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Aspergillus ibericus: a new species of section Nigri isolated from grapes

     Centro de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, Braga, 4710-057 Portugal

F. Javier Cabañes

     Department de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, España

Giancarlo Perrone

     Institute of Sciences of Food Production, CNR, Viale Einaudi 5, 70125 Bari, Italia

Gemma Castellá

     Departament de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona, 08193 Bellaterra, España

Armando Venâncio ¹

     Centro de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, Braga, 4710-057 Portugal

Giuseppina Mulè

     Institute of Sciences of Food Production, CNR, Viale Einaudi, 70125 Bari, Italia

Zofia Kozakiewicz

     CABI Bioscience UK Centre, Bakeham Lane, Egham, Surrey TW20 9TY, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 

As part of a study on the ochratoxin producing mycoflora of grapes, several Aspergillus strains were isolated and tested for their ochratoxin A (OTA) producing abilities. Aspergillus strains of the section Nigri, which did not produce detectable amounts of OTA but which had a similar morphology to A. carbonarius, were isolated from wine grapes and/or dried vine fruit in Portugal and Spain. These strains, however, have characters that allow morphological distinction from the other species in the section, particularly the conidia size (5–7 µm), which allows separation of the species from the two most common biseriate species in section Nigri: A. carbonarius (7–9 µm) and A. niger and its aggregate species (3–5 µm). The strains are described here as belonging to a new species, named A. ibericus. The validation of this new taxon is supported further by analysis of the ITS-5.8S rDNA and calmodulin gene sequences and by analysis of the amplified fragment length polymorphism (AFLP) patterns, which were consistent in separating these strains from other species in the section. A. ibericus strains do not produce OTA therefore they are interesting for biotechnological exploration because many metabolites with commercial value are produced by other species in the section.

Key words: A. carbonarius, A. niger, black aspergilli, fungi, ochratoxin A, systematics

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Aspergillus species within section Nigri are important in biotechnological processes as well as biodeterioration. Species such as A. niger have a GRAS status from the FDA due to its extensive commercial and industrial uses. Conversely species in the section have proved to be particularly significant in the production of ochratoxin A (OTA) (Abarca et al 1994, Horie 1995) in several food commodities, particularly in grapes and grape products (Abarca et al 2003; Battilani and Pietri 2002; Battilani et al 2003; Bau et al 2005a; Bellí et al 2004; Heenan et al 1998; Leong et al 2004; Magnoli et al 2003; Rosa et al 2002; Sage et al 2002, 2004; Serra et al 2003, 2005b; Tjamos et al 2004). A. carbonarius was highlighted as the main species responsible for mycotoxin production in grapes, with some researchers claiming that 100% of the strains have the ability to produce OTA under laboratory conditions (Bau et al 2005a, b; Leong et al 2004; Sage et al 2002, 2004; Serra et al 2003). OTA production also was found in other species, namely in A. niger but at a much lower rate (Abarca et al 2004).

Recent surveys on the mycoflora of distinct commodities revealed new species of the section unknown to science, with some having the ability to produce OTA. The taxonomy of the section was revised recently (Abarca et al 2004), and 15 taxa were accepted in the section in the last critical revision (Samson et al 2004): A. aculeatus, A. brasiliensis ined., A. carbonarius, A. costaricaensis, A. ellipticus, A. japonicus, A. foetidus, A. heteromorphus, A. homomorphus, A. lacticoffeatus, A. niger, A. piperis, A. sclerotioniger, A. tubingensis and A. vadensis. However only four species in the section are relatively common: the uniseriate species A. aculeatus and A. japonicus and the biseriate species A. carbonarius and A. niger aggregate (with its two molecular types, A. niger and A. tubingensis). The remaining taxa are rarely reported or known only from type isolates.

In surveys in Europe to characterize OTA-producing mycoflora of wine grapes and dried vine fruit, Aspergillus strains were isolated, identified, tested for OTA production and characterized with three molecular tools: ITS-5.8S rDNA and calmodulin gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Some strains initially identified as A. carbonarius were unable to produce detectable OTA (Abarca et al 2003; Bau et al 2005b; Serra et al 2005a, b). These strains exhibited other differences from A. carbonarius, both in morphological and molecular aspects.

In this report we describe a new black Aspergillus species, Aspergillus ibericus, and compare it with others in the section.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Fungal cultures.— – Origin of the strains analyzed are as indicated (TABLE I). All strains except the reference strains used for comparison were isolated from grapes or dried vine fruit in Europe. The strains are stored in the CABI culture collection (IMI) and in other culture collections (viz. Micoteca da Universidade do Minho culture collection [MUM] and Culture Collection of Institute of Sciences of Food Production, Bari [ITEM]).

SEM microscopy.— – SEM micrographs of conidia were obtained from CZ according to the method described by Kozakiewicz for Aspergillus conidia (1989) (viz. by rubbing a standard SEM aluminium stub across a growing colony, coating it with gold [SC502 Fisons Instruments Sputter Coater] and examining directly in a Leica Cambridge S360 microscope). For conidiophore observation the strains were grown on CZ medium for 10 d, 25 C in the dark. Agar blocks were cut and fixed on a 1% OsO₄ solution in sodium cacodylate buffer (pH 7.3) overnight. After fixation the solution was removed by suction and samples rinsed several times with distilled water. Samples were dehydrated through a graded series of ethanol (25–100%) and left to air dry. Mounted samples were coated with gold and examined immediately.

OTA assay.— – Strains were evaluated with a previously described HPLC screening method (Bragulat et al 2001). Briefly, the isolates were grown on CYA and incubated at 25 C, 30 C and 35 C for 5 d and 10 d. From each isolate and at each sampling time three agar plugs were removed from different points of the colony and extracted with 0.5 mL methanol. The extracts were filtered and injected into the HPLC. OTA detection, and quantification was made by a Waters LCM1 chromatograph with a fluorescence detector Waters 2475 (excitation wavelength: 330 nm/emission wavelength: 460 nm) and with a column C18 Spherisorb S5 ODS2, 250 x 4.6 mm. Twenty µL of each extract were applied. The mobile phase, with a flow rate of 1 mL/min, consisted of this linear gradient: acetonitrile, 57%; water, 41%, and acetic acid, 2%. The extracts with the same retention time as OTA (ca 6.8 min) were considered positive. Confirmation was made through derivatization of OTA in its methylester (Hunt et al 1980). The detection limit of the extraction procedure and the HPLC technique was 0.02 ng OTA and the quantification limit of HPLC technique with the extraction procedure was 0.05 µg/g for this mycotoxin.

Molecular methods.— – For ITS-5.8S rDNA sequencing analysis fungal DNA was extracted as described by Accensi et al (1999). The strains were inoculated in 1.5 mL Eppendorf tubes containing 500 µL of Sabouraud broth (2% glucose, w/v; 1% peptone w/v) supplemented with chloramphenicol (1 mg/L) and incubated overnight in an orbital shaker at 300 rpm and 30 C. Mycelium was recovered after centrifugation and washed with NaCl 0.9% (w/v), frozen in liquid nitrogen and ground to a fine powder with a pipette tip. The powder was incubated 1 h at 65 C in 500 µL extraction buffer (Tris-HCl 50 mM, EDTA 50 mM, SDS 3% and 2-mercaptoethanol 1%). The lysate was extracted with phenol : chloroform (1 : 1, v/v), 3 M NaOAc and 1 M NaCl. DNA was recovered by isopropanol precipitation. The pellet was washed with 70% (v/v) ethanol, dried under vacuum and resuspended in TE buffer (Tris-HCl 10 mM, EDTA 1 mM, pH 8). DNA was cleaned with Geneclean kit II (BIO 101 Inc., La Jolla, California), according to the manufacturer’s instructions. ITS rDNA and 5.8S rDNA were amplified as described by Gené et al (1996) with a Perkin Elmer 2400 thermal cycler. Primer pairs ITS5 and ITS4 were described by White et al (1990). The amplification process consisted of a predenaturation step at 94 C, 5 min, followed by 35 cycles of denaturation at 95 C/30 s, annealing at 50 C/1 min and extension at 72 C/1 min, plus a final extension of 7 min at 72 C. The molecular masses of the amplified DNA were estimated by comparison with the 100-bp DNA ladder (Bio-Rad Laboratories S.A, Barcelona, Spain) standard lane. The PCR product was purified with the GFX PCR DNA and gel band purification kit (Amersham Pharmacia Bio-tech, Uppsala, Sweden), following the supplier’s protocol. Purified PCR products were used as a sequencing template. The protocol BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Gouda, The Netherlands) was used for sequencing. Primers ITS5 and ITS4 described by White et al (1990) were used in the sequencing reaction. An Applied Biosystems mod. 3100 sequencer was used to obtain the DNA sequences. The sequences were aligned with Clustal X (1.81) of the multiple sequence alignment program (Thompson et al 1994). Adjustments for improvement were made by eye where necessary. Cladistic analyses with the neighbor joining method (Saitou and Nei 1987) were performed with the MEGA 2.1 computer program (Kumar et al 2001) with Kimura 2-parameter model, including transitions and transversions and with pairwise deletion for the treatment of the handling gaps/missing data. Confidence values for individual branches were determined by bootstrap analyses (1000 replications) and maximum parsimony. The nucleotide sequences determined in this study have been deposited at the GenBank database and are identified by accession numbers (TABLE I).

For DNA amplification and sequencing of the partial calmodulin gene and for fluorescent AFLP analysis, fungal strains were grown in shake culture (150 rpm) in Wickerham’s medium (40 g glucose, 5 g peptone, 3 g yeast extract, 3 g malt extract and water up to 1 L). About 40 mg of filtered, frozen and lyophilized mycelium from each strain were used for total genomic DNA extraction with the EZNA Fungal DNA Miniprep Kit (Omega Biotek, Doraville, Georgia). DNA was recovered and dissolved in sterile water. Concentrations of DNA were determined by gel electrophoresis, by measuring the ultraviolet-induced fluorescence emitted by ethidium bromide molecules intercalated into DNA and comparing the fluorescent yield of the samples with a standard.

Amplifications of the partial calmodulin gene were set up with 2.5 U of Taq Gold DNA polymerase (Applied Biosystems) in 100 µL reaction mixtures, containing 30 pmol of each outside primer, 12.5 µM of each deoxynucleoside triphosphate (Applied Biosystems), and 1 µL (approximately 10 ng) of fungal template DNA. All isolates were amplified with primers CL1 and CL2A (O’Donnell et al 2000). The reactions were performed under these PCR conditions: denaturation at 94 C for 10 min; 35 cycles of denaturation at 94 C for 50 s, annealing at 55 C for 50 s, extension at 72 C for 1 min; final extension at 72 C for 7 min, followed by cooling at 4 C until recovery of the samples. After PCR amplicons were purified with a centrifugal filter device (Millipore) and sequenced with Applied Biosystems BigDye Terminator cycle sequencing kit in a 9700 GeneAmp PCR system. All sequencing reactions were purified through Sephadex G-50 (Pharmacia) equilibrated in double-distilled water and analyzed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). The resulting regions sequenced of all the isolates were aligned by the Clustal method with the DNAMAN program (Lynnon BioSoft). The unique calmodulin sequences were deposited at the EMBL nucleotide sequence database (TABLE I).

For preparation of AFLP template we used the AFLP Microbial Fingerprinting Kit (Applied Biosystems-Perkin Elmer Corp., Foster City, California) according to manufacturer’s instructions. Approximately 10 ng of genomic DNA of each isolate was cut with EcoRI and MseI (New England Biolabs, Hitchin, Hertfordshire, United Kingdom) and the DNA fragments were ligated to double-stranded restriction site-specific adaptors from the kit. A preselective PCR (72 C 2 min, 20 cycles of 94 C 20 s, 56 C 30 s, 72 C 2 min and held at 4 C) was carried out in a 20 µL (final volume) mixture. For the selective PCR, 1.5 µL of a 1 : 20 dilution of the first PCR reaction was amplified in a 10 µL (final volume) mixture with selective primers. Two separate primer combinations were used: (i) EcoRI+AC and MseI+CC; (ii) EcoRI+AT and MseI+CG. EcoRI primers were labeled with fluorescent dye (Applied Biosystems). The PCR program for selective AFLP amplification was: one cycle of 94 C for 2 min, one cycle of 94 C for 20 s, 66 C for 30 s, and 72 C for 2 min; this cycle was followed by nine cycles in which the annealing temperature was lowered by 1 C at each cycle from 65 C to 57 C., after which 20 cycles of 94 C for 20 s, 56 C for 30 s, and 72 C for 2 min were performed, followed by a final extension step of 60 C for 30 min, then held indefinitely with a model 9700 GeneAmp PCR system.

After amplification 1 µL of reaction product was mixed with 20 µL formamide and 0.5 µL GeneScan-500 (ROX) size standard (Applied Biosystems), of 35–500 bp long. The mix was heated 2 min at 95 C and snap-cooled on ice. The product was separated by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). After electrophoresis the pattern was extracted with GeneScan collection version 3.1.2 software (Applied Biosystems) and the fingerprints were analyzed with Genotyper software (Applied Biosystems). DNA samples from the five A. ibericus strains were tested in triplicate, and DNA samples from other strains were tested in duplicate. DNA from three replicate cultures of five strains also was tested.

Peak height thresholds were set at 200. Genotyper software (Applied Biosystems) was set to medium smoothing. Bands of the same size in different individuals were assumed to be homologous and to represent the same allele. Bands of different sizes were treated with AFLP manager database developed by ACGT BioInformatica S.r.1. (via Principe Amedeo 347- 70100 BARI) and were exported in a binary format with "1" for the presence of a band/peak and "0" for its absence. For clustering fragments of 50–500 bp were analyzed with NTSYS software with the Dice similarity coefficient based on presence/absence of the bands and clustered by the unweighted pair group method (UPGMA) (Nei and Li 1979).

	TAXONOMY

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
Examination of morphological characters combined with the analysis of OTA production and molecular data revealed that the Aspergillus Section Nigri strains isolated do not match other closely related species of the section. Therefore a new species, A. ibericus, is proposed.

Aspergillus ibericus Serra, Cabañes et Perrone sp. nov.

Coloniae in agaro Czapekii celeriter crescentes, in septem dies 25 C, 38–43 mm diam attigentes, granulosae, superficie nigra, facie inferiore primo alba, deinde obscure ochraceae; exudata non conspicuosa. Conidiogenesis abundata. Capitula conidica globosa, in columnas aegre formatas fissantia, 500–600 µm diam. Stipites leves incolores, superne pallide ochracei, 1200–2000 µm longissimi, parie plerumque 14–20 µm crassis; vesiculae globosae 50–60 µm diam, per toto fertilibus. Aspergilla biseriata; metulis et phialidibus 30.0–40.0 x 5.0–7.5 µm, phialides 8.0–10.0 x 6.0–7.0 µm. Conidia nigra oculo nudo visa, globosa vel subglobosa, 5.0–7.0 µm, conspicue verruculosa in maturitate et spinis 1.0 µm projecti.

Colonies on Czapek agar growing rapidly, attaining 38–43 mm diam within 7 d at 25 C, granular, upper surface black (FIG. 1); reverse white, wrinkled, becoming dull yellow with age; exudates not conspicuous. Conidiogenesis abundant. Conidial heads globose (FIG. 2) splitting into poorly defined columns with age, 500–600 µm diam. Stipes smooth, thick-walled, 1200–2000 x 14–20 µm, uncolored, upper portion light yellowish-brown; vesicles globose, 50–60 µm, fertile over entire surface. Aspergilli, biseriate (FIG. 3), with lightly packed metulae and phialides; metulae 30–40 x 5.0–7.5 µm; phialides 8.0–10.0 x 6.0–7.0 µm. Conidia black when seen with the naked eye, globose to subglobose, 5.0–7.0 µm, conspicuously verruculose at maturity, with spines projecting up to 1.0 µm (FIGS. 4–6).

View larger version (152K): [in this window] [in a new window]   FIGS. 1–6. Aspergillus ibericus. 1. Colony grown in CZ (9 d). 2. Biseriate aspergilli of a 4 d old culture in CZ (bar = 10 µm). 3. Aspergilli at SEM (bar = 200 µm). 4. Conidia seen at Nomarski microscope (bar = 10 µm). 5. SEM picture of the conidia with variable ornamentation at different maturation stages (bar = 20 µm). 6. SEM picture of a mature conidia (bar = 2 µm).

Etymology. – ibericus, from the Iberian Peninsula.

Known distribution. – Iberian Peninsula, Europe.

Origin of strains. – HOLOTYPE. PORTUGAL. ALENTEJO, Évora. Vineyard at 38°33'38''N, 7°54'30''W, on healthy grapes, 4 Oct 2001, Rita Serra 01UAs294.

Additional specimens examined. – IMI 391430 (MUM 03.50), PORTUGAL. ALENTEJO, Évora, from healthy grapes, 25 Aug 2003, Rita Serra 03UAs89; IMI 391431 (MUM 03.51), DOURO, Pinhão, from healthy grapes, 1 Oct 2003, Rita Serra, 03UAs254.

IMI 391428 (CCFVB A1082. CCFVB: Collection of the Veterinary Faculty of Barcelona) SPAIN. From raisins collected in a Spanish market survey, Dec 2000, M.L. Abarca; CCFVB A1229. SPAIN. From raisins collected in a Spanish market survey, Oct 2001, F.J. Cabañes; CCFVB A1604. SPAIN. JEREZ de la Frontera vineyard, from healthy grapes, Aug 2003, F. J. Cabañes.

Colonies grown 7 d on CYA overgrew a 90 mm plate (TABLE II). Conidiogenesis is abundant. Conidial heads black, mycelium white, usually inconspicuous; exudates inconspicuous; reverse pale or dull yellow. When grown at 37 C on CYA, colonies were heavily overgrown and were identical to colonies grown at 25 C. When grown on G25N colonies attained (12–)15–18(–23) mm diam but otherwise were identical to those on CYA at 25 C. No growth or germination was observed at 5 C. Growth in CZ20 was 44–46 mm diam in 7 d, with abundant conidiogenesis. Colonies were identical to those on CYA at 25 C, apart from reverse, which was yellow. On M40Y colonies were overgrown (90 mm plates) in 7 d at 25 C, otherwise identical to those on CZ20. Colonies on MEA (36–)38–43(–51) mm in 7 d at 25 C; conidia black, mycelium inconspicuous or as a white basal felt; reverse uncolored; otherwise as on CYA.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

Molecular analyses.— – A phylogenetic tree (FIG. 7) was generated with the neighbor joining method. The section of DNA sequenced from A. carbonarius included 612 base pairs. The ITS1 region occupied nucleotides 57–240, the 5.8S rDNA gene from nucleotides 241–397 and the ITS2 from nucleotides 398–566. The section of DNA sequenced from A. ibericus included 614 base pairs. The ITS1 region occupied nucleotides 57–241, the 5.8S rDNA gene from nucleotides 242–398 and the ITS2 from nucleotides 399–567.

View larger version (16K): [in this window] [in a new window]   FIG. 7. Neighbor joining tree based on phylogenetic analysis of the ITS1-5.8S rRNA gene-ITS2 sequences. The numbers at branch points are the percentages of 1000 bootstrapped datasets that supported the specific internal branches. Species with GenBank numbers represent sequences obtained from GenBank.

We identified a significant number of differences when the ITS1-5.8S-ITS2 sequences of the A. ibericus strains were compared with the corresponding sequences of other black Aspergillus species. Dissimilarities between A. ibericus strains and A. niger IMI 050566 and A. ellipticus IMI 172283 in their ITS1-5.8S-ITS2 sequences were 3.7% (23 of 614 nucleotides) and 5.5% (34 of 614 nucleotides) respectively.

The PCR product of the partial calmodulin gene was a fragment of about 660 bps long for the analyzed strains. The alignment of the sequences of the four A. ibericus strains showed a high similarity 99–100%, only the strain IMI 391428 differs in the calmodulin sequences from the other three A. ibericus strains for eight nucleotide positions (FIG. 8). The cladogram indicates that there are four major clades in section Nigri. One of these clades includes two subclades in which the A. carbonarius and A. ibericus strains are clearly separated. In fact the A. ibericus subclade resulted in clear separation from the A. carbonarius reference strain (IMI 16136) with a similarity of 88% which led to a difference of 78–80 bps in the partial calmodulin sequences alignment between the A. carbonarius and A. ibericus strains. On the other hand the A. ibericus clade, although close to A. carbonarius, was clearly separated from the A. niger aggregate (similarity > 80%) and also from the other species of Aspergillus Section Nigri (similarity of 72%). Aspergillus flavus (NRRL 21882) was used as outgroup and showed a similarity of 67% with the Aspergillus section Nigri cluster.

View larger version (12K): [in this window] [in a new window]   FIG. 8. Homology tree obtained by comparison of partial calmodulin gene sequences. The dendrogram obtained clearly separated the four atypical strains (A. ibericus) from Aspergillus carbonarius strains and also from other closely related species.

View larger version (44K): [in this window] [in a new window]   FIG. 9. UPGMA dendrogram assessed from the comparison of AFLP fingerprints generated with primer A. Fragments of 50–500 bp are shown. The Dice similarity index was used for similarity calculation. Numbers on the tree indicate the percentage of similarity according to Dice. The dendrogram clearly differentiate the four atypical A. carbonarius strains (A. ibericus) from the type-strain of A. carbonarius (IMI 16136-ITEM 4503).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
A. ibericus is most similar phenotypically to A. carbonarius. Both species have black biseriate aspergilli with long stipes and relatively large conidia (> 5 µm) when compared to the species of the A. niger aggregate (3–5 µm). Of the 15 species in the section Nigri accepted by Samson et al (2004) in the last revision, only two taxa had conidia of more than 6 µm diam, namely A. carbonarius and the rare species A. ellipticus. Nevertheless the size, shape and ornamentation of A. ibericus conidia are different (FIGS. 10–15). A. carbonarius (FIG. 10) has the largest echinulate conidia of all the black aspergilli; A. ellipticus (FIG. 11) has elliptical conidia echinulate of 7–9(–10) µm; A. ibericus (FIGS. 12, 13) has smaller echinulate conidia of 5–7 µm diam.

View larger version (142K): [in this window] [in a new window]   FIGS. 10–15. SEM pictures of conidia of black Aspergillus species. 10. A. carbonarius (bar = 4 µm). 11. A. ellipticus (bar = 2 µm). 12, 13. A. ibericus (bar = 2 µm). 14, 15. A. niger aggregate (bar = 2 µm).

Regarding the distinction between A. niger aggregate and A. ibericus, apart from conidia size, the ornamentation of the conidia is a useful characteristic. A. niger aggregate conidia are verrucose (FIGS. 14, 15) (Kozakiewicz 1989, Varga et al 2000) and echinulate in A. ibericus (FIGS. 12, 13).

The taxonomy of this section has received much attention from molecular analysis and from extrolite profiles, particularly for mycotoxin production by the species (Samson et al 2004). A. ibericus strains isolated to date are unable to produce detectable OTA in culture media, unlike A. carbonarius, in which all strains are strong OTA producers (Bau et al 2005a, b; Cabañes et al 2002; Leong et al 2004; Sage et al 2002, 2004; Serra et al 2003, 2005b). Some researchers claim that certain A. carbonarius strains do not produce OTA (Battilani et al 2003, Rosa et al 2002), but this claim must be investigated further. Bau et al (2005a, b) did not confirm these findings, stating that 100% of the A. carbonarius strains tested produce OTA, apart from those here now described as A. ibericus.

A summary of the characters of A. ibericus compared with other species in section Nigri is listed (TABLE III). A. carbonarius and A. ibericus strains can be distinguished based on two characteristics: (i) conidia size, with A. ibericus having smaller conidia, 5–7 µm, compared with A. carbonarius, 7–9(–10) µm; (ii) secondary metabolite profile, with none of the A. ibericus strains tested producing OTA, while all A. carbonarius are recognized as good OTA producers. A. sclerotioniger is a recently described species isolated from coffee beans related to A. carbonarius (Samson et al 2004) but with relatively smaller conidia (5–6 µm). A. ibericus can be distinguished from A. sclerotioniger based on cultural traits, with A. sclerotioniger producing abundant yellow to red-brown sclerotia in CYA and MEA and by the secondary metabolite profile, because A. sclerotioniger produces OTA.

Calmodulin results give a similar confirmation of the uniqueness of A. ibericus relative to other members of section Nigri. Both genes analyzed (rDNA and calmodulin) for sequence identity revealed a higher homology of A. ibericus strains to A. carbonarius strains (99% and 88% respectively) than to other black aspergilli (FIGS. 7, 8). In other studies the calmodulin gene has been shown to be highly reliable for phylogenetic analysis on the Gibberella fujikuroi complex and Fusarium related species (O’Donnell et al 2000), a candidate gene for population genetic analysis (Geiser et al 2000) and useful to discriminate among the Aspergillus of section Nigri (Perrone et al 2004). The AFLP profiles evidenced that A. ibericus strains showed a similarity > 50% among them when analyzed by AFLP, while the homology to the other species was 20%; this means that they must belong to a separate new species within the section Nigri. AFLP analysis results, supported also by DNA sequence analysis of diagnostic genes, confirmed the validity of this technique in analyzing genetic relatedness among fungal species (Zeller et al 2003).

The three molecular techniques clearly differentiate these strains from A. carbonarius and together with the morphological and biochemical differences unequivocally underline that these strains represent a new species in section Nigri.

In grapes A. ibericus is not significant regarding OTA production because the strains do not produce detectable OTA in laboratory media and in grape juice-based medium. The species responsible for OTA production in grapes is A. carbonarius. In Portuguese vineyards A. ibericus strains were less frequently isolated compared with A. niger aggregate and A. carbonarius isolates. They were isolated from five out of 17 vineyards studied, colonizing 2–8% of the healthy berries in the samples. A. ibericus strains were not isolated from rotten berries. Nevertheless its pathogenic capabilities must be evaluated.

In other European vineyards from France, Greece, Italy, Spain (and also from Israel), A. ibericus strains were much less frequently isolated than A. niger aggregate and A. carbonarius strains (Bau et al 2005b). Furthermore in previous studies A. ibericus (IMI 391428) was not able to produce OTA at any temperature (Esteban et al 2004) or pH value (Esteban et al 2005) tested.

Aspergillus species in section Nigri are widely used in industrial processes and have several biotechnological applications (Roehr et al 1992). Because A. niger is a GRAS organism and recent studies have shown that some isolates are capable of producing OTA in low concentrations, a more careful study into commercially used strains is advocated. The fact that strains of A. ibericus do not produce detectable OTA in culture media is an advantage for further studies on its potential biotechnological applications.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the support of the EC, Quality of Life Programme (QoL), Key Action 1 (KA1) on Food, Nutrition and Health; contract number QLK1-CT-2001-01761, Wine-Ochra Risk. Rita Serra was supported by grant SFRH/BD/1436/2000 from Fundação para a Ciência e Technologia.

	FOOTNOTES

 
Accepted for publication February 25, 2006.

¹ Corresponding author. E-mail: avenan{at}deb.uminho.pt

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS TAXONOMY RESULTS DISCUSSION LITERATURE CITED

 
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———, Castellá G, Bragulat MR, Cabañes FJ. 2005b. DNA-based characterization of ochratoxin-A-producing and non-producing Aspergillus carbonarius strains from grapes. Res Microbiol 156:375–381.

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