Microbiological Research 159 (2004) 317322

Antibacterial activity from Penicillium

corylophilum Dierckx
Marley Garcia Silva
a,b
, Niege Arac-ari Jacometti Cardoso Furtado
b
,
Monica Tallarico Pupo
b
, Maria Jose Vieira Fonseca
b
, Suraia Said
b
,
Ademar Alves da Silva Filho
b
, Jairo Kenupp Bastos
b,
a
Faculdade de Filosoa, Ciencias e Letras de Formiga, Fundac-ao Educacional Comunitaria Formiguense, Avenida Dr.
Arnaldo de Senna, 328 CEP 35570 000 Formiga MG, Brazil
b
Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Departamento de Ciencias Farmaceuticas, Universidade de
Sao Paulo, Avenida do Cafe, s/n, CEP 14040-903, Ribeirao Preto SP, Brazil
Accepted 17 June 2004
Summary
A strain of Penicillium corylophilum isolated from Brazilian soil sample was submitted
to different culture conditions to investigate the production of secondary metabolites
with antimicrobial activity. The largest number of conidia was obtained after 5 days of
incubation in oat medium and the highest level of antimicrobial activity was produced
when the fungus culture was developed in the Czapek medium. The activity against
Staphylococcus aureus was found only in the chloroform extract from Czapek culture
broth, which also showed activity against Micrococcus luteus. Fumiquinozoline F was
isolated from the active chloroform extract by using chromatographic methods. The
minimal inhibitory concentration (MIC) values for M. luteus and S. aureus were 99 mg/
mL and 137 mg/mL, respectively.
& 2004 Elsevier GmbH. All rights reserved.
Introduction
Microorganisms have been traditionally used to
produce a variety of important substances for the
pharmaceutical and food industries. Hence, pri-
mary and secondary metabolites, such as peptides,
enzymes, organic acids and antibiotics produced by
lamentous fungi are used for these purposes
(Bennett, 1998; Demain, 2000).
The discovery and development of antibiotics
was one of the most signicant advances in
medicine in the 20th century. Nevertheless, many
antimicrobial agents that were used to treat a
variety of human infectious diseases are now
www.elsevier.de/micres
KEYWORDS
Fumiquinazoline F;
Antibacterial
activity;
Penicillium
corylophilum
0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2004.06.003

Corresponding author. Tel.: +55-16-602-4162; fax: +55-16-633-1941.

E-mail address: jkbastos@fcfrp.usp.br (J. Kenupp Bastos).
ineffective. Therefore, to ensure that effective
drugs will be available in the future, it is necessary
to improve the antimicrobial use patterns and to
devise strategies to identify new antibiotics
through previously unexplored targets (Smith and
Jarvis, 1999).
Natural products play an important role in the
discovery of leads for the development of drugs for
the treatment of human diseases and microbial
environment is an important source of novel active
agents (Newman et al., 2003). Many of this
products currently used are produced by microbial
fermentation, or are derived from chemical mod-
ication of a microbial product (Donadio et al.,
2002). In this regard, the fermentation process is an
important tool for production of secondary meta-
bolites that can not be isolated from plants and
animals, or synthesized by chemists because of the
ease of increasing production by environmental and
genetic manipulation (Demain, 2000).
Several secondary metabolites have been identi-
ed from Penicillium corylophilum, as well as their
biological activities. In this work, we determined
the best procedure for the production and extrac-
tion of antimicrobial metabolites from P. corylo-
philum and an alkaloid derived from tryptophan
with antimicrobial activity was isolated by using
chromatographic methods.
Material and methods
Microorganisms: P. corylophilum was isolated from
a soil sample collected in Sa o Carlos, state of Sao
Paulo, Brazil, and identied by Fundac-ao Tropical
de Pesquisas e Tecnologia Andre Toselo. The
fungus is stored as a conidial suspension on silica
gel (612 mesh, grade 40, desiccant activated) at
4 1C. The strains of Staphylococcus aureus ATCC
25923, Micrococcus luteus ATCC 9341, Pseudomo-
nas aeruginosa ATCC 27853 and Escherichia coli
ATCC 25922 were acquired from the ATCC.
Conidia production: Penicillium corylophilum
was submitted to four different media: oat agar
(Crotti et al., 1999), malt extract (Tzean et al.,
1992), potato dextrose agarPDA (Kim et al., 1990)
and Vogel medium (Vogel, 1956). The cultures were
incubated at 30 1C for 5 days. Conidia produced in
each culture were harvested with 2% of Tween 80
and counted in a Neubauer hemocytometer.
Production of secondary metabolites: The pro-
duction was carried out by inoculating 4 10
6
con-
idia/mL, obtained in oat medium, into the pre-
fermentative medium (Jackson et al., 1993) at
30 1C with shaking (120 rev/min) for 24 h. The
obtained mycelium mass was then inoculated into
three different fermentative media: Czapek (Atlas,
1995), Jackson (Jackson et al., 1993) and Vogel
(Vogel, 1956). Cultures were reincubated at 30 1C
and 120 rev/min for an additional 144 h. For
alkaloid production the fungus was grown in ve
Erlenmeyer asks containing 240 mL of pre-fermen-
tative medium in each and was transferred to
Erlenmeyer asks containing 480 mL of Czapek
medium.
Partition of the culture broth with organic
solvents and isolation of the fumiquinazoline F
from the chloroform extract: The culture broths
were separated by ltration, followed by three
times partition with chloroform and buthanol in
sequence. All the resulting organic solvents were
evaporated under vacuum and the remaining water
fractions of the culture broths were lyophilized.
The crude chloroform extract from Czapek culture
broth (3698 mg), the active one, was then sub-
mitted to column chromatography over 400 g of
silica gel 60 H (70230 mesh, Merck) and eluted
with hexane-ethyl acetate at increasing propor-
tions. The 7th fraction eluted with ethyl acetate
(1077 mg) was submitted to ash chromatography
(Still et al., 1978) over 11 g of silica gel 60
(230400 mesh), which was eluted with an isocratic
mobile phase of hexane-acetate 3:2. Afterwards,
the fourth obtained fraction (576 mg) was sub-
mitted to HPLC analysis. Instrumentations con-
sisted of a Shimadzu (SCL-10Avp, Japan)
multisolvent delivery system, Shimadzu SPD-
M10Avp Photodiode Array Detector, and an Intel
Celeron computer for analytical system control and
data collection and processing. Analytical chroma-
tography was carried out using isocratic gradient
(methanol/water/acetonitrile 11:1:8) in 25 min. A
CLC-ODS (M)4.6 250 mm
2
Shimadzu column was
used at a ow rate of 1.0 mL/min. The spectral
data from the detector were collected within
25 min over the 200400 nm range of the absorption
spectrum and the chromatograms were analyzed
and plotted at 281 nm. The alkaloid was collected
between 16.1 and 16.5 min.
General experimental procedures: A Bruker DRX-
400 spectrometer, operating at 400.13 MHz for
1
H
and 100.62 MHz for
13
C was used. All spectra were
run in CDCl
3
with TMS as internal standard. For a
HREIMS an Autospec-Micromass-EBE was used (Uni-
versidade Estadual de Campinas, UNICAMP).
Antimicrobial activity: The crude extracts were
investigated for antimicrobial activity by using the
agar diffusion method with Petri dish template
system inoculated with the assayed microorgan-
isms, followed by incubation at 37 1C for 24 h. For
this procedure, aliquots of the extracts, free of
M. Garcia Silva et al. 318
solvents, were solubilized (5.0 mg/mL) in 50%
dimethyl sulfoxide (DMSO, v/v) aqueous solution
and applied into the template holes made on the
medium surface. Negative controls were run for
DMSO aqueous solution, chloroform and buthanol.
Streptomycin (77.7 U, 50 mL, Sigma) for Gram-
negative bacteria (P. aeruginosa and E. coli),
Penicillin G (0.6075 U, 50 mL, Sigma) for M. luteus
and Penicillin G (1.215 U, 50 mL, Sigma) for S.
aureus were used as the positive controls. The
inoculum was prepared by culturing each organism
in Antibiotic N11 agar medium (Merck) for 24 h at
37 1C. The microorganisms were transferred to 0.9%
NaCl until they reached the turbidity equivalent to
0.5 McFarland standard. Each microorganism sus-
pension (0.5%) was added in Antibiotic N11 agar
medium, and distributed over the plates. The MIC
values of the isolated alkaloid were evaluated in
triplicate by microdilution broth method (Andrews,
2001). The alkaloid was solubilized in DMSO
(dimethyl sulfoxide) at 1 mg/mL, and was diluted
in Tryptone soya broth in the range of
147.536.8 mg/mL. The inoculum was adjusted to
each organism yielding a cell concentration of 10
3
colony forming units (CFU/mL). It was included
both inoculated wells, that controls the adequacy
of the broth to support the growth of the organisms
and uninoculated wells, that remains free of
antimicrobial agent to check the sterility of the
medium. Penicillin G (Sigma) was used as positive
control. The microplates (96-well) were incubated
at 37 1C for 24 h. After that, 40 mL of 2,3,5-
triphenyltetrazolium chloride (0.7%) in aqueous
solution were added to indicate the viability of
microorganisms (Leverone et al., 1996). The MIC
was determined as the lowest concentration of
drug able to totally inhibit microorganism growth.
Results
Determination of the best conditions for
conidial production
The largest number of conidia was obtained after 5
days of incubation in oat agar, Vogel, malt extract
and PDA media, in this order (Fig. 1).
Selection of fermentative medium for
antimicrobial activity production
Different fermentative media were investigated to
determine in which broth the antimicrobial activity
was produced. The highest level of antimicrobial
activity was produced by P. corylophilum when the
fungus culture was developed in the Czapek
medium, as observed for the results obtained by
the agar diffusion method (Table 1). The activity
was considered for inhibition zones wider than
12 mm since the template punches holes of
approximately 1011 mm into the agar surface.
The activity against S. aureus was found only in the
chloroform extract from Czapek culture broth.
Independently of the culture medium, the anti-
microbial activity was detected only in the chloro-
form extracts. Moreover, no extracts showed
activity against Gram-negative bacteria at the
concentration evaluated in this work.
Oat agar Vogel Malt extract PDA
0.0
2.5
5.0
7.5
Culture media
C
o
n
i
d
i
a

x

1
0
8
/
m
L
Figure 1. Determination of the best conditions for
conidial production by P. corylophilum.
Table 1. Antimicrobial activity of different extracts from cultures of Penicillium corylophilum
Microorganisms Extracts or standards Fermentative medium Inhibition zones (mm)
Micrococcus luteus Chloroformic Czapek 1470.577
Jackson 1470.577
Vogel 1370.577
Penicillin G 2670.577
Staphylococcus aureus Chloroformic Czapek 1370.577
Jackson 0970.577
Vogel 0870.577
Penicillin G 2070.577
Antibacterial activity from Penicillium corylophilum Dierckx 319
Identication and antimicrobial activity of
the alkaloid from Czapek chloroform extract
The chemical structure of the isolated compound
(Fig. 2) was identied from
1
H and
13
C Nuclear
Magnetic Ressonance (NMR), Attached Proton Test
(APT),
1
H
1
H Correlation Spectroscopy (COSY),
Heteronuclear Multiple Bond Coherence (HMQC),
Heteronuclear Multiple Quantum Coherence
(HMBC), and Mass Spectra (MS) spectral data in
comparison with previous published data (Takaha-
shi et al., 1995). The MS High Resolution showed
the [M
+
] peak m/z 358.14298. The
13
C NMR
spectrum of the compound showed 21 carbon
signals. The multiplicities of the carbons deter-
mined by APT led to the attribution of: 8 C, 11 CH,
1 CH
2
and 1 CH
3
. Among the quaternary carbons
two were attributed to amide carbonyls (d 168.9,
C-12 and 160.7, C-5). The
1
H NMR spectrum showed
two broad singlets at d 8.00 (1H, H-13) and d 5.83
(1H, H-16) indicating that these hydrogens could be
attached to nitrogens. Hence, these data suggested
an alkaloid type structure for the compound
(Fig. 2).
The
1
H NMR spectrum also showed nine hydrogen
signals between d 6.64 and 8.30. In the
1
H
1
H COSY
experiment the signals at d 7.35 (dd, J 8:1 and
0.8 Hz, H-9), d 6.87 (ddd, J 8:1; 7.1 and 0.8 Hz, H-
10), d 7.06 (ddd, J 8:1; 7.1 and 0.8 Hz, H-11) d
7.23 (dd, J 8:1 and 0.8 Hz, H-12) were coupled to
each other. The hydrogen at d 6.64 (d, J 2:5 Hz;
H-14) was coupled to the hydrogen at d 8.00 (H-13).
The chemical shifts of the carbons attached to
these hydrogens were attributed according to the
HMQC data (Table 2). These data allowed us to
propose an indolic moiety for the compound, which
was corroborated by the H-C correlations observed
in the HMBC experiment. The HMBC experiment
showed the correlation of the indolic carbon at
d109:6 (C-8a) with the hydrogens at d 3.66 (dd,
J 14:9 and 3.3 Hz, H-8
0
) and d 3.59 (dd, J 14:9
and 5.3 Hz, H-8
00
). In the HMQC experiment these
hydrogens were attached to the carbon at d 27.0.
The
1
H
1
H COSY experiment showed that these
hydrogens (H-8) are coupled to each other and to
another hydrogen at d 5.63 (dd, J 5:3 and 3.3 Hz,
H-7). The HMBC experiment showed the correlation
of both H-8 with C-7, C-8a and C-15. These data led
us to propose an alkaloid derived from tryptophan
for the isolated compound (Bergman and Bergman,
1985).
The
1
H
1
H COSY experiment also revealed that
the hydrogens at d 8.30 (ddd, J 8:1; 1.5 and
0.5 Hz, H-4), d 7.47 (ddd, J 8:1; 7.3 and 1.3 Hz,
H-3), d 7.71 (ddd, J 8:3; 7.3 and 1.5 Hz, H-2)
and d 7.53 (dd, J 8:3 and 1.5 Hz, H-1) were
coupled to each other. The aromatic carbons
chemical shifts were attributed on the basis of
HMQC data. These spectral data suggested an
anthranilic acid derivative moiety. It was corrobo-
rated by the correlation of the carbon at d 160.7
(C-5) with H-4 (d 8.30).
The remaining
1
H NMR signals were observed at d
1.29 (d, J 6:6 Hz; 3 H, H-19) and d 2.99 (q, J
Table 2.
13
C (100 MHz) and
1
H NMR (400 MHz) spectral
data for fumiquinozoline F (CDCl
3
)
Position C H
1 127.3 7.53 dd (8.3; 0.5)
2 134.6 7.71 ddd (8.3; 7.3; 1.5)
3 127.1 7.47 ddd (8.1; 7.3; 1.3)
4 126.9 8.30 ddd (8.1; 1.5; 0.5)
4a 120.3
5 160.7
6
7 55.6 5.63 dd (5.3; 3.3)
8 27.0 3.66 dd (14.9; 3.3)
3.59 dd (14.9; 5.3)
8a 109.6
8b 127.2
9 118.6 7.35 dd (8.1; 0.8)
10 121.6 6.87 ddd (8.1; 7.1; 0.8)
11 122.6 7.06 ddd (8.1; 7.1; 0.8)
12 111.1 7.23 dd (8.1; 0.8)
12a 136.0
13 8.00 br s
14 123.4 6.64 d (2.5)
15 168.9
16 5.83 br s
17 49.1 2.99 q (6.6)
17a 151.6
18
18a 147.1
19 19.3 1.29 d (6.6)
N
H
N
H
N
O
N
O
1
2
3
4
4a
5
6
8
8a 8b
9
10
11
12
12a
13 14
15
16
17
17a
18
18a
19
7
Figure 2. Structure of the fumiquinozoline F, isolated
from Czapek chloroform extract.
M. Garcia Silva et al. 320
6:6 Hz; 1 H, H-17), both coupled to each other as
observed in the
1
H
1
H COSY experiment. The HMBC
experiment showed the following correlations: H-
19 (d 1.29) correlated with a carbon at d 151.6 (C-
17a) and C-19 (d 19.3) correlated with H-16 (d
5.86). These data suggested an alanine moiety for
the compound. Therefore, the data allowed to
propose the structure of fumiquinozoline F as an
alkaloid derived from the linking of the aminoacids
tryptophan and alanine plus an anthranilic acid
unit.
Regarding the antimicrobial activity, the MIC
values of this compound against M. luteus and S.
aureus were of 99 mg/mL and 137 mg/mL, respec-
tively. Nevertheless, the penicillin G standard was
more active than the isolated compound for both
microorganisms (0.011 mg/mL and 0.023 mg/mL,
respectively).
Discussion
The expression of secondary metabolites might
depend on the culture conditions and the strains. P.
corylophilum is often isolated from temperate
climates (Malmstrom et al., 2000), but in this work
we investigated a strain isolated from Brazilian soil
sample, a tropical country.
According Gaden-Junior (2000), the metabolites
production is inuenced by medium composition,
nutrients availability and others aspects. Different
nitrogen and carbon sources may affect the synth-
esis of enzymes involved in primary and secondary
metabolism. Microorganisms are able to use a wide
variety of carbon and nitrogen sources. Never-
theless, many secondary metabolic pathways are
negatively affected by these sources favorable for
growth (Sanchez and Demain, 2002).
In the study of the secondary metabolites of the
P. corylophilum, Cutler et al. (1989) isolated the
3,7-dimethyl-8-hydroxy-6-metoxysocroman, from
the mycelia using shredded wheat medium, which
was maintained 12 days at 22 1C. By cultivation in
malt medium, strains of P. corylophilum produced
the alkaloid epoxyagroclavine I (Grabley et al.,
1992).
In this work, a two step culture was used for
antimicrobial active compounds production. In the
rst step, the microorganism was cultivated in pre-
fermentative medium, which is rich in nutrients to
increase vegetative biomass production. The har-
vested mycelium was then transferred to fermen-
tative medium for secondary metabolites
production. The fumiquinazolina F was isolated
from the chloroform extract of the Czapek fermen-
tative medium, after 144 hours of incubation. From
the three evaluated media, the Czapek one was the
best for antimicrobial metabolites production.
Fumiquinazoline F was originally obtained from a
strain of Aspergillus fumigatus, which was isolated
from the gastrointestinal tract of the marine sh
Pseudolabrus japonicus (Takahashi et al., 1995),
and it was also detected in Penicillium thymicola
(Larsen et al., 1998). It was the rst time that
Fumiquinazoline F was isolated from P. corylophi-
lum. Besides, it is the rst time that the anti-
microbial activity of this compound is been
described.
The fumiquinazolines belong to a class of
compounds possessing a wide range of biological
activities. For instance, fumiquinazolines A, B and
C from A. fumigatus showed moderate cytotoxicity
against the cultured P-388 lymphocytic cells (Nu-
mata et al., 1992). Fumiquinazolines H and I,
isolated from Acremonium sp. presented signicant
antifungal activity (Snider and Zeng, 2003).
Kariba et al. (2002), showed that extracts of
Schizozygia coffaeoides, containing indolines pre-
sented antifungal and antibacterial activities. The
berberine, benzylisoquinoline alkaloid, isolated
from Hydrastis canadensis, is active against many
Gram-positive and Gram-negative bacteria, as well
against fungi and parasites (Scazzocchio et al.,
2001).
Fumiquinazoline F is derived of tryptophan, plus
anthranilic acid and it showed value of MIC lower
than berberine, canadaline and canadine for S.
aureus (ATCC 25923) (Scazzocchio et al., 2001).
The identication of this class of alkaloid in
cultures of P. corylophilum opens new possibilities
for bioprospection of new compounds belonging to
this class with potential for the design of new
drugs.
Acknowledgements
The authors are grateful to CAPES for fellowship
and FAPESP for nancial support (Grants # 01/
14209-7 and # 99/09850-8) for nancial support.
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