RESEARCH LETTER

Stress induction of mycotoxin biosynthesis genes by abiotic factors

Markus Schmidt-Heydt1, Naresh Magan2 & Rolf Geisen1
1
Max Rubner Institute, Karlsruhe, Germany; and 2Applied Mycology Group, Cranfield Health, Cranfield University, Silsoe, Bedford, UK

Correspondence: Rolf Geisen, Max Rubner Abstract

Institute, Haid-und-Neu-Str. 9, Karlsruhe
76131, Germany. Tel.: 149 7216625450;
fax: 149 7216625453; e-mail:
rolf.geisen@mri.bund.de
Received 29 February 2008; accepted 28 March
2008.
First published online 29 May 2008.
DOI:10.1111/j.1574-6968.2008.01182.x
Editor: Jan Dijksterhuis
Keywords
trichothecenes; aflatoxin; Aspergillus
parasiticus ; Fusarium culmorum ; Penicillium
verrucosum ; ochratoxin.
Systematic expression analysis of mycotoxin biosynthesis genes by real-time PCR
and microarray was carried out to examine the relationship between growth and
general expression patterns in relation to single environmental factors such as
temperature, water activity (aw) and pH and water activity  temperature interac-
tions. For single parameters, one major peak of expression occurred close to
optimum growth conditions. However, a second minor peak was observed under
suboptimal growth conditions, when intermediate environmental stress was
imposed on Aspergillus parasiticus (afl genes), Penicillium verrucosum (ota genes)
and Fusarium culmorum (tri genes). This expression profile pattern was more
pronounced in relation to changes in temperature and aw than to pH. In a two-
factorial experimental design with temperature  aw regimes, again two peaks of
expression were observed for cluster genes after microarray analysis, one close to
those giving optimal growth and one under imposed stress conditions. Interest-
ingly, when the activity of single genes of the microarray data were plotted in
relation to the two parameters, again a two-peak expression profile became
obvious independently for both parameters. Expression of the mycotoxin bio-
synthesis genes was followed exactly by phenotypic mycotoxin production. This
expression profile appears to be generic across the mycotoxigenic fungi examined.

2006). However, only a few studies have systematically

Introduction
Mycotoxins are toxic secondary metabolites produced by
certain groups of filamentous fungi. The most important
mycotoxins are the aflatoxins, mainly produced by Aspergil-
lus flavus and Aspergillus parasiticus, the trichothecenes
produced by various Fusarium species, with Fusarium
culmorum being an important producer, and ochratoxin
A (OTA) synthesized by Penicillium verrucosum and other
Penicillium or Aspergillus species. The biosynthesis of myco-
toxins is highly regulated and it has been shown that
substrate (Skrinjar & Dimic, 1992), temperature (Belli
et al., 2004; Mitchell et al., 2004), water activity (Valero
et al., 2006) and pH (Arroyo et al., 2005) can have profound
effects. The environmental profiles and optima for the
synthesis of the studied mycotoxins on the phenotypic level
have been determined for OTA biosynthesis by P. verrucosum
(Arroyo et al., 2005; Cairns-Fuller et al., 2005; Pardo et al.,
2006) trichothecene biosynthesis by Fusarium (Llorens et al.,
2004; Hope et al., 2005; Ramirez et al., 2006) and aflatoxin
biosynthesis by Aspergillus (Nesci et al., 2003; Ribeiro et al.,
investigated the influence of these parameters on the expres-
sion of mycotoxin biosynthesis genes (Feng & Leonhard,
1998; Geisen, 2004; O’Callaghan et al., 2006; Schmidt-Heydt
& Geisen, 2007a; Jurado et al., 2008). These experiments
have shown that the effect of external factors on mycotoxin
biosynthesis is exerted at the level of transcription. In
addition to these general growth parameters, substances or
conditions that impose stress on the fungus also have an
influence on mycotoxin biosynthesis. Jayashree & Subrama-
nyam (2000) described the positive influence of oxygenic
stress on aflatoxin biosynthesis by A. parasiticus. The appli-
cation of suboptimal concentrations of some fungicides, e.g.
strobilurins, can have a potentiating effect on mycotoxin
biosynthesis (Ellner, 2005). Furthermore, for P. verrucosum,
suboptimal concentrations of preservatives, such as calcium
propionate and potassium sorbate, have been shown to lead
to an increase in OTA biosynthesis. In parallel, it was shown
that the otapksPV gene (the OTA polyketide synthase), a key
gene in the OTA biosynthesis pathway, is activated under
these stress conditions (Schmidt-Heydt et al., 2007). Stress

c 2008 Federation of European Microbiological Societies FEMS Microbiol Lett 284 (2008) 142–149
Published by Blackwell Publishing Ltd. All rights reserved
Stress induction of mycotoxin biosynthesis genes
activation was also demonstrated for the fum1 gene of
Fusarium verticillioides (Jurado et al., 2008).
The objectives of this study were to determine the
influence of abiotic factors, such as temperature, water
activity (aw) and pH, and temperature  aw interactions on
mycotoxin biosynthesis gene expression, mycotoxin produc-
tion and growth. This was done to evaluate whether a
generic expression profile could be established, which could
show the impact that environmental stress conditions have
on the triggering of mycotoxin biosynthesis gene induction
in three important mycotoxigenic species (A. parasiticus,
P. verrucosum and F. culmorum).
Materials and methods
Strains and culture conditions
The following strains from the culture collection of the Max
Rubner Institute have been used throughout the experi-
ments: A. parasiticus BFE096p as an aflatoxin producer,
F. culmorum BFE928 as a trichothecene producer and
P. verrucosum BFE808 as an OTA producer. The following
growth conditions were routinely used to support mycotox-
in biosynthesis – A. parasiticus BFE096p: incubation for 7
days at 30 1C on YES agar (20 g L 1 yeast extract, 150 g L 1
sucrose, 15 g L 1 agar); F. culmorum BFE928 and P. verruco-
sum BFE808: incubation for 7 days at 25 1C on YES agar.
Modifications of the growth media
The following modifications of the growth media and
growth conditions were used to measure the effect of
environmental factors (aw, temperature and pH) on gene
expression and mycotoxin biosynthesis in the analysed
strains: the pH in the YES medium was adjusted by adding
HCl or sodium hydroxide up to the desired value. The aw in
the YES medium was modified with the appropriate amount
of glycerol [aw/glycerol (mL) per 100 mL YES medium: 0.99/
10.8, 0.98/13.1, 0.95/19.9, 0.93/24.5]. For incubation at
different temperatures (15–30 1C), the inoculated plates
were placed in temperature-controlled cabinets.
Assessment of colony growth rate
For measuring the radial mycelial growth rate, the diameter
of the colony was measured in two directions at right angles
to each other. The increase in colony radius was divided by
the time to obtain the relative growth rates.
Isolation of RNA from samples
To perform microarray and real-time PCR experiments,
RNA was isolated using the RNAeasy Plant Mini kit (Qiagen,
Hilden, Germany). One gram of the mycelium was ground
FEMS Microbiol Lett 284 (2008) 142–149
143
using a mortar and pestle in liquid nitrogen. About 250 mg of
the resulting powder was used for isolation of total
RNA. The powder was resuspended in 750 mL lysis buffer,
mixed with 7.5 mL b-mercaptoethanol and 100 glass beads with
a diameter of 1 mm (B. Braun Biotech International GmbH,
Melsungen, Germany) in a 2-mL RNAse-free microreaction
tube. The extracts were mixed thoroughly and incubated for
15 min at 55 1C and 42 kHz in an S10H ultrasonic bath (Elma,
Singen, Germany). All further procedures were essentially the
same as suggested by the manufacturer of the kit.
cDNA synthesis
For cDNA synthesis, 8 mL of the DNAse I-treated total RNA
was used along with the Omniscript Reverse Transcription
kit (Qiagen). The reaction mixture was essentially as de-
scribed by the manufacturer and incubated at 37 1C for 1 h.
The cDNA was either directly used for real-time PCR or
stored at 20 1C.
Reverse transcriptase real-time PCR
The reverse transcriptase real-time PCR reactions to mea-
sure the expression of the otapksPV gene in P. verrucosum
were performed essentially as described in Schmidt-Heydt
et al. (2007).
Microarray experiments
For labelling of cDNA, 10–50 mg of the DNAse I-treated total
RNA was used according to the specifications of the manu-
facturer of the Micromax cDNA Direct Labelling kit (Perkin
Elmer Life and Analytical Sciences Inc., Boston). After
cDNA synthesis and labelling, the cDNA was purified with
a QiaQuick MinElute-Kit (Qiagen). The labelled and pur-
ified cDNA was brought to dryness in a Speed Vac concen-
trator (Savant Instruments, Farmingdale), resuspended in
60 mL hybridization buffer (Scienion, Berlin, Germany),
heated for 2 min at 95 1C and hybridized for 18 h at 42 1C
to the microarray using an automatic hybridization station
(Perkin Elmer). Following hybridization the array was
scanned with a confocal laser scanner system (Scanarray lite,
Perkin Elmer) at a resolution of 5 mm. Analysis of the results
was performed using SCANARRAY software (Perkin Elmer).
The results were normalized using the Lowess algorithm
(locally weighted scatter plot smoothing) and subtraction of
the background signal intensity. As control, the constitu-
tively expressed b-tubulin gene was used.
Quantitative determination of OTA by HPLC
Detection and quantitative determination of OTA from
fungal colonies was performed according to the method
described in the ISO 15141 standard (1998, www.iso.ch). For
this, 100 mg of the fungal colony was extracted under shaking
c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
144 M. Schmidt-Heydt et al.

(a1) (b1) (c1)

6 6
6 5 4
Colony growth
(cm week –1)
4 4
3
3 3
2
2 2
1 1 1
0 0 0
0.99 0.98 0.95 0.93 30 25 20 15 8 6 5 4
(a2) (b2) (c2)
100 100 100
Copy number otapks

10 10 10
PV × 1000

1
1 1
0.1
0.1 0.1
0.01

0 0 0
0.99 0.98 0.95 0.93 30 25 20 15 8 6 5 4

(a3) (b3) (c3)

10 10 10
(ng mL–1) × 1000

1
Ochratoxin A

1 1

0.1 0.1 0.1

0.01 0.01 0.01

0 0 0
0.99 0.98 0.95 0.93 30 25 20 15 8 6 5 4
Water activity Temperature pH

Fig. 1. Growth rate (a1, b1, c1), otapksPV gene expression level (a2, b2, c2) and OTA biosynthesis of Penicillium verrucosum (a3, b3, c3). To obtain
these data, P. verrucosum was grown on YES medium adjusted to the parameters given here. Only the indicated parameters were variable; all others
were kept constant. After 7 days of incubation, growth rate was determined by measuring colony diameter, otapksPV copy number was determined by
real-time PCR and OTA biosynthesis was measured by HPLC. All experiments were performed several times.

conditions in 500 mL chloroform at room temperature for Results

30 min. The residue was discarded and the chloroform was
evaporated to dryness in a vacuum concentrator (Savant
Instruments). The residue was then redissolved in 500 mL
methanol and subjected to HPLC analysis (20 mL) in a Pharma-
cia HPLC apparatus LKB 2150 (Pharmacia, Uppsala, Sweden).
A nucleosil 100-5 C18 CCV 250/4 column was used for
separation (Machery and Nagel, Düren, Germany) at a flow
rate of 1 mL min 1 with acetonitril/water/acetic acid (40 : 60 : 1,
v/v/v). The peak was determined with a fluorescence detector
(Shimazu RF551, Düsseldorf, Germany).
Reproducibility of the data
All experiments were carried out several times (at least three
times for the real-time PCR and twice for the microarray
experiments). The mean value is shown. The fact that a
generic expression profile is seen with several strains and
species and with data from several experimental set-ups
and methods indicates the reproducibility and significance
of the data.
Growth, gene expression and OTA biosynthesis
in relation to modifications of temperature,
water activity and pH
Growth and ochratoxin production by P. verrucosum at the
phenotypic and molecular level were monitored in relation
to changes in each of these single abiotic factors. Penicillium
verrucosum was grown for 7 days on YES medium at
different temperatures, aw levels or initial pH values. The
parameter values were in a range that permitted growth of
P. verrucosum. Growth rate was determined based on measure-
ments of colony diameter. In addition, expression of the
otapksPV gene was measured by real-time PCR and the OTA
produced via HPLC. The results are shown in Fig. 1. With
changes in aw there was a clear optimum for growth rate in
the range 0.99 0.98 aw. Below these values a linear decrease
occurred depending on aw. At 0.93 aw the growth rate was
about one-fifth of that under optimal conditions. Similar
behaviour was observed with changing temperature. The

c 2008 Federation of European Microbiological Societies FEMS Microbiol Lett 284 (2008) 142–149
Published by Blackwell Publishing Ltd. All rights reserved
Stress induction of mycotoxin biosynthesis genes
analysed strain of P. verrucosum showed optimum growth
between 25 and 30 1C. There was a decrease in the growth
rate until 15 1C. Highest growth was found at pH 6.
Penicillium verrucosum showed a reduction in growth under
slightly alkaline conditions, e.g. at pH 8, and a more
significant inhibition under acidic conditions.
The general expression profile of the otapksPV gene was
similar in relation to changes in these three abiotic parameters.
In each case two expression peaks were observed, albeit at
different intensities. The major peak of expression was within
the parameter range for optimal growth conditions. However,
this was never found at the same value at which optimal
growth occurred. For example, in the case of aw the major
gene expression peak was at 0.98, whereas optimal growth was
at 0.99. This was also true for temperature (30 1C for growth,
25 1C for otapksPV expression) and pH (pH 6 for growth, pH
8 for otapksPV expression). The minor peak of expression was
always located at conditions, which posed a mild stress to the
fungus. This stress was indicated by the reduced growth rate of
the fungus at that parameter value. Based on these results,
changes in aw and temperature had a greater impact on
otapksPV gene expression compared with pH. This expression
behaviour is nearly 1 : 1 reflected by the phenotypic produc-
tion of OTA. Furthermore, the major and minor peaks of OTA
production were found at the same parameter values as the
gene expression peaks.
This expression and production behaviour seems to be a
general phenomenon. The same experiments were per-
formed with F. culmorum and a very similar picture could
be observed, except that the optima and minima were
specific for this fungus (data not shown).
Microarray expression analysis of the mycotoxin
cluster genes in relation to combinations of the
two parameters, temperature and a w
In order to analyse the efficacy of combinations of the two
most effective environmental parameters (temperature, aw)
on the expression of different mycotoxin biosynthesis gene
clusters, a microarray analysis (Schmidt-Heydt & Geisen,
2007b) was performed with P. verrucosum (OTA)-,
F. culmorum (trichothecene)- and A. parasiticus (aflatoxin)-
related gene clusters. For this, the fungal species were grown
on YES medium (adjusted to pH 6 and to variable aw values)
for 7 days at different temperatures. Figure 2 shows the
results. Again, a general expression profile became clear with
all three species. Thus, two peaks of cluster gene expression
could be observed: one major peak close to parameter
combinations which supported optimal growth, and a
second minor peak under mild stress conditions, where only
slower growth was possible. These expression profiles re-
sembled the profiles from the experiments in which only a
single abiotic parameter was changed. It was also clear that
FEMS Microbiol Lett 284 (2008) 142–149
145
the two parameters had a significant influence on relative
expression of certain genes, as both parameters could either
activate or repress gene expression, when the other para-
meter was kept constant. However over a certain range
(20 1C) temperature seems to be the most important factor
in mycotoxin cluster gene regulation. With all three species,
gene expression appeared to be lowest at this temperature,
irrespective of the applied water activity. For A. parasiticus a
broad temperature range was tested (15–37 1C). No other
similar zone of lower expression could be observed. Gen-
erally, in these experiments the actual production of the
respective mycotoxins followed the expression of the genes
determined by microarray analysis (data not shown).
Relative expression of single genes determined
by microarray in relation to combinations of a w
and temperature
In order to analyse the influence of the above two parameters
on single gene activation, the microarray expression data of
two genes, the nor-1 gene of the aflatoxin pathway (Fig. 3a)
and the tri5 gene of the trichothecene pathway (Fig. 3b), were
investigated. During this analysis the same expression pattern
as with the real-time PCR experiments became obvious for
both genes, if one parameter was kept constant, e.g. one major
peak at near optimum parameters and a minor peak under
conditions which impose mild stress to the fungus. This was
true for the data at either variable temperatures but constant
water activity or vice versa. This profile, however, was found
only if the constant parameter is at a moderate value, which
means for the minor peak that stress is only imposed by one
factor. If, however, both factors are in a suboptimal range the
effect acts additively or synergistically and nearly completely
abolishes gene expression, albeit that growth was still possible
at the conditions analysed by the microarray. This was true for
the tri5 gene at a combination of 15 1C/0.93 (Fig. 3b) or for
the nor-1 gene at combinations of 15 or 17 1C and water
activities below 0.98 (Fig. 3a). Only two genes of the micro-
array analyses are shown as examples, although this general
expression profile was found for many genes of the clusters.
Discussion
Based on the results presented here, abiotic factors such as
temperature, aw and pH have a strong influence on the
expression of mycotoxin biosynthesis genes. This is in
agreement with the findings of several other authors (Keller
et al., 1997; Geisen, 2004; Price et al., 2005; O’Callaghan
et al., 2006; Jurado et al., 2008). In the present study the gene
expression profile always paralleled and preceded the phe-
notypic biosynthesis of the analysed mycotoxins. According
to the results obtained it seems to be a generic feature of
mycotoxin biosynthesis genes that different expression opti-
ma in relation to changing a single external parameter
c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
146
(a)
(b)
(c)
(temperature, aw, pH) exist. For P. verrucosum a clear
optimum for the growth rate could be defined, whereas for
gene expression a major and a minor optimum could be
identified for all three parameters tested. The major opti-
mum of gene activation was near the growth optimum and
c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Schmidt-Heydt et al.
Fig. 2. Microarray expression analysis of the OTA biosynth-
esis genes of Penicillium verrucosum (a), the trichothecene
biosynthesis genes of Fusarium culmorum (b) and the
aflatoxin biosynthesis genes of Aspergillus parasiticus (c)
after controlling the parameters temperature and water
activity. The graphs show the relative expression of the gene
clusters. The genes are indicated on the z-axis, the growth
conditions on the x-axis and the relative expression values
on the y-axis. The applied microarray has been described
(Schmidt-Heydt & Geisen, 2007b). The fungal species were
grown on YES plates for 7 days. Subsequently, mRNA from
each culture was isolated and subjected to microarray
analysis. The bars represent relative intensities of the
hybridized spots. These are average values from several
measurements.
the minor optimum occurred in an intermediate parameter
range, where growth was still possible but was restricted. The
latter conditions represent the imposition of abiotic mild
stress to the fungus (Magan, 2007). This general behaviour
was true for all three abiotic parameters analysed. All three
FEMS Microbiol Lett 284 (2008) 142–149
Stress induction of mycotoxin biosynthesis genes 147

(a) agreement with the results presented here. Both aw and

temperature have a similar influence on the regulation of
2000 mycotoxin biosynthesis genes, as the same expression pro-
1800 files arose in response to changes in these parameters. The
1600
impact of pH on gene expression followed the same pattern
but was not as pronounced. This might be due to the
Relative expression

1400
presence of a regulatory system which supports pH home-
1200
ostasis in the cell (Penalva & Arst, 2002; Arst & Penalva,
1000
2003). A correlation between environment (osmotic stress),
800
stress response and trichothecene biosynthesis was recently
600
shown at the molecular level (Ochiai et al., 2007).
400 0.99
The major peaks of gene activation near the optimum
200 0.98
a growth rates do not seem to be related to any imposed
0 0.95
abiotic stress, because the growth rate of the colony was
30°C
25°C 0.93 high. However, under these conditions nutrients may often
20°C
Temperature (°C)
15°C be exhausted rapidly (especially after growth on agar plates,
when diffusion of nutrients plays a role), which may in turn
(b) lead to nutrient-related stress effects. Previous studies have
shown that N-limitation can activate aflatoxin biosynthesis
6000
(Bennett & Christensen, 1983). The reason why the major
5000
expression peak does not completely match the highest
growth rate is not clear yet. At the optimal growth rate
Relative expression

4000 primary metabolism is a priority, resulting in a slightly later

3000
2000
1000
0 0.98
37°C
30°C
25°C
20°C
17°C
Temperature (°C)
Fig. 3. Selected microarray expression data of the single genes nor-1
(a) and tri5 (b) plotted in relation to the variable parameters temperature
and water activity. The typical two-peak expression profile can be found
several times if one parameter is kept constant. As examples for each
gene, two profiles, one at constant water activity and the other at
constant temperature, are marked in dark grey.
stresses (aw, pH, temperature) similarly activated the myco-
toxin biosynthesis genes. The connection between environ-
mental stress and mycotoxin biosynthesis was supported by
the fact that other stress factors, e.g. fungicides (Ellner,
2005) or preservatives (Schmidt-Heydt et al., 2007), can
lead to enhanced mycotoxin biosynthesis. Doohan et al.
(1999) also described an increase in the expression of the
tri5 gene, a key gene of the trichothecene biosynthesis
pathway, after application of fungicides, again indicating
the correlation between stress and mycotoxin biosynthesis.
Jurado et al. (2008) showed in a highly sophisticated study
that osmotic stress has a profound influence on induction of
the transcript level of the fum1 gene, despite the fact that
growth of F. verticillioides was retarded. This is in good
effect on switching on/off the gene clusters related to
mycotoxin production. The observations that abiotic stress
factors can strongly influence mycotoxin biosynthesis is
supported by the fact that at moderate conditions (e.g. 0.95
aw, 20 1C, pH 5) the expression of biosynthesis genes and
0.99
toxin production were lowest. This, however, does not mean
0.95 a that no mycotoxin is produced under these conditions, but
0.93 only at a reduced rate compared with the other conditions.
15°C The described influences of the variation of a single
abiotic parameter on OTA gene expression and biosynthesis
by P. verrucosum seems to be a generic phenomenon.
A highly similar expression profile and trichothecene
biosynthesis was found for F. culmorum (data not shown).
The imposition of two-factor abiotic stress parameters
using microarray analysis of cultures grown under the same
treatment conditions showed similar effects on gene expres-
sion. The main and minor peaks in expression were again
related to growth variables. This showed that a coordinated
activation of the whole or nearly the whole gene cluster took
place under near optimal growth conditions and under mild
stress conditions. Again, at moderate conditions the lowest
gene activity was detected. Interestingly, for all three species
tested the lowest gene activity was at 20 1C for all tested
values of aw, indicating a prominent influence of this
temperature on mycotoxin gene expression nearly irrespec-
tive of the actual aw. No other comparable zone of low
expression was found even in the case of A. parasiticus, for
which a broad temperature range has been analysed. The
reason for this significant reduced gene expression at 20 1C
is not yet clear. It is intriguing to speculate that these

FEMS Microbiol Lett 284 (2008) 142–149

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
148
conditions might be the most stress-free growth conditions
for the fungus. At these conditions, growth rate is moderate,
resulting in a reduction of nutrient utilization. However,
other reasons for this phenomenon cannot be ruled out.
The activated genes and levels of transcription were not
identical in the major peak compared with the minor peak.
This might reflect differences in the response to nutritional
stress (major peak) and abiotic stress (minor peak).
The fact that the microarray expression profile of the
respective gene cluster was similar for all three species and
for three different mycotoxins (OTA/P. verrucosum; tri-
chothecene/F. culmorum; aflatoxin/A. parasiticus) again
strongly indicates that this is a general phenomenon. The
patterns of single gene expression of the microarray data
obtained nicely matches and extends the findings of the real-
time PCR experiments. If one factor is kept constant the
same two-peak expression patterns become obvious by
changing the other factor, but only if the constant factor is
at a moderate value. This indicates that only moderate stress
conditions lead to induction of mycotoxin biosynthesis
genes. However, if both parameters are at suboptimal values,
they act either additionally or synergistically and toxin gene
expression ceases. These results are in good agreement with
a recent report of Baert et al. (2007), who showed that mild
stress leads to an increased production of patulin by
Penicillium expansum, whereas more severe abiotic stress
conditions reduced biosynthesis.
The microarray data of the aflatoxin biosynthesis genes
obtained in this study also confirm the data of OBrian et al.
(2007). These authors also found a strong influence of
temperature by microarray analysis on aflatoxin gene ex-
pression of A. flavus. They analysed gene expression at 28
and 37 1C. An expression optimum was found at 28 1C,
which is also supported by our results. However, an increase
of the temperature to 37 1C drastically changed aflatoxin
gene expression and no aflatoxin was produced under these
conditions. However, they used A. flavus whereas in this
analysis A. parasiticus was used, which may explain the
differences of expression at 37 1C.
Taken together, the results described imply that the
production of mycotoxins can be regarded as an adaptation
to imposed abiotic and other stress conditions by these
mycotoxigenic species. Whether the activation of mycotoxin
biosynthesis is the cause or the consequence of a stress
reaction cannot yet be concluded. The fact that this
two-peak pattern can be found with different species
(P. verrucosum, F. culmorum, A. parasiticus), different
methods (real-time PCR, microarray) and at different levels
(gene expression, mycotoxin biosynthesis) strongly supports
its biological significance.
From a food safety point of view, conditions which
impose an intermediate stress on mycotoxigenic species
during storage or transport must be avoided. Recent studies
c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Schmidt-Heydt et al.
on food preservatives such sorbate and propionate support
the stimulation of both gene expression and toxin produc-
tion by P. verrucosum (Schmidt-Heydt et al., 2007). Thus,
care is needed to ensure that where control strategies are
used complete inhibition of growth occurs, thus limiting the
potential for activation of the mycotoxin biosynthesis genes
and thus mycotoxin contamination.
Acknowledgements
This work was supported by the EU-project ‘Development
of cost-effective control and prevention strategies for emer-
ging and future foodborne pathogenic microorganisms
throughout the food chain’ (Pathogen Combat), FOOD-
CT-2005-07081 and by a project of the Landesstiftung
Baden-Württemberg. We would like to thank Nicole
Mischke, Doreen Roblick and Lars Uhlmann for skilful
technical assistance.
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