An ITS-RFLP method to identify black Aspergillus isolates responsible for OTA contamination in grapes and wine
P.V. Martínez-Culebras ⁎ , D. Ramón
Departamento de Medicina Preventiva y Salud Pública, Ciencias de la Alimentación, Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universitat de València, Vicent Andrès Estellès s/n 46100 Burjassot, Valencia, Spain
Dpto. de Biotecnología, Instituto de Agroquimica y Tecnología de los Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), P.O. 73, 46100, Burjassot, Valencia, Spain
Received 15 February 2006; received in revised form 2 June 2006; accepted 2 June 2006
Abstract
Ochratoxigenic mycobiota in grapes from representative wine regions in Valencia was identified. Black aspergilli were predominant among the differentAspergillusspp. isolated. Restriction digestion analysis of the ITS products was tested as a rapid method to identify isolates of black Aspergillus species from grapes. Restriction endonuclease digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI, distinguished five types of restriction fragment length polymorphism (RFLP) corresponding toAspergillus niger,Aspergillus tubingensis,Asper- gillus carbonariusandAspergillus aculeatusspecies. In addition, a new RFLP type in theA. nigeraggregate was identified. The fragments obtained by digestion with the endonucleaseNlaIII could be used to identify these new isolates. BlackAspergillusisolates were tested for their ability to produce OTA. Most of the isolates that produced ochratoxin A in YES medium belonged toA. carbonariusspecies. These results support evidence thatA. carbonariousgreatly contributes to OTA contamination in grapes and consequently in wine. The ITS-RFLP assay is proposed as a rapid and easy method to identify blackAspergillusspecies isolated from grapes, especially in studies that involve a large number of isolates.
© 2006 Elsevier B.V. All rights reserved.
Keywords:Black aspergilli; RFLP analysis; Identification; ITS; Grapes; Wine
1. Introduction
Ochratoxin A (OTA) is one of the most common naturally occurring mycotoxins, which is receiving increasing attention for its toxic effects and high incidence in a wide range of food commodities. It has been shown to be nephrotoxic, carcinogenic, immunotoxic, genotoxic and teratogenic and has been associated with Balkan Endemic Nephropathy (Krogh, 1978; Kuiper- Goodman and Scott, 1989; Abouzied et al., 2002). In fact, the International Agency for Research on Cancer classifies OTA in group 2B (possibly carcinogenic to humans) (IARC, 1993).
Unfortunately, the presence of OTA in blood from healthy humans confirms continuous and widespread exposure (Creppy, 1999).
In the European diet, wine and especially red wine, has been identified as the second major source of human exposure to OTA,
following cereals (Anonymous, 1998). The occurrence of OTA in wine and grape juice has been reported in different countries, but higher levels of OTAwere found in southern regions of Europe than in northern regions (Battilani et al., 2003; Majerus and Otteneder, 1996; Ospital et al., 1998; Otteneder and Majerus, 2000; Pietri et al., 2001; Lopez de Cerain et al., 2002). Consequently, the European Commission has considered it necessary to impose regulatory limits and has established 2μg/l as the maximum level of OTA in wine and grape products (EC No 123/2005).
In recent years, black Aspergillus species (section Nigri) have been described as the main source of OTA contamination in grapes and also in dried grapes worldwide (Da Rocha Rosa et al., 2002; Sage et al., 2002; Abarca et al., 2003; Battilani and Pietri, 2002; Serra et al., 2003; Bellí et al., 2004; Leong et al., 2004).
Black Aspergillus species producing OTA in grapes include Aspergillus carbonariusand species belonging to theAspergil- lus nigeraggregate. More recently, the ability of the uniseriate species belonging to the section Nigri, such as Aspergillus aculeatusandAspergillus japonicus, to produce OTA has been
⁎Corresponding author. Tel.: +34 963900022; fax: +34 963636301.
E-mail address:[email protected](P.V. Martínez-Culebras).
0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2006.06.023
reported but has yet to be confirmed (Battilani et al., 2003;
Dalcero et al., 2002). Some species, such asA. carbonariusand uniseriate species can easily be recognised by following the morphological criteria. In contrast, species included in the A. niger aggregate have always been extremely difficult to distinguish from each other morphologically. Molecular meth- ods (for an overview see Abarca et al., 2004) have led to an important progress in deciphering the relationships among these black aspergilli species; however, these techniques are imprac- tical for routine identification.
The internal transcribed spacer regions (ITS), non-coding and variable, and the 5.8S rRNA gene, coding and conserved, are useful for measuring close phylogenetic relationships in fungi (Lee and Taylor, 1992). Because ribosomal regions evolve in a concerted way, they display a low intraspecific polymorphism and a high interspecific variability, which has been proved useful for the identification of Aspergillus species (Accensi et al., 1999). The aim of this study is to test the value of the restriction analysis of the rRNA region spanning the 5.8S-ITS gene and the two ITS (from the now called 5.8S-ITS region) as a fast and easy method to identify black aspergilli species responsible for OTA contamination in grapes and wine.
2. Materials and methods 2.1. Samples and reference strains
During the 2004 season, a total of 22 vineyards were studied, located along the Mediterranean coast of Spain in the provinces of Alicante and Valencia and belonging to three different wine regions (D.O. Alicante, Utiel-Requena and Valencia). Nine grape varieties were included: Bobal, Cabernet Sauvignon, Garnatxa, Malvasia, Merlot, Merseguera, Moscatell, Semillon and Tempra- nillo. Samples were taken at the harvesting stage. At each vine- yard, 10 bunches were picked from 10 different plants located approximately along two diagonals crossing the vineyard. Each bunch was collected in a separate paper bag. Samples were sent to the laboratory as soon as they were collected and analysed within 24–48 h maximum.
Five berries were taken randomly from each bunch and directly plated onto plates of Dichloran Rose Bengal Chloram- phenicol medium (DRBC) (Pitt and Hocking, 1997). Plates were incubated at 25 °C for 7 days. In order to avoid the skin con- tamination of the berries, they were surface decontaminated using a 5% chlorine solution for 1 min followed by two rinses with sterile-distilled water. All fungi considered to represent different species were isolated and transferred to Malt Extract Agar (MEA) (Pitt and Hocking, 1997) plates for identification.
In order to study the OTA producing mycobiota of grapes, only the isolates belonging to Aspergillus and Penicillium genera were identified at species level. The remaining fungi were identified at genus level. Identification of isolates was achieved through macroscopic and microscopic observation with the aid of guidelines published (Klich, 2002; Samson et al., 2004a,b).
Identities of isolates were confirmed by 5.8-ITS sequencing.
Aspergillusspecies belonging to the sectionNigri, provided by the Spanish Type Culture Collection (CECT, Valencia, Spain)
and Centraalbureau voor Schimmelcultures (CBS, Utrecht, The Netherlands), were included as reference species (seeTable 1).
2.2. DNA preparation
All strains were grown on MEA medium for 6–8 days. My- celium was collected from the plates, frozen in liquid nitrogen and ground to a fine powder. DNA extractions were performed using 100 mg of powder and the commercial EZNA Fungal DNA kit (Omega bio-teck, Doraville, USA) according to the manufac- turer's instructions.
2.3. PCR reaction and DNA digestions
The 5.8S-ITS region was amplified by PCR using universal primers its5 and its4 (White et al., 1990). PCR reactions were performed in 100μl as the final volume, containing 100–200 ng of DNA, 50 mM KCl, 10 mM Tris–HCl, 80μM (each) dNTP, 1μM of each primer, 2 mM MgCl2and 1 U of DNA polymerase
Table 1
Reference strains of blackAspergillusspecies used in this study
Species Origin
A. aculeatus CECT 2968
CECT 20387
A. awamori CBS 101701
CECT 2907
A. brasiliensis CBS 101740
A. carbonarius CECT 20384
CECT 2086 CBS 11380
A. costariciensis CBS 115574 T
A. ellipticus CECT 20394
CECT 20395
A. ficuum CBS 555.65
A. foetidus CBS 101708
CECT 20388 CECT 20389 CECT 20396 CECT 20397
A. foetidusvaracidus CBS 128.528
A. foetidusvarpallidus CBS 114.52
A. helicotrix CECT 20390
A. hennebergui CECT 2801
A. heteromorphus CECT 20391
A. homomorphus CBS 101889
A. japonicus CECT 20386
A. lactcofeatus CBS101883T
A. niger CBS 101697
CECT 2088 CECT 20156 CECT 2090 CECT 2091 CECT 2700 CECT 20385 CECT 2948
A. piperis CBS 112811T
A. phoenicis CBS 136.52
A. pulverulentus CBS 558.65
A. tubingensis CECT 20392
CECT 20393
A. usamiivarshijo-usami CBS 191700
(Netzyme, Molecular Netline Bioproducts, N.E.E.D, SL, Valencia, Spain). The reaction mixtures were incubated in a thermalcycler (Techne TC-512) for 35 cycles consisting of 1 min at 95 °C, 1 min at 52 °C and 1 min at 72 °C.
PCR products were digested with the restriction enzymes HhaI, NlaIII and RsaI (MBI Fermentans, Vilnius, Lithuania).
PCR products and their restriction fragments were separated on 1 and 3% agarose gels, respectively, with 0.5× TBE buffer. After electrophoresis, gels were stained with ethidium bromide (0.5 mg ml−1), and the DNA bands visualised under UV light. Sizes were estimated by comparison with a DNA standard length (GeneRu- ler™100 pb DNA ladder, MBI Fermentans, Vilnius, Lithuania).
2.4. Sequencing analysis
PCR products were cleaned with the GeneClean II Puri- fication Kit (Bio 101, La Jolla, CA, USA) and directly se- quenced using the Taq DyeDeoxy terminator cycle sequencing Kit (Applied Biosystems, Falmer, Brighton, UK), according to the manufacturer's instructions in an Applied Biosystems auto- matic DNA sequencer (model 373A). The primers its5 and its4 were also used to obtain the sequence of both strands.
The 5.8S-ITS sequence from the isolate number 64 was previously obtained for phylogenetic analysis. 5.8S-ITS sequences available in the EMBL data library fromA. nigeraggregate species were included in the analysis. Sequences were included for the following accessions: AY373840, A. awamori; AY373850, A. foetidus; AF223852,A. niger; U65307,A. phoenicis; AJ223843, A. tubingensis. The 5.8S-ITS region sequences were aligned using the multiple-sequence alignment program CLUSTAL X. The genetic distances were calculated using the Jukes–Cantor model and the phylogenetic inference was obtained by the neighbour- joining (NJ) method (Saitou and Nei, 1987). The NJ tree and the statistical confidence of a particular group of sequences in the tree, evaluated by bootstrap test (1000 pseudoreplicates) (Hills and Bull, 1993), were performed using the computer program MEGA version 2.0 (Kumar et al., 2001). AF459734,A. carbonariusand
AJ279994, A. japonicus were designated as outgroups for the sequence analyses.
2.5. Extraction and detection of OTA from culture
Ochratoxin A was extracted using a variation of a simple method described previously (Bragulat et al., 2001). The isolates were grown on Yeast extract Sucrose agar (YES) (Pitt and Hocking, 1997) and incubated at 25 °C for 7 and 14 days.
Briefly, three agar plugs (diameter: 6 mm) were obtained from the inner, middle and outer areas of each colony of potential ochratoxin producers and were placed in a vial containing 900μl of methanol. After 60 min, the extracts were shaken and filtered (Millex® SLHV 013NK, Millipore, Bedford, MA, USA) into another vial and stored at 4 °C until chromatographic analysis.
The production of OTA was detected and quantified by high- performance liquid chromatography (HPLC) with fluorescence detection (λexc330 nm;λem460 nm) using a C18column (Luna 2, 5μm, 4.6 × 250 mm, Phenomenex, Jasco, Spain). The mobile phase (acetonitrile:water:acetic acid, 57:41:2) was pumped at 1.0 ml min−1. The injection volume was 20μl and the retention time was about 8 min. The detection limit of the analysis was 0.02 μg OTA g−1 of YES medium. The ochratoxin standard was obtained from A. ochraceus (Sigma-Aldrich, Steinheim, Germany). The standard solution was made in methanol (Merck, Darmstadt, Germany).
3. Results
3.1. Fungal contamination of grapes
Predominant mycobiota belonged to black Aspergillus species that constituted 84.8% of the total isolates. The incidence of other species belonging to the generaAspergillus(4.4%) and Penicillium (6.6%) was low compared to black Aspergillus species. AllAspergillusspp. andPenicilliumspp. considered to represent different species were isolated and identified by ITS sequencing. Within the genera Aspergillus, we identified As- pergillus flavus, Aspergillus nidulans, Aspergillus ochraceus, Aspergillus tamarii,Aspergillus terreus andAspergillus wenti.
From the genera Penicillium, seven species were identified.
They include Penicillium brocae, Penicillium citrinum, Peni- cillium commune,Penicillium chrysogenum,Penicillium glab- rum, Penicillium griseoroseum, Penicillium minioluteum and Penicillium sumatrense. Finally, isolates from the genera
Fig. 1. Ribosomal DNA restriction patterns exhibited by black Aspergillus isolates from grapes after digestion with the restriction endonucleasesHhaI, RsaI andNlaIII. Lanes M correspond to the 100 bp molecular weight marker (GeneRuler™100 pb DNA ladder, MBI Fermentans, Vilnius, Lithuania).
Table 2
Ribosomal restriction patterns and composite patterns or types exhibited by the blackAspergillusisolates analysed in the present study
Species Type Restriction patterns Number of isolates
Number of OTA + isolates HhaI NlaIII RsaI
A. niger N A A A 33 0
A. tubingensis T1 A A B 101 1
A. tubingensis-like T2 A B B 30 1
A. carbonarius C B A A 45 20
A. aculeatus A C C A 3 0
Alternaria,Botrytis,Cladosporium,Fusarium,Mucor,Rhyzo- pusand Trichodermawere also isolated although at very low frequencies (5.2%). They were identified at genus level.
3.2. Molecular characterization of the black Aspergillus isolates
Due to the high frequency of isolation and the potential for production of OTA (Abarca et al., 2004), the mean objective in this study was to identify black Aspergillus isolates. Identifi- cation was based on RFLPs of the 5.8S-ITS region. The its5 and its4 primers were used to amplify the ribosomal region, which includes the two non-coding ITS1 and ITS2, and the 5.8S rRNA gene. A total of 212 isolates of blackAspergilluswere analysed.
The size of all amplified PCR products was estimated to be 650 bp.
On the basis of ITS sequences the endonucleases HhaI, NlaIII andRsaI were used in the restriction analysis in order to differentiate among isolates. Their typical restriction patterns are shown inFig. 1. These individual profiles designated with the letters A–C, can be combined into composite restriction patterns or RFLP types (Table 2). Each one of the 212 isolates analysed was then assigned to its RFLP type. The restrictions patterns obtained for the different isolates were compared with those obtained from the reference strains of blackAspergillus species (Table 1).
For the 212 black Aspergillusisolates analysed, five RFLP types were observed (Table 2). The most common was type T1,
Fig. 2. Neighbour-joining tree based on nucleotide divergences, estimated according to Jukes–Cantor model, from the 5.8S-ITS sequences. The numbers on the nodes are the frequency (in percent) with which a cluster appears in a bootstrap test of 1000 runs. The phylogenetic tree shows the relationships between the new isolate of the RFLP type T2 and species belonging toA. nigeraggregate.
Fig. 3. Specific identification of isolates belonging to RFLP type T2 by digestion with the restriction endonucleaseNlaIII. Lane 1 corresponds to representative isolate of the RFLP type T2. Lanes 2–20 correspond to reference species of the blackAspergillusspecies. Lanes M correspond to the 100 bp molecular weight marker (GeneRuler™100 pb DNA ladder, MBI Fermentans, Vilnius, Lithuania).
represented by 101 isolates (47.6%). This RFLP type corresponded to A. tubingensis species. Type N included 33 isolates (15.6%) and corresponded toA. nigerspecies. Type C included 45 isolates (21.2%) that corresponded toA. carbonar- ius species. Type A included only 3 isolates (1.4%) that cor- respond toA. aculeatus species. Finally, type T2 included 30 isolates (14.2%) and did not correspond to any reference spe- cies. Previously, the ITS sequence for the isolate number 64 belonging to the type T2 was obtained and compared with those available in the NCBI Nucleotide Database. It was nearly identical to sequences ofA. tubingensisCBS 643.92 and CBS 127.49, except for a single nucleotide (G instead of T) at position 532. The phylogenetic relationship between isolate 64 and species belonging toA. niger aggregate is illustrated in a cluster analysis (Fig. 2). Isolate 64 representing type T2 clus- tered with other members of A. niger aggregate in a major cluster (100% bootstrap support). However, isolate 64 formed a very close group, with high (86%) bootstrap support, which includedA. tubingensisandA. phoenicisspecies, both of them showing identical ITS sequences. For this reason, isolates belonging to type T2 could be namedA. tubingensis-like spe- cies. Nucleotide (G) at position 532, present in the sequence of these isolates, affected the target of NlaIII endonuclease gen- erating a pattern with 4 fragments (390, 130, 60 and 50) in the case of these isolates and three (390, 130 and 110) for the rest of the black aspergilli isolates analysed. All the blackAspergillus strains listed inTable 1were subjected to digestion withNlaIII.
TheNlaIII target sequence at position 532 was only present in A. tubingensis-like species but was missing for the rest of the species analysed in this work (Fig. 3). We conclude, therefore, that the fragments resulting from digestion withNlaIII could be used as a method to identify these isolates.
3.3. Ochratoxigenic ability of black Aspergillus isolates
A total of 212 black aspergilli isolates, which had previously been identified by RFLP, were tested for their ability to produce OTA in YES medium (Table 2). Twenty-two isolates were shown to produce OTA (10.4%). Twenty of the ochratoxigenic isolates were classified as A. carbonarius (44. 4% of the 45 tested), while the remaining 2 were classified asA. tubingensis andA. tubingensis-like respectively. They mainly produced low amounts of OTA with a maximum concentration of 2.85μg g−1. OTA was not detected in cultures of the 33 isolates ofA. niger.
OTA was also not detected in cultures ofA. aculeatus.
4. Discussion
The main fungi isolated were black Aspergillus species.
Similar results were obtained by other researchers from the Mediterranean area (Bellí et al., 2004, 2005; Bau et al., 2005;
Medina et al., 2005). In addition to black aspergilli, other As- pergillusspecies, as well asPenicilliumspecies, were isolated and identified. Although some of theAspergillusspp. isolated have a well-known potential for producing mycotoxins such as aflatox- ins (A. flavus) and sterigmatocystin (Emericellaspp.) (Pitt and Hocking, 1997), given their low incidence they do not appear to
be a source of mycotoxins in this substrate. The incidence of species belonging toAspergillussectionCircumdati, which are traditionally considered to be ochratoxigenic, was very low. Only two isolates ofA. ochraceuswere isolated. Within thePenicillium spp. identified, there were some important mycotoxin producers, such asP. citrinum(citrinin) andP. chrysogenum(roquefortine C) (Pitt and Hocking, 1997). Given the low frequency of isolation, their potential for mycotoxins production is not a cause of concern.
Restriction digestion analysis of the ITS products was tested to assess its effectiveness as a rapid method to identify different isolates of blackAspergillusspecies from grapes. To do this, a large number of isolates, including collection strains of black aspergilli were checked. Five different RFLPs types were identified among black Aspergillus isolates by using the endonucleases HhaI, NlaIII and RsaI. By comparison with reference species, isolates analysed in this study were assigned to the species A. aculeatus, A. carbonarius, A. niger and A.
tubingensis, and a new type within theA. nigeraggregate was named as A. tubingensis-like (see below). The most common species wasA. tubingensis(47.6%) followed byA. carbonarius (21.2%),A. niger(15.6%),A. tubingensis-like (14.2%) andA.
aculeatus (1.4%). Identification of black aspergilli species involved in OTA production in grapes and wine has been difficult. Consequently, it has not been easy to determine species occurrence and distribution. Whereas A. carbonarius and the uniseriate species (A. aculeatusand A. japonicus) can be distinguished microscopically by vesicle and conidial size and ornamentation (Abarca et al., 2004), all the taxa in theA.
nigeraggregate are morphologically indistinguishable.Accensi et al. (1999) described the restriction endonucleaseRsaI as a useful tool to classify the isolates of theA. nigeraggregate into pattern N (corresponding to A. niger) or pattern T (corre- sponding to A. tubingensis) and it was used to determine the distribution of strains in theA. nigeraggregate (Accensi et al., 2001). The results from the present study support those by these authors, the target for endonucleaseRsaI was present in theA.
niger sequences but does not exist in the sequences of A.
tubingensis. Moreover, in this study two additional endonucle- ase enzymes (HhaI andNlaIII) were used to differentiate black aspergilli from grapes. On one hand, the endonuclease HhaI enable us to differentiate amongA. carbonarius,A. japonicus and species forming A. niger aggregate without the need for specific knowledge of fungal morphology. On the other hand, restriction NlaIII and RsaI can be used to differentiate three different RFLP types within blackAspergillusspecies belong- ing to the A. niger aggregate. Due to its simplicity, the three patterns found within A. niger aggregate (N, T1 and T2) are easier to recognise than the RFLP patterns described by other authors (Kusters van Someren et al., 1991; Varga et al., 1993, 1994; Parenicová et al., 2001).
Thirty isolates (type T2) could not be assigned to any reference species and a new RFLP type (RFLP T2) was de- scribed (Table 2). However, the 5.8S-ITS sequence for these isolates was nearly identical to sequences of A. tubingensis species. Phylogenetic relatedness among different species belonging to A. niger aggregate supports the sequence data indicating a close relationship between the new isolates
identified (type T2) and A. tubingensisspecies. Nevertheless, on comparing sequences, a target for the restriction enzyme NlaIII was found at position 532 of the sequences of the isolates T2, which was not present in the A. tubingensis se- quence. Neither was the target for NlaIII present in a large number of culture collection strains, even though tests were made of most blackAspergillusspecies currently recognised by Samson et al. (2004c). Although we prefer to collect more evidence of their taxonomic interrelationships, it is tempting to speculate that the blackAspergillusisolates belonging to type T2 may represent a new species in theAspergillussectionNi- gri. Additional morphological studies and sequence data from other genes are necessary in order to examine the taxonomic relatedness. The same ITS sequence was described byMedina et al. (2005)for two OTA-producing isolates, which they named asA. tubingensis. However, at least in the studies of occurrence and distribution, we prefer to differentiate these new isolates under the name ofA. tubingensis-like.
The screening method (Bragulat et al., 2001) used in this study was found to be good in checking OTA production in a large number of isolates.A. carbonariuswas the major ochratoxin A producing species and this result confirms previous findings that A. carbonariouscontributes to OTA contamination in grapes and consequently in wine (Battilani et al., 2003; Bau et al., 2005; Bellí et al., 2004, 2005). Two isolates from each,A. tubingensisand A. tubingensis-like were positive for OTA production. These data agree with those from Medina et al. (2005). One isolate that matched at 100% withA. tubingensisand 2 isolates that matched at 99% with the same species were positive for OTA production in that study. These isolates, classified by Medina et al. (2005) as A. tubingensis, clearly correspond to isolates identified as A. tubingensisandA. tubingensis-like respectively in the present study, which confirms that theA. tubingensisspecies is able to produce OTA. No OTA-producingA. nigerisolates were detected in the present work, which is also in agreement withMedina et al.
(2005). However, these results disagree with those ofAccensi et al. (2001), where all the OTA producing isolates were classified as pattern N (A. niger) and none of the isolates classified as pattern T (A. tubingensis) produced OTA. Apart from the different numbers of isolates tested and their origin, there are a number of possible reasons for the disagreement in results reported by different researchers. The difficult differentiation between A. niger and A. tubingensisbased on morphological characteristics may have led to misidentifications. In addition, laboratory practices used in extraction and detection of OTA (culture medium, incubation time and/or temperature) might also lead to different results. A consensus in laboratory practices in OTA extraction and pro- duction and also in identification techniques is needed in order to compare results reported by different researchers.
In conclusion, a rapid identification technique like the one described in the present study (by the digestion with only three restriction enzymes), enables easy and rapid identification of the black Aspergillus isolates derived from grapes and their assignation to different species. This kind of information will help to develop an understanding of the epidemiology and distribution of black aspergilli in grapes, where vast numbers of isolates have to be screened in a short time. Accurate iden-
tification of theAspergillusspecies in theNigrisection is also of great importance in determining toxicological risks because the toxic profile of each species could be different.
Acknowledgements
This research was supported by grants from the Generalitat Valenciana (GV2004-B-195) and the Spanish Government (AGL2005-00707/ALI). P.V.M.C. is supported by a Ramón y Cajal research contract, cofinanced by the Spanish Government and the University of Valencia. We thank Jose Vte. Gil, Teresa Lafuente and Ana Rosa Ballester for their help in HPLC analysis.
References
Abarca, M.L., Accensi, F., Bragulat, M.R., Castellá, G., Cabañes, F.J., 2003.Asper- gillus carbonariusas the main source of ochratoxin A contamination in dried vine fruits from the Spanish market. Journal of Food Protection 66, 504–506.
Abarca, M.L., Accensi, F., Bragulat, M.R., Castellá, G., Cabañes, F.J., 2004.
Taxonomy and significance of black aspergilli. Antonie van Leeuwenhoek 86, 33–49.
Abouzied, M.M., Horvat, A.D., Podlesny, P.M., Regina, N.P., Metodiev, V.D., Kamenova-Tozeva, R.M., Niagolova, N.D., Stein, A.D., Petropoulos, E.A., Ganev, V.S., 2002. Ochratoxin A concentrations in food and feed from a region with Balkan endemic nephropathy. Food Additives and Contaminants 19, 755–764.
Accensi, F., Cano, J., Figuera, L., Abarca, M.L., Cabañes, F.J., 1999. New PCR method to differentiate species in theAspergillus nigeraggregate. FEMS Microbiology Letters 180, 191–196.
Accensi, F., Abarca, M.L., Cano, J., Figuera, L., Cabañes, F.J., 2001.
Distribution of ochratoxin A producing strains in theA. nigeraggregate.
Antonie van Leeuwenhoek 79, 365–370.
Anonymous, 1998. Position paper on ochratoxin A. Codex Alimentarius Commission, Codex Committee on Food Additives and Contaminants, Thirty-first Session. The Hague, the Netherlands, 22–26 March 1999, CX/
FAC 99/14.
Battilani, P., Pietri, A., 2002. Ochratoxin A in grapes and wine. European Journal of Plant Pathology 108, 639–643.
Battilani, P., Pietri, A., Bertuzzi, T., Languasco, L., Giorni, P., Kozakiewicz, Z., 2003. Occurrence of ochratoxin A-producing fungi in grapes grown in Italy.
Journal of Food Protection 66, 633–636.
Bau, M., Bragulat, M.R., Abarca, M.L., Minguez, S., Cabañes, F.J., 2005.
Ochratoxin species from Spanish wine grapes. International Journal of Food Microbiology 98, 125–130.
Bellí, N., Pardo, E., Marín, S., Farré, G., Ramos, A.J., Sanchis, V., 2004.
Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes. Journal of Science of Food and Agriculture 84, 541–546.
Bellí, N., Mitchell, D., Marín, S., Alegre, I., Ramos, A.J., Magan, N., Sanchis, V., 2005. Ochratoxin A-producing fungi in Spanish wine grapes and their relationship with mereological conditions. European Journal of Plant Pathology 113, 233–239.
Bragulat, M.R., Abarca, M.L., Cabañes, F.J., 2001. An easy screening method for fungi producing ochratoxin A in pure culture. International Journal of Food Microbiology 71, 139–144.
Creppy, E.E., 1999. Human ochratoxicoses. Journal of Toxicology. Toxin Reviews 18, 273–293.
Dalcero, A., Magnoli, C., Hallak, C., Chiachiera, S.M., Palacio, G., Rosa, C.A.R., 2002. Detection of ochratoxin A in animal feeds and capacity to produce this mycotoxin byAspergillussectionNigri in Argentina. Food Additives and Contaminants 19, 1065–1072.
Da Rocha Rosa, C.A., Palacios, V., Combina, M., Fraga, M.E., De Oliveira Rekson, A., Magnoli, C.E., Dalcero, A.M., 2002. Potential ochratoxin A producers from wine grapes in Argentina and Brazil. Food Additives and Contaminants 19, 408–414.
Hills, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology 42, 182–192.
International Agency for Research on Cancer (IARC), 1993. Some naturally Occurring Substances: Food Items and Constituents. Heterocyclic Aromatic Amines and Mycotoxins, vol. 56. World Health Organization, Lyon, France.
Klich, M.A., 2002. Identification of CommonAspergillusSpecies. Centraalbur- eau voor Schimmelcultures, Utrecht, The Netherlands.
Krogh, P., 1978. Causal associations of mycotoxic nephropathy. Acta pathologica et Microbiologica Scandinavica. Section A. Supplement 269, 1–28 (Suppl).
Kuiper-Goodman, T., Scott, P.M., 1989. Risk assessment of the mycotoxin ochratoxin A. Biomedical Environmental Science 2, 179–248.
Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2001. MEGA2: molecular evolutionary genetic analysis software. Bioinformatics 17, 1244–1245.
Kusters van Someren, M.A., Samson, R.A., Visser, J., 1991. The use of RFLP analysis in classification of the blackAspergilli: reinterpretation ofAsper- gillus nigeraggregate. Current Genetics 19, 21–26.
Lee, S.L., Taylor, J.W., 1992. Phylogeny of five fungi like protoctistanPhy- tophthoraspecies inferred from the internal transcribed spacers of ribosomal DNA. Molecular Biology and Evolution 9, 636–653.
Leong, S.L., Hocking, A.D., Pitt, J.I., 2004. Occurrence of fruit rot fungi (Aspergillussection Nigri) on some drying varieties of irrigated grapes.
Australian Journal of Grape Wine Research 10, 83–88.
Lopez de Cerain, A., González-Peñas, E., Jiménez, A.M., Bello, J., 2002.
Contribution to the study of ochratoxin A in Spanish wines. Food Additives and Contaminants 19, 1058–1064.
Majerus, P., Otteneder, H., 1996. Detection and occurrence of ochratoxin A in wine and grape juice. Deutsche Lebensmittel-Rundschau 92, 388–390.
Medina, A., Mateo, R., López-Ocaña, L., Valle-Algarra, F.M., Jimenez, M., 2005. Study of Spanish grape mycobiota and ochratoxin A production by isolates ofAspergillus tubingensisand other members ofAspergillussection Nigri. Applied and Environmental Microbiology 4696–4702.
Ospital, M., Cazabeil, J.M., Betbeder, A.M., Tricard, C., Creppy, E.E., Medina, B., 1998. L'ochratoxine A dans les vins. Enologie 169, 16–19.
Otteneder, H., Majerus, P., 2000. Occurrence of ochratoxin A (OTA) in wines:
influence of the type of wine and its geographical origin. Food Additives and Contaminants 17, 793–798.
Parenicová, L., Skouboe, P., Frisvad, J., Samson, R., Rossen, L., Hoor- Suykerbyuk, M.T., Visser, J., 2001. Combined molecular and biochemical
approach identifiesAspergillus japonicusandAspergillus aculeatusas two species. Applied and Environmental Microbiology 67, 521–527.
Pietri, A., Betuzzi, T., Pallaroni, L., Piva, G., 2001. Occurrence of ochratoxin A in Italian wines. Food Additives and Contaminants 18, 647–654.
Pitt, J.I., Hocking, A.D., 1997. Fungi and Food Spoilage. Blackie Academic and Professional, London.
Sage, L., Krivobok, S., Delbos, E., Seigle-Murandi, F., Creppy, E.E., 2002.
Fungal flora and ochratoxin A production in grapes and musts from France.
Journal of Agricultural and Food Chemistry 50, 1306–1311.
Saitou, N., Nei, M., 1987. The neighbour-joining method: a new method for reconstruction phylogenetic trees. Molecular Biology and Evolution 4, 406–425.
Samson, R.A., E.S., Frisvad, J.C., 2004a.PenicilliumSubgenusPenicillium:
New Taxonomic Schemes, Mycotoxins and Other Extrolites. Studies in Mycology, vol. 49. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
Samson, R.A., Hoekstra, E.S., Frisvad, J.C., 2004b. Introduction to Food- and Airborne Fungi, 7th edn. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.
Samson, R.A., Houbraken, J.A.M.P., Kuijpers, A.F.A., Frank, J.M., Frisvad, J.C., 2004c. New ochratoxin A or sclerotium producing species in Aspergillus sectionNigri. Studies in Mycology 50, 45–61.
Serra, R., Abrunhosa, L., Kozakiewicz, Z., Venancio, A., 2003. BlackAsper- gillus species as ochratoxin A producers in Portuguese wine grapes.
International Journal of Food Microbiology 88, 63–68.
Varga, J., Kevei, F., Fekete, C., Coenen, A., Kozakiewicz, Z., Croft, J.H., 1993.
Restriction fragment length polymorphism in the mitochondrial DNAs of Aspergillus nigeraggregate. Mycological Research 97, 1207–1212.
Varga, J., Kevei, F., Vriesema, A., Debets, F., Kozakiewicz, Z., Croft, J.H., 1994.
Mitochondrial DNA restriction fragment length polymorphism in field isolates of theAspergillus nigeraggregate. Canadian Journal of Microbi- ology 40, 612–621.
White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, pp. 315–322.
