Simple screening method for molds producing intracellular mycotoxins in pure culture

Abstract

A simple screening method for molds producing the intracellular mycotoxins brevianamide A, citreoviridin, cyclopiazonic acid, luteoskyrin, penitrem A, roquefortine C, sterigmatocystin, verruculogen, viomellein, and xanthomegnin was developed. After removing an agar plug from the mold culture, the mycelium on the plug is wetted with a drop of methanol-chloroform (1:2). By this treatment the intracellular mycotoxins are extracted within seconds and transferred directly to a thin-layer chromatography plate by immediately placing the plug on the plate while the mycelium is still wet. After removal of the plug, known thin-layer chromatographic procedures are carried out. The substrate (Czapek yeast autolysate agar) and growth conditions (25 degrees C for 7 days) used by Penicillium taxonomists proved suitable for the production of the mycotoxins investigated when 60 known toxigenic isolates and 865 cultures isolated from foods and feedstuffs were tested with this screening method.

Full text

APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Feb.
1983,
p.
581-585
0099-2240/83/020581-05$02.00/0
Copyright
C
1983,
American
Society
for
Microbiology
Vol.
45.
No.
2
Simple
Screening
Method
for
Molds
Producing
Intracellular
Mycotoxins
in
Pure
Cultures
OLE
FILTENBORG,*
JENS
C.
FRISVAD,
AND
JYTTE
A.
SVENDSEN
Food
Technology
Laboratory,
The
Technical
University
of
Denmark,
DK-2800
Lyngby,
Denmark
Received
11
August
1982/Accepted
27
October
1982
A
simple
screening
method
for
molds
producing
the
intracellular
mycotoxins
brevianamide
A,
citreoviridin,
cyclopiazonic
acid,
luteoskyrin,
penitrem
A,
roquefortine
C,
sterigmatocystin,
verruculogen,
viomellein,
and
xanthomegnin
was
developed.
After
removing
an
agar
plug
from
the
mold
culture,
the
mycelium
on
the
plug
is
wetted
with
a
drop
of
methanol-chloroform
(1:2).
By
this
treatment
the
intracellular
mycotoxins
are
extracted
within
seconds
and
transferred
directly
to
a
thin-layer
chromatography
plate
by
immediately
placing
the
plug
on
the
plate
while
the
mycelium
is
still
wet.
After
removal
of
the
plug,
known
thin-layer
chromatographic
procedures
are
carried
out.
The
substrate
(Czapek
yeast
autoly-
sate
agar)
and
growth
conditions
(25°C
for
7
days)
used
by
Penicillium
taxono-
mists
proved
suitable
for
the
production
of
the
mycotoxins
investigated
when
60
known
toxigenic
isolates
and
865
cultures
isolated
from
foods
and
feedstuffs
were
tested
with
this
screening
method.
A
simple
screening
method
for
toxigenic
molds
in
pure
cultures
on
agar
substrates
has
previously
been
described
(16).
This
is
a
very
time-
and
resource-saving
method
compared
with
other
screening
methods
(2,
5,
23,
34),
and
it
has
already
proved
valuable
in
investigations
where
numerous
isolates
had
to
be
screened
(4,
17).
The
use
of
the
method
is,
however,
restrict-
ed
to
pronounced
extracellular
mycotoxins
such
as
aflatoxins,
citrinin,
kojic
acid,
mycophenolic
acid,
3-nitropropionic
acid,
ochratoxins,
patulin,
penicillic
acid,
PR-toxin,
T-2
toxin,
and
zeara-
lenone,
as
it
is
based
on
diffusion
of
the
myco-
toxin
into
the
substrate.
This
means
that
impor-
tant
mycotoxins
like
roquefortine
C,
penitrem
A,
sterigmatocystin,
and
several
others,
which
are
chiefly
intracellular
(see
reference
29),
can-
not
be
detected
with
this
screening
method.
To
overcome
this
limitation
without
losing
the
ad-
vantages
of
the
screening
method,
the
method
described
in
this
paper
was
developed.
MATERIALS
AND
METHODS
Fungi.
A
total
of
60
known
toxigenic
cultures
(from
culture
collections)
and
865
isolates
from
foods
and
feedstuffs
were
tested
with
the
screening
method.
The
taxonomy
of
the
cultures
from
culture
collections
is
not
treated
in
this
paper,
but
the
remaining
865
cul-
tures
were
identified
according
to
the
following
taxo-
nomic
treatments:
the
penicillia
according
to
the
meth-
ods
of
Pitt
(27),
Ciegler
et
al.
(7),
and
Frisvad
(17),
and
the
aspergilli
according
to
the
methods
of
Raper
and
Fennell
(30),
Samson
(32),
and
Christensen
(6).
The
cultures
were
maintained
on
malt
extract
agar
(27)
and
Czapek
yeast
autolysate
agar
(CYA)
(27)
at
0.5°C.
Substrates.
The
cultures
were
three point
inoculated
on
yeast
extract-sucrose
agar
(34),
yeast
extract-glu-
cose
agar
(16,
17),
CYA
(27,
34),
potato-glucose-yeast
extract
agar
(potato
extract
[Difco
Laboratories,
De-
troit,
Mich.],
4
g;
glucose,
20
g;
yeast
extract
[Difco],
5
g;
agar,
20
g;
water,
1
liter),
and
malt
extract
agar
(E.
Merck
AG,
Darmstadt,
Germany;
art.
5398)
(23).
The
cultures
were
incubated
at
25°C
for
7
days.
Mycotoxin
analysis.
One
or
more
agar
plugs
were
cut
out
of
a
mold
colony
(near
the
center)
with
a
flame-
sterilized
stainless
steel
tube
(inner
diameter,
0.4
cm).
The
plugs
were
removed
by
using
a
flame-sterilized
scalpel
or
needle.
By
means
of
a
syringe,
a
drop
of
extraction
liquid
was
placed
directly
on
the
mycelium
or
conidia.
While
still
wet,
the
mycelium
side
of
the
plug
was
gently
pressed
against
the
application
line
on
a
thin-layer
chromatography
(TLC)
plate
and
then
removed
immediately.
After
drying
the
application
spot,
the
procedure
could
be
repeated
with
other
plugs.
The
TLC
plates
(Merck
precoated
silica
gel
G,
art.
5721;
with
and
without
oxalic
acid
impregnation)
were
activated
for
2
h
at
110°C.
To
produce
oxalic
acid-impregnated
TLC
plates,
precoated
plates
were
dipped
in
an
8%
methanolic
solution
of
oxalic
acid
for
2
min
and
air
dried
overnight.
Combinations
of
chloroform
or
ethyl
ether
with
ethanol,
methanol,
or
acetone
were
compared
for
efficiency
in
mycotoxin
extractions.
The
most
efficient
of
these
solvent
mixtures
was
chloroform-methanol
(2:1
[vol/vol]),
so
this
mixture
was
used
in
the
screen-
ing
method.
Representatives
of
cultures
which
did
not
produce
the
expected
mycotoxins
were
analyzed
by
using
a
stomacher
extraction
technique
(17)
after
1
and
2
weeks
of
incubation.
The
following
standard
toxins
were
used
as
external
and
internal
standards:
sterigmatocystin
(Karl
Roth,
Karlsruhe,
Federal
Republic
of
Germany);
luteoskyrin
581

582
FILTENBORG,
FRISVAD,
AND
SVENDSEN
TABLE
1.
Procedures
for
detection
of
the
mycotoxins
included
in
the
screening
method
Mycotoxin
~~~~~~TLC
developing
TetnSb
Mycotoxin
systems"
(references)
Treatments
Brevianamide
A
TEF
(23),
CA
(41)
VIS
(41),
UV
(41)
Citreoviridin
TEF
(10),
CM
(37)
VIS
(37),
UV
(37)
Cyclopiazonic
acid
Cl'
(35),
EPA
(22)
EHRLICH
(18),
FeCI3
(18)
Luteoskyrin
AHW
(38),
TEF
(34)
UV
(34),
ANIS
(34)
Penitrem
A
HE
(12),
CA
(9)
ACIC3,
FeCI3
(9)
Roquefortin
C
CMA
(33),
CAP
(19)
Ce(SO4)2
(19),
EHRLICH
(40)
Sterigmatocystin
TEF
(34),
BMA
(34)
AICI3
(1),
ANIS
(34)
Verruculogen
TEF
(11),
CA
(11)
VIS
(11),
H2SO4
(11)
Viomellein
BMA
(8),
TEF
(31)
VIS
(8),
NH3
(8)
Xanthomegnin
BMA
(8),
TEF
(31)
VIS
(8),
NH3
(8)
a
Abbreviations:
TEF,
toluene-ethyl
acetate-90%o
formic
acid
(5:4:1
[vol/vol/vol]);
CA,
chloroform-acetone
(9:1
[vol/vol]
or
93:7
[vol/vol]);
CM,
chloroform-methanol
(9:1
[vol/vol]);
CI,
chloroform-isobutylmethylketone
(4:1
[vol/vol]);
EPA,
ethyl
acetate-2-propanol-28%
NH3
in
water
(20:15:10
[vol/vol/vol]);
AHW,
acetone-n-
hexane-water
(4:2:1
[vol/vol/vol]);
HE,
hexane-ethyl
acetate
(6:4
[vol/vol]);
CMA,
chloroform-methanol-28%
NH3
in
water
(90:10:1
[vol/vol/vol]);
CAP,
chloroform-acetone-propane-2-ol
(85:15:20
[vol/vol/vol]);
BMA,
benzene-methanol-acetic
acid
(24:2:1
[vol/vol/vol]).
b
Abbreviations:
VIS,
viewed
under
normal
visible
light;
UV,
viewed
under
UV
light
at
366
nm;
EHRLICH,
1%
(wt/vol)
p-dimethylbenzaldehyde
in
96%
ethanol
was
sprayed
on
the
TLC
plate,
and
the
plate
was
dried
under
a
hair
dryer
and
placed
over
HCI
fumes
for
10
min;
FeCl3,
1%
(wt/vol)
FeCl3
in
butane-1-ol;
ANIS,
0.5%
p-
anisaldehyde
(vol/vol)
in
ethanol-acetic
acid-concentrated
H2SO4
(17:2:1
[vol/vol/vol]);
AlCl3,
20%
(wt/vol)
AlC13
in
96%
ethanol;
Ce
(SO4)2,
1%
Ce
(SO4)2
(wt/vol)
in
6
N
H2SO4;
H2SO4,
50%
(vol/vol)
concentrated
H2SO4
in
water;
NH3,
exposure
to
NH3
vapors
for
1
min.
See
the
original
references
for
details
on
the
colors
of
the
mycotoxins
after
different
treatments.
c
On
oxalic
acid-treated
plates.
(Sigma
Chemical
Co.,
St.
Louis,
Mo.); penitrem
A
and
cyclopiazonic
acid
(from
L.
Leistner,
Bundesanstalt
fur
Fleischforschung,
Kulmbach,
Federal
Republic
of
Germany);
brevianamide,
xanthomegnin,
and
viomel-
lein
(from
A.
Ciegler,
Southern
Regional
Research
Center,
New
Orleans,
La.);
verruculogen
(from
R.
T.
Gallagher,
Ruakura
Agricultural
Research
Center,
Hamilton,
New
Zealand);
citreoviridin
(from
A.
E.
de
Jesus,
Council
for
Scientific
and
Industrial
Research,
Pretoria,
South
Africa);
and
roquefortine
C
(from
U.
L.
Diener,
Auburn
University,
Auburn,
Ala.,
and
H.-J.
Rehm,
University
of
Munster,
Federal
Republic
of
Germany).
In
the
prescreening,
all
mycotoxins
were
detected
by
using
toluene-ethyl
acetate-90%
formic
acid
(5:4:1)
(40)
and
external
standards.
Roquefortine
C
and
cyclo-
piazonic
acid
had
very
low
Rf
values
in
that
developing
system.
The
toxins
were
visualized
in
daylight
and
UV
light
at
366
and
254
nm
(UV366
and
UV254),
before
and
after
treatment
with
a
50%
solution
of
sulfuric
acid
(11).
The
production
of
any
particular
mycotoxin
was
confirmed
by
using
external
and
internal
standards
in
optimal
developing
systems,
with
toxins
visualized
as
listed
in
Table
1.
The
visualization
of
penitrem
A
with
AIC13
has
not
previously
been
reported.
It
was
performed
by
spray-
ing
the
TLC
plate
with
a
20%
(wt/vol)
solution
of
AIC13
in
96%
ethanol
(7)
and
heating
for
5
min
at
120°C.
The
toxin
was
bluish
green
in
daylight
and
reddish
brown
in
UV366.
Seventy
nanograms
of
penitrem
A
could
be
detected
on
the
TLC
plate.
RESULTS
AND
DISCUSSION
The
chemical
detection
of
the
individual
tox-
ins
was
never
disturbed
by
interfering
metabo-
lites,
in
spite
of
the
omission
of
sample
purifica-
tion
before
application
on
TLC,
as
described
in
the
present
method.
A
limited
number
of
other
metabolites
were
indeed
observed,
but
they
were
always
well
separated
from
the
mycotox-
ins.
In
a
few
cultures,
confirmation
of
the
toxins
was
questionable
due
to
weak
responses
on
the
TLC
plate.
In
these
cases,
the
amount
of
toxin
applied to
the
plate
was
increased
by
superim-
posed
application
of
three
plugs,
whereas
exten-
sion
of
extraction
time
did
not
improve
the
result.
Several
protein-lipid-separating
solvent
systems
were
tested
in
the
extraction
procedure.
The
chloroform-methanol
(2:1)
system
appeared
as
optimal
for
toxin
extraction
as
it
is
for
lipid
extraction
(20).
The
release
of
the
toxin
from
the
mycelium
effected
by
simply
adding
this
solvent
mixture
may
be
explained
by
the
polar
solvent
breaking
the
protein-lipid
bonds
in
membranes
by
denaturating
the
proteins,
with
the
less
polar
solvent
helping
to
dissolve
the
lipids
(20).
Different
substrates
were
used
in
optimizing
toxin
production.
Certain
important
variations
within
toxins
and
isolates
were
observed,
but
of
main
interest
was
that
the
substrate
CYA,
al-
though
not
always
the
best,
appeared
useful
for
all
isolates,
except
for
roquefortine
C
production
from
a
few
Penicillium
roquefortii
isolates.
To
include
these
isolates,
cultures
on
yeast
extract-
sucrose
agar
could
be
used.
The
incubation
time
needed
to
detect
toxin
production
was
less
than
the
specified
7
days
for
many
cultures,
but
this
APPL.
ENVIRON.
MICROBIOL.

SCREENING
FOR
INTRACELLULAR
MYCOTOXINS
583
TABLE
2.
Detection
of
mycotoxin
production
on
CYA
at
25°C
from
a
selected
number
of
known
toxigenic
mold
isolates
Mycotoxin
Mold
isolate
Strains
(references)"
Brevianamide
A
Penicillium
brevicompactum
P.
viridicatum
Citreoviridin
P.
citreoviride
P.
citrinum
P.
miczynskii
P.
pulvillorum
IMI
40225
(3)
NRRL
963
(41),
Purdue
66-68-2
(41),
Sp
931
(23)
CBS
920.70b,
NRRL
2046b,
NRRL
2579b
Sp
865
(23,
24)
Sp
340
(23,
24)
CSIR
1405
(25),
CSIR
1406
(25)
Cyclopiazonic
acid
Luteoskyrin
Penitrem
A
Roquefortine
C
Aspergillus
flavus
P.
camembertii
P.
crustosum
P.
patulum
P.
puberulum
P.
cyclopium
P.
viridicatum
P.
islandicum
P.
commune
P.
crustosum
P.
cyclopium
P.
granulatum
P.
martensli
P.
olivinoviride
P.
palitans
P.
commune
P.
crustosum
P.
cyclopium
P.
roquefortii
NRRL
3251
(18)
CBS
299.48
(22,
36),
ATCC
42009
(22,
36),
Sp
1133
(36)
Sp
607
(24)
CSIR
1082
(21),
CSIR
1399
(24)
Sp
524
(24)
Sp
603,
Sp
605,
Sp
608,
Sp
613
(24)
Sp
119
(24)
CBS
587.68b,
NRRL
1036
(14)
AUA
827
(40)
NRRL
968,
NRRL
1983,
NRRL
5186
(9),
Sp
458,
Sp
1191
(24)
NRRL
3476',
NRRL
3477
(9),
NRRL
6093
(39)
NRRL
2036
(9)
NRRL
2034
(9)
NRRL
958
(9)
NRRL
3468'
(9)
AUA
827
(40)
G.
Engel
6842
(13)
NRRL
6093
(39)
NRRL
849
(33),
Sp
860,
Sp
1066,
Sp
1077,
Sp
1079
(23,
24)
Sterigmatocystin
Verruculogen
Aspergillus
versicolor
P.
estinogenum
P.
piscarium
P.
simplicissimum
P.
verruculosum
CBS
600.65b,
Frank
H9,
H22,
519,
543
(26)
76S9FC9d
(28)
NRRL
A-14996
(=Sp
306)
(24)
Sp
863
(23)
NRRL
5881
(=ATCC
24640)d
(11,
15,
28)
P.
viridicatum
P.
viridicatum
NRRL
963,
NRRL
A-15402,
NRRL
A-15505,
NRRL
A-
19118
(8),
Purdue
66-68-2
(31)
NRRL
963,
NRRL
A-15402,
NRRL
A-15505,
NRRL
A-
19118
(8),
Purdue
66-68-2
(31),
Sp
931
(23)
a
The
cultures
are
listed
as
received
or
listed
in
culture
collection
catalogues.
b
Toxin
production
by
these
isolates
has
been
stated
in
personal
communications
to
us.
Furthermore,
these
species
are
generally
accepted
as
producers
of
the
toxins
mentioned
(29).
See
also
the
culture
collection
catalogues
from
the
Commonwealth
Mycological
Institute
(CMI,
1982),
the
American
Type
Culture
Collection
(ATCC,
1982),
and
the
Centraalbureau
voor
Schimmelcultures
(CBS,
1978).
c
Equals
P.
crustosum
(28).
d
Equals
P.
simplicissimum
(28).
period
was
necessary
to
detect
all
tested
toxi-
genic
cultures.
The
detection
limits
of
the
method
cannot
be
specified
quantitatively
from
this
investigation.
But,
as
indicated
in
Table
2,
it
proved
sufficient
compared
with
alternative
methods
(2,
5,
23,
34)
for
the
detection
of
toxin
production
from
all
60
tested
known
toxigenic
mold
isolates.
As
a
further
illustration
of
the
sensitivity
of
this
method,
the
results
of
the
screening
of
some
of
our
own
isolates
from
foods
and
feedstuffs
are
listed
in
Table
3.
Viomellein
Xanthomegnin
VOL.
45,
1983

584
FILTENBORG,
FRISVAD,
AND
SVENDSEN
TABLE
3.
Detection
of
mycotoxin
production
on
CYA
at
25°C
from
isolates
of
known
toxigenic
mold
species
from
foods
and
feedstuffs
No.
of
No.
Mycotoxin
Species
isolates
producing
investigated
the
toxin
Brevianamide
A
Penicillium
brevicompactum
28
28
P.
viridicatum
I
320
7
Citreoviridin
P.
miczynskii
8
8
Cyclopiazonic
acid
Aspergillus
flavus
5
5
P.
camembertii
24
24
P.
griseofulvum
12
12
P.
puberulum
15
15
Luteoskyrin
P.
islandicum
7
6
Penitrem
A
P.
crustosum
66
66
Roquefortine
C
P.
crustosum
66
66
P.
roquefortii
39
39a
Sterigmatocystin
Aspergillus
versicolor
38
38
Verruculogen
P.
simplicissimum
3
3
Viomellein
P.
viridicatum
I
320
320
Xanthomegnin
P.
viridicatum
1
320
320
P.
aurantiogriseum
300 188
a
Toxin
from
a
few
P.
roquefortii
isolates
was
only
detected
after
several
superimposed
applications
on
the
TLC
plate
or
in
cultures
on
yeast
extract-sucrose
agar.
The
sensitivity
of
the
method
appears
to
be
sufficient
as
far
as
the
majority
of
the
species
are
concerned,
since
all
tested
isolates
of
11
species
produced
the
expected
intracellular
tox-
ins.
However,
within
Penicillium
aurantiogri-
seum
(xanthomegnin)
and
Penicillium
viridica-
tum
I
(brevianamide
A)
the
toxins
could
not
be
detected
in
the
cultures
of
a
considerable
num-
ber
of
the
isolates
tested.
The
demonstration
of
isolates
not
producing
the
expected
toxins
was
checked
with
an
alternative
screening
method
(stomacher
extraction
technique
[17,
23]),
but
no
disagreement
was
observed.
In
other
words,
we
have
not
been
able
to
demonstrate
any
false-
negative
results
with
the
screening
method.
The
described
method
is
meant
for
the
screen-
ing
of
intracellular
mycotoxins,
but
it
has
often
proved
useful
for
the
screening
of
typical
extra-
cellular
mycotoxins
as
well.
However,
the
com-
bination
of
this
method
and
the
screening
meth-
od
for
extracellular
mycotoxins
(16)
is
necessary
to
achieve
sufficient
sensitivity
in
the
general
screening
of
molds
in
pure
culture
for
their
ability
to
produce
known
mycotoxins.
The
test
can
be
performed
in
connection
with
mold
iden-
tification
procedures,
since
identical
incubation
conditions
and
substrates
can
be
used
for
these
purposes.
This
offers
a
very
fast
and
simple
way
to
confirm
the
identity
of
a
mold
isolate
with
important
mycotoxicological
characteristics.
ACKNOWLEDGMENTS
We
thank
L.
Leistner,
A.
Ciegler,
R.
T.
Gallagher,
A.
E.
de
Jesus,
U.
L.
Diener,
H.-J.
Rehm,
D.
T.
Wicklow,
C.
W.
Hesseltine,
P.
Krogh,
A.
H.
S.
Onions,
G.
Engel,
H.
K.
Frank,
and
M.
E.
de
Menna
for
the
supply
of
cultures
and
standard
mycotoxins
used
in
this
study.
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VOL.
45,
1983