Aspergillus uvarum sp. nov., an uniseriate black Aspergillus species isolated from grapes in Europe

Black aspergilli (Aspergillus section Nigri; Gams et al., 1985) have a significant impact on modern society. Many species cause food spoilage, and several are used in the fermentation industry to produce hydrolytic enzymes, such as amylases or lipases, and organic acids, such as citric acid and gluconic acid (Varga et al., 2000). They can also be employed for genetic manipulation in the biotechnology industries, for example Aspergillus niger used under certain industrial conditions has been granted GRAS (Generally Regarded As Safe) status by the Food and Drug Administration of the US government. Although the main habitat of black aspergilli is soil, members of this section have been isolated from various other sources (Kozakiewicz, 1989; Abarca et al., 2004; Samson et al., 2004). Apart from their economical importance, black aspergilli are also important as ochratoxin-producing organisms which contaminate several agricultural products, including grape-derived products, coffee and cocoa (Cabañes et al., 2002; Samson et al., 2004; Battilani et al., 2006 and other papers in this special issue).

In spite of their importance, the taxonomy of this section is still not completely resolved and the species distribution in food is often not clearly reported. Black aspergilli are one of the more difficult groups to classify and identify and the taxonomy of Aspergillus section Nigri has been studied by many taxonomists (Abarca et al., 2004; Varga et al., 2007). Nuclear and mitochondrial DNA (mtDNA) polymorphisms and PCR-based techniques led to the recognition of at least two species within the A. niger species complex (A. niger and Aspergillus tubingensis) (Kusters-van Someren et al., 1991; Varga et al., 1994). Phylogenetic analyses of sequences of the internal transcribed spacers region (ITS), the 5.8S rRNA gene and the D1–D2 region of the 28S rRNA gene indicated that, in addition to A. niger and A. tubingensis, at least five other species belong to section Nigri: Aspergillus heteromorphus, Aspergillus ellipticus, Aspergillus carbonarius, Aspergillus japonicus and Aspergillus aculeatus (Varga et al., 2000; Parenicova et al., 2001). Several other black Aspergillus species have been described recently, including Aspergillus vadensis (de Vries et al., 2005), Aspergillus costaricaensis, Aspergillus piperis, Aspergillus lacticoffeatus and Aspergillus sclerotioniger (Samson et al., 2004), Aspergillus ibericus (Serra et al., 2006) and Aspergillus brasiliensis (Varga et al., 2007).

During a molecular survey of black Aspergillus isolates collected from 107 vineyards in different European countries within the EU project Wine-Ochra Risk (QLK1-CT-2001-01761), we discovered a group of uniseriate aspergilli morphologically very similar to the well-recognized species A. japonicus and A. aculeatus, but clearly separated by amplified fragment length polymorphism (AFLP) molecular analysis (Perrone et al., 2006). In this paper, we analyse the relationships of these isolates to other aspergilli using molecular and chemotaxonomic approaches, and describe these isolates as a novel species, Aspergillus uvarum sp. nov.

The strains of the novel species examined are listed in Table 1. The morphology of these strains was re-examined according to Raper & Fennell (1965) and Kozakiewicz (1989). The strains were maintained on malt extract autolysate (MEA) agar slants. The 28 Aspergillus section Nigri strains listed in Table 2 were used as reference strains for sequences and AFLP comparison analyses, and the type strain of Aspergillus flavus CBS 100927^NT was used as an outgroup.

Table 1. Aspergillus uvarum sp. nov. isolates from grapes examined

Table 2. Aspergillus section Nigri strains used in this study CBS, Centraalbureau voor Scimmelcultures, Utrecht, The Netherlands; JHC, James H. Croft's culture collection, Birmingham, UK; IMI, CABI Bioscience Genetic Resource Collection, Egham, UK; ITEM, Agri-Food Toxigenic Fungi Culture Collection, Institute of Sciences of Food Production, Bari, Italy; IBT, Center for Microbial Biotechnology, BioCentrum-DTU, Lyngby, Denmark; NRRL, Agricultural Research Service Culture Collection, National Center for Agricultural, Utilization Research, Peoria, IL, USA.

Morphological analysis.
For macromorphological observations, Czapek yeast autolysate (CYA), MEA agar, Czapek agar (CZA) (Samson et al., 2004) and G25N agar (Pitt, 1979) were used. Czapek agar with 20 % sucrose (CZ20) and malt and yeast extract agar with 40 % sucrose (M40Y) were made as described previously (Raper & Fennell, 1965). Growth rates were studied and compared with reference strains for 7 days on CYA at 5, 25 and 37 °C, on G25N, CZA and MEA at 25 °C in the dark. Cultures were also grown on CZ20 and M40Y for 7 days at 25 °C in the dark to assess growth on media with reduced water activity. For micromorphological observations, microscopic mounts were made in lactic acid from MEA colonies and a drop of alcohol was added to remove air bubbles and excess conidia. Scanning electron microscopy (SEM) was performed with uncoated frozen samples and micrographs consisted of 30 averaged fast scans (SCAN 2 mode) in a JEOL 5600LV scanning electron microscope equipped with an Oxford CT1500 cryostation.

Extrolite analysis.
Extrolites were analysed by HPLC using alkylphenone retention indices and diode array UV-VIS detection as described by Frisvad & Thrane (1987), with modifications as described by Smedsgaard (1997). Standards of ochratoxin A and B, aflavinine, asperazine, austdiol, kotanin, asterric acid, secalonic acid D, neoxaline and roquefortine D from the collection at BioCentrum-DTU (Danish Technical University) were used for comparison with the extrolites from the strains under study.

Isolation and analysis of nucleic acids.
Forty-four strains representative of the uniseriate black aspergilli isolated from grapes were analysed by sequence analysis together with 27 representative strains of section Nigri. Total nucleic acids were isolated as described previously (Leach et al., 1986). Fragments containing the region encoding the internal transcribed spacer 1 (ITS-1), 5.8S rDNA and internal transcribed spacer 2 (ITS-2) were amplified using primers ITS1 and ITS4 as described previously (White et al., 1990). Amplification of part of the β-tubulin gene was performed using the primers Bt2a and Bt2b (Glass & Donaldson, 1995; Samson et al., 2004). Amplifications of the partial calmodulin gene were set up as described previously (Perrone et al., 2004). Sequence analysis was performed with the Big Dye Terminator Cycle Sequencing Ready reaction kit for both strands, and the sequences were aligned with the MT Navigator software (Applied Biosystems). All the sequencing reactions were purified by gel filtration through Sephadex G-50 (Amersham Pharmacia Biotech) equilibrated in double-distilled water and analysed on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Representative ITS, β-tubulin and calmodulin sequences were deposited at the GenBank nucleotide sequence database (Table 1).

AFLP analysis.
Seventy-nine strains, 54 uniseriate Aspergillus isolates from grapes and 25 isolates belonging to different species of Aspergillus section Nigri, were analysed by AFLP analysis (Tables 1 and 2). Fungal strain growth and relative DNA isolation were done according to Perrone et al. (2006).

Fluorescent AFLP was performed as described in the AFLP microbial fingerprinting kit protocol (Applied Biosystems). Four separate primer combinations were utilized for the selective amplifications: EcoRI+AC and MseI+CC, EcoRI+AT and MseI+CG, EcoRI+AC and MseI+CA and EcoRI+G and MseI+CT. GeneScan-500 (ROX) was used as internal size standard (Applied Biosystems). The product was separated by capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). After electrophoresis, the pattern was extracted with the GeneScan version 3.1.2 software (Applied Biosystems) and the fingerprints were automatically analysed with the Genotyper software (Applied Biosystems). To test the reproducibility of the method, DNA of five strains was isolated from three replicate cultures and tested separately in triplicate. DNA of remaining strains was tested in duplicate.

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 size were treated as independent loci with two alleles. Data were analysed with the AFLP Manager database (ACGT BioInformatica) and were exported in a binary format with 1 for presence of a peak and 0 for its absence. For clustering, two different analyses were performed: fragments between 100 and 500 bp and fragments between 200 and 500 bp were analysed with NTSYSpc software (Exeter Software) by using the Dice similarity coefficient and clustered by the unweighted pair group method (UPGMA) (Nei & Li, 1979). A. flavus CBS 100927^NT was used as an outgroup in these experiments.

Analysis of sequence data.
The alignment of ITS regions, partial β-tubulin and calmodulin gene sequence data was performed using the software package BioNumerics (Applied Maths) and manual adjustments were made where necessary.

Phylogenetic trees were prepared by the neighbour-joining (NJ) method (Saitou & Nei, 1987) using the program NEIGHBOR of the PHYLIP package. Bootstrap values were calculated from 1000 replications of the bootstrap procedure using programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package (Felsenstein, 1985; 1995). For parsimony analysis, the PAUP* version 4.0 software was used (Swofford, 2000). Alignment gaps were treated as a fifth character state and all characters were unordered and of equal weight. Maximum-parsimony analysis was performed for all datasets using the heuristic search option with 100 random taxa additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the trees obtained was evaluated by 1000 bootstrap replications (Hillis & Bull, 1993). A. flavus CBS 100927^NT was used as an outgroup in these experiments.

Several uniseriate black Aspergillus strains were isolated from grape berries in the Mediterranean area, mainly from Italy, France and Israel. They were found to be related to A. japonicus, based on morphological data, and none of these A. japonicus-related isolates produced ochratoxin A. Morphologically, A. uvarum sp. nov. is most closely related to A. japonicus. However, both vesicle and conidium sizes are somewhat smaller in A. uvarum sp. nov. For example, vesicle size in A. uvarum sp. nov. ranges from 20 to 30 µm diameter, whereas for A. japonicus they average 20–30 µm, but can be as large as 45 µm. Conidial shape is similar, namely subglobose to globose, but A. japonicus conidia are mostly 3.0–4.5 µm, compared with 3.0–4.0 µm for A. uvarum sp. nov. A. aculeatus has much larger conidial heads with vesicles ranging from 22 to 55 µm in diameter and conidia which are ellipsoidal in shape.

However, it is growth rates, particularly on CYA at 37 °C, which clearly separate A. uvarum sp. nov. from A. japonicus and A. aculeatus. Growth of A. uvarum sp. nov. is much slower, with colony diameters after 7 days growth ranging from 16 mm for isolates A. uvarum sp. nov. IMI 388537 and IMI 388671 to 23 mm for isolates IMI 388523, IMI 388537 and IMI 388671. Colony diameters for A. japonicus CBS 114.51^NT and A. aculeatus IMI 211388^T were 30 mm after 7 days. These morphological and physiological differences, together with the extrolite and molecular information discussed below, indicate that A. uvarum sp. nov. is a novel uniseriate species within section Nigri.

Furthermore, these isolates could readily be distinguished from other uniseriate black aspergilli by AFLP analysis (Perrone et al., 2006). We examined the genetic relatedness of some selected isolates (Table 1) to other black aspergilli, analysing nucleotide sequences of partial rDNA (ITS-1, 5.8S and ITS-2), calmodulin and β-tubulin genes. The β-tubulin dataset included 551 characters, with 161 parsimony-informative characters. The NJ tree based on partial β-tubulin gene sequences is shown in Fig. 1.

(41K): Fig. 1. Phylogenetic tree based on β-tubulin sequence data of Aspergillus section Nigri. Numbers next to nodes are bootstrap values. Only values above 70 % are indicated. Bar, changes per nucleotide position.

The topology of the β-tubulin tree is the same as that of a parsimony tree constructed by the PAUP* program (length: 437 steps, consistency index: 0.6865, retention index: 0.8880). The ITS dataset included 475 characters with 72 parsimony-informative characters. The NJ tree based on ITS sequence data, shown in Supplementary Fig. S1 (available in IJSEM Online), has the same topology as the ITS parsimony tree (tree length: 168, consistency index: 0.9048, retention index: 0.9615). The calmodulin dataset included 666 characters, with 272 parsimony-informative characters. The topologies of the calmodulin NJ tree (Supplementary Fig. S2) and the parsimony tree were the same (tree length: 664, consistency index: 0.7229, retention index: 0.8999).

Although sequence analysis of the ITS region placed all of the uniseriate black aspergilli into the same clade, the β-tubulin and calmodulin sequence data both indicate the presence of three, closely related uniseriate taxa. In fact, these isolates formed a monophyletic clade supported by high bootstrap values on phylogenetic trees based on β-tubulin and calmodulin sequence data (Fig. 1 and Supplementary Fig. S2), despite a difference in topology of the two trees in the concordant branches which identified the closely related taxa A. aculeatus and A. japonicus. The grape-derived isolates were found to be closely related to the atypical A. aculeatus isolates CBS 114.80 and CBS 620.78. These isolates, together with recent isolates from Thai coffee beans, represent another novel species in section Nigri (P. Noonim, R. A. Samson and J. Varga, unpublished data).

The grape-derived isolates also formed a well-defined cluster on the UPGMA tree obtained from AFLP data (Supplementary Fig. S3) relative to four-primer combination assays. Our data indicate that these isolates are well separated from other black aspergilli, based on all molecular approaches used. Specific polymorphisms, within and between species, were observed by AFLP. Each primer combination consistently distinguished the 16 different species of Aspergillus species of section Nigri: similarity obtained among species analysed was less than 20 % (Supplementary Fig. S3). All the strains belonging to the same species shared more than 50 % of peaks. The Aspergillus uvarum cluster consists of a main group, including 48 strains clustered at 64 % similarity, and 6 strains (4958, 4959, 4961, 5287, 4997 and 4848), having 52 % similarity with the others (Supplementary Fig. S3).

Extrolite profiles of these isolates were characteristic and unique among the black aspergilli. The isolates of the new taxon proposed below produce secalonic acid D and F in common with A. aculeatus, but produced geodin, erdin, dihydrogeodin and asterric acid, which are not seen in any other black aspergilli. Geodin, erdin, dihydrogeodin and asterric acid are characteristic extrolites of Aspergillus terreus (Raistrick & Smith, 1936) and Penicillium glabrum (Mahmoodian & Stickings, 1964; Sato et al., 2005) and are related to trypacidin and monomethylsulochrin, produced by Aspergillus fumigatus (Turner, 1965; Larsen et al., 2007).

An examination of the extrolite profiles of the uniseriate black aspergilli showed that A. aculeatus produced secalonic acid D and F and physcion, A. japonicus isolates produced festuclavine and cycloclavin and the novel species produced secalonic acid D and F, asterric acid, erdin, geodin and dehydrogeodin. The isolate identified as an atypical A. aculeatus, CBS 114.80, produced okaramine A, B, H and I; Aspergillus brunneoviolaceus CBS 621.78^T produced okaramines, secalonic acid D and neoxaline and CBS 620.78 produced secalonic acid D and F, roquefortine D and some unique indole alkaloids. This is in agreement with the separation of these isolates suggested by molecular results (Fig. 1, Supplementary Figs S2 and S3). Thus both extrolite profiles and molecular data indicate that five species can be differentiated in the uniseriate black aspergilli. The isolates of A. aculeatus and A. japonicus examined and the new species described here were consistent in their production of extrolites, and thus can be easily differentiated chemically. Based on common production of secalonic acid D and F, the novel species (represented by ITEM 4834^T) appears to be more closely related to A. aculeatus than to A. japonicus.

Our findings were based on a polyphasic approach to the species concept in Aspergillus (Frisvad & Samson, 2004). A novel species is different from any other species in a diagnostic sense in both phenotypic and genotypic features. Here we have used morphological, physiological and chemotaxonomic features to characterize the phenotype and sequencing of three genes combined with AFLP results to characterize the isolates genotypically. Since the isolates were unique with regard to morphology, extrolite profiles and genotypic features, we propose the name Aspergillus uvarum sp. nov. for these isolates.

Description of Aspergillus uvarum Perrone, Varga et Kozakiewicz sp. nov. Fig. 2
Coloniae in agaro Czapekii celeritercrescentes in septem dies 25 °C, 90 mm diametro attigentes, granulosae, superficie brunnea-nigra, facie inferiore primo alba, deinde obscure ochraceae; exudata non conspicuosa, con solubis pigmentis ochraceis. Sclerotia producta, brunnea usque nigra, globosa vel verticale elongata. Conidiogenesis abundanta. Capitula conidica radiantia, in columnas aegre formatas fissantia, 200–300 µm diametro. Stipites leves incoloures, superne pallide ochracei, 500–1000 µm longissimi, parie plerumque 5–10 µm crassis; vesiculae globosae vel ellipticae 20–30 µm diametro, per toto fertilibus. Aspergilla uniseriata; phialides 5–7x2.5–3.0 µm. Conidia brunnea-nigra oculo nudo visa, globosa vel subglobosa, 3–4 µm, conspicue spinulosa in maturitate et spinis 0.5 µm projecti.

(138K): Fig. 2. Aspergillus uvarum sp. nov. IMI 388523T. (a) Colonies on CYA, (b) colonies on MEA, (c) colonies on oatmeal agar, (d–h) conidiophores, (i) conidia under light microscopy, (j) conidia as seen using SEM. Bars, 10 µm (e–h), 100 µm (d) and 5 µm (j).

Typus siccus in herb. IMI 388523^T (=CBS 127591^T=ITEM 4834^T=IBT26606^T) et ex-typus vivus, isolatus e botrys, Brundisium, Italia.

Description of Aspergillus uvarum Perrone, Varga et Kozakiewicz sp. nov.
Aspergillus uvarum (u.va'rum. L. masc. adj. uvarum of grapes).

Colonies grow rapidly on Czapek agar, attaining 90 mm diameter within 7 days at 25 °C, granular, upper surface brown-black; reverse white, wrinkled, becoming dull yellow with age with black colony centres; a soluble pale-yellow pigment is produced; exudate not conspicuous. Sclerotia produced in some fresh isolates, dark brown to black, globose to vertically elongated. Abundant conidiogenesis. Conidial heads radiate, splitting into poorly defined columns as they age, 200–300 µm in diameter. Stipes smooth, 500–1000x5–10 µm, uncoloured, upper portion light yellowish-brown; vesicles globose to elliptical, 20–30 µm, fertile over the entire surface. Aspergilli uniseriate; phialides 5–7x2.5–3.0 µm. Conidia brown–black when seen with the naked eye, globose to subglobose, 3–4 µm, conspicuously spinose at maturity with spines projecting 0.5 µm.

Colonies grown for 7 days at 25 °C on CYA overgrow a 90 mm plate. Sclerotia produced by some fresh isolates, usually coloured black. Abundant conidiogenesis. Conidial heads brown–black, mycelium white, usually inconspicuous; exudate inconspicuous; reverse pale or dull yellow with black colony centres; pale-yellow soluble pigment produced. When grown at 37 °C on CYA colonies attain (14) 16–22 mm diameter and are identical to colonies grown at 25 °C. When grown on G25N, colonies attain 13–17 mm diameter but otherwise are identical to those on CYA at 25 °C. No growth or germination is observed at 5 °C. On M40Y and CZ20 colonies are overgrown (90 mm plates) in 7 days at 25 °C, and are identical to those grown on CYA at 25 °C. Colonies on MEA are overgrown (90 mm plates) after 7 days at 25 °C; conidia brown–black, mycelium inconspicuous; reverse orange-brown, otherwise as on CYA.

Colony colours and texture.
On MEA conidiophores are abundantly produced, conidial areas are dark brown; sclerotia absent. Reverse on MEA light yellow, furrowed.

Sequence data.
All Aspergillus uvarum sp. nov. strains examined have high sequence similarity inside the group: 100 % for ITS, 99.5 % (four groups of individual variability) for the β-tubulin gene and 99.8 % (three groups of individual variability) for the calmodulin gene. The interspecific sequence divergences in the ITS region between these isolates and A. aculeatus, A. japonicus and Aspergillus homomorphus are 0.3, 0.5 and 6.5 %, respectively. In the β-tubulin dataset the differences are higher, in the range 10–18 % for A. aculeatus and A. japonicus and 25 % for A. homomorphus; while, in the calmodulin dataset, the differences are in the range 4–7 % for A. aculeatus and A. japonicus and 15.8 % for A. homomorphus.

Extrolites.
All isolates produced secalonic acid D and F, asterric acid, geodin, erdin and dihydrogeodin.

Holotype.
Dried colonies of IMI 388523 after 7 days growth on CZA, deposited in the Herbarium of CABI (UK Centre), Egham, UK, formerly International Mycological Institute. Ex-type culture: IMI 388523.

Known distribution.
Mediterranean basin, Europe.

We thank Filomena Epifani from Institute of Sciences of Food Production, CNR, Bari, Italy, for his valuable technical assistance, Paola Battilani (Italy), Armando Venancio (Portugal), Elefterios Tjamos (Greece), Amnon Licther (Israel), Vicente Sanchis (Spain) and Ahmed Lebrihi (France) for providing most of the A. uvarum sp. nov. strains used. This research was supported in part by a grant from the Italian Ministry of Education, University and Research (MIUR) Project 12818 - SIVINA - Individuazione di metodologie innovative prontamente trasferibili per migliorare la sicurezza dei vini rossi di qualità del Salento. B. T. was supported by a Bolyai research fellowship grant.