Wageningen Academic P u b l i s h e r s World Mycotoxin Journal, August 2009; 2 (3): 263-277 A reappraisal of fungi producing aflatoxins J. Varga1,2, J.C. Frisvad3 and R.A. Samson1 1CBS Fungal Biodiversity Centre, P.O. Box 85167, 3508 AD Utrecht, the Netherlands; 2Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, 6726 Szeged, Hungary; 3Center for Microbial Biotechnology, Department of Systems Biology, Building 221, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark; j.varga@cbs.knaw.nl Received: 1 September 2008 / Accepted: 15 January 2009 © 2009 Wageningen Academic Publishers Abstract Aflatoxins are decaketide-derived secondary metabolites which are produced by a complex biosynthetic pathway. Aflatoxins are among the economically most important mycotoxins. Aflatoxin B1 exhibits hepatocarcinogenic and hepatotoxic properties, and is frequently referred to as the most potent naturally occurring carcinogen. Acute aflatoxicosis epidemics occur in several parts of Asia and Africa leading to the death of several hundred people. Aflatoxin production has incorrectly been claimed for a long list of Aspergillus species and also for species assigned to other fungal genera. Recent data indicate that aflatoxins are produced by 13 species assigned to three sections of the genus Aspergillus: section Flavi (A. flavus, A. pseudotamarii, A. parasiticus, A. nomius, A. bombycis, A. parvisclerotigenus, A. minisclerotigenes, A. arachidicola), section Nidulantes (Emericella astellata, E. venezuelensis, E. olivicola) and section Ochraceorosei (A. ochraceoroseus, A. rambellii). Several species claimed to produce aflatoxins have been synonymised with other aflatoxin producers, including A. toxicarius (=A. parasiticus), A. flavus var. columnaris (=A. flavus) or A. zhaoqingensis (=A. nomius). Compounds with related structures include sterigmatocystin, an intermediate of aflatoxin biosynthesis produced by several Aspergilli and species assigned to other genera, and dothistromin produced by a range of non-Aspergillus species. In this review, we wish to give an overview of aflatoxin production including the list of species incorrectly identified as aflatoxin producers, and provide short descriptions of the ‘true’ aflatoxin producing species. Keywords: aflatoxin production, Aspergillus, sterigmatocystin 1. Introduction Aflatoxins are the most thoroughly studied mycotoxins. In the early sixties, toxicity of animal feeds containing contaminated peanut meal led to the death of more than 100,000 turkeys from acute liver necrosis (turkey X disease; Blout, 1961; Sargeant et al., 1961; Nesbitt et al., 1962; Van der Zijden et al., 1962). Scientists identified the toxin-producing fungus as Aspergillus flavus, and the toxic agents as a group of structurally related difuranocoumarins that were named as aflatoxins B1, B2, G1, and G2 based on their fluorescence under UV light (blue or green) and relative chromatographic mobility during thin-layer chromatography (Figure 1). Aflatoxin B1 is the most potent natural carcinogen known ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2008.1094 (Squire, 1981) and is usually the major aflatoxin produced by toxigenic strains. Apart from those mentioned above, over a dozen other aflatoxins including aflatoxins P1, Q1, B2a and G2a have been described, especially as mammalian biotransformation products of the major metabolites (Heathcote and Hibbert, 1978), while aflatoxin D1 was detected in ammoniated corn (Grove et al., 1984), and aflatoxin B3 as a metabolite of A. flavus (Heathcote and Dutton, 1969). Aflatoxin M1, a hydroxylated metabolite is found primarily in animal tissues and fluids (milk and urine) as a metabolic product of aflatoxin B1 (Figure 1). Many substrates support growth and aflatoxin production by aflatoxigenic moulds; natural contamination of cereals, 263 J. Varga et al. O H OCH3 OCH3 O O H H Aflatoxin B1 Aflatoxin B2 O O O O O H OCH3 O O O O O H O O O O H O OCH3 O O H O O H Aflatoxin G1 Aflatoxin G2 O O O OH OH O H O O O H Aflatoxin M1 OCH3 O O OCH3 H Sterigmatocystin Figure 1. Structures of the most important aflatoxins and sterigmatocystin. figs, oilseeds, nuts, tobacco, and a long list of other commodities occurs commonly (Bennett and Klich, 2003). These mycotoxins most frequently contaminate peanut, corn and cereals, but also occur in meat, milk (aflatoxin M1) and eggs of animals that have consumed contaminated feeds. Aflatoxin B1 exhibits hepatocarcinogenic and hepatotoxic properties, and is referred to as the most potent naturally occurring carcinogen. The International Agency for Research on Cancer has classified aflatoxin B1 as a group I carcinogen (IARC, 1982). The diseases caused by aflatoxin consumption are called aflatoxicoses. Acute aflatoxicosis is produced when moderate to high levels of aflatoxins are consumed. Acute episodes of disease symptoms may include haemorrhage, acute liver damage, oedema, alteration in digestion, absorption and/or metabolism of nutrients, and may result in death. Acute aflatoxicosis epidemics occurred in India in 1974, resulting in 397 recognised cases and 106 deaths (Krishnamachari et al., 1975), in Kenya in 1981 (Ngindu et al., 1982) and in 2004 and 2005 causing more than 150 deaths (Azziz-Baumgartner et al., 2006; Lewis et al., 2005). Acute hepatitis associated with consumption of mouldy grains has also been reported in other areas in Africa, India and Malaysia (Chao et al., 1991; Coulter et al., 1986; Lye et al., 1995; Patten, 1981). Chronic aflatoxicosis results from prolonged ingestion of low to moderate levels of aflatoxins. The effects are usually subclinical and difficult to recognise. Some of the common symptoms are impaired food conversion and slower rates of growth with or without the production of an overt aflatoxin syndrome. Chronic 264 aflatoxicosis results in cancer and immune suppression and other ‘slow’ pathological conditions (Eaton and Groopman, 1994). The liver is the primary target organ, with liver damage occurring when poultry, fish, rodents, and nonhuman primates are fed aflatoxin B1. There are substantial differences in species susceptibility. Aflatoxicosis has been observed in various animals including birds, dogs and other wild and domesticated animal species (Eaton and Groopman, 1994; Newman et al., 2007). Aflatoxins are decaketide-derived secondary metabolites which are produced by a complex pathway involving over 16 steps after the synthesis of the first stable intermediate, norsolorinic acid. In contrast to most polyketide synthases, the starter unit for aflatoxin biosynthesis is hexanoate, which is produced by a fatty acid synthase (Hicks et al., 2002; Hitchman et al., 2001). Sterigmatocystin, a related dihydrofuran toxin, is a late metabolite in the aflatoxin pathway and is also produced as a final biosynthetic product by a number of species such as Aspergillus versicolor and Emericella nidulans. Sterigmatocystin is both mutagenic and tumorigenic but is less potent than aflatoxin (Berry, 1988; Figure 1). Biosynthetic genes for aflatoxin pathway enzymes from A. flavus and A. parasiticus show high sequence similarity to the sterigmatocystin pathway genes of E. nidulans (Brown et al., 1996; Payne and Brown, 1998). In A. nidulans, the sterigmatocystin gene cluster is about 60 kbp long and comprises 25 genes, the transcription of which is regulated by a Zn(II)2Cys6 DNA binding protein encoded by the aflR gene. The functions of the gene products identified so far include the fatty acid synthase and polyketide synthase mentioned earlier, five monooxygenases, several reductases, dehydrogenases, a methyltransferase, and an esterase (Brown et al., 1996). The A. flavus and A. parasiticus aflatoxin gene clusters are about 70 kbp long, and consist of at least 24 different genes (Yu et al., 1995, 2004). The A. flavus cluster is 96% identical to that of A. parasiticus and 91% identical to that of A. nomius. Coding regions generally have 4-10% higher sequence identity than intergenic regions (Cary and Ehrlich, 2006). In the recent years, considerable efforts have been made to understand the genetics and molecular biology of aflatoxin biosynthesis (Bhatnagar et al., 2003; Chang et al., 2007; O’Brian et al., 2007; Yu et al., 2002, 2004, 2007). 2. Aflatoxin producing species The list of species that have been (incorrectly) reported to produce aflatoxins is very long; several species have been reported to be able to produce this metabolite (Tables 1 and 2). None of these species produce aflatoxins, and many of these names are not accepted as valid species in any case. The reports on aflatoxin-producing abilities of A. terreus could be due to the fact that territrems reveal blue fluorescence under long-wave ultraviolet (UV) light and have retention values similar to that of aflatoxin B1 on World Mycotoxin Journal 2 (3) A reappraisal of fungi producing aflatoxins Table 1. Aspergillus species incorrectly reported to produce aflatoxins. Species Aspergillus section Aspergillus A. glaucus Eurotium amstelodami Eurotium chevalieri E. intermedium Eurotium herbariorum Eurotium repens Eurotium rubrum Aspergillus section Candidi A. candidus Aspergillus section Circumdati A. ochraceus A. ostianus A. sulphureus Aspergillus section Cremei A. wentii Aspergillus section Flavi A. oryzae A. tamarii A. terricola Aspergillus section Fumigati A. fumigatus Aspergillus section Nidulantes A. versicolor Emericella nidulans Emericella rugulosa Aspergillus section Nigri A. niger A. ficuum, A. carbonarius, A. japonicus Aspergillus section Restricti A. restrictus Aspergillus section Terrei A. terreus Aspergillus section Zonati A. zonatus Reference Hanssen and Jung, 1973; Samajpati, 1979 Janicki et al., 1972; Abarca et al., 1997 Mabrouk and El-Shayeb, 1980; Kulik and Holaday, 1966; Leitao et al., 1989; El-Kady et al., 1994; Abarca et al., 1997 Kulik and Holaday, 1966; Leitao et al., 1989; El-Kady et al., 1994 Vázquez-Belda et al., 1995 Kulik and Holaday, 1966; Janicki et al., 1972; Leitao et al., 1987, 1989; Abarca et al., 1997 Abarca et al., 1997; Leitao et al., 1987, 1989; Kulik and Holaday, 1966 Abarca et al., 1997; Jayaraman and Kalyanasundaram, 1980; Samajpati, 1979 Van Walbeek et al., 1968; Reddy et al., 2004 Scott et al., 1967 Scott et al., 1970; Barr and Dawney, 1975 Schroeder and Verrett, 1969; Kulik and Holaday, 1966; De Waart et al., 1975 El-Hag and Morse, 1976; Adebajo, 1992; El-Kady et al., 1994; Abdel-Mallek et al., 1993; De Waart et al., 1975; Atalla et al., 2003; Drusch and Ragab, 2003; Basappa et al., 1967; Samajpati, 1979; Boller and Schroeder, 1966 Lalithakumari and Govindaswarmi, 1970; El-Kady et al., 1994; Goto et al., 1996, 1997; Klich et al., 2000 Moubasher et al., 1977 Sodhi et al., 1985; Abarca et al., 1997; Pepeljnjak et al., 2004 Masimango et al., 1977; Atalla et al., 2003 Janicki et al., 1972; Hanssen and Jung, 1973; Ahmed et al., 2005 Schroeder and Kelton, 1975 Kulik and Holaday, 1966; Janicki et al., 1972; Masimango et al., 1977; Glinsukon et al., 1979; Sodhi et al., 1985; Ibrahim et al., 1990; Waghray et al., 1988; Reddy et al., 2004 Masimango et al., 1977 Samajpati, 1979 Sripathomswat and Thasnakorn, 1981; Abarca et al., 1997; Atalla et al., 2003 El-Kady et al., 1994; Abdel-Mallek et al., 1993 TLC plates when developed in certain solvent systems (Ling et al., 1979). The early reports on aflatoxin production by Penicillia and Aspergilli outside section Flavi were rejected by Bösenberg and Becker (1972), Frank (1972), Hesseltine et al. (1966), Langone and van Vunakis (1976), Mislivec et al. (1968), Parrish et al. (1966), Rabie and Terblanche (1967), Rehm (1972), Scott (1965) and Wilson et al. (1968). One of the first reports to show that Aspergillus oryzae was able to produce aflatoxin was published by El-Hag and Morse (1976). However, the culture of A. oryzae they used was shown to be contaminated by an aflatoxin producing World Mycotoxin Journal 2 (3) A. parasiticus (Fennell, 1976). Despite the fact that this problem was solved, later others repeatedly reported that A. oryzae was able to produce aflatoxins. Floccose strains of A. flavus and A. nomius may superficially look like A. oryzae, so this macromorphological resemblance may have been the reason for later erroneous reports of aflatoxin production by this species. Since A. oryzae is a domesticated form of A. flavus, the former species will not be isolated from natural sources, except if they escape the soy sauce production plants and similar factories and contaminate 265 J. Varga et al. Table 2. Other species incorrectly reported to produce aflatoxins. Species Zygomycota Absidia butleri, Absidia glauca Cunninghamella echinulata Mucor sp. Mucor circinelloides, M. griseocyanus, M. mucedo Rhizopus sp. Rhizopus nigricans Syncephalastrum racemosum Ascomycota Alternaria cheiranthi Cephalosporium curticeps, C. rosea-griseum Cladosporium cladosporioides, C. sphaerospermum Penicillium sp. P. baarnense P. brevicompactum P. chrysogenum P. citrinum P. cyaneum P. cyclopium P. digitatum P. expansum P. frequentans P. funiculosum P. glaucum P. oxalicum P. puberulum P. raistrickii P. roquefortii P. variabile P. verrucosum P. wortmannii “P. citromyces strictum” Scopulariopsis brevicaulis Bacteria Streptomyces sp. Actinomycetes Reference Swelim et al., 1994 Swelim et al., 1994 Hanssen, 1969; Sodhi et al., 1985 Swelim et al., 1994 Kulik and Holaday, 1966; Van Walbeek et al., 1968 Swelim et al., 1994 Swelim et al., 1994 Swelim et al., 1994 Swelim et al., 1994 Swelim et al., 1994 Schneider et al., 1972; Lee et al., 1975; Sodhi et al., 1985; Kulkarni et al., 1986; Kraemer and Stussi, 1998 Janicki et al., 1972 Janicki et al., 1972 Swelim et al., 1994; Janicki et al., 1972 Kulik and Holaday, 1966; De Waart et al., 1975 Janicki et al., 1972 Janicki et al., 1972 Hanssen and Jung, 1973 Hanssen and Jung, 1973 Kulik and Holaday, 1966; De Waart et al., 1975 Swellim et al., 2001; Janicki et al., 1972 Hanssen and Jung, 1973 Swelim et al., 1994 Hodges et al., 1966; De Waart et al., 1975 Janicki et al., 1972 Swelim et al., 1994 Kulik and Holaday, 1966; De Waart et al., 1975 Ahmed et al., 2005 Janicki et al., 1972 Kulik and Holaday, 1966 Swelim et al., 1994 Mishra and Murthy, 1968 Koul, 1987 the immediate surroundings. A detailed account on this issue is given by Jørgensen (2007). Although sterigmatocystin is an intermediate of aflatoxin biosynthesis (Frisvad, 1989), only A. ochraceoroseus (Frisvad et al., 1999; Klich et al., 2000), and some Emericella species accumulate both sterigmatocystin and aflatoxin (Frisvad et al., 2004; Frisvad and Samson, 2004). Members of Aspergillus section Flavi (Aspergillus flavus species group according to Raper and Fennell, 1965) which includes the major aflatoxin producers, efficiently convert sterigmatocystin into 3-methoxysterigmatocystin and then into aflatoxins (Frisvad et al., 1999, 2004). The major source 266 of sterigmatocystin in foods is A. versicolor. This fungus is common on cheese, but may also occur on other substrates (Pitt and Hocking, 1997). In addition, sterigmatocystin is also produced by a high number of other Aspergillus species; it has been reported from species in sections Flavi, Aspergillus, Nidulantes, Usti, Terrei and Flavipedes. The production of sterigmatocystin has been confirmed in the following Aspergillus and Emericella species: A. versicolor, A. rambellii, A. ochraceoroseus, E. acristata, E. astellata, E. aurantiobrunnea, E. bicolor, E. cleistominuta, E. corrugata, E. dentata, E. discophora, E. echinulata, E. foeniculicola, E. foveolata, E. fructiculosa, E. heterothallica, E. lata, E. navahoensis, E. nidulans, E. olivicola, E. quadrilineata, World Mycotoxin Journal 2 (3) A reappraisal of fungi producing aflatoxins E. rugulosa, E. spectabilis, E. striata, E. variecolor, and E. venezuelensis (Ballantine et al., 1965; Chexal et al., 1975; Frisvad, 1985; Holzapfel et al., 1966; Rabie et al., 1977; Horie et al., 1979, 1989; Horie and Yamazaki, 1985; Yamazaki et al., 1980; Zalar et al., 2008), and a fungus identified as A. multicolor (Hamasaki et al., 1980). Another group of Aspergilli in section Usti, A. ustus and A. puniceus, are able to produce austocystins (Steyn and Vleggaar, 1974) and compounds related to sterigmatocystin, while A. granulosus can also produce a sterigmatocystin-related extrolite (Houbraken et al., 2007). Sterigmatocystin production could not be confirmed in other Aspergilli reported previously to produce this compound, for example, in Emericella unguis (Barnes et al., 1994; Mislivec et al., 1975), in A. egyptiacus (Moubasher et al., 1977), in Eurotium rubrum (E. herbariorum), E. repens, E. chevalieri, E. pseudoglaucum and E. amstelodami (trace amounts) (Abramson et al., 1983; Ahmed et al., 2005; Bukelskiene et al., 2006; El-Kady et al., 1994; Karo and Hadlok, 1982; Labuda and Tancinova, 2006; Sanchis et al., 1982; Schroeder and Kelton, 1975; Szebiotko et al., 1981), A. sydowii and A. aureolatus (Abdel-Mallek et al., 1993), A. japonicus (Begum and Samajpati, 2000) or Aspergillus togoensis = Stilbothamnium togoense (Wicklow et al., 1989). Production of sterigmatocystin by Penicillium species has not been reported, apart from an obscure reference to Penicillium luteum in Dean (1963). However, Wilson et al. (2002) claimed that P. camemberti, P. commune and P. griseofulvum produce sterigmatocystin. Perhaps they have mistaken sterigmatocystin for cyclopiazonic acid. However, sterigmatocystin production also occurs in the phylogenetically unrelated genera Monocillium (Ayer et al., 1981), Chaetomium (Barnes et al., 1994; Koyama et al., 1991; Sekita et al., 1981; Udagawa et al., 1979a,b), Humicola (Joshi et al., 2002) and Bipolaris species (Maes and Steyn, 1984; Rabie et al., 1976). As the strains of Farrowia and Achaetomiella (Holzapfel et al., 1966) reported to produce sterigmatocystin are regarded as belonging to Chaetomium (Cannon, 1986; Udagawa, 1980) sterigmatocystin production may have evolved only once in Chaetomium, but this is unlikely since at least eight species have been reported to produce sterigmatocystin in Chaetomium so far: C. caprinum, C. cellulolyticum, C. gracile, C. longicolleum, C. tetraspermum, C. thielavioideum, C. udagawae and C. virescens. Another precursor of aflatoxins, norsolorinic acid has also been incorrectly claimed to be produced by A. niger and A. ochraceus (Reddy et al., 2005). Apart from the different types of aflatoxins and sterigmatocystin another fungal metabolite related to aflatoxins, dothistromin, has also been identified (Bradshaw et al., 2002). This metabolite is produced by Dothistroma septosporum (= D. pini = Scirrhia pini) World Mycotoxin Journal 2 (3) (Baer et al., 1970), Cercospora arachidicola (Stoessl, 1984), C. ferruginea, C. fusca, C. microsora, C. rosicola, C. rubi, C. smilacis, other Cercospora species (Assante et al., 1977a,b), and Mycosphaerella laricina (Stoessl et al., 1990), all pathogens belonging in the ascomycete order Dothideales. D. septosporum causes red-band needle blight in a wide range of pine species, a disease that leads to needle death, premature defoliation and, in severe cases, tree death (Bradshaw and Zhang, 2006). In contrast to the situation in aflatoxin-producing fungi where 25 aflatoxin biosynthetic and regulatory genes are tightly clustered in one region of the genome, the dothistromin gene cluster of D. septosporum is fragmented. Three mini-clusters of dothistromin genes have been identified, each located on a 1.3 Mb chromosome and each grouped with nondothistromin genes (Zhang et al., 2007). The aflatoxin precursors averufin and averythrin were isolated from Cercospora smilacis together with dothistromin (Danks and Hodges, 1974; Assante et al., 1977b; Stoessl, 1984). Recent data indicate that all known aflatoxin producing species belong to three sections of the Aspergillus genus: sections Flavi, Ochraceorosei and Nidulantes. Among these, sections Nidulantes and Ochraceorosei are assigned to subgenus Nidulantes, while section Flavi belongs to subgenus Circumdati based on multilocus sequence based phylogenetic studies (Peterson et al., 2008). A tree based on phylogenetic analysis of β-tubulin sequence data depicting relationships of aflatoxin- and some of the sterigmatocystinproducing Aspergilli is shown in Figure 2. Aspergillus section Flavi Aspergillus section Flavi historically includes species with conidial heads in shades of yellow-green to brown, and dark sclerotia. Isolates of the so-called domesticated species, such as A. oryzae, A. sojae and A. tamarii are used in oriental food fermentation processes and as hosts for heterologous gene expression (Campbell-Platt and Cook, 1989). The economically most important aflatoxin producers belong to this section of the Aspergillus genus. Aflatoxins have been shown to be produced by A. 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), A. parvisclerotigenus (Saito and Tsurota, 1993, Frisvad et al., 2005), A. zhaoqingensis (Sun and Qi, 1991), A. flavus var. columnaris (Van Walbeek et al., 1968), A. minisclerotigenes and A. arachidicola (Pildain et al., 2008). Aflatoxin B2 was found as a minor extrolite in all aflatoxin B1 producing species, but as the only type of aflatoxin in A. flavus var. columnaris NRRL 5821 and IBT 12654 and in A. zhaoqingensis CBS 399.93. A. zhaoqingensis produced kojic acid, aspergillic acid, one aflatoxin (B2), and tenuazonic acid like most strains of A. nomius (unpublished data). Molecular data indicate that A. flavus var. columnaris 267 J. Varga et al. A. flavus CBS 100927T A. minisclerotigenes CBS 115635T 100 A. parvisclerotigenus CBS 121.62T A. parasiticus CBS 100926T 79 100 99 97 100 Section Flavi A. arachidicola CBS 117610T A. pseudotamarii CBS 766.97T A. nomius CBS 260.88T A. bombycis CBS 117187T A. ochraceoroseus CBS 550.77T A. rambellii CBS 101887T Section Ochraceorosei E. discophora CBS 469.88T E. nidulans CBS 589.65T 87 A. versicolor CBS 583.65T E. olivicola CBS 119.37T 87 99 Section Nidulantes E. venezuelensis CBS 868.97T E. astellata CBS 135.55T 100 A. ustus CBS 261.67T A. puniceus CBS 756.74T Section Usti Talaromyces flavus CBS 225.66T 0.05 Figure 2. Phylogenetic tree of aflatoxin and sterigmatocystin-producing Aspergillus species based on β-tubulin sequence data. Aflatoxin producers are set in bold type. Numbers above branches indicate bootstrap values; only values above 70% are indicated. and A. zhaoqingensis are synonyms of A. flavus and A. nomius, respectively (Pildain et al., 2008; data not shown). Apart from aflatoxins B and G, aflatoxin M1 and M2 have also been found to be produced by some A. flavus and A. parasiticus isolates (Ramachandra et al., 1975; Engel, 1978; Dutton et al., 1985; Pallavi et al., 1997; Lopez-Diaz et al., 1996). Aspergillus flavus is the most common species producing aflatoxins (Sargeant et al., 1961) and other economically important mycotoxins including e.g. cyclopiazonic acid (Dorner et al., 1983) and gliotoxin (Fox and Howlett, 2008), occurring in most kinds of foods in tropical countries. This species is very common on maize, peanuts and cottonseed, and produces only B-type aflatoxins. It has been estimated that only about 30-40% of known isolates produce aflatoxin. A. flavus populations are genetically and phenotypically diverse (Geiser et al., 2000) with some isolates producing conidia abundantly, produce large (L) sclerotia, and variable amounts of aflatoxins, while another type produces abundant, small (S) sclerotia, fewer conidia and high levels of aflatoxins (Cotty, 1989). Probst et al. (2007) found that the 2004 aflatoxicosis outbreak in Kenya was caused by S-type isolates of A. flavus which produced large amounts of aflatoxin B1. A related species, A. oryzae is atoxigenic and has been used as a source of industrial enzymes and as a koji (starter) mould for Asian fermented 268 foods, such as sake, miso, and soy sauce (Van den Broek et al., 2001). A. oryzae isolates carry various mutations in the aflatoxin biosynthetic gene cluster resulting in their inability to produce aflatoxins (Tominaga et al., 2006). Notably, the aflR gene is absent or significantly different in some A. oryzae strains compared to A. flavus (Lee et al., 2006). There were many reports indicating that certain A. flavus strains including micro-sclerotial strains, and strains listed as intermediate between A. flavus and A. parasiticus can also produce type G aflatoxins (Begum and Samajpati, 2000; Codner et al., 1963; Cotty and Cardwell, 1999; Hesseltine et al., 1970). Recent molecular investigations of these isolates revealed that they belong to two separate species, A. parvisclerotigenus and A. minisclerotigenes (Frisvad et al., 2005; Pildain et al., 2008). Many other isolates producing both aflatoxins B and G and bearing small sclerotia have been reported to date (Bayman and Cotty, 1993; Cotty and Cardwell, 1999; Egel et al., 1994; Frisvad et al., 2005; Saito and Tsurota, 1993). Further studies are necessary to assign these isolates to species. Aspergillus pseudotamarii (Ito et al., 2001) is another effective producer of B type aflatoxins but its importance as regards mycotoxin occurrence in foods is unknown. The closely related species A. tamarii is not able to produce World Mycotoxin Journal 2 (3) A reappraisal of fungi producing aflatoxins aflatoxins, despite several reports claiming this (Goto et al., 1996; Klich et al., 2000). Aflatoxins G1 and G2 are found in A. parasiticus, A. nomius, A. bombycis, A. parvisclerotigenus, A. minisclerotigenes, A. arachidicola and A. toxicarius. Aspergillus parasiticus occurs rather commonly in peanuts, but is apparently quite rare in other foods. It is more restricted geographically than A. flavus. A. parasiticus produces both B and G aflatoxins (Sargeant et al., 1963), and virtually all known isolates are toxigenic. This species also produces kojic acid and aspergillic acid. A. toxicarius, another species closely related to A. parasiticus also produces B- and G-type aflatoxins (Murakami et al., 1966; Murakami, 1971). Sequence analysis of multiple loci indicate that A. toxicarius is a synonym of A. parasiticus (Pildain et al., 2008). A. sojae is the domesticated variety of A. parasiticus, which can scarcely be distinguished from it except for its inability to produce aflatoxins (Chang et al., 2007; Rigó et al., 2002). The lack of aflatoxin-producing ability of some A. sojae isolates results primarily from an early termination point mutation in the pathway-specific AflR regulatory gene, which causes the truncation of the transcriptional activation domain of AflR and the abolition of interaction between AflR and the AflJ co-activator. In addition, a defect in the polyketide synthase gene also contributes to its inability to produce aflatoxins (Chang et al., 2007). Aspergillus nomius and A. bombycis are two related species also producing both aflatoxins B and G, whereas neither produces cyclopiazonic acid (Peterson et al., 2001; Table 3). A. bombycis was isolated from silkworm-rearing houses in Japan and Indonesia, whereas A. nomius is more widespread: it was originally isolated from mouldy wheat in the USA, and later from various substrates in India, Japan and Thailand. Peterson et al. (2001) observed cryptic recombination in A. nomius populations using multilocus sequence data. Recently, Olsen et al. (2008) have observed that A. nomius is an important producer of aflatoxins in Brazil nuts. A. minisclerotigenes was isolated from Argentinean peanuts with small sclerotia and produces aflatoxins B1, B2, G1, G2, aspergillic acid, cyclopiazonic acid, kojic acid, parasiticolides and several other extrolites (Pildain et al., 2008; Table 3). One of the strains listed by Hesseltine et al. (1970), NRRL A-11611 = NRRL 6444 also produced aflatoxins B1, B2, G1 and G2, aflatrem, aflavinines, aspergillic acid, cyclopiazonic acid, parasiticolides, kojic acid, paspaline, paspalinine and emindole SB and is conspecific with A. minisclerotigenes. A. minisclerotigenes also includes isolates assigned by Geiser et al. (2000) to A. flavus group II. Another species producing small sclerotia and both aflatoxins B and G is A. parvisclerotigenus (Frisvad et al., 2005). The type strain of this species (CBS 121.62 = NRRL A-11612 = IBT 3651 = IBT 3851) was isolated from peanuts (Arachis hypogea) in Nigeria, and has an extrolite profile very similar to that of World Mycotoxin Journal 2 (3) A. minisclerotigenes, but in contrast with the Argentinean strains, it also produces parasiticolides, and compound A 30461 (aspirochlorin = oryzaechlorin; Table 3). A. arachidicola was isolated from leaves of Arachis glabrata in Argentina, and produces aflatoxins B1, B2, G1 and G2, aspergillic acid, chrysogine, oryzaechlorin, parasiticolide, and extrolites NO2 and EPIF. All strains had a floccose colony texture, a conidium colour similar to A. flavus but, except for the production of chrysogine by most isolates, they exhibited extrolite profiles similar to those of A. parasiticus isolates (Pildain et al., 2008; Table 3). Members of Aspergillus section Flavi that produce aflatoxin B1 also produce kojic acid, and, except for A. bombycis and A. pseudotamarii, aspergillic acid (Table 3). 3-O-methyl-sterigmatocystin was found in all aflatoxin producers. Species examined that produced the G type aflatoxins usually do not produce cyclopiazonic acid and vice versa as suggested by Takahashi et al. (2004). However, some isolates of two recently identified aflatoxigenic species, A. parvisclerotigenus and A. minisclerotigenes are able to produce both extrolites in culture (Frisvad et al., 2005; Pildain et al., 2008). Members of section Flavi produce different combinations of aflatoxins, kojic acid, cyclopiazonic acid and aspergillic acid and only share the aflatoxins (B type) with species in the sections Ochraceorosei and Nidulantes. Furthermore, none of the strains that produced aflatoxins in Aspergillus section Flavi produced detectable amounts of sterigmatocystin, while sterigmatocystin was always accumulated together with aflatoxin B1 in aflatoxigenic Emericella species and in A. ochraceoroseus and A. rambellii (Frisvad et al., 2005). Aspergillus section Ochraceorosei This section was established by Frisvad et al. (2005), and includes species not able to grow at 37°C, producing yellow ellipsoidal conidia, biseriate conidial heads and long, smooth conidiophore stipes. This section includes two aflatoxigenic species, A. ochraceoroseus and A. rambellii, both isolated from soil in Ivory Coast. These are the only species known to accumulate aflatoxin B1 and sterigmatocystin simultaneously. 3-O-methylsterigmatocystin was also detected in these cultures. A. ochraceoroseus has previously been assigned either to Aspergillus sections Wentii, Cremei or Circumdati (Christensen, 1982; Kozakiewicz, 1989; Samson, 1979). However, phylogenetic analysis of sequence data from aflatoxin and sterigmatocystin genes aflR and nor-1/stcE, as well as ITS and b-tubulin genes of aflatoxin producing species indicated that A. ochraceoroseus was related more closely to the species in subgenus Nidulantes than to species from subgenus Circumdati (Cary et al., 2005; Klich et al., 2003). A. ochraceoroseus produced more aflatoxin B1 than E. venezuelensis and E. astellata, but less than members of section Flavi, whereas A. rambellii 269 J. Varga et al. Table 3. Morphological characteristics, occurrence and extrolites of aflatoxin producing species (modified after Frisvad et al., 2006). Section Species Flavi A. bombycis A. flavus A. nomius A. parasiticus Morphological characteristics Occurrence Extrolites produced Reference mostly biseriate, sclerotia not reported mostly biseriate, sclerotia large or small mostly biseriate, sclerotia small, bullet-shaped mostly uniseriate, sclerotia uncommon, large Japan, Indonesia aflatoxins B & G, kojic acid, aspergillic acid Peterson et al., 2001 Worldwide aflatoxins B1 & B2, kojic acid, cyclopiazonic acid, aspergillic acid, asperfuran, paspalinine, paspaline Codner et al., 1963 USA, Thailand, Japan, India, Brazil USA, Japan, Australia, India, South America, Uganda Nigeria aflatoxins B & G, kojic acid, aspergillic acid, tenuazonic acid, nominine Kurtzmann et al., 1987 aflatoxins B & G, kojic acid, aspergillic acid, paspalinine, paspaline Schroeder, 1966 A. parvisclerotigenus mostly biseriate, sclerotia small, spherical A. minisclerotigenes mostly biseriate, sclerotia small, spherical Argentina, USA, Australia, Nigeria A. arachidicola mostly biseriate, sclerotia small, spherical biseriate, sclerotia large, spherical Argentina A. pseudotamarii Ochraceorosei A. ochraceoroseus A. rambellii Nidulantes E. astellata E. olivicola E. venezuelensis 270 aflatoxins B & G, parasiticol, cyclopiazonic acid, kojic Frisvad et al., 2005 acid, 3-O-methylsterigmatocystin, versicolorins, a and b aflatrem, paspalinine, paspaline, aflavarin, aspirochlorin aflatoxins B & G, kojic acid, aspergillic acid, Pildain et al., 2008 paspalinine, paspaline, cyclopiazonic acid, aflavarins, paspalinin, paspaline, aflatrems, aflavinines aflatoxins B & G, kojic acid, aspergillic acid, Pildain et al., 2008 parasiticolides, chrysogine Japan, Argentina aflatoxin B1, kojic acid, cyclopiazonic acid biseriate, sclerotia absent Ivory Coast biseriate, sclerotia absent Ivory Coast aflatoxins B1 & B2, sterigmatocystin, Frisvad et al., 2005 3-O-methylsterigmatocystin, kotanin-like metabolites, wortmannin-like metabolites, indole alkaloids aflatoxins B1 & B2, sterigmatocystin, Frisvad et al., 2005 3-O-methylsterigmatocystin, versicolorins, averufin, norsolorinic acid, kotanin/desertorin-, wortmannin-, emerin/xanthocillin- and indole-like extrolites biseriate, ascomata Ecuador and Hülle-cells, ascospores stellate biseriate, ascomata Italy and Hülle-cells, ascospores stellate biseriate, ascomata Venezuela and Hülle-cells, ascospores stellate Ito et al., 2001 aflatoxin B1, arugosin A & B, asperthecin, shamixanthone, sterigmatocystin, terrein, variecoxanthone A, B & C aflatoxin B1, arugosin E, siderin, shamixanthone, sterigmatocystin, terrein, varitriols Frisvad et al., 2004 aflatoxin B1, sterigmatocystin, terrein, compounds with chromophores of the shamixanthone, emerin and desertorin type Frisvad and Samson, 2004 Zalar et al., 2008 World Mycotoxin Journal 2 (3) A reappraisal of fungi producing aflatoxins produces the greatest amounts of aflatoxin B1 ever observed, even more than the best producers in section Flavi (A. parasiticus, A. parvisclerotigenus and A. nomius; Frisvad et al., 2005). Aspergillus section Nidulantes Aspergillus section Nidulantes (A. nidulans species group) includes some species which are able to reproduce only asexually, and the anamorphs of Emericella species. Characteristics of this section include the production of conidial heads that are typically short columnar, and in the shades of dark yellow-green to bluish green, and Hülle cells that are usually globose to citriform in shape. Raper and Fennell (1965) assigned 18 species to this section. Samson (1979) added 10 more species. Emericella is a genus containing species of considerable interest because of the well elucidated genetics of E. nidulans and because some species produce penicillin (Dulaney, 1947a,b). Several other species of Emericella produce sterigmatocystin (El Khady and Abdel Hafez, 1981; Frisvad et al., 2004), while three species, E. astellata (Frisvad et al., 2004), E. venezuelensis (Frisvad and Samson, 2004) and E. olivicola (Zalar et al., 2008) have been found to be able to produce aflatoxins in this section. Emericella astellata and E. venezuelensis produce the typical short brown to yellow brown conidiophores and small vesicles covered with metulae with phialides in their upper part. E. astellata and E. venezuelensis grow rather slowly or not at all at 37 °C (0 and 0-9 mm after one week of incubation, respectively) in contrast to most other species of Emericella. E. astellata produces aflatoxin B1, arugosins, asperthecin, shamixanthone, sterigmatocystin, terrein and variecoxanthones, while E. venezuelensis produces aflatoxin B1, sterigmatocystin, terrein, and compounds with chromophores of the shamixanthone, emerin and desertorin type (Frisvad et al., 2004; Frisvad and Samson, 2004). Another aflatoxin producing Emericella species, E. olivicola grows well at 37 °C, and has stellate ascospores with relatively narrow equatorial crests (up to 2.3 μm). The thin-walled Hülle cells are filled with oil droplets (Zalar et al., 2008). This species produces numerous extrolites including arugosin E, siderin, shamixanthone, sterigmatocystin, terrein, varitriols, and aflatoxin B1, of which the latter was detected only in one of the two strains. In Emericella species in general, aflatoxins were always minor components compared to other extrolites. 3. Conclusions In this review a current overview of aflatoxin-producing species is provided. Since their discovery of these mycotoxins in the early sixties, several Aspergilli and other fungi have been claimed to be able to produce aflatoxins. However, most of these reports were incorrect. To date, aflatoxin producing abilities of 14 Aspergillus species have been confirmed, 3 of which belong to section Nidulantes, 2 World Mycotoxin Journal 2 (3) to section Ochraceorosei (both sections belong to subgenus Nidulantes; Peterson, 2008), while the remaining 11 species are assigned to section Flavi. Based on these findings, aflatoxin biosynthesis thus seems to have evolved at least twice within the Aspergillus genus. The situation is more complex in the case of sterigmatocystin, which has been found to be produced by several Aspergilli assigned to sections Nidulantes, Flavi and Ochraceorosei, but also by species in the phylogenetically unrelated genera Monocillium, Chaetomium, Humicola and Bipolaris. Production of sterigmatocystin may have evolved independently two or three times in Aspergillus and its teleomorphs, and at least four times in the unrelated genera listed above. Regarding the economical importance of aflatoxin producing species, A. flavus and A. parasiticus are the most frequently encountered aflatoxigenic species in various food products including peanuts, maize, cotton and spices. Recent data indicate that A. nomius may contribute to aflatoxin contamination of Brazil nuts (Olsen et al., 2008), and the significance of the newly described microsclerotial species, A. minisclerotigenes and A. arachidicola, needs further investigations. 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