MYCOSES 41, 183-189 (1998)
OCTOBER
Molecular diagnostics of clinical strains of filamentous
Basidiomycetes
Molekulare Diagnostik klinischer Stamme filamentoser
Basidiomyzeten
G. S. de Hoog and A. H. G. Gerrits van den Ende
Key words. Basidiomycetes, ribosomal DNA, small subunit (18s)rDNA, variable domain 9, internal transcribed spacer,
diagnostics, restriction fragment length polymorphism.
Schliisselworter.Basidiomyzeten, ribosomale DNA, small subunit (18s)rDNA, Variable Domane 9, internal transcribed
1 spacer, Diagnostik, RFLP.
Summary. Eleven clinical and veterinary strains
of filamentous Basidiomycetes were compared with
15 reference strains representing the orders
Aphyllophorales and Agaricales. The methods
used were restriction analysis of small subunit
(18s)(SSU)rDNA and internal transcribed spacers
(ITS) 1 and 2 and variable domain 9 (VS)-ITSl
sequencing. Six strains were found to belong to
the teleomorph genera Schizophyllum or Coprinus,
whereas five could not be identified unequivocally.
A rapid diagnostic overview is obtained with HaeIII
and HinfI digestion of the ITS region.
Zusammenfassung. Elf klinische und veteri-
nare Stamme filamentoser Basidiomyzeten wurden
untereinander und mit funfzehn Referenzstammen
der Ordnungen Aphyllophorales und Agaricales
verglichen. Angewandt wurden Restriktionsanaly-
sen der SSU rDNA und der ITS 1 und 2 und
Sequenzierung von Region V9-ITS 1. Sechs
Stamme wurden als Reprasentanten der teleomor-
phen Gattungen Schizophyllum oder Coprinus identi-
fiziert; die ubrigen fiinf konnten nicht mit
Sicherheit bestimmt werden. Eine rasche diagno-
stische Ubersicht wurde durch HaeIII und HinfI
Verdauung der ITS Region erreicht.
Centraalbureau voor Schimmelcultures CBS, Baarn, The
Netherlands
Correspondence: Prof. D r G.S. de Hoog, Centraalbureau
voor Schimmelcultures, PO Box 273, NL-3740 AG Baarn,
The Netherlands.
24, 1997
ACCEPTED:
Abbreviations: SSU, small subunit (18s);
ribosomal DNA (rDNA); V9, SSU variable
domain 9; ITS, internal transcribed spacer; RFLP,
restriction fragment length polymorphism.
Introduction
Of the fungi with clinical significance, only rela-
tively few are of basidiomycetous affinity. Among
these the heterobasidiomycetous yeasts are by far
the most important: species of CgJptococcus,
Malassezia and Trichosporon are among the classical
pathogens, or opportunists, known since the pre-
vious century [ 11. Homobasidiomycetes in culture
generally produce a whitish, flu@, filamentous
mycelium rather than yeast cells, often with more
or less differentiated arthroconidia. In the natural
environment (wood, soil, compost, etc.) they are
recognized by macroscopically visible fruit bodies.
Clinical strains identified thus far belong to two
orders of the Homobasidiomycetes, namely the
Aphyllophorales and the Agaricales.
Until recently, only a few cases had been pub-
lished in which a member of these orders was the
aetiological agent [2]. Most of them belonged to
the genera Schizophyllum (Aphyllophorales) or
Coprinus (Agaricales). A significant increase in the
number of cases has been noted during the past 5
years. Possibly, this is due to a growing awareness
that such hyphal thalli may indeed represent
agents of mycoses. Their mycelia may be rather
undiagnostic as they are morphologically similar
over larger taxonomic groups, and often lack key
features such as clamp connections and conidia.
184 G. S. DE HOOG& A. H. G. GERRITS
VAN DEN
For the production of fruit bodies, and for the
demonstration of septa1 ultrastructure, special
methods are required [3, 41 that are not routinely
availablc in clinical laboratories.
In the present article, the ribosomal gene of a
number of recent clinical isolates that were sus-
pected or proven to be of basidiomycetous affinity
is compared with reference strains. O n the basis
of partial small subunit (18s) (SSU) and internal
transcribed spacer (ITS) 1 sequencing and of
restriction analysis of SSU and ITS1 -2 amplicons,
a diagiio>ticsystem applicable in the clinical labor-
atorv is developed.
Materials and methods
D"I2Pxtrartion
The strains studied are listed in Table 1.
Approximately 1 cm' of mycelium of 30-day-old
cultures was transferred to a 2.0-ml Eppendorf
tube containing 300 pl of CTAB (cetyltrimethyl-
ammoniumbromide) buffer and about 80 mg of a
silica mixture (Silica Gel H, Merck 7736/Kieselgur
Celite 545, Machery, 2 : 1, w/w). Cells were dis-
rupted mechanically with a tight-fit sterile pestle
for z 1 min. Subsequently 200 pl of CTAB buffer
was added, vortexed and incubated for 10 rnin at
65 'C. After the addition of 500 p1 of chloroform,
the solution was centrifuged for 5 min at
14 000 r.p.m., and the supernatant transferred to
a new tube with 2 vols of cold 96% ethanol. DNA
was allowed to precipitate for 30 min at -20 "C
and then centrifuged again for 5 min at
14 000 r.p.m. Subsequently, the pellet was washed
with cold 70% ethanol. After drying at room
temperature, it was resuspended in 97.5 1-11 of T E
buffer plus 2.5 p1 of 20 U ml-' RNAse and incu-
bated for 5 rnin at 37 "C.
PCR amplification
Fragments of rDNA were amplified using the
primer pairs (for RFLP) NS1-NS24 (SSU) plus
ITS 1-ITS4 [5] and (for sequencing) NS 1-ITS4
in a reaction mixture containing 30 pl of distilled
water, 5 pl of PCR buffer, 10 pl of N buffer, 1 p1
of each primer (50 pmol), 1 pl of Ampliterm DNA
polymerase and 2 pl of fungal DNA. Forty cycles
were performed in a Bio-med 60 thermocycler:
94 'C for 60 s (delay); 94 "C for 60 s (denatur-
ation): 58 "C for 60 s (annealing); 72 "C for 120 s
(extension), with a final extension at 72 "C for 60 s.
ENDE
Restriction anahsis
The PCR products were digested with the follow-
ing enzymes: Hinyl, HaeIII, RsaI and DdeI, accord-
ing to the suppliers' instructions. Fragments were
electrophoresed in 1.5% agarose gels, in TBE
buffer (Tris-Borate-EDTA) at 3-6 V cm-' for 3 h
and stained with ethidium bromide. Biozym Low
Ladder was used as the marker. The gels were
analysed and photographed using the Image
Master System (Pharmacia). The lengths of the
resulting bands were measured using VDS
software version 2.0 and the data were transferred
to a Quattro Pro 5.0 matrix. Molecular weights
were compared with those present in a database
available at CBS.
Sequencing
The remaining primers and dNTPs were removed
with Microspin S-300 H R (Pharmacia). The
sequencing primers used were V9G (5'-TTA-
C GTC C CTGC C CTTTGTA-3') and 5.8G
(5'-AATGTGCGTTCAAAGATTCG-3'), span-
ning the V9 variable domain of the SSU rDNA
until the central region of the 5.8s rDiVA.
Sequencing was performed using a primer termin-
ating protocol in a Perkin-Elmer 9600 automatic
sequencer, using 25 PCR cycles as follows: 96 "C
for 10 s (denaturation), 50 "C for 5 s (annealing),
60 "C for 4 rnin (extension).
Sequence u l ~ n m e and
~ t tree construction
The sequences obtained with the two primers were
aligned and adjusted using the Align and DCSE
package [6]. Trees were calculated using the neigh-
bour joining method of the Treecon program [ 71.
Results
The SSU amplicon of the majority of the strains
had a length of about 1800 bp, as expected
(Table 1). In two strains of Schizophyllurn c o m ~ u n e
the amplicons were about 2 100 bp in length, indi-
cating the presence of an intron-like element.
Despite the insertion in the 18s ribosomal gene,
the three strains of this species showed identical
patterns when ITS amplicons were digested with
HaeIII or HinzI (Table 1). This indicates that these
strains are closely related and that the 18s gene
outside the insertion is likely to be identical or
nearly identical.
Restriction patterns of 1800-bp SSU rDNA
were identical or very similar in all strains analysed
when RsaI or DdeI was used (Table 1). With HinzI
four patterns were generated, and seven were
mycoses 41, 183-189 (1998)
rains examined with their SSU and I T S restriction patterns

Number Source NS1-NS24 HaeIII Hinz RsaI DdeI ITSlLITS4 HmIII Hi@ ha1 Ddel
~~

ommune CBS 405.96 2100 A A A 600 A A A A

ommune CBS 333.85 2100 B B B 600 A A B B
ommune CBS 340.81 1800 C C C 600 A A B A
c c
s FMR 5149 Air, landfill 1800 D C C 650 B B
s FMR 5150 Leaves, landfill 1800 D C C 650 B B c c
mpergilhta CBS 519.91(T) Skin ND 650 B B C C
mpergdkzta FMR 5148 Air, landfill 1800 C D C C 650 B B c c
aspmgilkzta FMR 5151 Air, landfill 1800 C D C C 650 B B C C
mpergilluta FMR 5153 Soil, landfill 1800 C D C C 650 B B C C
uerticilhta CBS 509.94 Nervous tissue 1800 C D C C 650 B B c c
aspergilhta CBS 106.97 Lung 1800 C D C C 650 B B c c
mpergilhta CBS 833.96 Lung [ 121 1800 C D C C 650 B B C C
s CBS 258.96 Sputum [ 131 1800 D D C C 650 C
candehbratn CBS 517.9(T) Dung 1800 D D C C 650 H
uerticillata FMR 3936(T) Spinal catheter 1800 E D C C 650 H
uerticillata FMR 5154 Soil, landfill 1800 E D C C 650 H
uerticilata FMR 5155 Air, landfill 1800 E D C C 650 H
uerticillata FMR 5156 Air, landfill 1800 E D C C 650 H
mycete CBS 237.96 E Y ~~ 4 1 1800 F E C C 630 E
CBS 800.95 Dolphin I800 C E D C 600 F
rysosporium CBS 129.27(T) soil 1800 G E C C 600 F
rysosporium CBS 255.65 soil 1800 G E C C 600 F
rysosporium CBS 363.65 soil 1800 G E C C 600 F
dimorphosporum CBS 419.70(T) Potato 1800 H E D C 650 G
ustn CBS 230.93 Eucalyptus 1800 F ? C? ? 600 D
species CBS 257.96 Liquor 1800 F D? C? C? 600 D

lbureau voor Schimmclcultures,Baarn; FMR,Facultad de Medicina, Rcus; T , type strain.

186 C . S. DE HOOG& A. H. G. GERRITS
VAN DEN
qenerated with HueIII. ITS restriction patterns
were particularly variable with HueIII and H i n f I ,
both generating seven different patterns.
In main traits, RFLP of 18s and ITS allows the
distinction of five groups, each containing more
than one strain (Table 1). Group 1 contains the
three strains identified as S. commune. Among these
are two clinical strains and one environmental
strain (Table 1). Some heterogeneity is found in
I T S restriction patterns. Group 2, including four
clinical strains, contains Coprinus cinereus and its
anamorph Hormogruphiellu aspergillatu plus one strain
identified as Hormogruphiella uerticillatu,which appar-
ently is a misidentification. The core of H. verti-
czllutu is found in group 3, which contains a single
clinical strain but as yet no teleomorph. Group 4
contains three strains of Phunerochuete chrysosporium
but no clinical strain. Digestion patterns of group
5, containing Bjerkundera adusta and a clinical strain,
were interpreted with difficulty as the length
differences between fragments were minute.
Five strains could not be unambiguously attri-
buted to any of the groups above. Among these
was Dzsporotrzchum dimorphosporum, which could not
be assigned to any of the remaining reference
teleomorphs. None of the clinical strains appeared
similar to this species. The type strain of
Honnogruphiellu cundelubrutu was found to resemble
group 3 but showed different patterns with HinfI
and DdeI. Three clinical strains could not be
identified with certainty. CBS 258.96 from sputum
was found to be intermediate between H. uspergil-
Lutu and H. uerticillatu. CBS 237.96 had three 18s
RFLP patterns in common with P. chrysosporium
but had clearly different ITS restriction profiles.
A strain from dolphin could not be identified
either.
A distance tree of the moderately variable V9
domain of SSU rDNA plus the partial 5.8s rDNA
gene is presented in Fig. 2, and a tree of the highly
variable ITSl spacer is presented in Fig. 3, both
based on the sequence alignment presented in
Fig. 1. The V9 domain as well as the SSU terminus
and the 5.8s gene could be aligned with confi-
dence. A tree using 199 SSU and 108 5.8s pos-
itions of all strains analysed is presented in Fig. 2.
The RYLP groups 2 and 3 cannot be clearly
separated from each other. The two sequenced
S. commune strains (group 1) are found together but
at a rather large distance. The three strains of
P. chrysosporium (group 4) are found to be very
similar in the 18s distance tree (Fig. 2) and show
\Tery little variation in ITSl sequences (Fig. 1). D.
dimorphosporum and B. adustu are found at rather
isolated positions (Fig. 2). The clinical strains that
showed ambiguous results with RFLP could not
be identified with any of the reference strains used.
ENDE
A finer resolution of the Coprinus-Hormogruphiellu
complex is achieved when the positions 189-455,
spanning the ITSl spacer, are taken into account
(Fig. 3); the remaining species could not be aligned
with sufficient confidence. There is a marked
distinction between C. cinereus-Hormogruphiella usper-
gillutu (RFLP group 2)) clearly representing a single
taxon, and the remaining strains (RFLP group
3). The latter group is significantly more
heterogeneous.
Discussion
In recent years it has become apparent that higher,
filamentous Basidiomycetes occasionally do occur
as causative agents of human and animal mycoses.
Teleomorph connections of clinical fungi have
been proven by a demonstration of fruit bodies
[4] and septa1 ultrastructure [3, 81. Aetiological
agents identified thus far belong to different orders
of the class Basidiomycetes: Aphyllophorales
(Phanerochuete, Schizophyllum) and Agaricales
(Coprinus). These taxonomic groups are broadly
recognized in the grouping made on the basis of
RFLP patterns: group 1 corresponds to the
Schizophyllum relationship (Aphyllophorales),
groups 2 and 3 to the Agaricales, and groups 4
and 5 to the Aphyllophorales. B. adustu and the
clinical strain CBS 257.96 were found at a larger
distance from all groups, including the aphyllopho-
ralean species P. chlysosporium. With the remaining
species, despite the fact that the taxonomic dis-
tances between groups are currently recognized as
being at the ordinal level, SSU RFLP patterns
generated using RsaI and DdeI were found identical
or similar in all groups except B. adustu. Patterns
generated with HznfI were found to differ accord-
ing to the taxonomic borderlines of the orders and
this enzyme is therefore a useful indicator of main
relationships.
Of the 11 clinical strains analysed, six were
attributed to known species: S. commune, C. cinereus
or H. uerticillata. Strains CBS 258.96 and 517.91
(type strain of H. cundelabrutu) were similar to
C. cinereus and thus might represent another
Coprinus species. Similarly, CBS 257.96 might rep-
resent another Bjerkandera species. The strains CBS
237.96 and 800.95 seem too remote to be associ-
ated with any of the reference taxa. None of the
clinical strains came close to D. dimorphosporum.
The number of Basidiomycetes involved in human
or animal mycoses is apparently larger than the
ones mentioned in the literature [9], but: on the
other hand, they are not randomly distributed
over the orders of Basidiomycetes. Some relatively
limited groups seem to possess more virulence
mycoses 41, 183-189 (1998)
 MOLECULAR
DIAGNOSTICS OF BASIDIOMYCETES187

1634 v9

I I . ;O .I
20
I.. ! . . I .
30
1
40
..I ....
CCGCCCGTCG GTAAGAN-CC N-ACTWATA AGCTTAGTGA -GGTCTTCGG ATCGGCTTTG GGGAGCCGGC AACGGCACC-
1 .
50
L l .
60
I ..{..I
70
..I..
80
1.. 1
TCATTGCTGA
. I
90

1 CBS 3 4 0 . 8 1 Schizophyllum comune

. .: . ..A:-.. - - T.G-.AT 0.. . . . . . - . . . . . . C . . . . . . . . . . . . . . . . . . :. . . . . . . 2 CBS 405.96 Schizophyllum commune
T . T C .GT G-.T.C-.A G..... . . G..G .C. . . . . . . . . . . . . . . . C :
G
. ..... 3 CBS 230.93 Bjerkandera adusts
. . . . . C.NC:GG --.T.G-.AT G . . . . . . . . . A.... C.. ..T ..GG.. . . . . . . . . . . . . . . . . . C :... .. 4 CBS 258.96 copprinus species
. . . . . . C ..T.--. G-.T &.AT 0. . . . . . . . . - . . .C... . .T.... . . . . . . . . . . . . . . . . . . . . C . 5 FMR 5150 coprinus cinereus
... C .CT G G . . ----.GT.ATG - . . . . . . . - . . . . .C... ..T...GG.. . . . . . . . . . . . . . . . . . .C . -. . . . . . . . 6 FMR 3936 Homogrsphiella vercicilleta
. . . . .T. . . . . G-.T.G-.AT 0.A . . . . . . . C... . .T.... . . . . . . . . . . . . . . . . . . . . . C : ........ 7 CBS 509.94 Hormogrephiella verticillata
. . . . . . . . . . .T - - . G-.T.G-.AT G.A . . . . . . . G..G. . .T . . . . .C . . . . . . . . . . . . . . . . . . . . C . -G . . . . . . . 8 CBS 257.96 Homographiella species
. . . . . . . . . . G-.T.G-.AT G.A.. . . . . C... . .T . . . . . . . . . . . . . . . . . . . . . . . C . - ........ 9 CBS 519.91 Homographidla a s p e r g i l l a t a
. . . . . .T. . ..T.--.. G-.T.G-.AT G . . . . . . . . - . . . .C... . T.... . . . . . . . . . . . . . . . . . . . . C .. 10 CBS 106.97 Homographiells s s p e r g i l l a t a
.......... G-.T.G-.AT 0.A . . . . . . . C... . .T...... . . . . . . . . . . . . . . . . . . C . -. . . . . . . . 11 CBS 833.96 Homographidle a s p e r g i l l a t e
............ G-.T.G-T.T 0.A . . . . . . . - . . . . .C.. ..T. .GG.. . . . . . . . . . . . . . . . . C . -. . . . . . . . 12 CBS 517.91 Homographidla csndelabreta
. . . . . . . . C .TT.--..GC-T.G-.AT 0 . . . . . . . . . A...G.. . .T . . . . .C. . . . . . . . . . . . . . . . ..C .-G..... . 13 CBS 237.96 Holobasidiomycetes
. . . . . . . C..CT.--.. -C.T.G-.AT G . . . . . . . . . G..G.. . .T.... C . . . . . . . . . . . . . . . . . . .C . -G . . . . . . . 14 CBS 800.95 Basidiomycetes
. . . . . . . . . . C..CT.--.. GC-T.G-.AT G.A . . . . . . . - . . C ..G.. . .T . . . . .C. . . . . . . . . . . . . . . . . . . C : G....... 15 CBS 255.65 Phanerochaece chrysosporium
......... T.C---GG.. GG.T.G-.AT G..... ... -..C...G.. ..T. . . .C. . . . . . . . . . . . . . . . . . C -G.. .... 16 CBS 363.65 Phanerochaete chrysosporium
. . . . . .T. G-.T.G-.AT 0.0 . . . . . . . G.. . .T . . . . .C. . . . . . . . . . . . . . . C . 17 CBS 129.27 Pheneroehaete chrysosporium
. . . . . . .T. . . . .

100
G-.T.G-.AT G.A . . . . . . . - . . . . . . . . . . .T . . . . .C. . . . . . . . . . . . . . . . . . . .C

110 120 130 140 150 160 170

-
. -G . . . . . . .

180
18 CBS 419.70 Disporotrichum dimorphosporum

.. I....) ..../....I ....I.... I ....)....I . . . . I . . . . ) . . . . I _ . . .1 . . . . ) . . . . 1 . _ . _ / . _I .. _ . _ I . . . . I

GAAGTTGATC FAACTTGGTC ATTTAGAGGA A G T W T C GTAACAAGGT TTCCGTAGGT GAACCTGCGG AAGGATCATT AACGAATCAA 1 CBS 340.81 Schizophyllum commune
. . . . . . . . . . . .A , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 CBS 405.96 Schizophyllum commune
. . .C..C ...................................................................... .T...G.TT- 3 CBS 230.93 Bjerkandera adusta
. . C..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T....A.C 4 CBS 258.96 Coprinus species
. . .C..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T T . . . . A.. 5 FMR 5150 Coprinus cinereus
. .c..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 FMR 3936 Bornogrephiella v e r t i c i l l a t a
. . . .c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .TT....A.. 7 CBS 509.94 Xomographiella v e r t i c i l l a t a
. .T..C... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .T...G.TTT 8 CBS 257.96 Homographiella species
. . . .c . . . ........................................ ......................... . T T . . . . A,. 9 CBS 519.91 Homographiella aspergillaca
. . . . C..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T T . . . . A.. 10 CBS 106.97 Homographidla a s p e r g i l l e t a
. . . .C..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .TT....A.. 11 CBS 833.96 Homographidle aspergillata
. . . .C..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A.C 12 CBS 517.91 Homographiella csndelabrata
A..C...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G
.. 13 CBS 237.96 Holobasidiomycetes
. . .C . . C . . . ..................................................................... .T...G.T-- 14 CBS 800.35 Basidianycetes
... C...G.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G.A: 15 CBS 255.65 Phaneroehaete chrysosporium
. .C...G.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G.A: 16 CBS 363.65 Phaneroehaete chrysosporium
. . .C...G.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G.A: 17 CBS 129.27 Phanerochaete chrysosporium
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G.A . 18 CBS 419.70 Disporotrichum dimaiphosporum

ITS1

190 200 210 220 230 240 250 260 270

.. 1,. .I ....I
ACAAGTTCAT CTTG-TTCTG ATCCTGTGCA CCTT-
......................
--T--GAAC--GG -..G.C -.G . GCTC G.AAGGG--C . . . . . GCA.0 .CTGT-.TCA TCCACT.TCA AC..C-- GT .CACTTT..
TTT-.G--CG T-G.-..G: G: . GCTC
T.T--GA-CG T. ..... G.C ..G...GCTC
TATG..GTC. .AG.-..G.- .. G...GCTC
T.T--GA-CG T-G -..G.C ..G...GCTC
.....GAACG --G.-..G.C .. G...GCTC
T.T--GA-CG T-G.-. G.C ..G...GCTC
~ ~~
T T--GA-CG T-G.-..G.C ..G...GCTC
T.T--GAL-CGT-0.-..G.C ..G...GCTC
TATG. GT-- T-G.-..G: .. G...CCTC
TTT--GAA.G G-G.T..G.- . .G...GCCT
TTTG . . .G - - --G:C.G.C ..G...GCC-
-.T--GAACG - - G .. G...GCCT
-.T--GAAC- -AG.-T.G: .. G...GCCT
-.T--GAAC- -AG.-T.G: .. G...GCCT
. .G.GGAG- -----T.G.-.. G...GCC-
. I . . 1 , . . I ... 1 I .I ...I . . . I . . . / . . .I . . . . / ... I . . . 1 . . . I .I
- -ATGTAGTCT CTAAAGCCTT CATGGGCGGC GGTTGACTAC GTCTACCICA 1 CBS 340.81 Schizophyllum commune
........ . . . . c . . . . . . . . . c......................... 2 CBS 405.96 Schizophyllum comime
3 CBS 230.93 Bjerkandera adusta
TT-A-GGAGC .... GCA.A .CCGT-.A.C TT.ATCTTT. C A C C - - - GT .CAC..TATG 4 CBS 258.96 coprinus species
CCCWGGAGC ..... GCA.0 .CCGT-.ACC TT.ATTTff. CACC---.GT .CAC..ACTG 5 FMR 5150 coprinus cinereus
.TC--GGAGC A....GCA.G .CCGC-.A.T TT.ATCTTT. CACC---.GT .CACCGACTG 6 FMR 3936 Homographidla v e r t i c i l l a t a
..CCGGGAGC . . . . . GCA.0 .CCGT-.ACC TT.ATTTCT. CACC--- GT .CAC .ACTG 7 CBS 509.94 Homographidla v e r c i c i l l a t a
G.AAGG&-C ..... GCA.G .CTGT-.TCA TCCACT.T-. AAC-TT. GT .CACTTT... 8 CBS 257.96 Bornogrephiella species
..CCGGGAGC . . . . . GCA.G .CCGT-.ACC TT.ATTTCT. CACC---.GT .CAC..ACTG 9 CBS 519.91 Homographidla aspergillata
..CCGGGAGC ..... GCA.G .CCGT-.ACC TT.ATTTCT. CACC---.GT .CAC..ACTG 10 CBS 106.97 Homographidla a s p e r g i l l a t a
..CCGGGAGC ..... GCA.G .CCGT-.ACC TT.ATTTCT CACC---.GT .CACACACTG 11 CBS 833.96 Homographidla aspergillata
.TC--GGAGG A....GCA.G .CCGC-.A.. TT.ATCTTT. CACC---.GT .CACCGACTG 12 CBS 517.91 Homographidla candelebrata
T.C---GAGG C....GCT.G CCTG-.TCA TCCATCTA-. -ACC---.GT .AACTTT.TG 13 CBS 237.96 Holobasidiomycetes
G.AA-GG--C ..... GCA.G .CTGG-.TCA TCCACT-T. AAC-TT. GT .CACTATCGC 14 CBS 800.95 Basidiomycetes
TTCGGGG--C ..... 0CA.A .CTG&.TCA TCCACT.R. AACC-T..GT CACTTG.TG 15 CBS 255.65 Phaneroehaete chrysosporium
TTCGGGG--C ..... 0CA.A .CTGG-.TCA TCCACT.TT. AACC-T..GT .CACTTG.TG 16 CBS 363.65 Phanerochaete chrysosporium
TTCGGGG--C . . . . . 0CA.A .CTGG-.TCA TCCACT.TT. AACC-T..GT .CACTTG.TG 17 CBS 129.27 Phanerochaete chrysosporium
G.AF-GG--C..... GCA.G ..CCC-TAC. ..ATCC.AC. TTACAC..GT .CA.CTA TG 18 CBS 419.70 Disporotrichum dimorphosponuo

280 290 300 310 320 330 340 350 360

1 ....1.... / ....I.... I ....[....I ..../ . . . . I
G AATGTAATCA TGGTCTTGAC AGACCCTAAA AAGTTAA--- 1 CBS340.81 Schizophyllum c o m n e
......................................................... ................................. 2 CBS 405.96 Schirophyllum comune
T.GGCC-GGC .TGTGGGTGC GT.CGCGCAC .T..AGGTGT C.GG.T..TG CTT.ATTACA AACGATTCAG TTTTAGAATG TCATACTTTG 3 CBS 230.93 Bjerkandera edusts
T.GA.CTGGA ..AC.CTCGC CC.TCTGAGC GGPAACAAGG .TTG.TGCGT CGCAAGGCCA GCTCTCTTTG AATTTCCAGG TCTATGTCAT 4 CBS 258.96 Coprinus species
T.GGCCTGGA ..CC.CTCGT CGCAAGGCGG ATGCG.GGCT T.CTGTCGCT T T C W . G A A GGCCGGCTTG CCATPAATTT CCAGGTCTAT 5 FMR 5150 coprinvs cinereus
T.GG.CTGGA ..CC.CCCGC CGCAAGGCGG ATGCGAGGGT T.CT.G . . . . GGGC.CCCCT CGAACTTCCA GGCTCTACGT CTTTTTACAC 6 fMR 3936 Homographidla vercicillata
..................................
T Psr-rTCCB CC CTPGT CGCPAGGCGG ATGCG.GGCT T.CTGTCGCT TTCG.A.GAA ~~ GGCCGGCTTG CCATAAATTT CCAGGTCTAT 7 CBS 509.94 Bornogrephiella v e r t i e i l l a t e
T,GGCC-^&C .TGTGGGTGZ GT.CGCGCAC .T ACGTGT C.GG.T..TG CTT.ACTACA AACGATTCAC TTTTAGMTG ICATACTRG 8 CBS 257.96 Bormographiella species
1 GGCCTISGA CC.CTCGT CGZAAXCGG ATGCG.GGCT T.CTGTCGCT TTCGAA.GAA GGCCGGCTTG CCATAAATTT CCAGGXTAT 9 CBS 519.91 Homographidla aspergillata
1,GGCCTGGA ..CC.CTCGT CGCARGGCISG ATGCG GGCT T.CTGTCGCC TTCGAA.GAA GGCCGGCPTG CCATAAATTT CCAGGTCTAT 10 CBS 106.97 Homographidla a s p e r g i l l e t a
T.GGCCTGGA ..CC.CTCGT CGCAAGGCGG ATGCG.GGCT T.CTGTCGCT TTCGAA.GAA GGCCGGCTIG CCATAAATTT CCAGGTCTAT 11 CBS 833.96 Homographidla e s p e r g i l l a t a
T.GG.CTGGA ..AC,CTCGC CTCACGGCGG ATGCGAGG.T T.G---CGTT CGCG.CCTIC CTC----GAA CPTCCAGGCT CTACGTC-TT 12 CBS 517.91 Homographidla csndelabreta
T.G..CGGGA .G.A.T-GGA GG.CAGTGAT GGCCGACAGT GT.GT.CGTT CTACGATCTT TACACACACA CTTTAAAAGT TTAGAATGTA 13 CBS 237.96 Aolobasidiomycetes
............
&TC.T&lCXTT GGCT.TGTGA
.......... CGGAGGCTGG CPTCGCGGC. G.T..TCGR GTA.CGCTGC CTTGCGTGTT TCTTACTAUL AACGCTTCAG 14 CBS 800.95 Basidiomycetes
.
~ ~~~

T . GG .CGGTA GAA.AGCGA. C. .T.. . .GC TGCT.GG .A GCCTT’.CT.TGTT.. .CTAC AAACGClTCA GTTTAAGAAT GTTTACCTGC 15 CBS 255.65 Phanerochaete chrysosporium
T.GG.CGGTA G.A.AGCGA. c. .c. . . .GC .TGCT.GG.A GCCTT. CT .T GTT.. .CTAC AAACGCTTCA GTTTAAGAAT GTTTACCTGC 16 CBS 363.65 Phaneroehaete chrysosporium
T.GG.CGGTA G.A.AGCGA. C..T....GC .TGCT.GG.A GCCTT.CT.1 GTTA..CTAC AAACGCTTCA GTTTAAGAAT GTTTACCTGC 17 CBSl 29.27 Phanerochaete chrysosporium
T.GG.CGGGT GGCT.GGCTC C....C.GGC .GTC...CCT .TGT.TAC.C .TACACTTTA TAGTTTCAGA ATGTAAACTG CGTATA- 18 CBS 419.70 Disporotrichum dimorphosporum

Figure 1. Alignment of partial SSU rDNA and ITSl sequences starting at position 1634 of the 5’ end with reference to S.cerevisiae. Schizophyllum
commune, CBS 340.81 is used as leading strand. Dots indicate nucleotides identical to this reference; dashes represent deletions necessary for
alignment. The upper lines mark the positions of the variable domain V9 of SSU rDNA, the ITSl spacer and the 5.8s rDNA gene.

factors than the remaining thousands of culturable are distinguishable with HinfI digestion, only
species of filamentous Basidiomycetes. Bjerkunderu being found as a fourth group (Table 1).
RFLP of the complete, PCR-amplified SSU ~ ~ ~ p o r is o ~an~as~ yet
c ~unclassified
~ m anamorph
ribosomal gene provides a fairly confident insight [lo]. HueIII generates more patterns, leading to
into the relationships of the fungi under study. differences within the orders. Earlier RFLP studies
The three groups, namely Schiz@hyllum (Aphyllo- [ 111 have shown that also in hyphomycetes of
phorales), Agaricales and other Aphyllophorales ascomycetous affinity HueIII has restriction sites in

mycoses 41, 183-189 (1998)

188 G. S. DE HOOG& A. H. G. GERRITS
VAN DEN ENDE

ITSl 5.8s

_I 383 350 400 410 420 430 440 450

I . 1 .’ 1 1 . I . . . : . .
1 1 .I.. 1
........................................................... --TACAACTTTCGACAACGG 1 CBS 340.81 Schizophyllum commune
............................................................... . . . . 2 CBS 4 0 5 . 9 6 Schizophyllum comune
CTATmCCCA ATTTATA--. ..................................................... AG... . 3 CBS 230.93 Bjerksndera adusta
TTACAAACCZ CAATTGAATG ATGCAGAATG TCGTCAATGG GCTCTAA--- ----GCCTATAAAACA---AA A . . . . . . AG . . . . 4 CBS 258.96 c a p r i m s species
GATTTCTTAC ACACCCCAAA CTGAATGTTA TGGAATGTCA TCTCAAGGCC TTGTGCCTAT AAA-C----CTA . . . . . . .AG . . . . . . 5 FMR 5150 c o p r i n u s cinereus
ACCCCAATAG TATGATGCAG AATGTAGTCA ATGGGCTTCA C A
.... ----GCCTATAAAACA---C TA. . . . . . ..AG.. . . 6 FMR 3936 Xormographiella v e r t i c i l l a t a
GATTTCTTAC ACACCCCAAA CTGAATGTTA TGGAATGTCA TCTCAAGGCC TTGTGCCTAT AAA-C----CTA . . . . . . . . . .AG . . . . . . 1 CBS 509.94 Homographidla verticillata
CTATPIpIC........................................... --..GCAATT TA........ TA. . . . . . . . AG . . . . . 8 CBS 257.96 Xormographiella species
GATTTCTTAC A C A C C C W CTGAATGTTA TGGAATGTCA TCTCAAGGCC TTGTGCCTAT M - C - - - - CTA . . . . . . . . . .AG . . . . . 9 CBS 519.91 Hormographiella a s p e r g i l l a t a
GATTTCTTAC ACACCCCAAA CTGAATGTTA TGGAATGTCA TCTCAAGGCC TTGTGCCTAT AAA-C----CTA . . . . . . . .AG . . . . . ia CBS 106.97 Homographiella a s p e r g i l l a t a
GATTTCTTAC ACACCCCAM. CTGAATGTTA TGGAATGTCA TCTCAAGGCC TTGTGCCTAT AAA-C----CTA . . . . . . . ..AG.. . . 11 CBS 833.96 Hormographiella a s p e r g i l l a t a
TACAWLCCC AATAGTATGA TATA------ GAATGTAGTC AATGGGCTTC TTA-GCCTAT AAAACA---C TA . . . . . . . . .TAG..... 12 CBS 511.91 Hormogrephiells c a n d e l a b r a t a
TTCTTGCGAT T T T W C G . .......... ...CATcTAI\ TA ................................ . . . . . . ..A.. . . . . . 13 CBS 231.96 Holobasidiomycetes
TTATAGAATG TCTTTATTTG CGGATAACGC AACTATA--- T A
.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AG . . . . . . 14 CBS 8ao.95 Baaidiomycetes
G ................... --..TATARC GCATTT................................. A TA . . . . . . . . . .AG . . . . . . 15 CBS 255.65 Phanerochaete chrysosporium
G ....................... TATAAC GCATTT----............................. A TA . . . . . . . . . .AG . . . . . . 16 CBS 363.65 Phanerochaete chrysosporium
G.. ..................... TATAAC GCATTT.--. ............................. A TA . . . . . . . . . AG.... . 17 CBSl 29.21 Phanerochaete chrysosporium
..................... AC G ......................... CATC TAT- . . . . . . . . . . . . . . . . . . . . . . . . 18 CBS 419.70 Disporotrichum dimorphosporum

5.8s

460 470 480 490 500 510 520 530 540

, :, ,...I.. . I , . . . / . . . ./ . . . . / . . _ _./. . , / . . . . I .._./._..I . ..I. . . / . . . ./ . . . . I . . . .I . . , . l
ATCTCTTGGC TCTCGCATC- GATGAA-GAA CG-CAGCGAA ATGC-GATAA GTAATG-TGA ATTGCACAA- TTCCAGT-G- AATULTC- 1 CBS 340.81 Schizophyllum commune
...... c.. . CNNCGC.. . . . .c.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . N . . . . .N.C 2 CBS 405.96 Schizophyllum commune
. . . . . . . . . . . . . . .C-ACC.T.. . . . .-.TA..A.TTG.T.. . . . . . . .C.A .N.. . . . . . . . . CA.- 3 CBS 230.93 Bjerkandera e d u s t a
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -.:.A . . ... ... ... . . . . . . 4 CBS 258.96 c o p r i n u s species
. . . . . . . . . . . . . . . . . . . . . . . . . T. . . . . . . . . . .A,: . . .. . .. . .. . . . . . 5 PMR 5150 coprinus cinereus
. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G.: ......... A . . . . . . C. 6 FMR 3936 Homographidla verticillata
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G..- . . . . . . .:T . . . . . . . . 7 CBS 509.94 Hormographiella v e r t i c i l l a t s
. . . . . . . . . . .. . . . . . . -CNG.... . . . . . . . . . . . . . . . . . . . . . . .G..A . . ... ... . .. . . . . . c. 8 CBS 251.96 Hormographiella species
. . . . . . .c . . . . . .A , .. . . . . . . . T. . . . . . . . . . . . . . . . . . . . . . .:T ...... 9 CBS 519.91 Hormographiella a s p e r g i l l a t a
. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .:T . . . . . . C 10 CBS 106.97 Hormographiella a s p e r g i l l e t a
. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .N.: . . . . . . .:T . . . . . . .C 11 CBS 833.96 Hormographielle a s p e r g i l l a t a
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. : . . . . .G..- . . . . . .. . .. . . . . . . 12 CBS 511.91 Homogrephielle candelabrata
. . . . . ....... . . . . . . ......... ......... . . . ... ... ... . . . . . c. 13 CBS 231.96 Holobasidiomycetes
. . . . .c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T: . . . . . .NC 14 CBS 800.95 Basidiomycetes
. . . . . . .. . . . . . . . . . . . . . . T. . . . . . . . . . . . . .G..- . . .. . .. . .. . . . . . 15 CBS 255.65 Phanerochaete chrysosporium
. . . . . . .. . . . . . . . . ......... ......... ........ .................. 16 CBS 363.65 Phanerochaete chrysosporium
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .G.: . . ... ... ... . . . . . . 17 CBS 129.27 Phanerocheete chrysosporium
. . . . . . . . . . .c.. . . . . . . . . . . . . . . . . . . . . . . . ..G..- . . . . . . .:T . . . . . . . . 18 CBS 419.70 Disporotrichum dirnorphosporm

Figure 1. (continued)

- 2%
CBS 405.96 Schnophyllum commune
relatively variable 18s rDNA domains, whereas
HinzI and Sau3A1 (with restriction sites identical
to those of DdeI) cut in conserved areas and thus
the resulting patterns are expected to be identical
CBS 517.91 Hormographiella candelabrata when the fungi are phylogenetically not too
2.3
remote. DdeI digestion patterns are nearly
CBS 519.91 Hormographiella aspergillata invariant, whereas with Hinf I and Sau3A1 three
and four groups, respectively, could be
CBS 106.97 Hormographiella aspergillata
CBS 419.70 Disporotrichum dimorphosporum distinguished.
Basic relationships of the strains studied are
CBS 363.65 Phanerochaete chrysosponum
CBS 129.27 Phanerochaete chrysosporium evaluated on the basis of a sequence comparison
CBS 255.65 Phanerochaete chrysosporium of the terminal portion of 18s rDNA that includes
5
the V9 variable domain. This could be sequenced
CBS 230.93 Bjerkandera adusta 5 in a single run together with ITSl when the
primer V9G was used. The number of informative
Figure 2. Distance tree of studied filamentous Basidiomycetes gen-
erated with the neighbour-joining algorithm using the Treecon sites, however, is rather low, and the groups
software package, based on unambigously aligned positions of SSU distinguished in the distance tree (Fig. 2) are mostly
r D N h BJerkandera adusta was used as outgroup. statistically insignificant having low bootstrap
values (not shown). The RFLP groups 2 and 3
could not be distinguished as strain FMR 5150
2%
proved to be a sister group to the remaining

i-
CBS 257 96 Hormographiella species
CBS 230 93 Blerkendera adusta
Figure 3. Distance tree of the Homographiella-Copinus complex
generated with the neighbour-joining algorithm using the Treecon
softtvarc package, based on unambiguously aligned positions of ITS1.
I3jrkandera adusfa was used as outgroup.
CBS 509.94 Hormographiella verticillata
FMR 5150 Coprinus cinereus
CBS 106.97 Hormographrella aspergillata
CBS 519.91 Hormographiella aspergillata
CBS 833.96 Hormographiella aspergillata
CBS 517.91 Hormographiella candelabrnta
FMR 3936 Hormographiella verticillata
CBS 258.96 Coprinus species
species. Figure 3 shows that RFLP group 2 prob-
ably comprises a single species, whereas group 3
is more heterogeneous. The resolution power of
morphology is insufficient in the Homobasidio-
mycetes as strain CBS 509.94 was incorrectly
identified as H. verticillata, and the remote,
Bjerkandera-like strain CBS 257.96 had been sup-
posed to be a Hormographiella on the basis of the
presence of arthroconidial elements.
The hypervariable ITS 1 spacer was sequenced

mycoses 41, 183--189 (1998)


in strains representing all groups found with
restriction analysis. Most sequences could hardly
be aligned. In contrast, the type of H. aspergillata,
from a clinical source, and C. cinereus proved to be
nearly identical (Fig. l), confirming the conclusion
presented by Gent: et al. [4] on the basis of nDNA
restriction analysis. H. uerticillata, represented by
clinical isolate FMR 3936 and three environmental
isolates (Table l), had similar 18s RFLP patterns
but was otherwise clearly different from H. aspergil-
lata with ITS1-2 RFLP (Table 1) and ITS1
sequencing (Fig. 3); probably another Coprinus
species is concerned.
The applied primers V9G and 5.8G worked
well in all fungal orders studied, despite the fact
that in some strains a base substitution was found
near the 5‘ ends of these primers. To increase
amplification confidence in filamentous
Basidiomycetes it is therefore recommended that
one base is replaced and three bases omitted at
the 5‘ end of these primers and that they are
replaced by the primers V9D (5’-TTAA-
GTCCCTGCCCTTTGTA-3’) and 5.8D
(5’-GTGCGTTCAAAGATTCG-3.
Acknowledgements
J. A. Stalpers is thanked for taxonomic advice,
Y. Graser for verifying the alignment and
K. Sterflinger for comments on the manuscript.
T. Hoeijmakers is acknowledged for generating
sequence data.
References
Espinel-Ingroff, A. (1996) History of medical mycology in
the United States. Clin. Microbiol. Rev. 9, 235-272.
Sigler, L. & Abbott, S. P. (1997) Characterizing and
conserving diversity of filamentous Basidiomycetes from
human sources. Microbiol. Cult. Coll. 13, 2 1-27.
Sigler, L., Maza, L. M. de la, Tan, G., Egger, K. N. &
mycoses 41, 183-189 (1998)
MOLECULAR
DIAGNOSTICS OF BASIDIOMYCETES 189
Sherburne, R. K. (1995) Diagnostic difficulties caused by
a nonclamped Schizophyllum commune isolate in a case of
fungus ball of the lung. 3. Clin. Microbiol. 33, 1979- 1983.
4 Gent, J., Guillambn, J., Pujol, I. & Ulfig, K. (1996)
Molecular characterization, relatedness and antifungal
susceptibility of the basidiomycetous Hormographiella
species and Coprinus cinereus from clinical and environmen-
tal sources. Antonie uan Leeuwenhoek 70, 49-57.
5 White, T. J., Bruns, T., Lee, S. & Taylor, J. (1990)
Amplification and direct sequencing of fungal ribosomal
RNA genes for phylogenetics. In: Innis, M. A., Gelfland,
D. H., Sninsky,J. J. & White, T. (eds) PCR Protocols. San
Diego: Academic Press, pp. 315-322.
6 De Rijk, P. & De Wachter, R. (1986)DCSE, an interactive
tool for sequence alignment and secondary structure
research. Comput. Appl. Biosci. 9, 735-740.
7 Van de Peer, Y., Nicolai, S., De Rijk, P. & De Wachter,
R. (1996) TREECON for Windows: a software package
for the construction and drawing of evolutionary trees for
the Microsoft Windows environment. Comput. Appl. Biosci.
10, 569-570.
8 Tintelnot, K., Dunckelmann, K., Ozel, M. & Hoog, G. S.
de (1997) Transmission electron microscopy (TEM) and
rDNA-RFLP for identification of filamentous
Basidiomycetes, isolated from human specimens, abstract.
13th Congress ISHAM, Parma, p. 78.
9 De Hoog, G. S. & Guarro, J. (eds) (1995) Atlas of Clinical
Fungi. Centraalbureau voor Schimmelcultures, Baarn and
Universitat Rovira i Virgili, Reus.
10 Stalpers, J. A. (1978) Identification of wood-inhabiting
Aphyllophorales in pure culture. Stud. Mycol. 16, 1-246.
11 De Hoog, G. S., Uijthof, J. M. J., Gerrits van den Ende,
A. H. G., Figge, M. J. & Weenink, X. 0. (1997)
Comparative rDNA diversity in medically significant
fungi. Microbiol. Cult. Coll. 13, 39-48.
12 Verweij, P. E., Kasteren, M. van, Nes, J. van de, Hoog,
G. S. de, Pauw, B, E. de & Meis, J. F. G. M. (1997) Fatal
pulmonary infection caused by the basidiomycete
Hormographiella nspegillata.J . Clin. Microbiol. 35,267 5- 2 67 8.
13 Nenoff, P., Friedrich, T., Schwenke, H., Mierzwa, M.,
Horn, L.-C. & Haustein, U.-F. (1997) Rare fatal simul-
taneous mould infection of the lung caused by Aspergillus
flauus and the basidiomycete Coprinus sp. in a leukemic
patient. J . Med. Kt. Mycol. 35, 65-69.
14 Bartz-Schmidt, K. U., Tintelnot, K., Steffen, M., Ozel,
M., Kirchhof, B. & Heimann, K. (1997) Chronic basidi-
omycete endophthalmitis after extracapsular cataract
extraction and intraocular lens implantation. Gaefe’s Arch.
Clin. Ex!. Ophthalmol. 234, 591-593.