FEMS Microbiology Letters 245 (2005) 353361

www.fems-microbiology.org

Discrimination of Aspergillus niger and other Aspergillus

species belonging to section Nigri by PCR assays
Amaia Gonzalez-Salgado a, Belen Patino b, Covadonga Vazquez b,
Ma Teresa Gonzalez-Jaen a,*
a

Departamento de Genetica, Facultad de Biologa, Universidad Complutense de Madrid, Jose Antonio Novais 2, 28040-Madrid, Spain
b
Departamento de Microbiologa III, Universidad Complutense de Madrid, Spain
Received 2 February 2005; received in revised form 15 March 2005; accepted 16 March 2005
First published online 25 March 2005
Edited by M.J. Bidochka

Abstract
Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coee and
derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce
ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section
is dicult and requires considerable expertise using conventional methods based on morphological features. In this work we
describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section
Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate:
A. niger and A. tubingensis. The species-specic primers have been designed on the basis of ITS (internal transcribed spacers of
rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specic and represent a good
tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.

2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
Keywords: Aspergillus niger; Section Nigri; Ochratoxin A; PCR; ITS

1. Introduction
Species of Aspergillus belonging to section Nigri [1]
are known as black Aspergilli and have a considerable
importance because of their ability to produced enzymes
and organic acids [2]. Aspergillus species also cause food
spoilage and produce mycotoxins [3], among which aatoxins and ochratoxin A are the most important.
Identication up to now is based on morphological
characteristics, such as conidial shape, color and size.

Corresponding author. Tel.: +34 913 944 830; fax: +34 913 944 844.
E-mail address: tegonja@bio.ucm.es (M.T. Gonzalez-Jaen).

Thom and Raper [4] and Raper and Fennell [5] divided
the black Aspergilli into 15 and 12 species, respectively.
In 1980, Al-Musallam [6] revised this classication and
recognized ve distinguishable species and the A. niger
aggregate, subdivided into seven varieties. Kusters-van
Someren using molecular methods (nuclear and mitochondrial DNA polymorphism [7]) separated the black
Aspergilli into A. japonicus, A. heteromorphus, A. ellipticus, A. carbonarius and the A. niger aggregate. The
A. niger aggregate would include, according to
Kusters-van Someren [8], two morphologically indistinguishable species, A. niger and A. tubingensis based on
RFLP analysis which was corroborated by other studies
[9,10].

0378-1097/$22.00
2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.
doi:10.1016/j.femsle.2005.03.023

354

A. Gonzalez-Salgado et al. / FEMS Microbiology Letters 245 (2005) 353361

Ochratoxin A (OTA) is a secondary metabolite produced by Aspergillus and Penicillium species. This mycotoxin is a potent nephrotoxin known also to be
immunosupressive, genotoxic and teratogenic towards
several animal species, and has been classied by the
International Agency for Research on Cancer as a possible carcinogen to humans (group 2B) [11]. OTA occurs
in various foodstus and beverages including a variety
of cereals, beans, groundnuts, spices, dried fruits, coee,
milk, wine and beer [1214], and its occurrence on several dietary products for human consumption is under
legal regulation.
Only two Aspergillus belonging to section Nigri are
known to produce OTA: A. carbonarius and A. niger
[1519], although OTA production by some A. japonicus
strains has been also reported [20]. These fungi are common in plant products and processed food in warm and
tropical climates. For this reason, the accurate identication of Aspergillus species in section Nigri is of great
importance in order to reduce OTA contamination risk.
PCR-based methods that target DNA are considered
a good alternative for rapid diagnosis because of their
high specicity and sensitivity which is particularly enhanced when multi-copy sequences are used to develop
species-specic primers [21]. ITS (internal transcribed
spacer) and IGS (intergenic spacer) regions of rDNA
units are present at 100300 copies per haploid fungal
genome and are considered highly variable regions.
The high variability provided by these regions is particularly useful when it is necessary to discriminate among
closely related species or at intraspecic level. Both ITS
and IGS regions have been used to carry out phylogenetic and population studies in lamentous fungi [22
27] and to develop specic PCR assays to identify
important mycotoxigenic species aecting commodities,
such as Fusarium or Aspergillus [2729].
The objective of this work was to develop specic
PCR assays to discriminate the main species included
in section Nigri, especially within the Aspergillus niger
aggregate, where both morphologically indistinguishable species dier in their ability to produce OTA. The
PCR assays were based on sequence information of
the multicopy ITS region.

2. Materials and methods

2.1. Fungal isolates and culture conditions
All the isolates used in this study, along with their
sources, are given in Table 1. The isolates were from different sources: the Spanish Type Culture Collection
(Spain), the Centraalbureau voor Schimmel Cultures
(CBS, The Netherlands), Agroindustrial Fungi and
Yeasts Collection (MUCL, The Belgian Co-ordinated
Collections of Micro-organisms) and isolates kindly

provided by Dr. V. Sanchs (University of Lleida, Spain)

and by Dr. Moretti (Agro-food Important Toxigenic
Fungi Culture Collection, Italy). The rest of the strains
were isolated from grapes, wheat and barley in our
laboratory.
Cultures were maintained on potato dextrose-agar
(PDA, Scharlau Chemie, Barcelona, Spain) at 4 C
and stored as spore suspension in 15% glycerol at
80
C. The isolates were cultured in 100 mL Erlenmeyer
asks containing 20 mL liquid medium Sabouraud
(Scharlau Chemie, Barcelona, Spain). Cultures were
inoculated with mycelial disks cut from the plates and
incubated at 25 C, 150 rpm. Mycelia from 2-day-old
cultures were harvested by ltration through Whatman
paper n 1 and kept at
80 C for DNA isolation.
2.2. DNA extraction and PCR amplication
Genomic DNA of the strains was obtained using
either the genomic DNA Extraction Kit (Genomix, Talent, Trieste, Italy), according to the manufacturers
instructions, or with the protocol described by Querol
[30].
All genomic DNAs used in this work were tested for
suitability for PCR amplication using primers ITS1
and ITS4 [31], which amplify the ITS region in Aspergillus. The PCR reaction was performed in an Eppendorf
Mastercycler Gradient (Eppendorf, Hamburg, Germany) using between 10 pg to 10 ng of genomic DNA.
The amplication program used was described by Henry
[22]. The amplication products were isolated using the
High Pure PCR Product purication Kit (Roche, Germany) and were sequenced using the ABI PRISM
DNA Sequencer (Applied Biosystems, Foster City,
USA) according to the manufacturers instructions in
the Genomic Unit of the University Complutense of
Madrid (Spain). All the strains were sequenced in both
directions. Sequences were analysed and aligned by the
Clustal method using the program DNASTAR (Lasergene, Wisconsin, USA).
PCR assays were carried out using primer ITS1 [31]
in all cases combined with a species-specic primer:
NIG (5 0 CCGGAGAGAGGGGACG GC 3 0 ) for A. niger, JAP (5 0 GAGAAGATTGGGGGTCGAGG 3 0 )
for A. japonicus, HET (5 0 GGAAAATGGTTGGAGAGGTCG 3 0 ) for A. heteromorphus and ELL (5 0
CCCGGGATGGGGGACGG 3 0 ) for A. ellipticus.
PCR reactions were performed in an Eppendorf Mastercycler Gradient (Eppendorf). The PCR amplication
protocol used for A. niger was as follows: 1 cycle of 4
min 30 s at 95 C, 25 cycles of 30 s at 95 C (denaturalization), 25 s at 66 C (annealing), 40 s at 72 C (extension) and nally 1 cycle of 5 min at 72 C. In the case of
A. japonicus, A. heteromorphus and A. ellipticus the PCR
program was the same that for A. niger except for the
annealing temperatures which were 62, 65 and 65.5 C,

A. Gonzalez-Salgado et al. / FEMS Microbiology Letters 245 (2005) 353361

355

Table 1
Fungal strains analysed indicating, origin, species, host and the occurrence of PCR amplication product with primers: ITS1-NIG, ITS1-HET, ITS1JAP and ITS1-ELL
Strain

Origin

Species

CECT 2091
CECT 20157
CECT 2574
CECT 2775
CECT 20156
T.MV.A16
C.AL.A37
Z.MA.A29
B.ME.A28
Z.GA.A29
Z.MA.A27
B.ME.A26
T.TT.A6
B.ME.A33
T.MV.A14
B.ME.A34
Z.GA.A22
Z.GA.A23
B.ME.A30
TR 11(A)
TR 18 1
TR 19(C)
TR 20(A)
TR 20 1
TR 20 2
TR 20 3
TR 21 5
TR 21 9
TR 22 1
TR 22 2
TR 22 4
TR 22 18
TR 23(A)
TR 23 3
TR 23 17
TR 25 1
TR 26 4
TR 26 7
TR 27 14
TR 43(A)
TR 45 1
TR 45 2
TR 45 3
TR 55(A)
TR 55(B)
TR 55 1
TR 55 2
TR 55 3
TR 55 4
TR 55 8
TR 58 1
TR 58 2
TR 58 3
TR 58 6
TR 58 9
TR 58 12
TR 62 2
TR 62 6
CB 2.5 1
CB 2.44 1
CB 2.44 2
TB 2 1

Canada
Canada

A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.

Zamora (Sp)
Zamora (Sp)
Valladolid (Sp)
Leon (Sp)
Valladolid (Sp)
Valladolid (Sp)
Leon (Sp)
Zamora (Sp)
Leon (Sp)
Zamora (Sp)
Leon (Sp)
Valladolid (Sp)
Valladolid (Sp)
Leon (Sp)
Bilbao (Sp)
Burgos (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Palencia (Sp)
Palencia (Sp)
Palencia (Sp)
Palencia (Sp)
Palencia (Sp)
Palencia (Sp)
Palencia (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Burgos (Sp)
Burgos (Sp)
Burgos (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Valladolid (Sp)
Valladolid (Sp)
Trevino (Sp)
Logrono (Sp)
Logrono (Sp)
Soria (Sp)

niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
niger
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis

Host

ITS1-NIG

ITS1-HET

ITS1-JAP

Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
Barley
Barley
Barley
Wheat

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

ITS1-ELL

(continued on next page)

A. Gonzalez-Salgado et al. / FEMS Microbiology Letters 245 (2005) 353361

356
Table 1 (continued)
Strain

Origin

Species

Host

ITS1-NIG

ITS1-HET

ITS1-JAP

ITS1-ELL

TB 2 3
TB 3 1
TB 3 2
TB 5 1
TB 5 2
OTAI
T.TT.A5
ZD.MF.ZD.A9
T.TT.A11
T.TT.A2
T.TT.A1
T.TT.A7
Asp Q
A1
A2
A3
A4
CBS 117.55

Soria (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)
United Kingdom
Zamora (Sp)
Valladolid (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Zamora (Sp)
Logrono (Sp)
Puerto Real (Sp)
Puerto Real (Sp)
Puerto Real (Sp)
Puerto Real (Sp)
Brazil

A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.
A.

Wheat
Wheat
Wheat
Wheat
Wheat

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+

+
+
+
+

+
+
+

MUCL 13578
ITEM 4158b
ITEM 4685b
ITEM 4687b
MUCL 31303
CBS 482.62
CBS 707.79
168a
229a
CECT 2086
CECT 2093
CECT 2969
CBS 589.68
CL.1
UCO.1
BO.1
CECT 2906
a
b

Italy
Portugal
Spain
Costa Rica
Costa Rica
Costa Rica
Spain
Spain

USA
Valladolid (Sp)
Valladolid (Sp)
Valladolid (Sp)

tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
tubingensis
heteromorphus

A. japonicus
A. japonicus
A. japonicus
A. japonicus
A. ellipticus
A. ellipticus
A. ellipticus
A. carbonarius
A. carbonarius
A. carbonarius
A. ochraceus
A. ochraceus
A. ochraceus
Cladosporium sp
Alternaria consortiale
Botrytis sp
Penicillium verrucosum

Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Grapes
Culture
contaminant
Grapes
Grapes
Grapes
Soil
Soil
Soil
Grapes
Grapes

Grapes
Grapes
Grapes

Strains supplied by Dr. Sanchis (University of Lleida, Spain).

Strains supplied by Dr. Moretti (CNR, Bari, Italy).

respectively. All amplication reactions were carried out

in volumes of 25 lL containing 3 lL (10 pg10 ng) of
template DNA, 1.25 lL of each primer (20 lM), 2.5
lL of 10 PCR buer, 1 lL of MgCl2 (50 mM), 0.25
lL of dNTPs (100 mM) and 0.2 lL of Taq DNA polymerase (5 U/lL) supplied by the manufacturer (Ecogen,
Barcelona, Spain). PCR products were separated in 2%
agarose ethidium bromide gels in 1 X TAE buer (Trisacetate 40 mM and EDTA 1.0 mM). The DNA ladder
Real escala n 2 (Durviz, Valencia, Spain) was used
as molecular size marker.
2.3. RFLP analysis
Aliquots of 10 lL of PCR amplication reactions obtained with ITS1/NIG primers were digested overnight
with RsaI (AfaI) (Amersham Pharmacia Biotech, Buckinghamshire, England) at 37 C in a total volume of 40
lL. The restriction fragments were separated by electro-

phoresis in 2.5% agarose gels. The DNA ladder Real

escala n 2 (Durviz, Valencia, Spain) was used as
molecular size marker. At least two independent experiments were performed.

3. Results
The ITS1-5.8S-ITS2 sequences of several isolates of
A. niger, A. tubingensis, A. japonicus, A. ellipticus, A. heteromorphus, A. carbonarius and other related Aspergillus
species were obtained and aligned together with other
sequences of Aspergillus species available in GenBank.
Fig. 1 shows the alignment of ITS1-5.8S-ITS2 sequences
of representative strains of the species A. carbonarius
(AJ876878), A. niger (AJ876876), A. tubingensis (AJ876877), A. heteromorphus (AJ876879), A. japonicus
(AJ876880), A. ellipticus (AJ876881) obtained in our
laboratory. Four specic primers, NIG, JAP, HET

A. Gonzalez-Salgado et al. / FEMS Microbiology Letters 245 (2005) 353361

TCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGTCCTTTGGG CCCAACCTC
-------------------------------------------------- ---------------------------------------------------------- --------18 S
----------------------------------------------C---G------------------------------------------------------C---G----------------------------------------------------------G---------

60

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

CCACCCGTGTCTATTGTACC TGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCCGGGG
---T---------------C-----------------------------------------T-----------A----C---------------------------------------------------CC-----A-----------------------C---------------------------CC---------------------------TTCG------- -----------------CC-----A---------------------------------------

120

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

GGGCATCTCTGCCCCTCGGGCCCGTGCCCGCCGGAGACACCAACACGAACACTGTCTGAA
----GC---------C----------------------C---------G-C------------GC---------C----------------------C---------G-----------------------------------------------------------------C-----------------------------------TG -------------------------------------------------------------------------

180

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

ATCGTGAAGTCTGAGTCGATTGTTTTCAATCAGTTAAAACTTTCAACAATGGATCTCTTG
-G----C---------------AA-G----------------------------------G----C---------------AA-G---------------------------------5.8 S
---A-----------------C---------------------------------------------------------------------------------------------------A-----------------C--------------------------------------

240

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

GTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAATGTGAATTGCAGAATTCA
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

300

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

GTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTG
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

360

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

TCCGAGCGTCATTGCTGCCCTCAAGCCCGGCTTGTGTGTTGGGTCGCCGTCCCCC TGTC
------------------------------------------------------T--C-------------------------------------------------------T--C-5.8 S
----------------------------------------------T--------CC-------------T--C-----C-----C---G-- -------C--- -C-----C
-------------------------------------------------------A-CC-

420

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

TGGGGGGACGGGCCCGAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGC
C----------------------------------------------------------C----------------------------------------------------------G--------------------------------------------------------------G------TCG-GA-A-A----------- ----G---------------------------------------------------------------------------

480

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

TTTGTCACATGCTCTGTAGGATTGGCCGGC GCCTGCCGACAACT CCAACCTTTTTTTC

------------------------------T----------GTT-T------A--C---------------------------------T----------GTT-T------A-------------CC----C------CC------------------ CTC-------A- -----------CC-----A-G--CCC------G---T----T -G-CCC----C--CT
--------CC-----------CC------------------GTT-T--------------

540

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

CAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAG
----------------------------------------------------------------------------------------------------------------------28 S
--------------------------------------------------------------A-------------------------------------------------------------------------------------------------------------------

600

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

GAA
----28
S
-------

603

A.
A.
A.
A.
A.
A.

carbonarius
niger
tubingensis
heteromorphus
japonicus
ellipticus

5.8 S

357

Fig. 1. Alignment of ITS1-5.8S-ITS2 sequences in six representative strains of A. carbonarius (229), A. niger (CECT 2091), A. tubingensis (T.TT.A2),
A. heteromorphus (CBS 117.55), A. japonicus (MUCL 13578) and A. ellipticus (MUCL 31303) and the location of primers ITS1 (underlined), NIG
(shadowed in pale gray), HET (double underlined), JAP (thick-underlined) and ELL (shadowed in dark gray). A dash represents the same nucleotide.
An empty space indicates a missing nucleotide. The restriction target of RsaI is indicated in bold.

and ELL, were designed on the basis of the sequence

alignment mentioned above. The position of the primers
and 5.8 gene are indicated using as reference the begin-

ning of ITS1 sequence for each isolate. In A. niger, the

primer NIG was located within the ITS2-rDNA at the
position +374 and the 5.8S gene was located between

358

A. Gonzalez-Salgado et al. / FEMS Microbiology Letters 245 (2005) 353361

positions +184 and +342. In A. japonicus, primer JAP

was located at position +466 and the 5.8S gene was located between positions +175 and +333. In A. heteromorphus primer HET was located at position +483
and the 5.8S gene was located between positions +182
and +340. In A. ellipticus, primer ELL was located at
position +376 and the 5.8S gene was located between
positions +185 and +343.
All the Aspergillus strains listed in Table 1 were tested
for amplication using the primer pair ITS1/NIG. A single fragment of about 420 bp was amplied only when
genomic DNA from the A. niger aggregate strains was
used (Fig. 2). Similarly, PCR amplications of genomic
DNA from all the strains indicated in Table 1 were performed using the primers ITS1/JAP, ITS1/HET and

ITS1/ELL (Figs. 35, respectively). A single fragment

of about 520, 540 and 420 bp was only obtained when
genomic DNA from A. japonicus (Fig. 3), A. heteromorphus (Fig. 4) and A. ellipticus (Fig. 5) strains was used.
Control amplications of the genomic DNA with primers ITS1 and ITS2 were positive for all the strains
analysed.
A. niger and A. tubingensis only diered in three
nucleotides. One of these nucleotides, located at position
+45 of the A. niger ITS1 sequence, aected the target of
RsaI endonuclease generating a pattern with two fragments in the case of A. niger (345 and 76 bp) and one
in A. tubingensis (420 bp) (Fig. 6). All the A. niger and
A. tubingensis strains listed in Table 1 were subjected
to digestion with RsaI, presenting the patterns described

Fig. 2. PCR amplication using primers ITS1/NIG and DNA from A. niger aggregate strains, lanes 110: CECT 2574, CECT 2091, CECT 2775,
T.MV.A14, B.ME.A26, TR55-B, ZD.MF.ZD.A9, TR11-A, T.TT.A1 and T.TT.A2, lane 11: Non-template control, lanes 1215: Aspergillus section
Nigri: A. carbonarius (168), A. japonicus (MUCL 13578), A. ellipticus (MUCL 31303) and A. heteromorphus (CBS 117.55), respectively, lane 16: A.
ochraceus (CECT 2093), lane 17: Penicillium verrucosum (CECT 2906), lane 18: Cladosporium sp. (CL.1) and lane 19: Alternaria consortiale (UCO.1).
M: DNA marker.

Fig. 3. PCR amplication using primers ITS1/JAP and DNA from Aspergillus japonicus strains, lane 14: MUCL 13578, ITEM 4158, ITEM 4685,
ITEM 4687, lanes 59: Aspergillus section Nigri: A. niger (CECT 2574), A. tubingensis (OTAI), A. carbonarius (229), A. heteromorphus (CBS 117.55)
and A. ellipticus (MUCL 31303), respectively, lane 10: A. ochraceus (CBS 589.68), lane 11: Penicillium verrucosum (CECT 2906), lane 12:
Cladosporium sp. (CL.1) and lane 13: Alternaria consortiale (UCO.1), lane 14: Non-template control. M: DNA marker.

A. Gonzalez-Salgado et al. / FEMS Microbiology Letters 245 (2005) 353361

359

Fig. 4. PCR amplication using primers ITS1/HET and DNA from Aspergillus heteromorphus strain, lane 1: CBS 117.55, lanes 26: Aspergillus
section Nigri: A. niger (CECT 2574), A. tubingensis (OTAI), A. carbonarius (168), A. japonicus (MUCL 13578) and A. ellipticus (MUCL 31303),
respectively, lane 7: A. ochraceus (CECT 2969), lane 8: Penicillium verrucosum (CECT 2906), lane 9: Cladosporium sp. (CL.1) and lane 10: Alternaria
consortiale (UCO.1), lane 11: Non-template control. M: DNA marker.

Fig. 5. PCR amplication using primers ITS1/ELL and DNA from Aspergillus ellipticus strains, lane 13: MUCL 31303, CBS 707.79, CBS 482.62,
lanes 48: Aspergillus section Nigri: A. niger (CECT 2574), A. tubingensis (OTAI), A. carbonarius (229), A. heteromorphus (CBS 117.55) and A.
japonicus (MUCL 13578), respectively, lane 9: A. ochraceus (CBS 589.68), lane 10: Penicillium verrucosum (CECT 2906), lane 11: Cladosporium sp.
(CL.1) and lane 12: Alternaria consortiale (UCO.1), lane 13: non-template control. M: DNA marker.

in Fig. 6 for the two species. The RsaI target sequence

was only missing in A. tubingensis and it was present
in the rest of the species analysed in this work (Fig. 1).

4. Discussion
Specic PCR assays have been developed in this
study for detection of A. niger, the main OTA producer
in section Nigri, as well as for the rest of the species included in this section, except A. carbonarius, for which a
specic PCR assay has been also developed and is described elsewhere [32].
The set of specic primers described in this work have
been designed on the basis of ample ITS sequence com-

parisons of several strains of Aspergillus species (Fig. 1)

and taking into account the phylogenetic and taxonomic
analyses reported previously by other authors [68]. The
specicity of the assays was tested on a number strains
of Aspergillus species as well as on other fungi commonly associated with grapes, cereals or coee, such as
Penicillium, Cladosporium or Alternaria species. The
strains were obtained from Collections or isolated from
the main commodities colonized by OTA-producing
Aspergillus strains. In the case of A. heteromorphus,
however, only one strain could be found in collections.
The lack of strains of this species could be due to the difculty of taxonomic recognition of species within this
section or to its absence on substrates or hosts related
with agro-food products. The PCR assay developed

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A. Gonzalez-Salgado et al. / FEMS Microbiology Letters 245 (2005) 353361

Fig. 6. RFLP patterns obtained after a digestion with RsaI of the PCR
amplications using primers ITS1/NIG and DNA from A. niger
aggregate strains. Lanes 13: A. niger CECT 2574, CECT 2775 and
T.MV.A14, respectively, lanes 46: A. tubingensis TR-22 1, T.TT.A1
and T.TT.A2, respectively. M: DNA marker.

for this species might facilitate its recognition, contributing to determining its presence, distribution and relative
importance in diverse substrates.
The PCR assays described in this work represent an
advantage in terms of time of analysis and specicity
in comparison with the conventional methods of identication and the more laborious molecular methods
based on AFLP proles [33], SSCP of the PCR-IGS
[34], or on secondary metabolite proles [25] reported
so far for Aspergillus. PCR assays reported for species
of the group (A. japonicus and A. carbonarius) [32,35]
are useful to provide complementary or conrmation
tests to assist correct identication.
The PCR assay with primers ITS1/NIG allowed discrimination of the two species of the A. niger aggregate
(A. niger and A. tubingensis) from the rest of the species
included in the section. This pair of primers amplied a
product including the target sequence for RsaI endonuclease which allowed distinction of A. niger and A.
tubingensis by a simple digestion of the PCR amplication product with RsaI. This endonuclease has been also
used to discriminate A. tubingensis from isolates of the
A. niger aggregate by digestion of the amplication fragment produced using primers ITS1/ITS2 [9]. However,
in this work, the other Aspergillus species of section Nigri were not tested but all of them showed the same sequence as A. niger for the RsaI target sequence, GTAC,
(Fig. 1). Our assay would provide a more accurate identication of A. niger isolates since the amplication frag-

ment used for digestion was specic to the A. niger

aggregate.
The detection limit of ITS amplication product, dened as a clearly visible product on agarose gels containing ethidium bromide, has been estimated between 1 and
10 pg of DNA template in Fusarium [21,36]. We found
similar detection levels (10 pg) with all sets of primers
when serial dilutions of genomic DNA of the dierent
Aspergillus strains were used as template for PCR amplication (data not shown). The sensitivity of our PCR assay based on ITS sequences was, therefore, more
sensitive than primers based on single copy genes, estimated between 0.1 and 1 ng of DNA template per reaction [21].
The specic assays reported in this work together
with other assays developed for other OTA-producing
Aspergillus species such as A. carbonarius and A. ochraceus [32,35] provides a simple and ecient tool for early,
rapid, sensitive and accurate detection of the main
OTA-producing Aspergillus species contaminating food
and raw products in order to prevent OTA from entering the food chain.

Acknowledgement
This work was supported by the Spanish MCyT
(AGL 2004-07549-C05-05/ALI). A. Gonzalez-Salgado
was supported by a Gobierno Vasco fellowship.

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