High genetic variability in non-aflatoxigenic A. flavus strains by using Quadruplex
Giuseppe Criseo⁎, Cosimo Racco, Orazio Romeo
Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Italy
A B S T R A C TA R T I C L E I N F O
Received 11 October 2007
Received in revised form 3 April 2008
Accepted 24 April 2008
nor-1, omt-A, ver-1 and aflR genes
Aflatoxin non-producing A. flavus strains
Aflatoxigenic Aspergillus flavus isolates always show, by using a multiplex PCR-system, four DNA fragments
specific for aflR, nor-1, ver-1, and omt-A genes. Non-aflatoxigenic A. flavus strains give variable DNA banding
pattern lacking one, two, three or four of these genes. Recently, it has been found and reported that some
aflatoxin non-producing A. flavus strains show a complete set of genes. Because less is known about the
incidence of structural genes aflR, nor-1, ver-1 and omt-A in aflatoxin non-producing strains of A. flavus, we
decided to study the frequencies of the aflatoxin structural genes in non-aflatoxigenic A. flavus strains
isolated from food and feed commodities.
The results can be summarized as following: 36.5% of the examined non-aflatoxigenic A. flavus strains
showed DNA fragments that correspond to the complete set of genes (quadruplet pattern) as found in
aflatoxigenic A. flavus. Forty three strains (32%) showed three DNA banding patterns grouped in four profiles
where nor-1, ver-1 and omt-A was the most frequent profile. Twenty five (18.7%) of non-aflatoxigenic A.
flavus strains yielded two DNA banding pattern whereas sixteen (12%) of the strains showed one DNA
banding pattern. In one strain, isolated from poultry feed, no DNA bands were found. The nor-1 gene was the
most representative between the four aflatoxin structural assayed genes. Lower incidence was found for aflR
Our data show a high level of genetic variability among non-aflatoxigenic A. flavus isolates that require
greater attention in order to design molecular experiment to distinguish true aflatoxigenic from non-
aflatoxigenic A. flavus strains.
© 2008 Elsevier B.V. All rights reserved.
Aflatoxins are among the most potent mutagenic, teratogenic, and
carcinogenic natural compounds occurring in foods and feeds.
Aspergillus flavus Link is the most common causal fungus that
produces aflatoxins (Bennett and Klich, 2003). However, a significant
portion of the naturallyoccurring isolates of A. flavus lack the ability to
produce aflatoxin. In fact, aflatoxin biosynthesis pathway may become
unstable in these fungi; furthermore large differences in the levels of
aflatoxins produced were showed (Cary and Ehrlich, 2006).
Genetic studies suggest that the genes involved in the aflatoxin
biosyntheticpathwayareclustered(Bhatnagaretal., 2006). In general,
the aflatoxin gene cluster in A. flavus consists of 25 genes spanning
approximately 70 kb. The non-aflatoxigenic A. flavus isolates in many
cases have large deletions of a part or the entire aflatoxin gene cluster
(Chang et al., 2005).
Aflatoxin biosynthesis in A. flavus is strongly dependent on the
activities of regulatory proteins and enzymes encoded by four genes
named aflR, nor-1, ver-1 and omtA. By using specific PCR-based
methods, the aflatoxigenic A. flavus isolates always show the complete
gene set, whereas non-aflatoxigenic isolates lacking one, two, three or
four PCR products indicating that the genes do not exist in these
strains or that the primer binding sites changed. Interestingly, some A.
flavus strains show a complete set of genes but do not produce
aflatoxins (Criseo et al., 2001). In previous studies only a few non-
aflatoxigenic A. flavus strains were examined for molecular diversity
(Geisen, 1996; Shapira et al., 1996; Criseo et al., 2001).
In this study, we evaluate the presence and the frequencies of the
PCR products corresponding to amplification of aflR, nor-1, ver-1 and
omtA genes in non-aflatoxigenic A. flavus strains isolated from food
and feed commodities.
2. Materials and methods
134 non-aflatoxin producing strains of A. flavus isolated from food,
feed and officinal plants were used in this work (Table 2). A. flavus
International Journal of Food Microbiology 125 (2008) 341–343
⁎ Corresponding author. Department of Microbiological, Genetic and Molecular
Sciences, University of Messina, Salita Sperone, 31 98166 Messina, Italy. Tel.: +39
0906765195; fax: +39 090392733.
E-mail address: email@example.com (G. Criseo).
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aflatoxigenic strain MAM 13 and A. flavus non-aflatoxigenic strain
MAM 03 (collection of the Department of Microbiological, Genetic and
Molecular Sciences—University of Messina—Italy) were used as
positive and negative control strains respectively.
2.2. Determination of aflatoxin production
Aflatoxin production was carried out according to conventional
methods as previously described (Criseo et al., 2001).
2.3. Isolation of fungal DNA and Quadruplex PCR reaction
DNAextraction fromfungal strains was performed according tothe
method described by Yelton et al. (1984).
Quadruplex PCR was performed with primers designed from
specific DNA sequences derived from nor-1, ver-1, omt-A and aflR
genes of Aspergillus parasiticus (Table 1).
A typical PCR was carried out under the following condition: 5 μl of
genomic DNA were used as template (2 μg ml−1), 0.5U EuroTaq
polymerase (Euroclone, Pero-Milan, Italy), 1×reaction buffer, 2.5 mM
MgCl2, 200 μM each dNTP and 7.5 pmol each primer in a total reaction
cycle; and 94 °C, 30 s; 65 °C, 30 s; 72 °C, 30 s for the 34 left.
with ethidium bromide and compared to the DNA size marker (2-log
DNA Ladder 0.1–10.0 kb., BioLabs) by using Kodak digital science 1D
image analysis software (Eastman Kodak, Rochester, NY, USA).
All non-aflatoxigenic A. flavus strains examined, except for the
MAM 13 strain utilized as a positive control, do not produce aflatoxins
testedusing conventionalmethods.MultiplexPCR amplificationof the
aflatoxin structural genes used in this study gave the expected DNA
fragments (Criseo et al., 2001); amplicons of 400 bp, 537 bp, 797 bp
and 1032 bp were obtained with nor-1, ver-1, omt-A and aflR genes
respectively. In fact, the aflatoxigenic strain MAM 13 showed a
quadruplet pattern, indicating the presence of the four genes involved
in aflatoxin biosynthetic pathway, whereas all non-aflatoxigenic
examined strains and the reference strain MAM 03 yield a different
DNA banding patterns with a number of bands ranging from zero to
four (Table 2 and Fig. 1).
As showed in Table 3, an omogenous group of non-aflatoxigenic A.
flavus strains, rappresenting more than 36.5%, showed DNA fragments
that correspond to the complete set of genes. A second group
constituted by forty three (31.4%) of non-aflatoxigenic A. flavus strains
showed three DNA banding pattern clustered in four profiles: nor-1,
ver-1 and omt-A was the most frequent profile (12.7%) followed by
nor-1, omt-A, aflR (11.2%); nor-1, ver-1 andaflR (5.2%) andver-1, omt-A
and aflR (2.3%) (Table 3). A Third group constituted by twenty five
(18.7%) of non-aflatoxigenic A. flavus strains yielded two DNA banding
pattern grouped in five characteristic profiles (Table 3). Finally, a
fourth group constituted by sixteen strains (11.9%) gave two profiles
with one DNA fragment specific for nor-1 and aflR genes respectively.
No single DNA fragment was detected for ver-1 or omt-A genes. The
frequencies of banding pattern and respective profiles are showed in
Fig. 1. In one strain, isolated from poultry feed, no DNA bands were
Sequences of the oligonucleotide primers used for Quadruplex PCR amplification
Primer sequence (5′Y 3′) Target gene and
nor-1; 501–900 400
omt-A; 301–1098 797
Origin and genetic pattern of non-aflatoxin producing strains of A. flavus isolated from
food and feed commodities
Poultry feed (pellet)
Fig. 1. Frequencies of three (A), two (B) and one (C) genes pattern in examined non-
aflatoxigenic A. flavus strains.
G. Criseo et al. / International Journal of Food Microbiology 125 (2008) 341–343
found. The nor-1 gene was the most representative (88%) between the
four aflatoxin structural assayed genes followed by ver-1 and omt-A
that were found with the same frequencies (70.1%). Lower incidence
(61.9%) was found for aflR gene (Table 3).
Member of the A. flavus group are widespread in the most part of
the world and they are capable of growing on, and producing
aflatoxins in, a wide range of foods and feeds. However, not all strains
of A. flavus produce aflatoxins.
The present work reports, for the first time, the incidence of the
aflatoxin structural genes nor-1, ver-1, omt-A and aflR, in non-
aflatoxigenic A. flavus strains. It shows a high molecular diversity in
non-aflatoxigenic A. flavus isolates when the gene set (nor-1, omt-A,
ver-1 and aflR) is assayed by molecular techniques. In the last few
years, several PCR-basedsystemshave been developed to discriminate
between aflatoxin-producing and non-producing A. flavus strains
(Geisen, 1996; Shapira et al., 1996; Zachova et al., 2003; Färber et al.,
1997). However, these methods do not permit an unequivocal
differentiation among these strains based on genetic approach. In
fact, in a previous study, Criseo et al. (2001) found that some A. flavus
non-aflatoxigenic strains showed the same quadruplet banding
pattern of the aflatoxigenic strains in a multiplex PCR-system.
Recently, some Authors have suggested a nested-PCR approach
based on the amplification of aflR sequence to detect aflatoxigenic
Aspergilli in pure and mixed culture systems (Manonmani et al.,
2005). These authors used as control strains fungi belonging to the
genus Penicillium, Rhizopus, Fusarium avoiding any false positives, but
none A. flavus non-producing strains have been used in this study. Our
(61.9%) shows the aflR gene. This could impair the use of aflR gene to
identify aflatoxigenic Aspergilli. A PCR-restriction fragment length
analysis of aflR gene was proposed for differentiation and detection of
A. flavus and A. parasiticus in maize (Somashekar et al., 2005). In our
study, we found that 51 of aflatoxin non-producing A. flavus strains
lacking the aflR PCR amplicon because in these strains the aflR gene
has been lostor mutations occurs within the primer binding sites. This
could be due to the location of the aflatoxin gene cluster in the
telomeric region of A. flavus, that would facilitate gene loss as well as
recombination, DNA inversions, partial deletions, translocations and
other genomic rearrangements (Carbone et al., 2007). In these cases,
accurate species-level identification of the fungus, by using RFLP
analysis, could be not reliable.
Furthermore, several research groups have adopted reverse
transcription PCR technique (RT-PCR) to detect a mRNA specific for
an aflatoxin biosynthetic gene and differentiate aflatoxin-producing
from non-producing strains of A. flavus (Mayer et al., 2003; Scherm
et al., 2005; Degola et al., 2007).
In this study we found that 36.5% of the assayed strains have the
same genetic pattern primarily found in aflatoxigenic strains. In these
cases, the mRNAs could be also detected by RT-PCR as showed in
Degola et al. (2007). Furthermore, the detection of transcripts of the
some aflatoxin genes not always correlated to the actual toxicity of
each strain (Scherm et al., 2005).
To date, it seems that molecular methods by targeting only some
selected genes of the aflatoxin biosynthetic pathway do not permit an
unequivocal discrimination between aflatoxigenic and non-aflatoxi-
genic A. flavus strains due the high genetic variability observed within
A. flavus isolates. In fact, in non-aflatoxigenic A. flavus strains that
showthecompletegeneset,non-aflatoxigenicitycanbe due todefects
on various molecular levels such as post transcriptional level and/or
protein level. This fact makes it difficult to develop molecular
techniques to distinguish true aflatoxigenic from non-aflatoxigenic
A. flavus strains. Therefore, accurate detection of aflatoxigenicity can
be carried out by using immunological or cultural methods.
We are grateful to an anonymous reviewer for helpful comments
on previous drafts of this manuscript.
Bennett, J.W., Klich, M., 2003. Mycotoxins. Clinical Microbiology Reviews 16, 497–516.
Bhatnagar, D., Cary, J.W., Ehrlich, K., Yu, J., Cleveland, T.E., 2006. Understanding the
genetics of regulation of aflatoxin production and Aspergillus flavus development.
Mycopathologia 162, 155–166.
Carbone, I., Ramirez-Prado, J.H., Jakobek, J.L., Horn, B.W., 2007. Gene duplication,
modularity and adaptation in the evolution of the aflatoxin gene cluster. BMC
Evolutionary Biology 7, 111.
relationships and evolutionary implications. Mycopathologia 162,167–177.
Chang, P.K., Horn, B.W., Dorner, J.W., 2005. Sequence breakpoints in the aflatoxin
biosynthesis gene cluster and flanking regions in non-aflatoxigenic Aspergillus
flavus isolates. Fungal Genetics and Biology 42, 914–923.
Criseo, G., Bagnara, A., Bisignano, G., 2001. Differentiation of aflatoxin-producing and non-
producing strains of Aspergillus flavus group. Letters in Applied Microbiology 33,
Degola, F., Berni, E., Dall'Asta, C., Spotti, E., Marchelli, R., Ferrero, I., Restivo, F.M., 2007. A
multiplex RT-PCR approach to detect aflatoxigenic strains of Aspergillus flavus.
Journal of Applied Microbiology 103, 409–417.
Färber, P., Geisen, R., Holzapfel, W.H., 1997. Detection of aflatoxigenic fungi in figs by a
PCR reaction. International Journal of Food Microbiology 36, 215–220.
Geisen, R., 1996. Multiplex polymerase chain reaction for the detection of potential
aflatoxin and sterigmatocystin producing fungi. Systematic and Applied Micro-
biology 19, 388–392.
Manonmani, H.K., Anand, S., Chandrashekar, A., Rati, E.R., 2005. Detection of aflatoxigenic
fungi in selected food commodities by PCR. Process Biochemistry 40, 2859–2864.
Mayer, Z., Bagnara, A., Färber, P., Geisen, R., 2003. Quantification of the copy number of
nor-1, a gene of the aflatoxin biosynthetic pathway by real-time PCR, and its
correlation to the cfu of Aspergillus flavus in foods. International Journal of Food
Microbiology 82, 143–151.
Scherm, B., Palomba, M., Serra, D., Marcello, A., Migheli, Q., 2005. Detection of
transcripts of the aflatoxin genes aflD, aflO, and aflP by reverse transcription-
polymerase chain reaction allows differentiation of aflatoxin-producing and non-
producing isolates of Aspergillus flavus and Aspergillus parasiticus. International
Journal of Food Microbiology 98, 201–210.
Shapira, R., Paster, N., Eyal, O., Menasherov, M., Mett, A., Salomon, R.,1996. Detection of
aflatoxigenic molds in grains by PCR. Applied and Environmental Microbiology 62,
Somashekar, D., Rati, E.R., Chandrashekar, A., 2005. PCR-restriction fragment length
analysis of aflR gene for differentiation and detection of Aspergillus flavus and Asper-
gillus parasiticus in maize. International Journal of Food Microbiology 93,101–107.
Yelton, M.M., Hamer, J.E., Timberlake, W.E., 1984. Transformation of Aspergillus nidulans by
Zachova, I., Vytrasova, J., Pejchalova, M., Cervenka, L., Tavcar-Kalcher, G., 2003. Detection
of aflatoxigenic fungi in feeds using the PCR method. Folia Microbiologica 48,
Frequency of single genes in non-aflatoxigenic A. flavus strains examined
N° strains %
nor-1 ver-1omtA aflR
MAM 13 = positive control.
MAM 03 = negative control.
+ = PCR amplification signal present.
− = PCR amplification signal absent.
G. Criseo et al. / International Journal of Food Microbiology 125 (2008) 341–343