated from Pinus wood (Table 1) but could not be
matched with H. dematioides or H. merioides either.
Physiological patterns of available strains of dothideaceous teleomorphs are presented in Table 3.2. Most carbon and nitrogen sources are assimilat- ed, with the exception of D-glucosamine, methyl-o~- glucoside, galactitol, citrate and methanol, which are utilized weakly or not at all. Variation of diag- nostic value was obtained in growth responses to D- galactose, L-sorbose, D-arabinose, L-rhamnose, su- crose, c~,a-trehalose, melibiose, inulin, most higher alcohols and sugar acids, ethylamine and L-lysine.
The analyzed species of Kabatiella resemble the previous set of strains by being unable to assimilate
46
Table 3.2. G r o w t h r e a c t i o n s a n d o t h e r t e s t s o f Hormonema a n d D o t h i d e a c e a e w i t h Hormonema a n a m o r p h s .
• . ,= ~. , ~- ~ ~ ~ . t--
"= ~ ~ 8 "~ "~ ~ "~
D - g l u c o s e + + + + + + + + + + + + + + + +
D - g a l a c t o s e w w - w w - + - + + + - + - - +
L - s o r b o s e w w w + w - + - + + + w + + + +
D - g l u c o s a m i n e . . . . . . . . . . . . . . . .
D - r i b o s e + + + + + - + + w w + w w + + w
D - x y l o s e + + + + + + + + + + + + + + + +
L - a r a b i n o s e + + + + + + + - + + + + + + + +
D - a r a b i n o s e w w . . . . w w + + + w + + - +
L - r h a m n o s e + + + + + w + + + + + + + - - +
s u c r o s e + + + + + + + + + + + + + + - +
m a l t o s e + + + + + + + + + + + + + + + +
c~,c~ t r e h a l o s e + + + + + + + + + + + + + + - +
m e t h y l ~ - g l u c o s i d e - w - - - w + w - + . . . . . .
c e l l o b i o s e + + + + + + + + + + + + + + + +
s a l i c i n + + + + + + + + w w + + + + + +
a r b u t i n + + + + + + + + + + + + + + + +
m e l i b i o s e + + + + + + + + + + + + + + + +
l a c t o s e + + w + - + + w + + + + + + + +
r a f f i n o s e + + + + + + + + + + + + + + + +
m e l e z i t o s e + + + + + + + + w + + + + + + +
i n u l i n w w . . . . . w - w - + w - - w
s o l u b l e s t a r c h + + + + + + + + + + + + + + - w
g l y c e r o l + w w w w w - + + + + + + + + +
m e s o - e r y t h r i t o l + + + + w - + w + + + + + + - +
r i b i t o l + + + + w w + w w + + + + + w +
x y l i t o l + + + + + + + w + + + + + + + +
L - a r a b i n i t o l + + + + + w + - w + + + + + - +
D - g l u c i t o l + + + + + + + w + + + + + + - +
D - m a n n i t o l + + + + + + + + + + + + + + - +
g a l a c t i t o l . . . . . . . . . w . . . . . .
m y o - i n o s i t o l + + + + + - - - + + + + + + - -
g l u c o n o 6 - 1 a c t o n e + + + w w w + w + - + + + + - w
D - g l u c o n a t e + w - - - w w + + + + + + + - w
D - g l u c u r o n a t e + + w w w w + - + - + + + - - w
D - g a l a c t u r o n a t e + + + + - - + - + + + + + + w +
D L - l a c t a t e w w w + w - + w . . . . . . . .
s u c c i n a t e + + + w - w + + - - + + + + - +
c i t r a t e + + + w + + + + . . . . . . . .
m e t h a n o l . . . . . . . . . . . . . . . .
e t h a n o l + + + w + - + - w w - w + w - -
n i t r a t e + + + + + + + + + + + + + + + +
n i t r i t e + + + + + + + + + + + + + + + +
e t h y l a m i n e + + + + + + + + w - + + + + + +
Table 3.2. C o n t i n u e d .
47
• d ~ ~ ~ ~ ~ ~.
i
L - l y s i n e + + + + + - + + w w + + - + - +
c a d a v e r i n e + + + + + + + + + w + + + + + +
c r e a t i n e w - w - w - - w - + + + w w + w
c r e a t i n i n e w - w - w - - w - + + + w w + w
2 % M g C 1 2 + + + + + + + + + + + + + + + +
5 % M g C I 2 + + + + + + + + + + + + + w w +
1 0 % M g C 1 z + + + + + + + + w w + w - w - w
2 % N a C l + + + + + + + + + + + + + + + +
5 % N a G 1 + + + + + + + + w w + w w + - w
1 0 % N a C I w + w w - - - + . . . . . . . .
0 . 0 1 % c y c l o h e x i m i d e . . . . + . . . . . . . . . . .
0 . 0 5 % c y c l o h e x i m i d e . . . . . . . . . . . . . . . .
m y c o s e l . . . . . . . . . . . . . . . .
a c i d p r o d u c t i o n w w + + + w + w + w w w + - - w
u r e a s e t e s t + + + + + + + + . . . . . . . .
s t a r c h p r o d u c t i o n ( l u g o l ) - + . . . . . . . . . . . . . .
s t a r c h p r o d u c t i o n ( m e l z e r ) + + . . . . . . . . . . . . . .
f e r m e n t a t i o n . . . . . . . . . . . . . . . .
D B B w w - w . . . . . . + + + - - -
g e l a t i n . . . . . . . + n d n d n d n d n d n d n d n d
2 5 ° C + + + + + + + + + + + + + + + +
3 0 ° C + + w + + - w - w + + + + w w w
3 5 ° C . . . . . . . . . . w - w - - -
4 0 ° C . . . . . . . . . . . . . . . .
M E A 3 . 6 3 . 7 4 . 7 5 . 1 5 . 1 1 . 9 3 . 6 2 . 4 0 . 7 1 . 4 2 . 3 2 . 0 1 . 3 3 . 6 1 . 5 1 . 3
S G A 5 . 4 0 . 7 4 . 1 5 . 0 6 . 0 0 . 6 4 . 7 2 . 0 0 . 7 1 , 2 1 .1 0 . 7 1 . 2 3 . 0 1 . 2 0 . 9
a m y l a s e + + + + - - + + + + + + + + - +
methyl-o~-glucoside and DL-lactate. Diagnostic cri- teria are particularly found in assimilation of meli- biose, galactitol, D-gluconate, citrate and L-lysine.
D i s c u s s i o n
Aureobasidium pullulans shows presence of ure- ase, assimilation of glucuronate and inositol, ab- sence of fermentation and presence of extracellular DNAse (Hagler & A h e a r n 1981). Extracellular starch is produced in some strains of H. dernatioides (Table 3). In yeast taxonomy all these characters are
regarded as indicative of basidiomycete relation- ship (Golubev & Semenova 1988). DBB reactions of the fungi studied (Table 3), cell wall lysis with 1,3- f3-glucanase, the absence of xytose and fucose from cell wall hydrolyzates (Yurlova & Sinatskaya 1993), 2-layered cell wall ultrastructure and simple septal pores (Durrell 1968; Crang & Pechak 1977; Simon 1984) all point to an ascomycetous affinity. The val- ue of these character sets to establish broad taxo- nomic affinities of anamorphic strains should thus be applied with caution outside the yeasts.
Prillinger et al. (1990) supposed an affinity of Au- reobasidium to Schizosaccharomycetales, but this
48
Table 4. I n t r a - s p e c i f i c v a r i a t i o n o f Aureobasidium pullulans a n d Horrnonema dematioides: p o s i t i v e vs . n e g a t i v e g r o w t h r e a c t i o n s .
Aureobasidium pullulans: 105.22 626.85 123.37 298 .56 584.75 146.30 701 .76
G a l a c t i t o l + + + . . . .
C i t r a t e + + + + + + -
1 0 % N a C 1 + + + + + + -
1 0 % M g C I z + + + + + + -
G e l a t i n w w - + + + +
G r o w t h a t 37 ° C + + . . . . .
H o r m o n e m a dematioides: 215 .50 116 .29 719 .76 750.71 128.64
L a c t o s e + + w + -
D - G l u c o n a t e + w - - w
D - G a l a c t u r o n a t e + + + + -
S u c c i n a t e + + + w -
1 0 % N a C 1 w + w w -
S t a r c h p r o d u c t i o n w + - - -
seems unlikely because of known bitunicate Asco- mycete connections in related fungi, viz. the genera Discosphaerina (Van der Aa 1975; Sivanesan 1984), Sydowia (Butin 1964) and Pringsheimia and Doth- iora (Froidevaux 1972). Given the close similarities of the Hormonema anamorphs of these Ascomy- cetes with Aureobasidium and Hortaea in thallus development and karyology (Takeo & De Hoog 1991) and coenzyme-Q systems (Yamada et al. 1989), it is supposed here that all these anamorphs
are related to a single ascomycete family, Dothidea- ceae.
In nature, members of this family reproduce by ascomata or by either pycnidial or sporodochial anamorphs. The anamorph-genera Aureobasidium and Hormonema can be regarded as poorly differ- entiated cultural states of these fungi. In some cases the various types of asexual propagation have been attributed separate anamorph names. An overview of known connections is presented in Table 2. Spe-
Table 5. P h y s i o l o g i c a l k e y c h a r a c t e r s d i f f e r e n t i a t i n g b e t w e e n Aureobasidium pullulans, H o r m o n e m a dematioides, H. prunorum a n d H .
merioides.
A . p u l l u l a n s H . d e m a t i o i d e s H . m e r i o i d e s H . p r u n o r u r n *
D - G l u c o s a m i n e w ( - ) * * - - -
D - R i b o s e + ( - ) + - +
L - A r a b i n o s e + + + -
D - A r a b i n o s e w ( + ) - / w - w
methyl-a-Glucoside + ( w ) - ( w ) w w
I n u l i n w ( + ) - / w - w
meso-Erythritol + + - w
L - A r a b i n i t o l + + w -
myo-Inosi tol + + - -
D - G l u c o n a t e + ( w ) - ( + / w ) w w
D - G a l a c t u r o n a t e + + ( - ) - -
E t h a n o l + + ( - ) - -
L - L y s i n e + + - +
D a i l y g r o w t h r a t e M E A 1 .7 -3 .5 3 .6 -5 .1 1.9 2 .4
* B a s e d o n t y p e s t r a i n only .
** B e t w e e n b r a c k e t s : o n e s t r a i n d e v i a t i n g ; s l a s h : s e v e r a l s t r a i n s d e v i a t i n g .
49
cies of Aureobasidium, having synchronous coni- diation, are connected to the acervular genus Kaba- tiella (Hermanides-Nijhof 1977) or Selenophoma (Ramaley 1992). Species of Hormonema are con- nected to the pycnidial genera Sarcophoma (Van der Aa 1975) and Dothichiza (Robak 1952) or the acervular genus Kabatina (Ramaley 1992). Aureo- basidium synanamorphs of Kabatiella have strictly synchronous conidiation; groups of 3-35 conidia are produced on each hyphal cell (Table 6). K. mi- crosticta is remarkable in producing well-differen- tiated, erect conidiophores with saucer-shaped heads bearing 20-35 conidia (Hermanides-Nijhof 1977), in addition to creeping conidium-bearing hy- phae.
From the point of view of their ecology, several groups can be distinguished within the fungi studied (Table 1). Aureobasidium pullulans is particularly found in the phyllosphere (Dickinson 1967; Fokke- ma 1971), where it is one of the first fungi in the suc- cession of colonization (H0gg & Hudson 1966). An important factor may be the osmotic tension of the substrate. Several of our strains were isolated from the surface of sugar-containing fruits (Table 1). Par- ticularly overripe fruits are colonized (Cooke 1959, 1962). The species is an important component of the flora in aphid honeydew (Deshpande et al. 1992). The frequent isolation ofA. pullulans in surface wa- ters in late summer probably originates from air- carried flowers and fruits (Slfivikovfi et al. 1992). It is regularly found in somewhat salty environments, such as salt marsh soil, halophytes (Mushin &
Booth 1987) and intertidal zones (Torzilli et al. 1985). In vitro the species tolerates raised salt levels (Table 3). A similar ability and supposed ecology has been found in Hortaea werneckii (De Hoog & Gerrits-van den Ende 1992). Pringsheimia and Dothiora species are plant pathogens and do not grow with salt levels over 5% (Table 3). The same holds true for strains belonging to black yeast gen- era of supposed herpotrichiellaceous relationship, such as Exophiala (Kane & Summerbell 1987; De Hoog & Haase 1993), which are not found in osmot- ic substrates either.
The morphologically and culturally similar Ka- batiella species are pathogens of herbaceous plants. Hormonema dernatioides, including the anamorph of Sydowia polyspora, occurs as an opportunistic pathogen on, for example, weakened conifers (Funk 1985), where it occurs as an endophyte. Acid rain might therefore be a promotive factor for its occurrence. Helander & Rantio-Lehtim~iki (1990) showed that the competitive ability of Hormonerna in phyllosphere flora was not significantly affected by simulated acid rain. Aureobasidium pullulans occurs on conifer leaves as a common phyllosphere fungus rather than a pathogen (Sherwood & Car- roll 1974), where it has an antagonistic effect on the H. dematioides (Ranta 1990). An increase ofA. pul- lulans at the expense of other phylloplane orga- nisms was also noted after heavy metal pollution (Mowl & Gadd 1985). The genera Pringsheimia, Plowrightia and Dothidea are host-specific patho- gens on various kinds of plants (Froidevaux 1972).
Table 6. Types of conidiogenesis in Kabatiella, Aureobasidium and Hormonema species.
Young cells synchronous* Young cells percurrent Mature cells percurrent
Kabatiella
lini 3 - 5
microstica 5-35
zeae 4 - 7
Aureobas id ium
pullulans 2-14
H o r m o n e m a dematioides 1- 2
merioides 1- 2 p runorum 1- 2
Dothidea, Pringsheimia 1
m
m
+ +
+ + + +
+ +
* No of conidia, cell ~.
50
The type of conidiation defines the genus Kaba- tiella unambiguously, but in other members of the Dothideaceae this feature does not seem to be of prime importance. Discosphaerina fulvida is the te- leomorph of Kabatiella (Aureobasidium ) lini (Siva- nesan 1990) with synchronous conidiation, but the related species D. miribelii has a Hormonema-like anamorph with percurrent conidiation (Van der Aa 1975). Hortaea werneckii mostly produces conidia on annellated zones, but in some strains conidia are found in spiral arrangement (Nishimura & Miyaji 1984; De Hoog & Gerrits-van den Ende 1992).
The vegetative morphology of Aureobasidium pullulans is extremely variable. The preponderance of any stages in the life cycle can be influenced by nitrogen sources (Ramos & Garcfa Acha 1975) but also by carbon sources (our data). Therefore the type of conidiogenesis has been used as a diagnostic feature, rather than general morphology (Herma- nides-Nijhof 1977). The species initially shows syn- chronous conidiation, although the number of coni- dia produced may be very small. Synchronous coni- dium initials break through the mother cell wall (Crang & Pechak 1977). Later these loci remain productive, showing percurrent conidiation as in H. dematioides (Table 6). Consequently, the difference in conidiogenesis between A. pullulans and H. de- matioides is based mainly on whether 2-14 (A. pul- lulans) or 1-2 (H. dematioides) conidiogenous loci are present on each expanding hyphal cell. Since strains with two loci per cell do exist, this character is insufficient as sole diagnostic feature.
The physiological data of the strains tested corre- spond well with those given by Dennis & Buhagiar (1973), Elinov et al. (1989) and Sugai & Veiga (1988). Assimilation of monosaccharides by CBS 584.75 was found identical to results published by Dobberstein & Emeis (1989). In contrast to the ob- servation of Wynne & Gott (1956), no significant fermentation was detected in A. pullulans. In their nutritional physiology, A. pullulans and H. dema- tioides are very similar (Table 3), underlining the close taxonomic relationship of the two species. They are distinguished from other black yeasts by assimilation of soluble starch (Cooke & Matsuura 1963), lactose and citrate (Dennis & Buhagiar 1973), which are not utilized by most members of
Herpotrichiellaceae (De Hoog, unpublished re- suits). A. pullulans and H. dematioides are distin- guished by growth reactions to D-glucosamine, D- arabinose, methyl-~-glucoside and D-gluconate (Tables 3 and 5). The presence of urease activity was shown earlier by Federici (1982). Most strains of A. pullulans show good or weak gelatin liquefac- tion, while H. dematioides liquifled gelatin weakly or not at all. Federici (1982) reported that about 50 % of his strains were gelatin-negative, but Dennis & Buhagiar (1973) found them all positive. Colo- nies of A. pullulans showed less expansion growth than those of H. dernatioides; this was used as one of the main criteria for species distinction by Herma- nides-Nijhof (1977). However, expansion growth is greatly influenced by environmental conditions. Suspensions made in carbon or in nitrogen base and plated out on MEA medium resulted in totally dis- similar colonies, the differences being strain-de- pendent. None of the differences between A. pullu- lans and H. dematioides is exclusive and sufficient to distinguish the species; the intra-specific varia- tion of the two species is larger than their inter-spe- cific variation (Tables 4 and 5). Nevertheless signif- icant differences are known to exist in 18S RNA se- quences (J.M.J. Uijthof, W.A. Untereiner, pers. comm.).
Diagnostic problems are particularly significant with strains isolated from other sources than the ec- ological niches of the species. A. pullulans has been implicated in divergent opportunistic mycoses, such as infection of the mandible after removal of an impacted molar (Koppans et al. 1991), an infect- ed lymph node (Wynne & Gott 1956), otomycosis (Haley 1950), keratitis (Jones & Christensen 1974), onychomycosis (Vieira 1961), cutaneous phaeohy- phomycosis (Ciferri & Ashford 1929; Marill et al. 1973; Vermeil et al. 1971), subcutaneous granuloma (Coskey et al. 1983; Sarrat et al. 1973), localized sys- temic infection (Salkin et al. 1986), pulmonary in- fection with dissemination (Akagi et al. 1958) and generalized sepsis in immunocompromized pa- tients (Kaczmarski et al. 1986; Girardi et al. 1993). A cutaneous infection has also been described in H. dematioides (Coldiron et al. 1990). H. dematioides tends to be slightly more psychrophilic than A. pul- lulans. The two strains of the latter species that were
able to grow at 37°C had been isolated from hu- mans. It may be supposed that for both species the only requirement needed for successful infection is an ability to grow at 35-38 ° C, since otherwise A. pullulans is able to survive inside animal tissue (Bulman & Stretton 1974). The non-specificity of clinical pictures demonstrates that the pathogenic- ity of such strains is low (Ajello 1978), the symptoms varying with the portal of entry and condition of the host.
Hermanides-Nijhof (1977) introduced a var. mel- anogenurn for strains of A. pullulans that rapidly turn black due to chlamydospore formation. This property is highly variable. Blackening was mostly stimulated when D-galactose, L-sorbose or gluco- no-fi-lactone was used as sole source of carbon. Cer- nfikovfi et al. (1980) interpreted hyaline strains hav- ing lost their ability to produce melanin, concom- itantly with loss of monophenol monooxygenate ac- tivity. Horvath et al. (1976) found loss of pigment after repeated subculturing on aromatic com- pounds. Chlamydospore formation seems inappro- priate to delineate a taxonomic entity.
The type strains of Hormonema prunorum and H. merioides are recognizable by numerous physio- logical characters (Tables 3 and 5). H. prunorum is unambiguously characterized by no growth with L- arabinose, while H. merioides is the only species that does not assimilate L-lysine. Both species differ from H. dematioides and A. pullulans by not grow- ing with D-galacturonate and ethanol (Table 5).
In their general nutritional pattern, the fungi classified in Dothiora and Pringsheimia are similar to the Horrnonema/Sydowia species studied (Table 3). In both groups, D-galactose, D-glucosamine, methyl-o~-glucoside, galactitol and methanol are mostly poorly assimilated. Dothiora europaea grows very slowly in liquid medium, and in its poor assimilation of sugar alcohols there is a marked sim- ilarity to Horrnonerna merioides, the main differ-
51
ence being found in disaccharide assimilation. Striking differences between the two groups are found in growth responses to inulin and citrate.
Sigler (1990) described a Hormonerna-(Hypho- zyma-)like synanamorph of the coelomycete Eleutheromyces subulatus (Tode:Fr.) Fuckel. It is easily distinguished physiologically from the spe- cies complex described above by being unable to as- similate lactose, melibiose, xylose, melezitose, me- thyl-o~-glucoside, glucitol and glycerol.
On the basis of diagnostically usable differences between analyzed taxa, a key for physiological identification to the species is presented below. With this key, we have tried to identify a number of strains listed in 'Fable 1. Strains 347F and 378F showed percurrent conidiogenesis and were initial- ly identified as Hormonema species; conidium pro- duction in 11,16, 18, Fl116 and 238 was synchronous and hence these strains were regarded as being close to Aureobasidium pullulans. These tentative identifications matched with the key characters. However, strain 8 deviated from A. pullulans by not growing with methyl-c~-glucoside and lactose, strains 346F and 378F deviated from H. dematioides by assimilation of methyl-o~-glucoside, and 16 was the only isolate that was consistently tolerant of cy- cloheximide.
Acknowledgements
The authors are indebted to M. Helander for donat- ing cultures and to W. Zijlstra, M. Zembib and H.R van Barreveld for technical assistance. T. van den Berg-Visser is acknowledged for typing manuscript and tables. E.J. Hermanides-Nijhof, W. Gams and H.A. van der Aa are thanked for comments on the manuscript, and C.A.N. Jacobs-van Oorschot for correction of the English text.
52
Physiological key to Aureobasidium, Hormonema and related fungi 1.
I a. Xylose - lb. Xylose + 2a. Citrate - 2b. Citrate + 3a. D-Glucosamine + 3b. D-Glucosamine - 4a. L-Sorbose + 4b. L-Sorbose - 5a. Urease + 5b. Urease - 6a. methyl-c~-Glucoside + 6b. methyl-~-Glucoside - 7a. Sucrose-, c~,c~-trehalose- 7b. Sucrose + , c~,u-trehalose + 8a. L-Rhamnose - 8b. L-Rhamnose + 9a. L-Lysine - 9b. L-Lysine + or w 10a. myo-lnositol - 10b. myo-Inositol + 1 la. Succinate + 11 b. Succinate - 12a, Melibiose - 12b. Melibiose + 13a. 10% NaCI + 13b. 10% NaCI- 14a. Ethanol - 14b. Ethanol + 15a. Ribose + , L-arabinose- 15b. Ribose-, L-arabinose + 16a. methyl-~-Glucoside + (rarely w) 16b. methyl-a-Glucoside - (rarely w) 17a. Galactitol - 17b. Galactitol +
Eleutheromyces subulatus 2 3
12 4 5
Kabatiella zeae Kabatiella lini
A ureobasidium pullulans 6
Pringsheimia euphorbiae 7
Dothiora europaea 8
Dothiora cannabinae 9
Pringsheimia srnilacis 10
Dothiora rhamni-alpinae 11
Pringsheimia sepincola Pringsheimia chamaecyparidis
13 14
Kabatiella microsricta Kabatiella caulivora
15 16
Hormonema prunorum Hormonema merioides
A ureobasidium pullulans 17
Hormonema dematioides Selenophoma mahoniae
1 In case of Weak assimilation, both alternatives should be tried. 2 Based on type strain only,
References
Ajello L (1978) The black yeasts as disease agents: historical per- spective. In: The black and white yeasts, Proe. 4th Int. Conf. Mycoses, Pan Amer. Health Organ. Sci. Publ. 356:9-16
Akagi M, Fujino T, Hayashi S, Yamamoto Y & Akabe T (1958) A case of pulmonary infection caused by a black yeast-like fun- gus. Med. J. Osaka Univ. 8:585-599
Bulman RA & Stretton RJ (1974) Lesions experimentally pro- duced by fungi implicated in extrinsic allergic alveolitis. J. Hyg. 73:369-374
Butin H (1964) (Jber zwei Nehenfruchtformen von Sydowia po- lyspora (Bref. et v. Tav.) Mtiller. Sydowia 17:114-118
Cern~ikovfi M, Kockovfi-Kratochvflov~ A, Suty L, Zemek J & Kunifik B (1980) Biochemical similarities among strains of Aureobasidium pullulans (De Bary) Arnaud. Folia Microbiol. 25:68-73
Ciferri R & Ashford KB (1929) Two strains of Pullularia pullu- lans (de Bary) Berkhout isolated from the human skin. Puerto Rico J. Publ. Health Trop. Med. 5: 188-195.
Coldiron BM, Wiley EL & Rinaldi MG (1990) Cutaneous phae- ohyphomycosis caused by a rare fungal pathogen, Hormon- ema dematioides: successful treatment with ketoconazole. J. Am. Acad. Dermatol. 23:363-367
Cooke WB (1959) An ecological life history of Aureobasidium pullulans (De Bary) Arnaud. Mycopath. Mycol. Appl. 12:1-45
Cooke WB (1962) A taxonomic study in the 'Black Yeasts'. My- copath. Mycol. Appl. 17:1-43
Cooke WB & Matsuura G (1963) Physiological studies in the black yeasts. Mycopath. Mycol. Appl. 21:225-271
Coskey P J, Mehregan AH & Rippon JW (1983) Wood implanta- tion with phaeomycotic contamination. J. Am. Acad. Derma- tol. 8:428-429
Crang RE & Pechak DG (1977) Aureobasidium pullulans: fine structure and development. J. Coatings Technol. 50:36-42
De Hoog GS & Gerrits-van den Ende B (1992) Nutritional pat- tern and eco-physiotogy of Hortaea werneckii, agent of hu- man tinea nigra. Antonie van Leeuwenhoek 62:321-329
De Hoog GS & Haase G (1993) Nutritional physiology and se- lective isolation of Exophiala dermatitidis. Antonie van Leeu- wenhoek 64:17-26
Dennis C & Buhagiar RWM (1973) Comparative study of Aure- obasidium pullulans, A. prunorum sp. nov. and Trichosporon pullulans. Trans. Br. Mycol. Soc. 60:567-575
Deshpande MS, Rale VB & Lynch JM (1992) Aureobasidium pullulans in applied microbiology: a status report. Enzyme Mi- crobiol. Technol. 14:514-527
Dickinson CH (1967) Fungal colonization of Pisum leaves. Can. J. Bot. 45:915-927
Dobberstein J & Emeis CC (1989) [3-Xylanases produced by Au- reobasidium pultulans CBS 58475. Microbiol. Biotechnol. 32: 262-268
Durrell LW (1968) Studies of Aureobasidium pullulans (De Ba- ry) Arnaud. Mycopath. Mycol. Appl. 35:113-120
Elinov NP, Yurlova NA & Bogdanova TV (1989) Physiological and biochemical characteristics of some Aureobasidium spe- cies. Mikol. Fitopatol. 23:425-430
Federici L (1982) Extracellular enzymatic activities in Aureoba- sidium pullulans. Mycologia 74:738-743
Fokkema NJ (1971) The effect of pollen in the phyllosphere of rye on colonization by saprophytic fungi and on infection by Helminthosporium sativum and other leaf pathogens. Neth. J. P1. Path. 77, Suppl. 1:1-60
Froidevaux L (1972) Contribution fi l'6tude des Dothioracdes (Ascomyc6tes). Nova Hedwigia 23:679.734
Funk A (1985) Foliar fungi of Western trees. Can. For. Service, Victoria
Girardi LS, Malowitz R, Tortora GT & Spitzer ED (1993) Aureo- basidium septicaemia. Clin. Infect. Dis. 16:338-339
Golubev VI & Semenova SA (1988) Identification of taxonomic affinity among asporogenic yeasts. Mikol. Fitopatol. 22: 396- 399
Hagler AN & Ahearn DG (1981) A rapid DBB test to detect basidiomycetous affinity of yeasts. Int. J. Syst. Bact. 31: 204- 208
Haley LD (1950) Etiology of otomycosis. I. Mycologic flora of the ear. Arch. Otolaryngol. 52:202-207
Helander ML & Rantio-Lehtim~iki AH (1990) Effects of water- ing and simulated acid rain on quantity of phyllosphere fungi of birch leaves. Microb. Ecol. 19:119-125
Hermanides-Nijhof EN (1977) Aureobasidium and allied gen- era. Stud. Mycol. 15:141-177
53
Hogg BM & Hudson HJ (1966) Micro-fungi on leaves of Fagus sylvatica. I. The micro-fungal succession. Trans. Br. Mycol. Soc. 49:185-192
Horvath RS, Brent MM & Cropper DG (1976) Paint deteriora- tion as a result of growth of Aureobasidium pullulans on wood. Appl. Environm. Microbiol. 32:505-507
Jones BR & Christensen GR (1974) Pullularia corneal ulcer. Arch. Ophthalmol. 92:529-530
Kaczmarski EB, Liu Yin JA, Tooth JA, Love EM & Delamore IW (1986) Systemic infection with Aureobasidium pullulans in a leukemic patient. J. Infect. 13:289-291
Kane J & Summerbell RC (1987) Sodium chloride as an aid in identification of Phaeoannellomyces werneckii and other medically important dematiaceous fungi. J. Clin. Microbiol. 25:944-946
Koppans HS, Olsen I, Stuge U & Sandven P (1991) Aureobasidi- um infection of the jaw. J. Oral Pathol. Med. 20:191-195
Marill FG, Liautaud M, Liautaud B, Vodov J & Mariat F (1973) Dermatitie verruqueuse ~t Aureobasidium pullulans (De Ba- ry). Bull. Soc. Fr. Mycol. M6d., N. S6r. 2:73-74
Mowl JL & Gadd GM (1985) Effect of vehicular lead pollution on phylloplane microflora. Trans. Br. Mycol. Soc. 84:685-689
Mushin TM & Booth T (1987) Fungi associated with halophytes of an inland salt marsh, Manitoba, Canada. Can. J. Bot. 65: 1137-1151
Nishimura K & Miyaji M (1984) Hortaea, a new genus to accom- modate Cladosporium werneckii. Jap. J. Med. Mycol. 25:139- 146
Prillinger H, D/Srfler C, Laaser G, Eckerlein B & Lehle L (1990) Ein Beitrag zur Systematik und Entwicklungsbiologie h6he- rer Pilze: Hefe-Typen der Basidiomyceten. Tell I: Schizosac- charomycetales, Protomyces-Typ. Z. Mykol. 56:219.250
Ramaley AW (1992) Tectacervulus mahoniae, Kabatina maho- niae, and Selenophoma mahoniae, three new fungi on Maho- nia repens. Mycotaxon 43:437-452
Ramos S & Garcfa Acha I (1975) A vegetative cycle of Pullularia pullulans. Trans. Br. Mycol. Soc. 64:129-135
Ranta HM (1990) Effect of simulated acid rain on quantity of epiphytic microfungi on Scots pine (Pinus sylvestris L.) nee- dles. Environm. Pollut. 67:349-359
Robak H (1952) Dothichiza pityophila (Cda.) Petr., the pycnidial stage of a mycelium of the type Pullularia pullulans (de B.) Berkhout. Sydowia 6:361-362
Salkin IE Martinez JA & Kemna ME (1986) Opportunistic in- fection of the spleen caused by Aureobasidium pullulans. J. Clin. Microbiol. 23:828-831
Sarrat H, Mathe H, Marchand JP & Faye I (1973) Agents 6tiolo- giques rares de mycoses sous-cutandes. Bull. Soc. Path. Exot. 66:615-620
Sherwood M & Carroll G (1974) Fungal succession on needles and young twigs of old-growth Douglas fir. Mycologia 66: 499- 506
Sigler L (1990) Occurrence of a yeast-like synanamorph in the fungicolous coelomycete Eleutheromyces subulatus. Crypt. Bot. 1:384-389
54
Simon L (1984) Etude ultrastructurale des diffdrentes r6gions de l'hyphe chez Aureobasidium pullulans (De Bary) Arnaud. Crypt. Mycol. 5:323-344
Sivanesan A (1984) The bitunicate Ascomycetes. J. Cramer, Va- duz, 701 pp.
Sivanesan A (1990) CMI Descriptions of Pathogenic Fungi and Bacteria No. 982: Discosphaerina fulvida. Mycopathologia 109:4344
Sl~ivikov~i E, Vadkertiov~i R & Kockov~i-Kratochvflovg A (1992) Yeasts isolated from artificial lake waters. Can. J. Microbiol. 38:1206-1209
Sugai JK & Veiga LA (1988) Induction of the xylitol dehydroge- nase of Pullularia pullulans. Can. J. Microbiol. 34:107-111
Takeo K & De Hoog GS (1991) Karyology and hyphal characters as taxonomic criteria in Ascomycetous black yeasts and relat- ed fungi. Antonie van Leeuwenhoek 60:35-42
Torzilli AP, Vinroot S & West C (1985) Interactive effect of tem- perature and salinity on growth and activity of a salt marsh isolate of Aureobasidium pullulans. Mycologia 77:278-284
Van der Aa HA (1975) The perfect state of Sarcophoma miribe- lii. Persoonia 8:283-289
Van der Walt JP & Yarrow D (1984) Methods for the isolation, maintenance, classification and identification of yeasts. In: Kreger-van Rij NJW (Ed) The Yeasts, a Taxonomic Study, 4th Ed (pp. 45-104) Elsevier, Amsterdam
Vermeil C, Gordeff A, Leroux M-J, Morin O & Bouc M (1971) Blastomycose ch61oidenne ~ Aureobasidium pullulans (De Bary) Arnaud en Bretagne. Mycopath. Mycol. Appl. 43:35-39
Vieira JR (1961) Onicomycose por Aureobasidium pullulans (De By.) Arnaud. Proc. Int. Congr. Trop. Med. Malaria 4: 768- 777
Wynne ES & Gott CL (1956) A proposed revision of the genus Pullularia. J. Gen. Microbiol. 14:512-519
Yamada Y, Sugihara K, Van Eijk GW, Roeijmans HJ & De Hoog GS (1989) Coenzyme Q systems in ascomycetous black yeasts. Antonie van Leeuwenhoek 56:349-356
Yurlova NA & Simatskaya IA (1993) Cell wall chemical compo- sition and structure of genus Aureobasidium. Mikol. Fitopa- tol. 27 (in press)
