SGM Journals
Eukaryotic Micro-Organisms

Two novel aflatoxin-producing Aspergillus species from Argentinean peanuts

María B. Pildain, Jens C. Frisvad, Graciela Vaamonde, Daniel Cabral, Janos Varga, Robert A. Samson

  • 1Faculty of Ciencias Exactas y Naturales, Pab. II, Lab. 69, University of Buenos Aires, CP EHA1428, Buenos Aires, Argentina
  • 2Center for Microbial Biotechnology, BioCentrum-DTU, Building 221, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
  • 3CBS Fungal Biodiversity Centre, PO Box 85167, 3508 AD Utrecht, The Netherlands
  • 4Department of Microbiology, Faculty of Sciences, University of Szeged, PO Box 533, H-6701 Szeged, Hungary
  • Correspondence
    Robert A. Samson
    samson{at}cbs.knaw.nl

    • International Journal of Systematic and Evolutionary Microbiology 2008; 58(3):725–735 · https://doi.org/10.1099/ijs.0.65123-0

    View at publisher PubMed

    Abstract

    Two novel species from Aspergillus section Flavi from different species of Arachis (peanuts) in Argentina are described as Aspergillus arachidicola sp. nov. and Aspergillus minisclerotigenes sp. nov. Their novel taxonomic status was determined using a polyphasic taxonomic approach with phenotypic (morphology and extrolite profiles) and molecular (β-tubulin and calmodulin gene sequences) characters. A. minisclerotigenes resembles Aspergillus flavus and Aspergillus parvisclerotigenus in producing aflatoxins B1 and B2, cyclopiazonic acid, kojic acid and aspergillic acid, but in addition it produces aflatoxins G1 and G2, aflavarins, aflatrem, aflavinines, parasiticolides and paspaline. This species also includes several isolates previously assigned to A. flavus group II and three Australian soil isolates. A. arachidicola produces aflatoxins B1, B2, G1 and G2, kojic acid, chrysogine and parasiticolide, and some strains produce aspergillic acid. The type strain of A. arachidicola is CBS 117610T =IBT 25020T and that of A. minisclerotigenes is CBS 117635T =IBT 27196T. The Mycobank accession numbers for Aspergillus minisclerotigenes sp. nov. and Aspergillus arachidicola sp. nov. are respectively MB 505188 and MB 505189 ().

    • The GenBank/EMBL/DDBJ accession numbers for the β-tubulin and calmodulin gene sequences of the strains examined in this study are shown in Fig. 1⇑ and Supplementary Fig. S1.

    • A neighbour-joining phylogenetic tree based on calmodulin gene sequences and colour versions of Figs 2⇑ and 3⇑ are available as supplementary material with the online version of this paper.

    INTRODUCTION

    Aflatoxins are the most potent natural carcinogens known (JECFA, 1997), affecting all vertebrate animal species, including humans. Four compounds are commonly produced in foods, aflatoxins B1, B2, G1 and G2, but other bio-transformed aflatoxins may occur, for example in milk, such as aflatoxins M1 and M2 (Cole & Cox, 1981). These mycotoxins have been shown to be produced by Aspergillus flavus, A. parasiticus (Codner et al., 1963; Schroeder, 1966), A. nomius (Kurtzman et al., 1987), A. pseudotamarii (Ito et al., 2001), A. bombycis (Peterson et al., 2001), A. toxicarius (Murakami, 1971; Murakami et al., 1982; Frisvad et al., 2004) and A. parvisclerotigenus (Saito & Tsurota, 1993, Frisvad et al., 2004) in Aspergillus section Flavi, by A. ochraceoroseus (Frisvad et al., 1999, Klich et al., 2000) and A. rambellii (Frisvad et al., 2005) in Aspergillus section Ochraceorosei and in Aspergillus section Nidulantes or the ascomycete genus Emericella by Emericella astellata (Frisvad et al., 2004) and E. venezuelensis (Frisvad & Samson, 2004a).

    The most important aflatoxin producers from a public health point of view are members of Aspergillus section Flavi, in particular A. flavus and A. parasiticus. Originally, several isolates of A. parasiticus were misidentified as A. flavus [for example NRRL 2999, 3000 and 3145 (Hesseltine et al., 1966; Applegate & Chipley, 1973); corrected to A. parasiticus by Hesseltine et al., 1970], and therefore G-type aflatoxins were connected with A. flavus. Like A. pseudotamarii and ‘A. flavus’ NRRL 3251, A. flavus has later been reported to produce only B-type aflatoxins (Dorner et al., 1984; Klich & Pitt, 1985; Bennett & Papa, 1988; Ito et al., 2001; Ehrlich et al., 2004; Frisvad et al., 2005), while A. parasiticus, A. toxicarius, A. nomius and A. bombycis can produce both B- and G-type aflatoxins (Ehrlich et al., 2004; Frisvad et al., 2005). There are many reports to indicate that certain A. flavus strains, microsclerotial strains, and strains listed as intermediate between A. flavus and A. parasiticus can also produce G-type aflatoxins (Codner et al., 1963; Hesseltine et al., 1970; Cotty & Cardwell, 1999). Many aflatoxin B- and G-producing strains have been reported to produce small sclerotia, but they do not obviously belong to A. parvisclerotigenus (Saito & Tsurota, 1993; Cotty & Cardwell, 1999; Bayman & Cotty, 1993; Egel et al., 1994; Frisvad et al., 2005). Isolates in section Flavi producing small sclerotia apparently produce the same mixture of indoloditerpene alkaloids, whether they produce only B-type aflatoxins (NRRL 3251) or both B- and G-type aflatoxins [CBS 121.62 (=NRRL A-11612) and Nigerian and Indonesian strains] (Tanaka et al., 1989). There is phylogenetic evidence that Aspergillus flavus sensu lato may consist of several species (Geiser et al., 1998, 2000; Chang et al., 2006). However, this is difficult to evaluate, as most strains examined in those studies were generally not deposited in major culture collections. In order to find out whether such B+G-type aflatoxin producers belonged to one or more species, we surveyed various Arachis species in Argentina because such wild specimens of Arachis could be expected to harbour a more diverse mycobiota than domesticated peanuts (Arachis hypogaea), which have been examined in depth by many authors (e.g. Austwick & Ayerst, 1963). Isolates representing A. flavus group II as defined by Geiser et al. (1998, 2000) and soil isolates from Australia kindly provided by J. I. Pitt (CSIRO, North Ryde, Australia) were also included in the analyses.

    We have used a polyphasic taxonomic approach in order to determine the taxon delimitation (Frisvad & Samson, 2004b; Varga et al., 2007; Houbraken et al., 2007, Samson et al., 2007a, b). For the phenotypic analyses, macro- and micromorphology, extrolite profiles and growth temperatures were studied. For the phylogenetic analyses, β-tubulin and calmodulin gene sequences were used.

    METHODS

    Isolates.

    The strains examined listed in Table 1⇓ were cultures from the CBS (CBS Fungal Biodiversity Centre, Utrecht, Netherlands), NRRL (NCAUR Culture Collection, Peoria, IL, USA) or IBT (at BioCentrum-DTU, Kgs. Lyngby, Denmark) collections or they were freshly isolated from seeds and leaves of cultivated peanut (Arachis hypogaea L.) and leaves of autochthonous peanut species [Arachis villosa Benth., Arachis correntina (Burkart) Krapov. & W. Gregory, Arachis glabrata Benth. and Arachis burkartii Handro] from Argentina. The Argentinean strains were chosen based on being representatives of known vegetative compatibility groups (VCGs) of A. flavus and from different hosts and agroecological zones within Argentina (Vaamonde et al., 1995; Novas & Cabral, 2002; Pildain et al., 2003, 2004).

    Table 1.

    Aspergillus isolates examined

    Morphology and extrolite profiles.

    For macromorphological observations, isolates were grown on Czapek yeast autolysate (CYA), malt extract agar (MEA), Czapek agar (CZA), yeast extract sucrose (YES) agar, oatmeal agar (OA) and creatine sucrose agar (CREA) (Samson et al., 2004). Aspergillus flavus and parasiticus agar (AFPA; Pitt et al., 1983) was used to determine the production of aflatoxins on agar medium. The strains were inoculated at three points and incubated at 25 °C in the dark for 7 days and/or at 37 and 42 °C on CYA. For micromorphological observations, microscope mounts were made in lactic acid from MEA colonies and a drop of alcohol was added to remove air bubbles and excess conidia. Extrolites were analysed by HPLC using alkylphenone retention indices and diode array UV-VIS detection according to Frisvad & Thrane (1993), as modified by Smedsgaard (1997).

    Genotypic analysis.

    The cultures used for molecular studies were grown in 2 ml malt peptone (MP) broth, containing 10 % (v/v) malt extract (Brix 10) and 0.1 % (w/v) bacto peptone (Difco), in 15 ml tubes. The cultures were incubated at 25 °C for 7 days in light/darkness. DNA was extracted from the cells using the Masterpure yeast DNA purification kit (Epicentre Biotechnologies) according to the instructions of the manufacturer. A fragment of the 5′ portion of the β-tubulin gene was amplified using primers bt2a and bt2b (Glass & Donaldson, 1995), while a segment of the calmodulin gene was amplified using primers cmd5 and cmd6 as described by Hong et al. (2006). The amplified DNA fragments were purified using a QIAquick PCR purification kit (Qiagene). DNA sequences were determined using a BigDye Terminator v3.1 cycle sequencing kit (ABI) and an ABI 3100 DNA sequencer. Both strands of each fragment were sequenced.

    DNA sequences were edited with the dnastar computer package and an alignment of the sequences was performed using the clustal w program (Thompson et al., 1994). The neighbour-joining (NJ) method was used for the phylogenetic analysis. For NJ analysis, the data were first analysed using the Tamura–Nei parameter distance calculation model with gamma-distributed substitution rates, which were then used to construct the NJ tree with mega version 3.1 (Kumar et al., 2004). To determine the support for each clade, a bootstrap analysis was performed with 1000 replications.

    Phylogenetic analysis of sequence data was also performed using paup* 4.0b10 (Swofford, 2000). Alignment gaps were treated as a fifth character state, uninformative characters were excluded and all characters were unordered and weighted equally. Maximum-parsimony (MP) analysis was performed for all datasets using the heuristic search option. To assess the robustness of the topology, 1000 bootstrap replicates were run by maximum-parsimony (Hillis & Bull, 1993). Other measures including tree length, consistency index and retention index (CI and RI, respectively) were also calculated. Sequences were deposited at GenBank under accession numbers listed in Fig. 1⇓ and Supplementary Fig. S1 (available in IJSEM Online).

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree based on β-tubulin sequence data of Aspergillus section Flavi. Numbers above branches are bootstrap values. Only values above 70 % are indicated. Bar, 2 substitutions per 100 nucleotide positions.

    RESULTS

    Morphological analysis

    In our survey, six species from Aspergillus section Flavi were isolated from Argentinean wild peanut species: A. caelatus, A. flavus, A. tamarii and A. parasiticus and two taxa related to A. parvisclerotigenus and A. parasiticus. All Aspergillus isolates analysed by microscope examination exhibited conidial heads in shades from yellow–green to brown and had similar colony characteristics and growth rates on all media analysed in this study, and they all grew very fast at 37 °C. These are typical morphological features associated with Aspergillus section Flavi (Raper & Fennell, 1965). The strains from Argentinean peanuts were compared to ex type and authentic strains of species in Aspergillus section Flavi (see Table 1⇑) and could be divided into two groups. One group of isolates (represented by CBS 117626) was similar to A. tamarii and was characterized by dark-brown conidia with conspicuously roughened to tuberculate thick walls and colonies with a dark-brown reverse on AFPA. The only species with such characteristics included A. tamarii, A. pseudotamarii and A. caelatus. The remaining strains had light to dark yellow–green conidia and less conspicuously roughened conidia. They also had a cadmium orange- or cream-coloured reverse on AFPA.

    Isolates of A. flavus have been reported to produce two types of sclerotia, small (S) and large (L) (Cotty, 1989). In our study, we found 16 strains (represented by CBS 117620, CBS 117633–117635 and CBS 117639) with small sclerotia, which were similar to A. parvisclerotigenus CBS 121.62T. One A. parasiticus isolate produced sclerotia of intermediate size (CBS 117618), while A. flavus IBT 27177, CBS 117622, CBS 117630 and CBS 117733 produced large sclerotia.

    DNA analysis

    For the molecular analysis, two regions of the genome were analysed, namely parts of the calmodulin and β-tubulin genes of the isolates. For the analysis of part of the β-tubulin gene, 510 characters were analysed. Among the 128 polymorphic sites, 74 were found to be phylogenetically informative. The NJ tree based on partial β-tubulin genes sequences is shown in Fig. 1⇑. The topology of the tree is the same as one of the more than 105 MP trees constructed by the paup program (tree length, 173 steps; CI, 0.8844; RI, 0.9564).

    The calmodulin dataset included 520 characters, with 80 parsimony informative characters. The topology of the NJ tree (Supplementary Fig. S1) was the same as one of the more than 105 MP trees (length, 229; CI, 0.8603; RI, 0.9290).

    Most of the sequenced Argentinean isolates fell into one of two main clades, represented by A. flavus and A. parasiticus. Isolate CBS 117616 was related to A. caelatus (Horn, 1997), which we could also confirm by its morphology. A. sojae, A. toxicarius and A. terricola var. americanus were found to belong to the A. parasiticus clade. Four isolates (CBS 117610–117612 and CBS 117615) from Arachis glabrata leaves formed a well-defined clade related to A. parasiticus on the trees based on β-tubulin and calmodulin data (Fig. 1⇑ and Supplementary Fig. S1). However, these isolates have internal transcribed spacer (ITS) sequences identical to those of A. parasiticus (data not shown). Another six Argentinean isolates from Arachis hypogaea seeds formed a well-defined clade related to A. flavus and A. parvisclerotigenus on trees based on β-tubulin and calmodulin sequence data (Fig. 1⇑ and Supplementary Fig. S1). These results are in agreement with our morphological and extrolite results. Furthermore, these Argentinean isolates belong to the same VCG as described by Pildain et al. (2004, 2005). This clade also includes four isolates assigned to A. flavus group II by Geiser et al. (1998, 2000) and three isolates collected from soils from Australia, all producing small sclerotia. Our calmodulin and β-tubulin sequence data indicate that A. oryzae, A.thomii, A. kambarensis, A. fasciculatus and A. subolivaceus are very closely related to A. flavus.

    Extrolites

    In our extrolite study of 34 strains from Argentinean peanuts, we found that the strains which were identified as A. flavus produced kojic acid (100 %), aspergillic acid (100 %), cyclopiazonic acid (82 %), aflatoxins B1 and B2 (74 %), oryzaechlorin (44 %) and flavimine (94 %) (Table 2⇓). A single strain of A. tamarii from Argentinean peanuts produced kojic acid and oryzaechlorin. Four strains of A. parasiticus from Argentinean peanuts produced aflatoxins B1, B2, G1 and G2, aspergillic acid, kojic acid and parasiticolides, one strain (IBT 27180) produced oryzaechlorin, one strain (IBT 27194) produced paspaline and paspalinine and one strain (CBS 117618) produced aflavinines and other sclerotial metabolites.

    Table 2.

    Production of mycotoxins and other extrolites by selected species in Aspergillus section Flavi based on HPLC-DAD analyses

    Domesticated species, A. oryzae and A. sojae, and species with yellow conidia, Petromyces alliaceus and A. lanosus, are not included. A, Kojic acid; B, aflatoxin B1; C, aflatoxin G1; D, cyclopiazonic acid; E, aspergillic acid; F, asperfuran; G, parasiticolides; H, chrysogine; I, aflavarins; J, paspalinin and paspaline; K, aflatrems and aflavinines; L, nominine.

    Sixteen strains from Argentinean peanuts with small sclerotia produced aflatoxins B1, B2, G1 and G2 (100 %), aflatrem (88 %), aflavarins (38 %), aflavinines (dihydroxyaflavinine, monohydroxyaflavinine, monohydroxyisoaflavinine and aflavinine) (100 %), aspergillic acid (100 %), cyclopiazonic acid (100 %), kojic acid (100 %), parasiticolides (100 %) and paspaline, paspalinine and emindole SB (100 %). This extrolite profile is very similar to that of A. parvisclerotigenus, but the Argentinean strains did not produce parasiticolides. Furthermore, A. parvisclerotigenus produced the compound A 30461 (oryzaechlorin). One of the strains listed by Hesseltine et al. (1970), NRRL A-11611 (=NRRL 6444), also produced aflatoxin B1, B2, G1 and G2, aflatrem, aflavinines, aspergillic acid, cyclopiazonic acid, parasiticolides, kojic acid, aspergillic acid, paspaline, paspalinine and emodin SB and is very similar to the eight Argentinean strains (Table 2⇑).

    Four isolates (CBS 117610–117612 and CBS 117615) produced aflatoxins B1, B2, G1 and G2 (100 %), aspergillic acid (33 %), chrysogine (67 %), oryzaechlorin (17 %), parasiticolide (50 %), an extrolite with parasiticolide chromophore (50 %), extrolite NO2 (100 %) and extrolite EPIF (100 %). All strains had a floccose colony texture and a conidium colour similar to that of A. flavus but, except for the production of chrysogine by most isolates, they exhibited extrolite profiles characteristic of A. parasiticus. Chrysogine production was also observed in A. cf. nomius NRRL 3353, a strain that had formerly been characterized as being an atypical A. flavus (Hesseltine et al., 1970). These strains had a conidial ornamentation between A. parasiticus and A. flavus, in agreement with isolates determined as ‘o-type’ by Feibelman et al. (1998) and Kumeda et al. (2003).

    DISCUSSION

    The isolates representing two new taxa are related to either A. parasiticus or A. parvisclerotigenus. The isolates proposed here as Aspergillus arachidicola sp. nov. are not as dark green (Raper & Fennell, 1965) as A. parasiticus. The conidiophores are regularly biseriate, but uniseriate conidial heads are also produced. However, conidium shape and ornamentation and other microscopical characteristics of A. arachidicola overlap with those of A. parasiticus. The production of aflatoxins B and G and lack of CPA production are similar in these two species, but the production of chrysogine and the negative results on AFPA of A. arachidicola are valuable characters for distinguishing these two species.

    A. flavus is different from A. arachidicola by its yellowish-green colony colour (Raper & Fennell, 1965) and the inability to produce type-G aflatoxins. Typical A. flavus isolates produce aflatoxin B1, CPA and aspergillic acid (Samson et al., 2004). Both species have roughened stipes, but A. flavus usually has longer stipes (more than 1000 μm), and also have smooth or finely roughened conidia.

    Aspergillus minisclerotigenes sp. nov. is proposed as a new taxon for isolates with typical small sclerotia which came from peanut plants or peanut fields from Argentina, Australia, Nigeria and Texas. Some of these isolates have been described as A. flavus group II by Geiser et al. (1998, 2000). These isolates resemble A. parvisclerotigenus (CBS 121.62T) on the basis of morphological characteristics and extrolite production, but differ by producing parasiticolide, while A. parvisclerotigenus produces the compounds A 30461 and speradine A, not detected in A. minisclerotigenes. Large amounts of parasiticol, sterigmatocystin and O-methylsterigmatocystin were also detected in A. parvisclerotigenus, but not in A. minisclerotigenes. On the other hand, A. minisclerotigenes was more effective than A. parvisclerotigenus in producing sclerotial metabolites (aflavinines, aflatrems, paspalinine, paspaline, aflavarins). However, all microsclerotial strains previously allocated to A. flavus appear to produce all these sclerotial indole metabolites (Tanaka et al., 1989). It has been shown that the biosynthesis of sclerotial metabolites and aflatoxin is regulated by the gene veA, which is necessary for sclerotial formation in an isolate producing small sclerotia (Duran et al., 2007). A more detailed phenotypic study of more isolates representing A. parvisclerotigenus is necessary to determine whether there are more phenotypic differences between the two taxa that produce small sclerotia. However, our sequence data clearly show that the two microsclerotial species are genetically different.

    Taxonomy

    Latin diagnosis of Aspergillus arachidicola Pildain, Frisvad & Samson sp. nov. MB 505189

    Coloniae in agaro MEA dicto post 7 dies 25 °C 6–6.5 cm diametro, velutinae, olivaceae vel olivaceo-brunneae; reversum viridi-luteum. Conidiophora uniseriata, stipes hyalinus, asperulatus, (250–)400–600(–1000)×(6.5–)9–10 μm; vesiculae globosae vel subglobosae, (23–)28–50 μm diametro; metulae 9.5–13.5×5–6.5 μm; phialides 7–11×3–6.5 μm. Conidia globosa vel subglobosa, echinulata, viridula, (3.5–)4.5–5(–6.5) μm. Sclerotia absentia.

    Typus siccus in herb. CBS 117610 et ex-typus vivus, isolatus Arachis glabrata, Corrientes provincia Argentina.

    Description of Aspergillus arachidicola Pildain, Frisvad & Samson sp. nov. MB 505189

    Aspergillus arachidicola (a.ra.chi.di.co′la. N.L. n. arachidicola inhabitant of Arachis).

    Colonies on YES, MEA, OA and CYA attain a diameter of 6–6.5 cm in 7 days at 25 °C; growing rapidly on CYA at 37 °C, with a diameter of 6–7 cm (Fig. 2⇓; a colour version of this figure is available as Supplementary Fig. S2). Colony surface velvety with abundant conidial heads, olive to olive brown en masse (Kornerup & Wanscher, 1978). Reverse greenish yellow without diffusible pigments. Sclerotia not observed. Conidial heads uniseriate or biseriate. Stipes hyaline, finely roughened, variable in length, mostly (250–)400–600(−1000) μm; diameter just below vesicles (6.5–)9–10 μm. Vesicles globose to subglobose, (23–)28–50 μm in diameter, fertile upper 75 % of their surface; metulae 9.5–13.5×5–6.5 μm; phialides 7–11×3–6.5 μm. Conidia globose to subglobose, echinulate, greenish, (3.5–)4.5–5(–6.5) μm. Isolates grow well at 25, 37 and 42 °C.

    Figure image not available in archive
    Fig. 2.

    Aspergillus arachidicola sp. nov. CBS 117610T. (a–c) Colonies on CYA at 25 °C (a), CYA at 37 °C (b) and AFPA (c) after 7 days. (d–h) Conidiophores; (i) conidia. Bars, 10 μm. A colour version of this figure is available as Supplementary Fig. S2.

    Extrolites: strains of A. arachidicola produce kojic acid, aflatoxins B1, B2, G1 and G2 and parasiticolide, and some strains produce chrysogine.

    The type strain, CBS 117610T =IBT 25020T, was isolated from an Arachis glabrata leaf, Corrientes province, Argentina.

    Latin diagnosis of Aspergillus minisclerotigenes Vaamonde, Frisvad & Samson sp. nov. MB 505188

    Coloniae in agaro MEA dicto 6–7 cm diametro post 7 dies 25 °C, velutinae vel floccosae, mycelium vegetatovum album et conidiophora sparsa et densum stratum sclerotiorum fuscorum formantes. Conidia aggregata dilute viridia vel griseo-viridia; reversum aurantiacum vel brunneum. Sclerotia copiosa, obscure brunnea vel atra, 150–300 μm diametro. Conidiophora biseriata, stipes hyalinus, asperatus, 1200–2000×11–21 μm; vesiculae globosae vel subglobosae, 35–50 μm diametro, metulae 11–14×3–5 μm; phialideas 6–10×3–5 μm. Conidia subglobosa vel ellipsoidea, (2–)3–4(–6) μm diametro, dilute viridia, levia vel echinulata.

    Typus siccus in herb. CBS 117635, et ex-typus vivus, isolatus Arachis hypogaea, Córdoba provincia Argentina.

    Description of Aspergillus minisclerotigenes Vaamonde, Frisvad & Samson sp. nov. MB 505188

    Aspergillus minisclerotigenes (mi.ni.scle.ro.ti′ge.nes. N.L. part. adj. minisclerotigenes producing small sclerotia).

    Colonies on YES, MEA, OA and CYA attain a diameter of 6–7 cm after 7 days at 25 °C and also on CYA at 37 °C. Colony surface velvety and, on OA and MEA, colony surface floccose, consisting of white vegetative mycelium and sparse conidial heads and dense felt of dark sclerotia (Fig. 3⇓; a colour version of this figure is available as Supplementary Fig. S3). Conidial structures light-greyish green en masse (Kornerup & Wanscher, 1978). Colony reverse greyish orange to brownish orange on YES, yellowish brown to light brown on MEA and OA and brown on CYA. Exudate droplets are not observed. Sclerotia 150–300 μm in diameter. Conidial heads normally biseriate, but uniseriate heads sometimes occur. Conidiophore stipes 1200–2000×11–21 μm, hyaline, coarsely roughened. Vesicles globose to subglobose, 35–50 μm in diameter. Metulae 11–14×3–5 μm, phialides 6–10×3–5 μm. Conidia ellipsoidal, subglobose (2–)3–4(–6) μm diameter, pale green, smooth walled to echinulate. Isolates grow well at 25, 37 and 42 °C.

    Figure image not available in archive
    Fig. 3.

    Aspergillus minisclerotigenes sp. nov. CBS 117635T. (a–c) Colonies on CYA at 25 °C (a), MEA (b) and YES agar (c) after 7 days. (d–g) Conidiophores; (h) conidia; (i) sclerotia. Bars, 10 μm (a–h) and 200 μm (i). A colour version of this figure is available as Supplementary Fig. S3.

    Extrolites: aspergillic acid, kojic acid, cyclopiazonic acid, aflatoxins B1, B2, G1 and G2, parasiticolides, paspaline and paspalinine, aflavarin, aflavinines and aflatrem. Aspergillic acid produced on AFPA.

    The type strain CBS 117635T =IBT 25032T (dried culture) was isolated from Arachis hypogaea, Córdoba province, Argentina.

    Acknowledgments

    We are indebted to D. Geiser (Pennsylvania State University, University Park, USA) and J. I. Pitt (CSIRO, North Ryde, Australia) for providing us with Aspergillus isolates for this study.

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