INTERNATIONAL JOURNAL
OF SYSTEMATIC BACTERIOLOGY,
OCt. 199.5, p. 826-831
0020-7713/95/$04.00+0
Copyright 0 1995, International Union of Microbiological Societies
Vol. 45, No. 4
Genome Comparisons in the Yeastlike Fungal
Genus Galactomyces Redhead et Malloch
MAUDY T. SMITH,'" A. W. A. M. DE COCK,l G. A. POOT,' AND H. Y. STEENSMA'
Centraalbureau voor Schimmelcultures Yeast Division, 2628 BC Dew, and Institute of
Molecular Plant Sciences, Leiden University, 2333 A L Leiden, The Netherlands
The G+C contents of the DNAs of 41 strains belonging to the genus Galactomyces Redhead et Malloch were
determined by the thermal denaturation method. Melting profiles revealed that the DNAs of these strains are
heterogeneous. Four groups were recognized on the basis of this heterogeneity. However, DNA similarity
values, which were calculated by using DNA-DNA reassociation kinetics, revealed that the strains could be
divided into six subgroups. Strains belonging to the same subgroup exhibited high levels of DNA similarity (84
to 100%).The members of two subgroups, correspondingto Galactomyces citri-aurantii and Galactomyces reessii,
exhibited low levels of DNA similarity with the members of the other subgroups (20 to 27%).The members of
the four remaining subgroups, which contained only strains previously identified as Galactomyces geotrichum,
exhibited intermediate levels of reassociation (41 to 59%). Some combinations of phenotypic characteristics
correlated with the subgroups; a key based on phenotypic characteristics that can be used to distinguish the
subgroups is presented.
In 1986, de Hoog et al. (3) revised the ascomycetous anamorph genus Geotrichum Link: Fr. and its teleomorphic genera GalactornycesRedhead et Malloch and Dipodascus Lagerh.
Delimiting species on the basis of morphological and physiological criteria, de Hoog et al. recognized in these genera 4, 2,
and 13 species, respectively. In this study de Hoog et al. compared nuclear genomes as well as phenotypic features. When
DNA-DNA reassociation experiments were performed, the results generally confirmed the phenotypic groups. However, discrepancies were observed in some taxa. High, intermediate,
and low levels of DNA-DNA reassociation were found among
strains which had been assigned to Galactomyces geotrichum
(Butler et Petersen) Redhead et Malloch on the basis of phenotypic properties. Similar discrepancies were observed previously in other yeasts and yeastlike fungi (1, 9, 11-13, 15, 18,21,
25).
It is genzrally accepted that levels of reassociation greater
than 80% indicate that taxa are conspecific (6, 8, 10, 18).
Intermediate and low values, however, cannot be interpreted
unambiguously. Correlations with interfertility data (11-13,20,
23, 26) and nutritional characteristics (27) and/or relationships
with ecological parameters (15, 22) have been invoked to justify the description of taxa as species or varieties.
In the case of G. geotrichum, some of the strains that exhibited low and intermediate levels of DNA-DNA reassociation
were found to react in mating experiments and were considered conspecific. However, in some combinations most asci
were abortive, and the viability of ascospores was not tested
(3).
To explain the conflicting observations obtained with G.
geotrichum strains, an extensive revision in which numerous
strains were used was undertaken to evaluate the heterogeneity of the members of this complex. Strains of the other two
Galactomyces species, Galactomyces citn-aurantii Butler and
Galactomycesreessii (van der Walt) Redhead et Malloch, were
also included in this study. G + C content data for 41 strains
and DNA-DNA reassociation values and some relevant physiological characteristics of 57 strains are presented below.
This study was part of a revision of the related genera Geotrichum, Dipodascus, and Galactomyces.
MATERIALS AND METHODS
Cultures examined. The strains which we studied and their origins are shown
in Table 1.The strains were identified as strains of G. geotiichum as described by
de Hoog et al. (3) unless they could be identified as either G. citri-auruntii or G.
reessii.
Physiology and cultural characteristics. The abilities of strains to assimilate
carbon sources were determined by using standard methods (24). Cultures were
grown in liquid media in tubes for 4 weeks at 25°C and were shaken continuously
at 30 rpm. Utilization of nitrogen compounds was determined by the auxanographic method after 1 week.
To measure growth, a heavy inoculum from a 1- to 7-day-old culture was
streaked onto GPYA (4% glucose, 0.5% peptone, 0.5% yeast autolysate, 2%
agar) in petri dishes, which were incubated at 25°C for 7 days. Growth was
measured from the center to the edge of each streak.
Isolation of DNA. Nuclear DNA was isolated from cultures grown in 1 liter of
YM broth (Difco Laboratories, Detroit, Mich.) for 1 day at 25°C on a rotary
shaker at 150 rpm. Cells collected in a Biichner funnel were washed twice with
demineralized water and saline EDTA (0.15 M sodium chloride, 0.01 M EDTA,
pH S.O), resuspended in 5 to 10 rnl of 0.27 M sodium phosphate buffer (pH 6.8)
containing 9 M urea and 0.9% (wt/vol) sodium dodecyl sulfate, and passed three
times through a cell in a French press at 1,150 atm (1.16 X lo" Pa). The disrupted
cells were centrifuged for 20 min at 9,000 X g. The DNA was purified from the
supernatant by hydroxyapatite column chromatography on a Bio-Rad Bio-Gel
HTP or Fluka "high resolution" column as described by Britten et al. (2). The
DNA fraction collected was finally dialyzed against 0.1 X SSC (1X SSC is 0.15 M
NaCl plus 0.015 M trisodium citrate, pH 7.0). If necessary, the DNA was precipitated with cold ethanol and redissolved in a minimal volume 0.1X SSC.
Ratios ofA,,, toA,,, of 1.86 ? 0.05 and ratios ofA,,, toA,,, of 0.5 ? 0.05 were
used to determine the quality of the DNA prepared.
Determination of the G+C content. The G + C content was determined at least
twice for each strain by the thermal denaturation (T,) method of Marmur and
Doty (14) by using 0.1X SSC and was calculated from the following formula of
Owen et al. (16): G + C content = 2.08 T,, - 106.4. Melting profiles were
obtained with a Perkin-Elmer recording spectrophotometer equipped with a
thermoprogrammer. DNA of Candida purapsilosis CBS 604' (T = type strain)
(T, in 0.1X SSC, 70.6"C) was included in every determination as a reference.
The G + C values were calculated from the T, values on the melting curves, as
well as from the peaks in the first derivatives of the T, profiles.
DNA-DNA complementarity. The levels of DNA-DNA reassociation were
determined spectrophotometrically by using the procedures described by Seidler
and Mandel (19) and modified by Kurtzman et al. (12). The optimal reassociation temperature (T, - 25"C), 55"C, was determined by using the method of
Kurtzman et al. (12). Reassociation experiments were performed at least three
times.
* Corresponding author. Mailing address: Centraalbureau voor
Schimmelcultures Yeast Division, Julianalaan 67, 2628 BC Delft, The
Netherlands. Phone: (31)15-782394. Fax: (31)15-782355.
826
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G E N O M E CO MPA RISO N S IN THE G E N U S GALACTOMYCES
VOL. 45. 1995
827
T ABLE 1. Strains examined in this study
G + C content
(mol%) as
determined
by Tm
methodh
Value(s)"
G. geotrichum, neotypc strain of Geotrichum candidum, homothallic,
soil, Puerto Rico
Soil, Puerto Rico
CBS 773.71, mating type of CBS 775.71
CBS 773.71, mating type of CBS 774.71
38.5
34.7, 42.1
7.4
39
38.4
39.5
39.8
34.1, 41.9
34.9, 42.5
35.3, 42.9
7.8
7.6
7.6
42
38
44
Milk, United States
Probable type strain of Oidium humi, Institut Pasteur
Bulb of Hyacinthus orientalis, The Netherlands
Authentic strain of Oospom fragans var. minuta, Musu sp.
Possible type strain of Oidium nubilum
Decaying fruit of Lycopersicon esculentum
Authentic strain of Oospora 1acti.s var. parasitica, fruit of Lycopersicon
esculentum, United States
Authentic strain of Oidium asteroides
Fruit of Durio zibethinus
Oopora lactis var. exuberans, white slime flux in Populus alba, Germany
Authentic strain of Geotrichum matelense var. chapmanii
Human nail, The Netherlands
Geotrichum javanense, yoghurt, Italy
Geotrichum versijorme
Possibly a subculture of CBS 149.26
Authentic strain of Geotrichum matelense var. matelense
Fly in petroleum
Sent by R. Ciferri as Geotrichum pulmoneum
Endomyces lacris, Germany
Human tongue, Germany
Human sputum, The Netherlands
Germinating grain of Ilordeum vulgare, The Netherlands
Camembert cheese
Soil polluted with oil, Germany
Soil, Senegal
Geotrichum novakii, fruit of Prunus persica, Egypt
Geotrichum redaellii
Fruit of Lycopersicon esculentum, France
Industrial contaminant, The Netherlands
Brie cheese, France
Drosophila sp., Cameroun
Drosophila sp., Cameroun
Trichosporon inu linum
Unknown
Fruit. The Netherlands
42.6
43.1
42.6
43.2
40.0, 43.1
42.9
42.0
41.5
3.1
43.0
40.8
37
24
35
35
31
32
32
40.4
42.0
41.4
40.3
37.3, 40.4
40.2
41.4
37.6, 40.5
3.1
43.0
42.2
43.5
41.7
43.1
42.4
42.6
43.2
41.8
42.7
40.6
39.8
40.9
43.4
37.2, 41.1
39.7
39.7, 41.7
41 .5
42.2
42.1
42.7
42.0
43.4
40.9
39.9
38.0, 42.7
39.8
41.5
43.0
38.0, 42.6
27
37
30
29
34
28
29
27
24
36
35
33
25
33
32
29
37
30
31
25
35
16
30
36
36
28
34
33
Paper pulp, France
Beta vulgaris, The Netherlands
Geotrichum pseudocandidum, stomach of elk, Francc
Wheat field soil, Germany
39.0
41.4
41.0
40.8
35.3, 41.0
37.5, 43.7
36.7, 43.4
36.7, 43.0
5.7
6.2
6.7
6.3
32
30
21
23
Citnis limonium, Argentina, mating type A1
G. citri-aurantii, soil of orange orchard, Zimbabwe, mating type A1
G. citri-aurantii, soil of orange orchard, California, mating type A2
Citrus paradisi, mating type A1
Citrus puradisi, mating type A2
Soil, Israel, from E. E. Butler, mating type A1
Soil, Israel, from E. E. Butler, mating type A2
Soil, Florida, from E. E. Butler, mating type A1
Soil, Florida, from E. E. Butler, mating type A2
37.9
38.9
36.7
38.4
39.2
32.7,
34.3,
32.4,
33.9,
34.5,
39.7
42.0
40.0
41.3
40.6
7.0
7.7
7.6
7.4
6. I
44
46
34
64
36
27
25
24
41
Endomyces reessii, cold-water retting of Hibiscus cannahinus
From E. GuCho
Dominican Republic, from lnstitut Pasteur
Soil, Costa Rica, from E. E. Butler
Soil, Costa Rica, from E. E. Butler
36.5
36.8
40.5
32.3
29.9, 41.9
30.2, 42.3
33.9, 45.9
31.6. 44.1
12.0
12.1
12.0
12.5
32
28
25
24
28
Strain"
G. geotrichum sensu stricto
CBS 772.717"
CBS 773.71"
CBS 774.71"
CBS 775.71"
G. geotrichum rou A
CBS 109.12'
CBS 110.12"
CBS 121.22d
CBS 122.22"'
CBS 114.23
CBS 115.23
CBS 116.23"
F P
CBS 149.26d
CBS 176.28"
CBS 178.30T"
CBS 180.33''
CBS 181.33
CBS 182.33T"
CBS 193.34Td
CBS 194.35
CBS 195.35
CBS 224.48
CBS 267.51
CBS 178.53r"
CBS 184.56"
CBS 185.56
CBS 240.62
CBS 187.67
CBS 178.71"
CBS 476.83d
CBS 557.83
CBS 279.84T"
CBS 299.84
CBS 607.84"
CBS 615.84"
CBS 606.85
CBS 607.85
CBS 624.8STd
CBS 357.86''
CBS 144.88
G. geotrichum rou B
CBS 820.71"
CBS 267.79'id
CBS 626.83
G. geotrichum group C
strain CBS 866.68
G. citri-aurantii
CBS 228.38"
CBS 175.89'
CBS 176.89T"
CBS 604.85
CBS 605.85"
Butler 564 B
Butler 564 C
Butler 594 A
Butler 594 B
G. reessii
CBS 179.60rfi
CBS 295.84)
CBS 296.84'
Butler 474"
Butler 492
"'
Origin and/or other information
CBS, Centraalbureau voor Schimmelcultures.
'Standard deviations were 51.0 mol% (n = 2 to 4).
' Differences between the peaks in the derivatives of the DNA T,, profiles.
" Strains
used in intraspecific reassociation experiments.
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G + C content (mol%)
as determined by
first derivatives
Difference'
2.9
3.9
2.0
4.1
3.0
Growth
rate
(mmi7
days)
SMITH ET AL.
828
INT. J. SYST.BACTERIOL.
3
0,007
- g
0
\o
N
Y
0,0018
c.l
CI
8
0,005-
2
0
8
e
2
-3
0,0013--
cd
e
si
0,003-
-2
o,ooo8--
Q
0,001-
l - ' - I-
0,4
-0,001
40
50
60
70
80
90
I
0,0003
V-
40
I
I
I
I
I
50
60
70
80
90
Temperature C
A
'
3
2
k
T
F
r
A
B
O
0
FIG. 1. (A) Melting curve for C. parapsilosis CBS 604T DNA in 0.1X SSC.
(B) First-derivative graph of the melting curve shown in panel A.
RESULTS
Nuclear DNA base composition. The melting profiles of the
C. parapsilosis reference strain (strain CBS 604T) and some
Galactomyces strains are shown in Fig. 1 through 4. The melting profile of C. parapsilosis is representative of the melting
profiles of most yeasts. The graphic representation of the first
derivative is a narrow Gaussian curve with a single peak. Only
a single G + C content can be calculated from this curve, and
this value is virtually the same as the value derived from the
melting profile. The graphs of the first derivatives of the Galactomyces strains, however, were broad and contained two
peaks in most cases, which indicated heterogeneity.
The G + C contents of 41 Galactomyces strains are shown in
Table 1. Calculating values from the T, values on the melting
curves resulted in single values, which were the average G + C
contents of the DNAs examined (Table 1). Calculating the
values from the first derivatives allowed us to determine the
G + C contents of the individual peaks, which represented major fractions of the DNA (Table 1). The G + C content range of
the first fraction was roughly 31 to 39 mol%, and the G + C
content range of the second fraction was 40.0 to 44.0 mol%. In
detailed examinations we distinguished four categories, in
which the two fractions differed by approximately 4, 6, 8, and
12 mol% G+C. In some cases only one peak was observed, but
in these cases the curves were as broad as the curves for those
strains that had two peaks 4 mol% G + C apart. Presumably,
T
T
7
-O'ooM 40I
0
Temperature C
O
0,0025
id
8
8
.fl
2
0,0015
I
/
/
0,0015
-2
Q
0,0305
0,0005
I
"
80
'
90
B
two peaks were present in these cases as well, but the resolution of the curves was not sufficient to reveal them.
Levels of DNA relatedness within groups. Levels of DNA
similarity calculated from reassociation rates are shown in Fig.
5. The strains used in the reassociation experiments are indicated in Table 1. We performed various experiments with
members of each of the four groups that exhibited different
peak distances.
Strains belonging to the group whose members produced a
single peak or exhibited a 4 mol% G + C peak difference, referred to as G. geotrichum group A, exhibited levels of DNA
similarity ranging from 82 to 100%. High levels of similarity
(90 to 100%) were also found among strains belonging to the
G. reessii group (12 mol% G + C peak difference). Two subgroups were distinguished among the strains with G + C peak
differences of approximately 8 mol%. The four strains belonging to one subgroup, referred to as G. geotrichum sensu stricto
(since it included the type strain of this species), exhibited
levels of DNA similarity ranging from 84 to 100%. The six
strains belonging to the second subgroup, referred to as G.
citri-aurantii (because it included the complementary mating
type strains of this species), exhibited levels of similarity of 91
to 100%. Three of the four strains belonging to the group
whose members exhibited G + C peak differences of 6 mol%,
formerly identified as G. geotrichum, exhibited levels of similarity ranging from 99 to 100%; this subgroup was referred to
N
-1
70
1
'
0,0035
/
60
FIG. 3. (A) Melting curve for C. geotrichum sensu stricto strain CBS 772.71
DNA in 0.1X SSC. (B) First-derivative graph of the melting curve shown in panel
A.
0
I
50
'
\
"
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VOL.
GENOME COMPARISONS IN THE GENUS GALACTOMYCES
45, 199s
used in yeast classification was assimilated. D-Galactose, Lsorbose, D-xylose (of six pentoses tested), glycerol, and D-glucitol, as well as ethanol, DL-lactate, and succinate, were utilized
by all strains. Utilization of ribitol, D-mannitol, glucono-&-lactone, D-gluconate, and citrate was variable. All but nine strains,
including the type strain of G. reessii and two complementary
mating type strains of G. citri-aurantii, were able to grow in
vitamin-free medium. Variation in the ability to grow at 35 and
37°C was observed. The growth rates ranged from 16 to 64
mm/7 days (Table 1). Salient phenotypic features are summarized in Table 2.
41:
27
'.
829
.*25
DISCUSSION
When we determined the base compositions of the DNAs of
Galactomyces strains, we found that the DNAs were heterogeneous. Normally, the nucleic acid bases are more or less
randomly distributed throughout the DNA in most yeasts, and
the first derivative of the melting profile is a Gaussian curve
(Fig. 1). However, in all Galactomyces strains we found relatively broad first derivatives of the melting curves with (commonly) two peaks (Fig. 2 through 4). The distances between
the peaks were identical for the strains belonging to the same
group and mostly different for strains belonging to different
groups.
Heterogeneity of DNAs has been described previously in
basidiomycetes as well as in ascomycetes (3, 5, 7, 17, 18). In
some of these reports the authors indicated that the heterogeneity was due to a significant portion of repetitive DNA (ribosomal DNA), which was visible as a shoulder on the peak (7),
or to mitochondria1 DNA (18). The possibility that the heterogeneity observed in Galactomyces strains was due to a large
amount of mitochondrial DNA was eliminated by the results of
studies of mitochondrial DNA in which CsCl ultracentrifugation and restriction analysis were used (data not shown). In
Galactomyces strains, the heights of the two peaks in first
derivatives were always more or less the same, suggesting that
there were equal amounts of two DNA fractions with different
G + C contents, as might be expected, for example, with a
hybrid of two parents whose genomes have different G + C
contents. We could not find any previous report of the phenomenon observed in this study. de Hoog et al. (3) calculated
the G+C contents of Galactomyces strains from the first derivatives and found shoulders on the melting curves, but only
the G + C content of the highest peak was given in their paper.
FIG. 5. Relationships of Galuctomyces groups based on levels of DNA similarity. Distances are not proportional to levels of relatedness. The standard
Values are averdeviations for levels of reassociation within groups were 3%.
ages of at least two determinations. The standard deviations for levels of reassociation between groups were 5 6 % .
as G. geotrichum group B. The remaining strain was referred to
as G. geotrichum group C.
Levels of DNA relatedness between groups. Three to six
strains from each group except G. geotrichum group C which
contained only one isolate, were used in the reassociation
experiments. There was little reassociation between G. reessii
or G. citri-auruntii strains and members of the other groups
(Fig. 5). The values obtained ranged from 20 to 25% for levels
of reassociation between G. reessii strains and members of
groups G. geotrichum A, B, and C and G. geotrichum sensu
stricto and from 24 to 27% for levels of reassociation between
G. citri-auruntii strains and members of the four groups mentioned above. The level of mutual reassociation was 23%.
Intermediate levels of reassociation ranging from 41 to 59%
were found when we compared G. geotrichum groups A, B, and
C and G. geotrichum sensu stricto (Fig. 5).
Physiological and cultural characteristics. All 57 Galuctomyces strains representing the six groups defined on the basis
of levels of DNA similarity assimilated only a limited number
of carbon sources. None of the six disaccharides, two trisaccharides, two polysaccharides, and three glycosides commonly
TABLE 2. Salient phenotypic characteristics of Galactomyces strains representing six groups based on levels of DNA relatedness
No. of strains used in:
Taxon
G. geotrichum sensu stricto
G. geotrichum group A
G. geotrichum group B
G. geotiichum group C
G. citri-uurmtii
G. reessii
Physiological and
expansion growth
expt
4
35
3
1
9
5
Growth at:
Growth on:
30°C
tion expt
4
21
3
1
3
4
-
-
-
-
-
-
+
+ V
+ +
+ v + +
- v + +
+ +
+ v + - +
-
-
-
v
-
v
h
+
V
-
+
35°C
37°C
-
-
+
v
?
?
'' Amount of growth from the center to the edge of the streak.
"
-, negative; +, positive; V, variable; ?, not determined.
Mean 5 standard deviation.
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-
-
Difference between
the peaks in the
derivative of the
DNA T, profile
(mol%)
Growth rate
(mmi7 days)"
7.8 2 0.2'
4.0 2 0.3
6.2 -t 0.2
6.3 5 0.0
7.3 -+ 0.6
12.0 % 0.1
38-44
16-37
23-32
2 1-2s
33-64
25-32
830
INr. J.
SMITH ET AL.
A study of the nature of Guluctomyces DNA heterogeneity is in
progress, The G + C contents of some Galuctomyces strains
reported previously by Gukho et al. (4) were determined by the
buoyant density method and were similar to the G + C contents
obtained from T,, graphs.
Our data confirm that the genus Guluctomyces is diverse.
Taking into account the heterogeneity in the DNA compositions and DNA reassociation values, we divided the strains into
six distinct groups. However, cultural and physiological characteristics did not clearly correlate with the groups which we
found. In particular, G. geotn'chum groups B and C could not
be differentiated by the phenotypic characteristics which we
studied, whereas the remaining groups could be distinguished
only by a combination of phenotypic characteristics.
The members of two groups, G. reessii and G. citri-uurantii,
exhibited low levels of reassociation with the members of all
other groups. This confirms the specific status of these two
species. G. reessii can be distinguished on the basis of physiological features alone (Table 2). Moreover, the two G + C content peaks of this organism are farther apart than the peaks
in any other group. The G. citri-uuruntii strain group contains
the complementary mating type strains of this species, as
well as isolates which were previously assigned to G. geotrichum (3). All of these isolates originated from similar sources,
soil under Citrus trees at different locations. However, the
physiology of these organisms hardly differs from the physiology of the other G. geotrichum strains, and de Hoog et al. (3)
observed positive mating reactions with the mating type strains
of G. geotn'chum sensu stricto despite the low levels of DNADNA reassociation between members of these groups. The
members of G. geotrichum sensu stricto and G. geotrichum
groups A, B, and C exhibited intermediate levels of reassociation. These organisms may be considered conspecific (G.
geotrichum strains), differing at an infraspecific level. The
group A and B strains were positive in mating reactions
with test strains belonging to G. geotrichum sensu stricto (3).
A possible explanation for the low or intermediate levels
of reassociation that correlated with apparent interfertility is
that the groups are in an early stage of species differentiation
in which nucleotide sequences of groups have diverged, but
genomes are still similar enough to allow crossing, at least in
the laboratory and up until the development of mature ascospores. Another possible explanation is that the observed mating reactions resulted from induction of sexual reproduction in
one strain by another. Therefore, we are continuing to study
these organisms by performing restriction analyses of mitochondrial DNA and PCR with random 10-mer primers to determine whether hybridization has occurred among members
of these groups in nature. Moreover, the interfertility of representative strains of every group will be examined in our
laboratories in all possible combinations to determine whether
real crossings (i.e., exchanges of genetic information) have
taken place.
To distinguish the six Guluctomyces groups, we created the
following key based on physiological and cultural characteristics:
1. a. Growth without vitamins negative
2
b. Growth without vitamins positive
3
2. a. Growth on ribitol and D-mannitol
negative
b. Growth on ribitol and D-rnannitol
positive
G. reessii
G. citri-aurantii
3. a. Growth on glucono-S-lactone
negative
b. Growth on glucono-6-lactone
positive
SYST.
BACTERIOL.
G. geotrichum
group B,
G. geotrichum
group C
4
4. a.
Growth rate at 25°C on GPYA 16
to 37 mm/7 days
G. geotrichum
group A
b.
Growth rate at 25°C on GPYA 38
to 44 mm/7 days
G. geotrichum
sensu stricto
ACKNOWLEDGMENTS
We thank E. E. Butler for providing cultures from his own collection
and G. S. de Hoog for critically reading the manuscript. We thank
Elma de Wit and Edwin Hornung for technical assistance.
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