Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi

Most aspergilli that produce aflatoxin are members of Aspergillus section Flavi, however isolates of several Aspergillus species not closely related to section Flavi also have been found to produce aflatoxin. Two of the species, Aspergillus ochraceoroseus and an undescribed Aspergillus species SRRC 1468, are morphologically similar to members of Aspergillus section Circumdati. The other species have Emericella teleomorphs (Em. astellata and an undescribed Emericella species SRRC 2520) and are morphologically distinctive in having ascospores with large flanges. All these aflatoxin-producing isolates were from tropical zones near oceans, and none of them grew on artificial media at 37 C. Aflatoxins and sterigmatocystin production were quantified by high-pressure liquid chromatography (HPLC) and confirmed by HPLC-mass spectrometry (LC-MS) detection. Phylogenetic analyses were conducted on these four species using A. parasiticus and Em. nidulans, (which produce aflatoxin and the aflatoxin precursor sterigmatocystin, respectively) for comparison. Two aflatoxin/sterigmatocystin biosynthesis genes and the beta tubulin gene were used in the analyses. Results showed that of the new aflatoxin-producers, Aspergillus SRRC 1468 forms a strongly supported clade with A. ochraceoroseus as does Emericella SRRC 2520 with Em. astellata SRRC 503 and 512.     

Taxonomic comparison of three different groups of aflatoxin producers and a new efficient producer of aflatoxin B1, sterigmatocystin and 3-O-methylsterigmatocystin, Aspergillus rambellii sp. nov

Accumulation of the carcinogenic mycotoxin aflatoxin B, has been reported from members of three different groups of Aspergilli (4) Aspergillus flavus, A. flavus var. parvisclerotigenus, A. parasiticus, A. toxicarius, A. nomius, A. pseudotamarii, A. zhaoqingensis, A. bombycis and from the ascomycete genus Petromyces (Aspergillus section Flavi), (2) Emericella astellata and E. venezuelensis from the ascomycete genus Emericella (Aspergillus section Nidulantes) and (3) Aspergillus ochraceoroseus from a new section proposed here: Aspergillus section Ochraceorosei. We here describe a new species, A. rambellii referable to Ochraceorosei, that accumulates very large amounts of sterigmatocystin, 3-O-methylsterigmatocystin and aflatoxin B1, but not any of the other known extrolites produced by members of Aspergillus section Flavi or Nidulantes. G type aflatoxins were only found in some of the species in Aspergillus section Flavi, while the B type aflatoxins are common in all three groups. Based on the cladistic analysis of nucleotide sequences of ITS1 and 2 and 5.8S, it appears that type G aflatoxin producers are paraphyletic and that section Ochraceorosei is a sister group to the sections Flavi, Circumdati and Cervini, with Emericella species being an outgroup to these sister groups. All aflatoxin producing members of section Flavi produce kojic acid and most species, except A. bombycis and A. pseudotamarii, produce aspergillic acid. Species in Flavi, that produce B type aflatoxins, but not G type aflatoxins, often produced cyclopiazonic acid. No strain was found which produce both G type aflatoxins and cyclopiazonic acid. It was confirmed that some strains of A. flavus var. columnaris produce aflatoxin B2, but this extrolite was not detected in the ex type strain of that variety. A. flavus var. parvisclerotigenus is raised to species level based on the specific combination of small sclerotia, profile of extrolites and rDNA sequence differences. A. zhaoqingensis is regarded as a synonym of A. nomius, while A. toxicarius resembles A. parasiticus but differs with at least three base pair differences. At least 10 Aspergillus species can be recognized which are able to biosynthesize aflatoxins, and they are placed in three very different clades.     

Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the internal transcribed spacer regions and the 5.8 S rRNA gene

Phenotypic features and sequences of the internal transcribed spacer (ITS) regions and the 5.8 S rRNA gene of type or neotype strains and other isolates of the 17 species currently assigned to Aspergillus section Circumdati and some potentially related species were analyzed. Parsimony analysis of sequence data indicated that Aspergillus section Circumdati is paraphyletic. Aspergillus campestris, A. lanosus, and A. dimorphicus with A. sepultus were found to be more closely related to Aspergillus sections Candidi, Flavi, and Cremei, respectively. These results were also supported by phenotypic data. A. robustus and A. ochraceoroseus were found not to be related to any of the species examined. Species of the proposed revised Aspergillus section Circumdati formed two main clades, which could also be distinguished based on phenotypic methods. Phylogenetic analysis of sequence data of other isolates assigned to species of the revised section indicates that either some of these isolates were misidentified or species concepts of A. ochraceus, A. melleus, and A. petrakii should be reconsidered.     

Evolutionary relationships within Aspergillus section Flavi based on sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene

Sequences of the intergenic transcribed spacer regions and the 5.8S rRNA gene (455 nucleotides) of type strains or representative isolates of 23 species and subspecies either currently assigned to Aspergillus subgenus Circumdati section Flavi or other closely related sections, were analyzed. Parsimony analysis of sequence data indicated that species of Aspergillus section Flavi form distinct clades. The three main clades identified based on sequence data could also be distinguished based on colony color, and their ubiquinone systems. The 'A. flavus' clade includes species characterized with Q-10(H(2)) as their main ubiquinone, conidial colors in shades of green, and dark sclerotia. The 'A. tamarii' clade involves species with ubiquinone system Q-10(H(2)), and conidia in shades of olive to brown, while the 'A. alliaceus' clade consists of species with Q-10 ubiquinone system, and conidia in shades of ocher. The synnematous species A. coremiiformis was found to be closely related to species in the 'A. tamarii' clade. A. thomii and A. terricola var. americana were found to be related to the 'A. flavus' clade in spite of producing brownish colonies. Three species, A. nomius, A. avenaceus, and A. leporis were found to form separate lineages not closely related to any of the main clades identified. It is suggested that A. clavatoflavus and A. zonatus be excluded from Aspergillus section Flavi. Phylogenetic analysis of partial 26S rRNA gene sequences (564 nucleotides) supported our findings.     

Functional and phylogenetic analysis of the Aspergillus ochraceoroseus aflQ (ordA) gene ortholog

Within the Aspergillus parasiticus and A. flavus aflatoxin (AF) biosynthetic gene cluster the aflQ (ordA) and aflP (omtA) genes encode respectively an oxidoreductase and methyltransferase. These genes are required for the final steps in the conversion of sterigmatocystin (ST) to aflatoxin B(1) (AFB(1)). Aspergillus nidulans harbors a gene cluster that produces ST, as the aflQ and aflP orthologs are either non-functional or absent in the genome. Aspergillus ochraceoroseus produces both AF and ST, and it harbors an AF/ST biosynthetic gene cluster that is organized much like the A. nidulans ST cluster. The A. ochraceoroseus cluster also does not contain aflQ or aflP orthologs. However the ability of A. ochraceoroseus to produce AF would indicate that functional aflQ and aflP orthologs are present within the genome. Utilizing degenerate primers based on conserved regions of the A. flavus aflQ gene and an A. nidulans gene demonstrating the highest level of homology to aflQ, a putative aflQ ortholog was PCR amplified from A. ochraceoroseus genomic DNA. The A. ochraceoroseus aflQ ortholog demonstrated 57% amino acid identity to A. flavus AflQ. Transformation of an O-methylsterigmatocystin (OMST)-accumulating A. parasiticus aflQ mutant with the putative A. ochraceoroseus aflQ gene restored AF production. Although the aflQ gene does not reside in the AF/ST cluster it appears to be regulated in a manner similar to other A. ochraceoroseus AF/ST cluster genes. Phylogenetic analysis of AflQ and AflQ-like proteins from a number of ST- and AF-producing Aspergilli indicates that A. ochraceoroseus might be ancestral to A. nidulans and A. flavus.     

Aflatoxin and cyclopiazonic acid production by a sclerotium-producing Aspergillus tamarii strain.

The production of aflatoxins B1 and B2 by Aspergillus tamarii (subgenus Circumdati section Flavi) is reported for the first time. The fungus was isolated from soil collected from a tea (Camellia sinensis) field in Miyazaki Prefecture, Japan. Three single-spore cultures, NRRL 25517, NRRL 25518, and NRRL 25519, were derived from subcultures of the original isolate 19 (MZ2). Each of these single-spore cultures of A. tamarii produced aflatoxins B1 and B2 and cyclopiazonic acid, as well as black, pear-shaped sclerotia. The demonstration of aflatoxin production by A. tamarii is examined in connection with A. tamarii phylogenetic relationships, chemical ecology, and potential use in food fermentations.     

Biogeography of Aspergillus species in soil and litter

Based on counts of Aspergillus species reported in over 250 studies of microfungi from soils and litter, chi-square analyses were conducted on species occurrence in five biomes and five latitude ranges to determine variations from expected distributions. There was no overall trend in distribution of the members of the entire genus by biome, however, individual sections of the genus appeared to have distinct distribution patterns. Most members of sections Aspergillus, Nidulantes, Flavipedes and Circumdati occurred at greater than expected frequencies in desert soils. There was no distinct pattern of species occurrence for forest, wetland, or cultivated soils, although members of section Nidulantes were quite rare in cultivated soils. Most species occurred at or below expected frequencies in grassland soils. Members of the genus tended to occur at greater than expected frequencies at latitudes in the subtropical/warm temperate zone between 26 and 35 degrees. Most species occurred at expected frequencies in the lower latitudes, and below expected frequencies in latitudes greater than 35 degrees.     

Genetic regulation of aflatoxin biosynthesis: From gene to genome

Aflatoxins are notorious toxic secondary metabolites known for their impacts on human and animal health, and their effects on the marketability of key grain and nut crops. Understanding aflatoxin biosynthesis is the focus of a large and diverse research community. Concerted efforts by this community have led not only to a well-characterized biosynthetic pathway, but also to the discovery of novel regulatory mechanisms. Common to secondary metabolism is the clustering of biosynthetic genes and their regulation by pathway specific as well as global regulators. Recent data show that arrangement of secondary metabolite genes in clusters may allow for an important global regulation of secondary metabolism based on physical location along the chromosome. Available genomic and proteomic tools are now allowing us to examine aflatoxin biosynthesis more broadly and to put its regulation in context with fungal development and fungal ecology. This review covers our current understanding of the biosynthesis and regulation of aflatoxin and highlights new and emerging information garnered from structural and functional genomics. The focus of this review will be on studies in Aspergillus flavus and Aspergillus parasiticus, the two agronomically important species that produce aflatoxin. Also covered will be the important contributions gained by studies on production of the aflatoxin precursor sterigmatocystin in Aspergillus nidulans.     

Assessment of toxigenic fungi on Argentinean medicinal herbs

This work was performed to determine the incidence of toxigenic fungi and their mycotoxins on 152 dried medicinal and aromatic herbs, belonging to 56 species, which are used as raw material for drugs. International methodologies for fungal enumeration and identification were applied as well as TLC and HPLC techniques for toxins detection. The 52% out of 152 samples were contaminated with species from Aspergillus genus, 27% belonging to the Flavi section and 25% to the Circumdati section. The 16% of the total samples was contaminated with species from Fusarium genus. Aspergillus flavus and A. parasiticus (Flavi section), were the predominant species isolated, 50% out of 40 isolates were toxigenic. Aflatoxin concentrations ranged from 10 to 2000 ng/g. Only 26% of isolates from the Circumdati section (A. alliaceus, A. ochraceus and A. sclerotiorum) produced ochratoxin A in low concentrations between 0.12 and 9 ng/g. From a total of 29 strains of Fusarium spp., 27.5% were Fusarium verticillioides and F. proliferatum, which produced fumonisin Bland fumonisin B2 ranged from 20 to 22000 microg/g and from 5 to 3000 microg/g respectively. The remaining species, F. equiseti, F. oxysporum, F. semitectum, F. compactum, F. sombucinum and F. solani were able to produce neither group A and B trichothecenes nor zearalenone. The incidence of A. ochraceus and Fusarium spp. and their toxigenic capacities on medicinal herbs were studied for the first time in Argentina. It would be important to look for natural contamination to define acceptability Limits which allow the control of sanitary quality of medicinal herbs used as phytotherapic medicines in several countries.     

Colonization of wounded peanut seeds by soil fungi: selectivity for species from Aspergillus section Flavi

Soil is a source of primary inoculum for Aspergillus flavus and A. parasiticus, fungi that produce highly carcinogenic aflatoxins in peanuts. Aflatoxigenic fungi commonly invade peanut seeds during maturation, and the highest concentrations of aflatoxins are found in damaged seeds. A laboratory procedure was developed in which viable peanut seeds were wounded and inoculated with field soil containing natural populations of fungi, then incubated under different conditions of seed water activity and temperature. Densities of Aspergillus section Flavi in soil used for inoculating seeds were low relative to the total numbers of filamentous fungi (<1%). Aspergillus species from section Flavi present in soil included A. flavus morphotypes L and S strains, A. parasiticus, A. caelatus, A. tamarii and A. alliaceus. Wounding was required for high incidences of fungal colonization; viability of wounded seeds had little effect on colonization by Aspergillus species. Peanut seeds were colonized by section Flavi species as well as A. niger over broad ranges of water activity (0.82-0.98) and temperature (15-37 C), and the highest incidences of seed colonization occurred at water activities of 0.92-0.96 at 22-37 C. A. parasiticus colonized peanut seeds at lower temperatures than A. flavus, and cool soil temperatures relative to temperatures of aerial crop fruits might explain why A. parasiticus is found mostly in peanuts. Other fungi, dominated by the genera Penicillium, Fusarium and Clonostachys, colonized seeds primarily at water activities and temperatures suboptimal for section Flavi species and A. niger. Eupenicillium ochrosalmoneum frequently sporulated on the conidial heads of section Flavi species and showed specificity for these fungi. The inoculation of wounded viable peanut seeds with soil containing natural populations of fungi provides a model system for studying the infection process, the interactions among fungi and those factors important in aflatoxin formation.     

Phylogenetic analysis of Aspergillus species using DNA sequences from four loci

DNA sequences were determined for beta tubulin (BT2), calmodulin (CF), ITS and lsu rDNA (ID) and RNA polymerase II (RPB2) from ca. 460 Aspergillus isolates. RPB2 and rDNA sequences were combined and analyzed to determine relationships in the genus and in the family Trichocomaceae. Eupenicillium species form a statistically supported clade with origins among the Aspergillus clades. A. crystallinus, A. malodoratus and H. paradoxus are members of the Eupenicillium clade. A. zonatus, A. clavatoflvus and W. spinulosa occur in a clade along with Hamigera sp. Other than these exceptional species, Aspergillus species and sections occur on three strongly supported clades that descend from a polytomy. Section Versicolores as a monophyletic group includes only A. versicolor and A. sydowii and is superfluous. The other sections were retained but modified. All four loci were used in genealogical concordance analysis of species boundaries. Fennellia flavipes and F. nivea are not conspecific with their supposed anamorphs A. flavipes and A. nivea. Synonymies were found for some species and more than 20 undescribed taxa were identified in genealogical concordance analysis. Newly discovered taxa will be described elsewhere. Possibly paralogous gene fragments were amplified with the BT2 primers in sections Nidulantes, Usti and Nigri. Use of nonhomologous sequences in genealogical concordance analysis could lead to false conclusions and so BT2 sequences were not used in analysis of those sections.     

Species identification of Aspergillus section Flavi isolates from Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecular approaches

AIMS: Section Flavi is one of the most significant sections in the genus Aspergillus. Taxonomy of this section currently depends on multivariate approaches, entailing phenotypic and molecular traits. This work aimed to identify isolates from section Flavi by combining various classic phenotypic and genotypic methods as well as the novel approach based on spectral analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF ICMS) and to evaluate the discriminatory power of the various approaches in species identification. METHODS AND RESULTS: Aspergillus section Flavi isolates obtained from Portuguese almonds were characterized in terms of macro- and micromorphology, mycotoxin pattern, calmodulin gene sequence and MALDI-TOF protein fingerprint spectra. For each approach, dendrograms were created and results were compared. All data sets divided the isolates into three groups, corresponding to taxa closely related to Aspergillus flavus, Aspergillus parasiticus and Aspergillus tamarii. In the A. flavus clade, molecular and spectral analyses were not able to resolve between aflatoxigenic and nonaflatoxigenic isolates. In the A. parasiticus cluster, two well-resolved clades corresponded to unidentified taxa, corresponding to those isolates with mycotoxin profile different from that expected for A. parasiticus.     

The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism

Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.     

Relationship between soil densities of Aspergillus species and colonization of wounded peanut seeds

Soil is a reservoir for Aspergillus flavus and A. parasiticus, fungi that commonly colonize peanut seeds and produce carcinogenic aflatoxins. Densities of these fungi in soil vary greatly among fields and may influence the severity of peanut infection. This study examined the relationship between soil density of Aspergillus species and the incidence of peanut seed colonization under laboratory conditions. Viable peanut seeds were wounded and inoculated with 20 soils differing in composition and density of Aspergillus species and were then incubated for 14 days at 37 degrees C (seed water activity = 0.92). The effect of soil density of individual section Flavi species (A. flavus strains L and S, A. parasiticus, A. caelatus, and A. tamarii), section Nigri, and A. terreus on the incidence of seed colonization was best expressed as a function of exponential rise to maximum. Exponential curves often rose to maximum percentages of seed colonization by section Flavi species that were well below 100% despite high species densities in some soils. Competition primarily among section Flavi species may explain the reduced incidences of seed colonization. An average of two or fewer propagules of each Aspergillus species in the soil at the wound site was required for colonization of 20% of peanut seeds. Other fungal species were capable of invading peanut seeds only when soil densities of sections Flavi and Nigri species were low.     

DNA fingerprinting analysis of Petromyces alliaceus (Aspergillus section Flavi)

The objective of this study was to evaluate the ability of the Aspergillus flavus pAF28 DNA probe to produce DNA fingerprints for distinguishing among genotypes of Petromyces alliaceus (Aspergillus section Flavi), a fungus considered responsible for the ochratoxin A contamination that is occasionally observed in California fig orchards. P. alliaceus (14 isolates), Petromyces albertensis (one isolate), and seven species of Aspergillus section Circumdati (14 isolates) were analyzed by DNA fingerprinting using a repetitive sequence DNA probe pAF28 derived from A. flavus. The presence of hybridization bands with the DNA probe and with the P. alliaceus or P. albertensis genomic DNA indicates a close relationship between A. flavus and P. alliaceus. Twelve distinct DNA fingerprint groups or genotypes were identified among the 15 isolates of Petromyces. Conspecificity of P. alliaceus and P. albertensis is suggested based on DNA fingerprints. Species belonging to Aspergillus section Circumdati hybridized only slightly at the 7.0-kb region with the repetitive DNA probe, unlike the highly polymorphic hybridization patterns obtained from P. alliaceus and A. flavus, suggesting very little homology of the probe to Aspergillus section Circum dati genomic DNA. The pAF28 DNA probe offers a tool for typing and monitoring specific P. alliaceus clonal populations and for estimating the genotypic diversity of P. alliaceus in orchards, vineyards, or crop fields.     

Exploration of members of Aspergillus sections Nigri, Flavi, and Terrei for feruloyl esterase production

The ability of members of Aspergillus sections Nigri, Flavi, and Terrei to produce feruloyl esterases was studied according to their substrate specificity against synthetic methyl esters of hydroxycinnamic acids. Type A feruloyl esterases (FAEA), induced during growth on cereal-derived products, show a preference for the phenolic moiety of substrates that contain methoxy substitutions, as found in methyl sinapinate, whereas type B feruloyl esterases (FAEB) show a preference for the phenolic moiety of substrates that contain hydroxyl substitutions, as occurs in methyl caffeate. All the strains of Aspergillus section Nigri (e.g., A. niger and A. foetidus) were able to produce feruloyl esterases with activity profiles similar to those reported for FAEA and FAEB of A. niger when grown on oat-spelt xylan and sugar beet pulp, respectively. The two genes encoding these proteins, faeA and faeB, were identified by Southern blot analysis. The strains of Aspergillus sections Flavi (e.g., A. flavus, A. flavo-furcatus, and A. tamarii) and Terrei (e.g., A. terreus) were able to produce type A and type B enzymes. faeA was revealed in genomic DNA of these strains, and FAEA was determined by immunodetection in cultures grown in oat-spelt xylan. In addition, type B enzymes, not related to faeB, were efficiently induced by oat-spelt xylan and exhibited very original activity profiles on sugar beet pulp. This work confirms that the members of the genus Aspergillus are good feruloyl esterase producers.     

In vitro studies on the potential for biological control on Aspergillus section Flavi by Kluyveromyces spp

AIMS: Antagonist activity of Kluyveromyces spp. isolates on Aspergillus section Flavi was studied. METHODS AND RESULTS: The screening of isolates were made through studies of growth at different water activities and temperatures, index of dominance (I(D)), ecological similarity, antifungal activity and impact on aflatoxin B1 accumulation. High optical density was obtained at 25 and 30 degrees C and 48 h of incubation. Cell growth decreases with decrease in water activity. The predominant interaction was mutual intermingling at a(w) = 0.982 and 0.955, while at a(w) = 0.999 and 0.937 mutual inhibition for contact was exhibited. All isolates were catabolically identical to Aspergillus section Flavi and compete by nutritional source. At high water activities yeasts showed inhibitory activity on Aspergillus strains, inhibition percentages varied between 75 and 100%. The isolates Y9, Y14, Y16, Y22, Y25 and Y33 showed antifungal activity and inhibitory activity on aflatoxin B1 accumulation at all water activities assayed from all Aspergillus section Flavi strains. CONCLUSIONS: The data show that the isolates selected in a wide range of environmental conditions could exert their roll like biological control agents for Aspergillus section Flavi in storage maize ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolates of Kluyveromyces spp. may have practical value in the postharvest control of storage maize.     

The isolation of a fungal metabolite which exhibits antimicrobial synergy with sterigmatocystin

A metabolite, which acted synergistically with sterigmatocystin to inhibit the growth of several Gram positive bacteria, has been isolated from several strains of Aspergillus nidulans. Preliminary characterization of this metabolite indicated that it was a high-molecular-weight glycoprotein. Dihydrosterigmatocystin, aflatoxin B, and aflatoxin G_t all failed to exhibit antimicrobial synergy with the glycoprotein. Thus, the synergy with the glycoprotein was specific for sterigmatocystin and the presence of the unsaturated bifuran moiety was shown to be essential to the antimicrobial action of the synergy.     

Molecular and physiological aspects of aflatoxin and sterigmatocystin biosynthesis by Aspergillus tamarii and A. ochraceoroseus

Until recently, only three species (Aspergillus flavus, A. parasiticus and A. nomius) have been widely recognized as producers of aflatoxin. In this study we examine aflatoxin production by two other species, A. tamarii and A. ochraceoroseus, the latter of which also produces sterigmatocystin. Toxin-producing strains of A. tamarii and A. ochraceoroseus were examined morphologically, and toxin production was assayed on different media at different pH levels using thin layer chromatography and a densitometer. Genomic DNA of these two species was probed with known aflatoxin and sterigmatocystin biosynthesis genes from A. flavus, A. parasiticus and A. nidulans. Under the high stringency conditions, A. tamarii DNA hybridized to all four of the A. flavus and A. parasiticus gene probes, indicating strong similarities in the biosynthetic pathway genes of these three species. The A. ochraceoroseus DNA hybridized weakly to the A. flavus and A. parasiticus verB gene probe, and to two of the three A. nidulans probes. These data indicate that, at the DNA level, the aflatoxin and sterigmatocystin biosynthetic pathway genes for A. ochraceoroseus are somewhat different from known pathway genes. Received: 21 May 1999 / Received revision: 17 November 1999 / Accepted: 3 December 1999     

Enzyme reactions and genes in aflatoxin biosynthesis

Aflatoxins are highly toxic and carcinogenic substances mainly produced by Aspergillus flavus and Aspergillus parasiticus. Sterigmatocystin is a penultimate precursor of aflatoxins and also a toxic and carcinogenic substance produced by many species, including Aspergillus nidulans. Recently, the majority of the enzyme reactions involved in aflatoxin/sterigmatocystin biosynthesis have been clarified, and the genes encoding the enzymes have been isolated. Most of the genes constitute a large gene cluster in the fungal genome, and their expression is mostly regulated by a product of the regulatory gene aflR. This review will summarize the enzymatic steps and the genes in aflatoxin/sterigmatocystin biosynthesis.