toxins
Article
Initial Spore Density Has an Influence on Ochratoxin
A Content in Aspergillus ochraceus CGMCC 3.4412 in
PDB and Its Interaction with Seeds
Caiyan Li 1 , Yanmin Song 1 , Lu Xiong 1 , Kunlun Huang 1,2 and Zhihong Liang 1, *
1
2
*
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and
Nutritional Engineering, China Agricultural University, Beijing 100083, China; [email protected] (C.L.);
[email protected] (Y.S.); [email protected] (L.X.); [email protected] (K.H.)
The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture,
Beijing 100083, China
Correspondence: [email protected]; Tel.: +86-136-6105-1184
Academic Editor: Richard A. Manderville
Received: 8 June 2016; Accepted: 7 February 2017; Published: 21 April 2017
Abstract: The morphology and secondary metabolism of Aspergillus spp. are associated with initial
spore density (ISD). Fatty acids (FA) are involved in the biosynthesis of aflatoxins (AF) through
Aspergillus quorum sensing (QS). Here, we studied how ochratoxin A (OTA) was regulated by
spore density in Aspergillus ochraceus CGMCC 3.4412. The results contribute to understanding the
role of spore density in morphogenesis, OTA biosynthesis, and host–pathogen interactions. When
A. ochraceus was grown in Potato Dextrose Broth (PDB) media at different spore densities (from
101 to 106 spores/mL), more OTA was produced when ISD were increased, but a higher level of
ISD inhibited OTA biosynthesis. Seed infection studies showed that peanuts (Arachis hypogaea)
and soybeans (Glycine max) with high FA content were more susceptible to OTA production when
infected by A. ochraceus and reactive oxygen species (ROS)-induced OTA biosynthesis. These results
suggested that FA was vital for OTA biosynthesis, and that oxidative stress was closely related to
OTA biosynthesis in A. ochraceus.
Keywords: quorum sensing; density effect; Ochratoxin A; fatty acids; oxidative stress
1. Introduction
Ochratoxin A (OTA) is a mycotoxin found in a variety of agricultural commodities, which include
cereal grains and dried fruits. It is produced mainly by A. ochraceus, A. carbonarius, and A. niger [1].
OTA has been shown to be carcinogenic in animals. The kidney is the main target organ [2,3]. It has
been recognized as a Group 2B carcinogen by International Agency for Research on Cancer Collection
Center (IARC) [4], although OTA-related carcinogenicity has not been determined conclusively to
occur in humans [5,6].
Among the OTA-producing fungi, A. ochraceus is the most common in tropical regions [7], and
Penicillium verrucosum predominates in temperate regions [8]. Other OTA producing strains include A.
carbonarius and A. alliaceus [9,10].
The OTA biosynthetic pathway has not yet been determined in detail [11], although the process
of OTA biosynthesis has been analyzed [12]. These studies show that the first steps involve the
pentaketide pathway [13]. The same study indicated that chlorination was a penultimate biosynthetic
step in OTA biosynthesis [14]. The modulating factors that are involved in OTA biosynthesis are still
not totally clear. The genes pks, p450-B03, and p450-H11 are coregulated during OTA biosynthesis, and
they are likely to be engaged in a cluster that presides over a multistep enzymatic reaction [15].
Toxins 2017, 9, 146; doi:10.3390/toxins9040146
www.mdpi.com/journal/toxins
Toxins 2017, 9, 146
2 of 11
Quorum sensing (QS) is a density-dependent process that can regulate several behaviors in
bacteria, such as secretion of virulence factors, biofilm formation, competence, and bioluminescence,
and the proliferation and secondary metabolism of fungi are regulated by QS [16,17]. There is very little
information on QS in filamentous fungi [18]. However, A. flavus reproduces in a density-dependent
manner [17,19]. As the population transitions to a high cell density, the reverse phenotype is observed
to have reduced sclerotia production and increased conidiation. In addition to development, secondary
metabolism is also regulated in a density-dependent manner, where the pattern of aflatoxin (AF)
biosynthesis mirrors that of sclerotia production: much higher production occurs at low population
density [19,20].
Several molecules and genes have been found to be important in QS of A. flavus. Chief among these
are oxylipins, a group of oxygenated polyunsaturated fatty acids, which are molecular signals across
the fungal, plant and animal kingdoms. Fungal oxylipins originated from oleic acid (18:1), linoleic
acid (18:2), and linolenic acid (18:3) after an O2 molecule is added to polyunsaturated fatty acids [21].
Lipoperoxidative signalling is essential for the regulation of mycotoxin biosynthesis, conidiogenesis,
and sclerotia formation in A. nidulans, A. flavus and A. Parasiticus [15].
Oxidative stress is also regarded as a trigger of many metabolites in all organisms; previous
studies showed a direct link between oxidative stress and AF formation [22]. Reactive oxygen species
(ROS) can be observed when cells start to metabolize, and overproduction leads to the action of
oxidative stressors that are present in the environment [23]. To eliminate the underlying serious
assemblage of ROS, cells form enzymatic and nonenzymatic systems. To remove the ROS, cells
require the involvement of superoxide dismutase (SOD) and catalase (CAT), which are the most
important antioxidant enzymes [22]. Collectively referred to as precocious sexual inducer (PSI)
factors in Aspergillus spp., oxylipins are known to regulate the balance between asexual and sexual
development in Aspergillus spp. including A. flavus [24].
In this study, our objective was to do a preliminary research of quorum sensing in A. ochraceus,
which included how spore densities affected morphology, OTA production, and the expression of genes
that were involved in OTA biosynthesis. We found that OTA was regulated by density-dependent
mechanisms, and that, after increasing the cell density (from 101 to 103 spores/mL), more OTA
was produced. However, a higher concentration of ISD (from 104 to 106 spores/mL) inhibited
OTA biosynthesis, which suggested that lipid modifiers have an effect on these density-dependent
phenomena. Seed infection studies showed that peanuts and soybeans with high fatty acids (FA)
content were more susceptible to OTA accumulation when infected by A. ochraceus and ROS-induced
OTA biosynthesis.
2. Results
2.1. Spore Density-Dependent OTA Production in Potato Dextrose Broth (PDB) Media
To investigate if the ISD affects development and OTA production in A. ochraceus, 101 , 102 , 103 ,
105 , and 106 spores/mL were inoculated into PDB media. Increasing the spore density (from 101
to 106 spores/mL) led to the formation of different sizes of mycelial pellets. Cultures grown with low
cell densities (101 , 102 , and 103 spores/mL) produced fewer, but larger mycelial pellets than using high
spore densities (105 and 106 spores/mL) (Figure 1A). The phenomenon suggested that there is a link
between proliferation of biomass and density in A. ochraceus.
We observed an OTA production, density-dependent phenomenon. When the ISD increased
progressively (from 101 to 103 spores/mL), an increasing amount of OTA was detected in PDB media
after six days. OTA production reached the maximum level when cultured at the ISD of 103 spores/mL,
and then OTA decreased significantly. At 104 spores/mL, OTA production was reduced significantly
compared to production of 103 spores/mL (Figure 1B), which suggested inhibition of OTA production
in the high-density PBD culture. Growth that is accelerated in PDB media with large ISD may result
in a reduction of intracellular oxygen, and then a decrease in oxidative stress. The less oxidative
104 ,
Toxins 2017, 9, 146
3 of 11
2017, 9, 146
3 of 10
stressToxins
finally
resisted the OTA production. In addition, mycelium dry weights increased continuously
1
6
whenincreased
ISD 2017,
was9,continuously
increased
from
10 ISD
to 10wasspores/mL
(Figure
which
showed(Figure
that the
growth
6 spores/mL
Toxins
146
3 of 10in the
when
increased from
1011B),
to 10
1B),
which
high-density
PDB
not
be inhibited.
showed that
theculture
growthwas
in the
high-density
PDB culture was not be inhibited.
increased continuously when ISD was increased from 101 to 106 spores/mL (Figure 1B), which
showed that the growth in the high-density PDB culture was not be inhibited.
Figure
1. Spore
density-dependent
morphological
transitions
ochratoxin
A
Figure
1. Spore
density-dependent phenomenon
phenomenon ininmorphological
transitions
and and
ochratoxin
A
(OTA)
production
in A.
ochraceus
broth (PDB)
(PDB)media.
media.
images
(OTA)
production
in A.
ochraceusininpotato
potato dextrose
dextrose broth
(A) (A)
images
showshow
the the
Figure 1. Sporephenomena
density-dependent
morphological
and
A
density-dependent
phenomena
six days
ofin incubation;
(B) transitions
mycelial
growth
and production
OTA
density-dependent
afterafter
sixphenomenon
days
of incubation;
(B) mycelial
growth
andochratoxin
OTA
(OTA)
production
in
A.
ochraceus
in
potato
dextrose
broth
(PDB)
media.
(A)
images
show
the
◦
by A. ochraceus
CGMCC
3.4412
that was
in PDB
on (180
a shaker
(180
by A.production
ochraceus CGMCC
3.4412
that was
cultured
in cultured
PDB media
on amedia
shaker
rpm)
at rpm)
28 Catin the
density-dependent
phenomena
after initial
six days
ofdensity
incubation;
mycelial
OTA
1 1052, 103growth
4 10and
5, and
2 , 103(B)
106 The
the dark
six days
sporeof
of
dark28
for°Csixindays
withfor
initial
sporewith
density (ISD)
101 , 10(ISD)
, 10104 , , 10
, and, 10
106, spores/mL.
production
by
A.
ochraceus
CGMCC
3.4412
that
was
cultured
in
PDB
media
on
a
shaker
(180
rpm)
at
spores/mL.
The mycelium
dry weights
were
six days.
All results
were
the mean
±
mycelium
dry weights
were measured
after
sixmeasured
days. Allafter
results
were the
mean ±
standard
deviation
1, 102, 103, 104, 105, and 106
28
°C
in
the
dark
for
six
days
with
initial
spore
density
(ISD)
of
10
standard deviation (SD) of three measurements mixed from three independent samples.
(SD) of three measurements mixed from three independent samples.
spores/mL. The mycelium dry weights were measured after six days. All results were the mean ±
standardof
deviation
(SD) of three
measurements
from
three independent samples.
2.2. Influence
Spore Densities
on Transcript
Levelsmixed
of OTA
Biosynthesis-Related
Genes
2.2. Influence of Spore Densities on Transcript Levels of OTA Biosynthesis-Related Genes
To determine
how
ISD affects
OTA production,
expression
of OTA biosynthesis-related
genes
2.2. Influence
of Spore
Densities
on Transcript
Levels of OTA
Biosynthesis-Related
Genes
To determine how ISD affects OTA production, expression of OTA biosynthesis-related genes
were examined by quantitative reverse transcription PCR (qRT-PCR) in mycelia that were initiated
To1determine
how ISD affects
OTA
production, expression
of OTAin
biosynthesis-related
genes
werewith
examined
by quantitative
reverse
transcription
PCR (qRT-PCR)
mycelia that were
initiated
10 , 102, 103, 104, 105, and 106 spores/mL at 28 °C for six days. In A. ochraceus, the pks transcript
1
2
3
4
5
6
◦
were
examined
by
quantitative
reverse
transcription
PCR
(qRT-PCR)
in
mycelia
that
were
initiated
with level
10 , 10
,
10
,
10
,
10
,
and
10
spores/mL
at
28
C
for
six
days.
In
A.
ochraceus,
the
pks
transcript
3
increased at first (Figure 2), reached a peak of 10 spores/mL, and then descended while the
1, 102, 103, 104, 105, and 106 spores/mL at 28 °C for
six about
days. In
A.and
ochraceus,
the
pks
transcript
with
10
3spores/mL
levelspore
increased
at increased
first (Figure
2), reached
a peak of 103was
spores/mL,
then descended
while the
density
(Figure
1B); the accumulation
2.5-fold
higher
at 10
3 spores/mL, and then descended
3
level
increased
at
first
(Figure
2),
reached
a
peak
of
10
while
the than
4
sporethan
density
at 10 increased
spores/mL.(Figure 1B); the accumulation was about 2.5-fold higher at 10 spores/mL
3spores/mL
spore
density
increased
(Figure
1B);
the
accumulation
was
about
2.5-fold
higher
at
10
4
at 10 spores/mL.
than at 104spores/mL.
Figure 2. High ISD repressed the expressions of OTA biosynthesis genes in A. ochraceus. qRT-PCR
(quantitative reverse transcription PCR) was used to analyze expressions of OTA biosynthesis genes
Figure
2. p450-b03,
High
ISD
repressed
the
of
OTA
biosynthesis
genes
A.
ochraceus.
qRT-PCR
Figure
2. High
ISD
repressed
theexpressions
expressions
of 3.4412
OTA biosynthesis
genes
in in
A.for
ochraceus.
(pks,
p450-h11)
by A. ochraceus
CGMCC
cultured in PDB
media
six days qRT-PCR
with ISD
(quantitative
reverse
transcription
PCR)
was
used
to
analyze
expressions
of
OTA
biosynthesis
(quantitative
reverse
transcription
PCR)
was
used
to
analyze
expressions
of
OTA
biosynthesis
genes
1
2
3
4
5
6
of 10 , 10 , 10 , 10 , 10 , and 10 spores/mL. The relative expressions were quantified by the expressiongenes
(pks, p450-b03,
p450-h11)
by ochraceus
A.are
ochraceus
CGMCC
3.4412
cultured
in PDB media
with
ISDISD of
(pks, level
p450-b03,
by Data
A.
CGMCC
3.4412
cultured
in
mediafor
forsix
sixdays
days
with
of thep450-h11)
Ao18s
gene.
the means
of three
separate
experiments.
1, 10
2, 10
3, 1054, 105, and6106 spores/mL. The relative expressions were quantified by the expression
1
2
3
4
of
10
10 , 10 , 10 , 10 , 10 and 10 spores/mL. The relative expressions were quantified by the expression
level
of Ao18s
the Ao18s
gene.
Data
are
themeans
means
ofp450-B03
three separate
transcription
level
ofare
p450-H11
andof
genesexperiments.
both achieved peaks at 101 spores/mL,
levelThe
of the
gene.
Data
the
three
separate
experiments.
and then decreased, which did not mirror closely that of the pks gene under the same growth
The transcription
leveltheir
of p450-H11
and p450-B03
peaks
101process
spores/mL,
conditions;
this indicated
non-involvement
with genes
pks in both
OTAachieved
biosynthesis
in at
this
and
The
transcription
level
of
p450-H11
and
p450-B03
genes
both
achieved
peaks
at
101 growth
spores/mL,
and
then
decreased,
which
did
not
mirror
closely
that
of
the
pks
gene
under
the
same
that they have a different manner of regulation. Nevertheless, we noticed that cultures with a high
and then
decreased,
which didtheir
not mirror
closely that
of the
genebiosynthesis
under the same
growth
conditions;
conditions;
this indicated
non-involvement
with
pks pks
in OTA
in this
process
and
this indicated
their
non-involvement
with
pks
in
OTA
biosynthesis
in
this
process
and
that
that they have a different manner of regulation. Nevertheless, we noticed that cultures with a they
high have
a different manner of regulation. Nevertheless, we noticed that cultures with a high initial density
Toxins 2017, 9, 146
4 of 11
restrained
the 9,
expression
of both p450-H11 and p450-B03 genes simultaneously. Both genes4 also
Toxins 2017,
146
of 10 can
be regulated during growth at different spore densities when the ISD increased gradually (from 102
density restrained
expression of
both p450-H11
and p450-B03
simultaneously.
Both
to 106initial
spores/mL);
pks gene the
transcription
behaved
in a similar
fashion.genes
However,
the highest
levels
1
genes
also
can
be
regulated
during
growth
at
different
spore
densities
when
the
ISD
increased
were observed at 10 spores/mL, where p450-H11 and p450-B03 transcript levels were 2.2- and 7.3-fold
2 to 106 spores/mL); pks gene transcription behaved in a similar fashion. However,
(from10
3 spores/mL.
highergradually
than at 10
the highest levels were observed at 101 spores/mL, where p450-H11 and p450-B03 transcript levels
3 spores/mL.
were
2.2- and 7.3-fold
higher than
at 10Talk
2.3. Oxylipin-Mediated
Host-Fungus
Cross
2.3. examined
Oxylipin-Mediated
Host-Fungus
Talk to produce OTA in live seeds. Here, we assessed the
We
the capacity
of A.Cross
ochraceus
effect of spore
density on
production.
Surface-sterilized
infected
with
suspensions
We examined
the OTA
capacity
of A. ochraceus
to produce OTAseeds
in livewere
seeds.
Here, we
assessed
the
of 103effect
spores/mL
106on
spores/mL
of A. ochraceus
at the same
It was evident
that seeds of
of spore and
density
OTA production.
Surface-sterilized
seedstime.
were infected
with suspensions
3 spores/mL
peanuts
and
soybeans,and
which
a highofoil
more
susceptible
to evident
infection
byseeds
A. ochraceus
of 10
106 have
spores/mL
A.content,
ochraceuswere
at the
same
time. It was
that
of
peanuts
andmaize(Zea
soybeans, which
high oil content, were
more
susceptible
to infection
by A. and
compared
with
mays)have
andawheat(Triticum)
because
the
surface color
of peanuts
ochraceus
compared
with maize(Zea
mays)
andIn
wheat(Triticum)
the surface
of peanuts
soybeans
change
more significantly
(Figure
3A).
addition, OTAbecause
biosynthesis
was color
significantly
higher
3 spores/mL,
and soybeans
change than
more in
significantly
In addition,
OTA
biosynthesispeanut
was significantly
in peanuts
and soybeans
maize and(Figure
wheat3A).
(Figure
3B, at 10
= 326 ng/mL;
3 spores/mL, peanut = 326
higher
in peanuts
and
soybeans
than in maize
and
wheat
(FigureAt
3B,10
at3 10
soybean
= 359
ng/mL;
maize
= 8 ng/mL;
wheat
= 11
ng/mL).
spores/mL,
OTA production
ng/mL; soybean = 359 ng/mL; maize = 8 ng/mL; wheat = 11 ng/mL). At 103 spores/mL, OTA
was much greater compared to seeds with 106 spores/mL,
which
suggested
that
OTA
production
production was much greater compared to seeds with 106 spores/mL, which suggested that OTA
was inhibited
in seeds with a high density of spores. Conidial numbers increased with increasing cell
production was inhibited in seeds with a high density of spores. Conidial numbers increased with
density.
ISD
of
seeds
with ISD
103 of
spores/mL
fewer
conidia
than
the ISD
increasing cell
density.
seeds with consistently
103 spores/mLproduced
consistently
produced
fewer
conidia
thanof
theseeds
6
6
at 10 ISD
spores/mL
in
both
peanuts
and
corn
seeds
(Figure
3C).
Conidial
production
with
ISD
of 103
of seeds at 10 spores/mL in both peanuts and corn seeds (Figure 3C). Conidial production with
6 spores/mL
3 spores/mL
spores/mL
decreased
63%
and 67%63%
compared
seedswith
at 10seeds
on peanuts
and soybeans,
ISD of 10
decreased
and 67%with
compared
at 106 spores/mL
on peanuts
and
soybeans,
(p ≤ 0.05).the
However,
levels of conidiation
and theofamount
of OTA
respectively
(p respectively
≤ 0.05). However,
levels ofthe
conidiation
and the amount
OTA biosynthesis
biosynthesis
were
low
on maize
and wheat,
regardlessthe
of ISD
whether
was 103 spores/mL
or 106
were low
on maize
and
wheat,
regardless
of whether
wasthe
103ISD
spores/mL
or 106 spores/mL
spores/mL
(Figure
4B).
be that
the A.3.4412
ochraceus
CGMCC
strain has lostability
the in
(Figure
4B). It may
be that
theItA.may
ochraceus
CGMCC
strain
has lost3.4412
the density-sensing
density-sensing ability in maize and wheat.
maize and wheat.
(A) growth
on seeds;
OTAproduction
production on
on seeds.
seeds. Colonization
of of
seeds
as reflected
by by
FigureFigure
3. (A)3. growth
on seeds;
(B)(B)
OTA
Colonization
seeds
as reflected
conidiation
levels:
Wild
type
CGMCC
3.4412
was
grown
on
seeds
for
six
days
at
28
°C;
(C)
conidia
◦
conidiation levels: Wild type CGMCC 3.4412 was grown on seeds for six days at 28 C; (C) conidia
were counted from three replicates that contained 10 seeds each.
were counted from three replicates that contained 10 seeds each.
Toxins 2017, 9, 146
Toxins 2017, 9, 146
5 of 10
5 of 11
2.4. Effects of A. ochraceus Infection on MDA
2.4. Effects of A. ochraceus Infection on MDA
Lipid peroxidation was estimated by measuring malonic dialdehyde (MDA) levels (Figure 4).
MDALipid
levelsperoxidation
were higher was
in soybeans
than
peanuts, maize,
or dialdehyde
wheat. In soybeans,
the formation
estimated
byinmeasuring
malonic
(MDA) levels
(Figure of
4).
6 spores/mL.
MDA levels
was 4.0
U/mg
protein
at 103 spores/mL
and 1.5maize,
U/mgor
protein
In contrast,
were
higher
in soybeans
than in peanuts,
wheat.atIn10
soybeans,
the formation
of
3
6
there
was
less
MDA
formed
in
peanuts
maize
and
wheat,
and
there
was
a
small
reduction
in
MDA
MDA was 4.0 U/mg protein at 10 spores/mL and 1.5 U/mg protein at 10 spores/mL. In contrast,
formation
withMDA
ISD offormed
106 spores/mL
compared
withwheat,
103 spores/mL
peanuts,
maize,
and wheat.
there was less
in peanuts
maize and
and thereinwas
a small
reduction
in MDA
6
formation with ISD of 10 spores/mL compared with 103 spores/mL in peanuts, maize, and wheat.
Figure 4. Effect of ISD on lipid peroxidation in seeds infected by A. ochraceus.
Figure 4. Effect of ISD on lipid peroxidation in seeds infected by A. ochraceus.
2.5. Effects of A. ochraceus Infection on ROS Activity and Antioxidant Enzymes
2.5. Effects of A. ochraceus Infection on ROS Activity and Antioxidant Enzymes
A. ochraceus induced ROS accumulation, which was determined quantitatively with
A. ochraceus induced ROS accumulation, which was determined quantitatively with
dichloro-dihydrofluorescein diacetate (H2DCFDA), and the results suggested that A. ochraceus infection
dichloro-dihydrofluorescein diacetate (H2DCFDA), and the results suggested that A. ochraceus
led to a continuous and significant increase in ROS content. The accumulation of ROS in peanuts,
infection led to a continuous and significant increase in ROS content. The accumulation of ROS in
soybeans, maize, and wheat was increased compared to the control after being infected by A. ochraceus,
peanuts, soybeans, maize, and
wheat was increased compared to the control after being infected by
and the ROS content at 103 spores/mL was higher than that at 106 spores/mL in peanuts and
A. ochraceus, and the ROS content at 103 spores/mL was higher than that at 106 spores/mL in peanuts
soybeans. On the contrary, the ROS content in maize and wheat decreased, and there was no significant
and soybeans. On the contrary, the ROS
content in maize and wheat decreased, and there was no
difference between ROS content at 103 spores/mL and 106 spores/mL. In wheat, the ROS content of
significant
difference between ROS content at 103 spores/mL and 106 spores/mL.
In wheat, the ROS
3
10 spores/mL did not change significantly, and it decreased with ISD at 106 spores/ mL (Figure 5A).
content of 103 spores/mL did not change significantly, and it decreased with ISD at 106 spores/ mL
The activity of the antioxidant enzymes SOD, CAT, and peroxidase (POD) in seeds of peanuts,
(Figure 5A).
soybeans, maize, and wheat decreased significantly after six days of incubation with A. ochraceus
The activity of the antioxidant enzymes SOD, CAT, and peroxidase (POD) in seeds of peanuts,
(Figure 5B–D). The activity of CAT, SOD, and POD all significantly decreased in infected seeds
soybeans, maize, and wheat decreased significantly after six days of incubation with A. ochraceus
compared to activity in the control. These antioxidant enzymes displayed significantly lower activity
(Figure 5B–D). The activity of CAT, SOD, and POD all significantly decreased in infected seeds
than under control conditions. However, the activity of CAT enzymes was higher than that in control
compared to activity
in the control. These antioxidant enzymes displayed significantly lower activity
with ISD of 106 spores/mL in maize. POD activity was not significantly different at 103 spores/mL
than under control conditions. 6However, the activity of CAT6 enzymes was higher than that in
compared to those detected at 10 spores/mL. SOD activity at 10 spores/mL then decreased to levels
control with ISD of 106 spores/mL
in maize. POD activity was not significantly different at 103
lower than that observed at 103 spores/mL in peanuts and soybeans. However, the activity of CAT
spores/mL compared to those detected at 106 spores/mL. SOD activity at 106 spores/mL then
showed no significant changes. In peanuts and soybeans, the reverse trend was observed, with higher
decreased to 6levels lower than that 3observed at 103 spores/mL in peanuts and soybeans. However,
activity at 10 spores/mL than at 10 spores/mL.
the activity of CAT showed no significant changes. In peanuts and soybeans, the reverse trend was
observed, with higher activity at 106 spores/mL than at 103 spores/mL.
Toxins
Toxins 2017, 9, 146
66 of
of 10
11
Figure 5. (A) The accumulation of reactive oxygen species (ROS) in seeds after being infected by
A.
ochraceus.
content was measured
H2DCFDA
fluorescence;
(B) the
of antioxidant
Figure
5. (A) ROS
The accumulation
of reactivebyoxygen
species
(ROS) in seeds
afteractivity
being infected
by A.
enzyme
superoxide
dismutase
(SOD)
(U/mg
protein)
at
different
times
of
incubation
in
seeds
infected
ochraceus. ROS content was measured by H2DCFDA fluorescence; (B) the activity of antioxidant
by A. ochraceus;
(C) the
activity of(SOD)
antioxidant
peroxidase
(POD)
(U/mg
protein) at in
different
enzyme
superoxide
dismutase
(U/mgenzyme
protein)
at different
times
of incubation
seeds
times of incubation in seeds infected by A. ochraceus and (D) the activity of antioxidant enzyme catalase
infected by A. ochraceus; (C) the activity of antioxidant enzyme peroxidase (POD) (U/mg protein) at
(CAT) (U/mg protein) at different times of incubation in seeds infected by A. ochraceus. The results are
different times of incubation in seeds infected by A. ochraceus and (D) the activity of antioxidant
the mean ± SD of three determinations from three separate experiments.
enzyme catalase (CAT) (U/mg protein) at different times of incubation in seeds infected by A.
ochraceus. The results are the mean ± SD of three determinations from three separate experiments.
3. Discussion
3. Discussion
OTA was more productive when ISD increased, although higher densities of ISD restrained
OTAOTA
biosynthesis.
phenomenon
observed
in potato
dextrose
agarof(PDA)
and malt
was moreThis
productive
when was
ISD also
increased,
although
higher
densities
ISD restrained
extract
agar (MEA)This
media.
QS is a density-dependent
that results
a coordinated
OTA
biosynthesis.
phenomenon
was also observedphenomenon
in potato dextrose
agar in
(PDA)
and malt
response
from
the
population
[20].
A.
flavus
reproduces
in
a
density-dependent
manner
where
low
extract agar (MEA) media. QS is a density-dependent phenomenon that results in a coordinated
population
densities
are
characterized
by
increased
sclerotia
production
and
reduced
conidiation.
response from the population [20]. A. flavus reproduces in a density-dependent manner where low
As the population
transitions
to high cell
thesclerotia
reverse production
phenotype and
is observed
reduced
population
densities
are characterized
by density,
increased
reducedwith
conidiation.
sclerotia
production
and
increased
conidiation
[19,20].
However,
the
phenomenon
of
morphological
As the population transitions to high cell density, the reverse phenotype is observed with reduced
transformation
from and
sclerotia
to conidiation
was[19,20].
not observed
when
ochraceus wasofcultured
in PDA
sclerotia
production
increased
conidiation
However,
theA.phenomenon
morphological
and
MEA
(Figure
6).
Sclerotia
production
might
be
related
to
culture
media.
The
phenomenon
transformation from sclerotia to conidiation was not observed when A. ochraceus was culturedwas
in
observed
when(Figure
A. flavus6).was
cultured
in glucose
minimal
medium
(AOBOX,
Beijing,
Based
PDA
and MEA
Sclerotia
production
might
be related
to culture
media.
TheChina).
phenomenon
on a observed
previous when
study, A.
theflavus
transcription
of p450-H11
andminimal
p450-B03medium
genes mirrored
closely
that
of the
was
was cultured
in glucose
(AOBOX,
Beijing,
China).
pks
gene
under
the
same
conditions,
which
suggested
the
involvement
of
the
abovementioned
Based on a previous study, the transcription of p450-H11 and p450-B03 genes mirrored closelythree
that
genes
in OTA
[15],conditions,
but in our which
experiments,
the the
twoinvolvement
genes did not
mirror that of
of
the pks
genebiosynthesis
under the same
suggested
of closely
the abovementioned
the pks
geneininOTA
this process.
It is probable
more
genes thatthe
are two
involved
a density-dependent
three
genes
biosynthesis
[15], but that
in our
experiments,
genesindid
not closely mirror
will
be
discovered.
that of the pks gene in this process. It is probable that more genes that are involved in a
Studies of the interactions
of A. ochraceus with seeds have also implicated the role of fatty acids and
density-dependent
will be discovered.
oxidative
stress
on
OTA
production.
this study,
weseeds
demonstrated
seeds enriched
as
Studies of the interactions of A. In
ochraceus
with
have also that
implicated
the role in
of FA,
fattysuch
acids
peanuts
and
soybeans,
were
more
susceptible
to
OTA
accumulation
when
infected
by
A.
ochraceus.
It
is
and oxidative stress on OTA production. In this study, we demonstrated that seeds enriched in FA,
generally
known and
that seeds
of plants
as susceptible
maize, peanuts,
and walnuts
that are
rich in
oils areby
more
such
as peanuts
soybeans,
weresuch
more
to OTA
accumulation
when
infected
A.
ochraceus. It is generally known that seeds of plants such as maize, peanuts, and walnuts that are rich
Toxins 2017, 9, 146
7 of 11
Toxins 2017, 9, 146
7 of 10
susceptible
to AFsusceptible
productiontowhen
infected bywhen
AF-producing
Aspergillus
spp. [25].
FA is essential
for
in oils are more
AF production
infected by
AF-producing
Aspergillus
spp. [25].
AF
biosynthesis
[26].
Here,
we
proved
that
FA
is
also
vital
for
OTA
biosynthesis.
FA is essential for AF biosynthesis [26]. Here, we proved that FA is also vital for OTA biosynthesis.
Oxidative
Oxidative stress
stress is
is also
also aa trigger
trigger of
of many
many metabolites
metabolites in
in organisms.
organisms. ROS
ROS is
is important
important in
in fungal
fungal
development
[27,28].
In
A.
parasiticus,
ROS
can
control
sclerotium
formation
[29].
In
A.
flavus,
development [27,28]. In A. parasiticus, ROS can control sclerotium formation [29]. In A. flavus, the
the
biosynthesis
connection
with
various
stages
of fungal
development
[30],[30],
and
biosynthesis of
ofmycotoxins
mycotoxinshas
hasa aclose
close
connection
with
various
stages
of fungal
development
oxidative
stressstress
is essential
in AF production
[31]. Our
results
that oxidative
stress andstress
OTA
and oxidative
is essential
in AF production
[31].
Our indicated
results indicated
that oxidative
production
in
A.
ochraceus
were
linked
tightly.
We
noticed
that
increased
levels
of
ROS
had
a
close
and OTA production in A. ochraceus were linked tightly. We noticed that increased levels of ROS had
connection
with increased
levels of OTA
biosynthesis
in A. ochraceus
it infected
different
seeds.
a close connection
with increased
levels
of OTA biosynthesis
inwhen
A. ochraceus
when
it infected
Further
investigations
of
how
ROS
triggers
OTA
production
may
help
to
control
the
biosynthesis
different seeds. Further investigations of how ROS triggers OTA production may help to control the
of
mycotoxins.
biosynthesis
of mycotoxins.
Figure 6. Morphology of A. ochraceus under different spore densities in malt extract agar (MEA) and
potato dextrose
dextrose agar
agar (PDA).
(PDA).
4. Conclusions
The results of
of this
this study
study suggested
suggestedthat
that when
when A.
A. ochraceus
ochraceus was
was grown
grown in
in PDB
PDB at different spore
1
6
densities (from 10 to 10 cells/mL),
cells/mL),OTA
OTA production
production was
was more
more when ISD increased, but higher
densities inhibited
inhibitedOTA
OTAbiosynthesis.
biosynthesis.Our
Ourstudy
study
fungal
infection
in seeds
showed
peanuts
densities
of of
fungal
infection
in seeds
showed
that that
peanuts
and
and
soybeans
that
are
rich
in
FA
are
more
susceptible
to
OTA
accumulation
when
infected
by
A.
soybeans that are rich in FA are more susceptible to OTA accumulation when infected by A. ochraceus.
ochraceus.
These
results
that FA for
wasOTA
important
for OTA
biosynthesis.
Meanwhile,
These
results
indicated
thatindicated
FA was important
biosynthesis.
Meanwhile,
oxidative
stress and
oxidative
stress in
and
production
in A.tightly.OTA
ochraceus were
linked
tightly.OTA
produced
by A.
OTA
production
A. OTA
ochraceus
were linked
produced
by A.
ochraceus may
be related
to
ochraceus
may
be
related
to
FA
and
oxidative
stress.
Therefore,
antioxidants
could
prevent
the
FA and oxidative stress. Therefore, antioxidants could prevent the production of OTA in agricultural
production ofUnderstanding
OTA in agricultural
commodities.
common
mechanisms
of
commodities.
the common
mechanismsUnderstanding
of biosynthesis the
in different
mycotoxins
would
biosynthesis
in
different
mycotoxins
would
promote
the
development
of
suitable
strategies
to
promote the development of suitable strategies to control several mycotoxins simultaneously.
control several mycotoxins simultaneously.
5. Materials and Methods
5. Materials and Methods
5.1. Fungal Strain and Culture Conditions
5.1. Fungal
Strain strain
and Culture
Conditions
A. ochraceus
CGMCC
3.4412 was purchased from the Institute of Microbiology, Chinese
Academy
of Sciences,
China.
was cultured
PDA medium
(AOBOX, Chinese
Beijing,
A. ochraceus
strainBeijing,
CGMCC
3.4412The
wasstrain
purchased
from theonInstitute
of Microbiology,
◦
China)
andofincubated
28 C. To
examThe
OTA
production,
A. ochraceus
CGMCC
3.4412
was inoculated
Academy
Sciences,atBeijing,
China.
strain
was cultured
on PDA
medium
(AOBOX,
Beijing,
◦ C in dark conditions for 6 days to obtain a heavy sporulation culture. The
and
cultured
on
PDA
at
28
China) and incubated at 28 °C. To exam OTA production, A. ochraceus CGMCC 3.4412 was
spores
wereand
resuspended
in PDA
sterileatdistilled
contained
20. aSpore
concentration
inoculated
cultured on
28 °C inwater
dark that
conditions
for 0.05%
6 daysTween
to obtain
heavy
sporulation
7 spores/mL. The
was
counted
using
a
haemacytometer,
and
the
final
concentration
was
adjusted
to
10
culture. The spores were resuspended in sterile distilled water that contained 0.05% Tween 20. Spore
spore
suspensions
diluted
when
necessary. Sporeand
suspensions
(5 mL) were kept
45 mL Potato
concentration
was were
counted
using
a haemacytometer,
the final concentration
wasinadjusted
to 107
Dextrose
Broth
Beijing, China)
thenecessary.
mixture was
shaken
(180 rpm)
28 ◦were
C in
spores/mL.
The(AOBOX,
spore suspensions
were media,
diluted and
when
Spore
suspensions
(5 atmL)
the
dark.
kept in 45 mL Potato Dextrose Broth (AOBOX, Beijing, China) media, and the mixture was shaken
(180 rpm) at 28 °C in the dark.
Toxins 2017, 9, 146
8 of 11
5.2. Determinations of Fungal Dry Weights and OTA Content
To determine dry weights, mycelia grown in 50 mL media were harvested by two layers of filter
paper, and then washed by sterilized water; before obtaining a constant weight, mycelia also should be
freeze-dried. For extraction of OTA from media, 1 mL of medium from each flask were extracted with
an equal volume of chloroform. The chloroform fraction was evaporated under nitrogen. The residues
were redissolved in 1 mL methanol. Samples were stored at −80 ◦ C until analyzed quantitatively for
mycotoxins by an ELISA kit.
5.3. Extraction of RNA and Preparation of cDNA
Mycelia were harvested and frozen immediately at −80 ◦ C. To extract the RNA, the mycelia were
ground in liquid nitrogen in a pre-cooled mortar and pestle.
Total RNA was extracted from the ground mycelia using an RNeasy Plant kit. cDNA was prepared
using cDNA Quantscript RT kit.
5.4. qRT-PCR Analysis of Gene Transcript Accumulation
qRT-PCR was conducted in a Bio-Rad C1000TM Thermal Cycler (Hercules, CA, USA) installed
with CFX96TM software (CFX96 system, Bio-Rad, Hercules, CA, USA) using the primers listed in
Table 1 and replicated three times in a 25 µL reaction volume. Real Master Mix (SYBR green) was used.
The PCR amplification program was as follows: one cycle of 95 ◦ C for 10 min followed by 39
cycles of denaturation (15 s at 95 ◦ C), annealing (15 s at 58 ◦ C), and extension (30 s at 72 ◦ C), with
a final elongation of 72 ◦ C for 1 min. The amplification was followed by a melting curve analysis
program with a temperature range of 65–95 ◦ C. 18S ribosomal RNA was used an endogenous control.
The gene expression values were normalized to those of reference gene Ao18s. The relative gene
expression was evaluated using the 2−∆∆CT method, where ∆∆CT = (CT, Target − CT, Ao18s) Infected
−(CT, Target − CT, Ao18s) Control.
Table 1. PCR primers used in this study.
Primer
Pks-F
Pks-R
P450-B03-F
P450-B03-R
P450-H11-F
P450-H11-R
Ao-18s-F
Ao-18s-R
Sequence
TTCTCTGCGCTTCTCACATC
AACATCATAAGAGGTCAACA
CTCGGTGACATCAGGGGTATC
AGCGTATTCAGTCACTCATTCAGA
AGAACGGGATGCCAAAACAGTGAG
AAGAATGCGAGGGATGGGATAACC
ATGGCCGTTCTTAGTTGGTG
GTACAAAGGGCAGGGACGTA
Annealing Temperature
◦C
60
60 ◦ C
60 ◦ C
60 ◦ C
64 ◦ C
64 ◦ C
60 ◦ C
60 ◦ C
Reference
[11]
[11]
[11]
[11]
[11]
[11]
[15]
[15]
5.5. Peanut, Bean, Wheat and Maize Seed Infection Studies and OTA Analysis from Seeds
Infection studies and OTA analysis of peanuts, soybeans, wheat, and maize seeds were carried
out as described previously [17]. First, seeds were surface sterilized by dipping them in a beaker that
contained 0.05% sodium hypochlorite in sterile water for 3 min. Then, seeds were washed by sterile
distilled water, 70% ethanol, and sterile distilled water again. This was followed by complete drainage,
and then the seeds were placed in a Petri dish until the time of infection. All steps were performed
aseptically in a biosafety hood.
For the infection experiment, 200 g of seeds was immersed in 200 mL of sterile distilled water
(control) or sterile distilled water with fungal conidia in a 500 mL flask while shaking for 30 min at
200 rpm. Seeds were placed in Petri dishes that were lined with moist filter paper to maintain high
humidity and incubated for 6 days at 28 ◦ C in dark conditions. Then, 200 g of seeds per replicate
was transferred to a humidity chamber and incubated as described previously. Seeds were collected
in tubes with the addition of 3 mL of 0.01% Tween 80 (vol/vol, water) after 6 days, and vortexed
Toxins 2017, 9, 146
9 of 11
vigorously. One milliliter was removed from each sample, and 3 mL of chloroform was added, followed
by shaking for 10 min at 200 rpm. Samples were allowed to stand for an additional 10 min at room
temperature, vortexed briefly, and centrifuged for 5 min at 8000 rpm to collect the organic lower phase.
The chloroform extract was evaporated to dryness under nitrogen. The residues were redissolved in
1 mL methanol.
5.6. Measurement of ROS, MDA, and Antioxidant Enzymes
The ROS content was measured by H2DCFDA fluorescence, according to the ROS kit. The ROS
kit and MAD, SOD, POD, CAT were all purchased from Nanjing Jiancheng Bioengineering Institute,
Nanjing, China [32], in relation to MDA, SOD, POD, CAT.
Acknowledgments: This study was supported by the National Natural Science Foundation of China (31671947).
Author Contributions: Caiyan Li and Zhihong Liang conceived and designed the experiments; Caiyan Li
performed the experiments, Yanmin Song and Lu Xiong were responsible for statistical analysis and data analysis;
Caiyan Li wrote the manuscript; and Zhihong Liang and Kunlun Huang corrected the written English. All authors
have read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
ISD
FA
AF
QS
OTA
CGMCC
IARC
ROS
SOD
CAT
MDA
POD
Initial spore density
Fatty acids
Aflatoxin
Quorum sensing
Ochratoxin A
China General Microbiological Culture
International Agency for Research on Cancer Collection Center
Reactive oxygen species
Superoxide Dismutase
Catalase
Malonic dialdehyde
Peroxidase
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Bui-Klimke, T.R.; Wu, F. Ochratoxin A and human health risk: A review of the evidence. Crit. Rev. Food
Sci. Nutr. 2015, 55, 1860–1869. [CrossRef] [PubMed]
Evaluation of certain mycotoxins in food. In Fifty-Sixth Report of the Joint FAO/WHO Expert Committee on Food
Additives; World Health Organization: Geneva, Switzerland, 2002; Volume 906, i–viii, pp. 1–62.
Duarte, S.C.; Pena, A.; Lino, C.M. Human ochratoxin A biomarkers-From exposure to effect. Crit. Rev. Toxicol.
2011, 41, 187–212. [CrossRef] [PubMed]
Boeniger, M.; Combes, R.D.; Dragani, T.A.; Fitzgerald, J.; Hardin, B.D.; Hayes, R.B.; Kadlubar, F.; Kinlen, L.J.;
Kriek, E.; Lynge, E. Iarc Monographs Program on the Evaluation Of Carcinogenic Risks To Humans.
IARC Monog. Eval. Carcinog. 1993, 7, 13–33.
Fazekas, B.; Tar, A.; Kovacs, M. Ochratoxin a content of urine samples of healthy humans in Hungary.
Acta Vet. Hung. 2005, 53, 35–44. [CrossRef] [PubMed]
Petzinger, E.; Ziegler, K. Ochratoxin A from a toxicological perspective. J. Vet. Pharmacol. Ther. 2000, 23,
91–98. [CrossRef] [PubMed]
Pardo, E.; Marin, S.; Ramos, A.; Sanchis, V. Occurrence of ochratoxigenic fungi and ochratoxin A in green
coffee from different origins. Food Sci. Technol. Int. 2004, 10, 45–49. [CrossRef]
Pitt, J.I. Toxigenic fungi: Which are important? Med. Mycol. 2000, 38, 17–22. [CrossRef] [PubMed]
Bau, M.; Bragulat, M.R.; Abarca, M.L.; Minguez, S.; Cabañes, F.J. Ochratoxigenic species from Spanish wine
grapes. Int. J. Food Microbiol. 2005, 98, 125–130. [CrossRef] [PubMed]
Toxins 2017, 9, 146
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
10 of 11
Bayman, P.; Baker, J.L.; Doster, M.A.; Michailides, T.J.; Mahoney, N.E. Ochratoxin production by the
Aspergillus ochraceus group and Aspergillus alliaceus. Appl. Environ. Microbiol. 2002, 68, 2326–2329. [CrossRef]
[PubMed]
O’Callaghan, J.; Stapleton, P.C.; Dobson, A.D. Ochratoxin A biosynthetic genes in Aspergillus ochraceus are
differentially regulated by pH and nutritional stimuli. Fung. Genet. Biol. 2006, 43, 213–221. [CrossRef]
[PubMed]
Varga, J.; Rigo, K.; Teren, J.; Mesterhazy, A. Recent advances in ochratoxin research - II. Biosynthesis, mode
of action and control of ochratoxins. Cereal Res. Commun. 2001, 29, 93–100.
Harris, J.P.; Mantle, P.G. Biosynthesis of ochratoxins by Aspergillus ochraceus. Phytochemistry 2001, 58, 709–716.
[CrossRef]
Lee, S.E.; Park, B.S.; Bayman, P.; Baker, J.L.; Choi, W.S.; Campbell, B.C. Suppression of ochratoxin biosynthesis
by naturally occurring alkaloids. Food Addit. Contam. 2007, 24, 391–397. [CrossRef] [PubMed]
Reverberi, M.; Punelli, F.; Scarpari, M.; Camera, E.; Zjalic, S.; Ricelli, A.; Fanelli, C.; Fabbri, A.A.
Lipoperoxidation affects ochratoxin A biosynthesis in Aspergillus ochraceus and its interaction with wheat
seeds. Appl. Microbiol. Biotechnol. 2010, 85, 1935–1946. [CrossRef] [PubMed]
Albuquerque, P.; Casadevall, A. Quorum sensing in fungi—A review. Med. Mycol. 2012, 50, 337–345.
[CrossRef] [PubMed]
Brown, S.H.; Scott, J.B.; Bhaheetharan, J.; Sharpee, W.C.; Milde, L.; Wilde, L.; Wilson, R.A.; Keller, N.P.
Oxygenase coordination is required for morphological transition and the host-fungus interaction of
Aspergillus flavus. Mol. Plant-Microbe Interact. 2009, 22, 882–894. [CrossRef] [PubMed]
Raina, S.; Vizio, D.D.; Keshavarz, T. Quorum sensing in filamentous fungi. J. Biotechnol. 2010, 150, 76.
[CrossRef]
Brown, S.H.; Zarnowski, R.; Sharpee, W.C.; Keller, N.P. Morphological transitions governed by density
dependence and lipoxygenase activity in Aspergillus flavus. Appl. Environ. Microbiol. 2008, 74, 5674–5685.
[CrossRef] [PubMed]
Affeldt, K.J.; Brodhagen, M.; Keller, N.P. Aspergillus oxylipin signaling and quorum sensing pathways
depend on g protein-coupled receptors. Toxins 2012, 4, 695–717. [CrossRef] [PubMed]
Singh, A.; Del Poeta, M. Lipid signalling in pathogenic fungi. Cell Microbiol. 2011, 13, 177–185. [CrossRef]
[PubMed]
Reverberi, M.; Zjalic, S.; Ricelli, A.; Punelli, F.; Camera, E.; Fabbri, C.; Picardo, M.; Fanelli, C.; Fabbri, A.A.
Modulation of antioxidant defense in Aspergillus parasiticus is involved in aflatoxin biosynthesis: A role for
the ApyapA gene. Eukaryot. Cell 2008, 7, 988–1000. [CrossRef] [PubMed]
Mecocci, P.; Polidori, M.C. Oxidative stress in human aging and in age-related diseases. Free Radic. Res. 2002,
36, 21–23.
Calvo, A.M.; Gardner, H.W.; Keller, N.P. Genetic connection between fatty acid metabolism and sporulation
in Aspergillus nidulans. J. Biol. Chem. 2001, 276, 25766–25774. [CrossRef] [PubMed]
Severns, D.E.; Clements, M.J.; Lambert, R.J.; White, D.G. Comparison of Aspergillus ear rot and aflatoxin
contamination in grain of high-oil and normal-oil corn hybrids. J. Food Prot. 2003, 66, 637–643. [CrossRef]
[PubMed]
Yan, S.J.; Liang, Y.T.; Zhang, J.D. Autoxidated linolenic acid inhibits aflatoxin biosynthesis in Aspergillus
flavus via oxylipin species. Fung. Genet. Biol. 2015, 81, 229–237. [CrossRef] [PubMed]
Lambeth, J.D. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 2004, 4, 181–189.
[CrossRef] [PubMed]
Lara-Ortiz, T.; Riveros-Rosas, H.; Aguirre, J. Reactive oxywgen species generated by microbial NADPH
oxidase NoxA regulate sexual development in Aspergillus nidulans. Mol. Microbiol. 2003, 50, 1241–1255.
[CrossRef] [PubMed]
Chang, P.K.; Bennett, J.W.; Cotty, P.J. Association of aflatoxin biosynthesis and sclerotial development in
Aspergillus parasiticus. Mycopathologia 2002, 153, 41–48. [CrossRef] [PubMed]
Shimizu, K.; Keller, N.P. Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling
pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics 2001, 157,
591–600. [PubMed]
Toxins 2017, 9, 146
31.
32.
11 of 11
Narasaiah, K.V.; Sashidhar, R.B.; Subramanyam, C. Biochemical analysis of oxidative stress in the production
of aflatoxin and its precursor intermediates. Mycopathologia 2006, 162, 179–189. [CrossRef] [PubMed]
Nanjing Jiancheng Bioengineering Institute. Available online: http://www.njjcbio.com (accessed on
18 March 2016).
© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).