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Characterization of aflatoxin-producing fungi outside of Aspergillus section Flavi

     USDA, ARS, Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, Louisiana 70124

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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.

Key words: aflR, Aspergillus astellatus, Aspergillus flavus, Aspergillus ochraceoroseus, Aspergillus parasiticus, beta tubulin, Emericella astellata, Emericella nidulans, stcE

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Aflatoxin, a highly toxic secondary metabolite, contaminates a number of crops causing large economic losses (CAST 2002, Cary et al 2000). The agriculturally important species producing aflatoxin are Aspergillus flavus and A. parasiticus. Both are members of Aspergillus section Flavi. Several other members of this section also produce aflatoxin, including A. nomius, A. pseudotamarii and A. bombycis (Peterson et al 2001). The genes of the biosynthetic pathway of aflatoxin in A. flavus and A. parasiticus are similar and well characterized (Yu et al 2004, Bhatnagar et al 2003, Cary et al 2000). Sterigmatocystin is structurally and biosynthetically similar to aflatoxin and in fact is an intermediate compound in the aflatoxin biosynthetic pathway (Brown et al 1996). Sterigmatocystin is produced by a number of fungi including the well known species Emericella nidulans and Aspergillus versicolor (Frisvad and Samson 1991).

In recent work with A. ochraceoroseus, an aflatoxin-producing species outside Aspergillus section Flavi, we found that the genes involved in aflatoxin production by this species were similar to those of known aflatoxin and sterigmatocystin producers but somewhat more homologous to those of the Em. nidulans sterigmatocystin pathway than to those of the A. parasiticus aflatoxin pathway (Klich et al 2003), especially with respect to organization of the genes within the cluster. A. ochraceoroseus apparently did not acquire the aflatoxin biosynthetic pathway through horizontal transfer of the gene cluster. Another species closely related to A. ochraceoroseus, SRRC 1468, as well as two aspergilli with Emericella teleomorphic states, Em. astellata and an undescribed Emericella (SRRC 2520), also have been reported to make aflatoxin although at levels much lower than observed with A. parasiticus (unpublished data in our lab, R.A. Samson and J.C. Frisvad pers comm, Frisvad et al 2004). Formal descriptions of the undescribed taxa are being written (Robert Samson, CBS, pers comm). Discovery of these new aflatoxin-producers led to the current project in which we are examining these novel producers with known aflatoxin and sterigmatocystin producers for common morphological and ecological characters and comparing them phylogenetically. We ultimately hope to determine whether these aflatoxin-producing species share common characteristics that could help us understand how the ability to produce aflatoxin evolved.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Strain selection.— – Species morphologically similar to known aflatoxin-producers A. ochraceoroseus SRRC 1432 (= CBS 550.79) and an undescribed Emericella species, SRRC 2520 (= CBS 868.97 referred to hereafter as Emericella SRRC 2520) were examined for the initial screening process. Species similar to A. ochraceoroseus included A. alliaceus SRRC 9 (= NRRL 4181), A. albertensis SRRC 501 (= NRRL 20602), Aspergillus sp. SRRC 1468 (= ATCC 42001, listed therein as Aspergillus ochraceoroseus but found to differ from the type in a number of morphological characteristics and levels of mycotoxin production and referred to hereafter as Aspergillus SRRC 1468) and A. lanosus SRRC 500 (= NRRL 3648). Species similar to Emericella SRRC 2520 included Em. variecolor SRRC 505 (= NRRL 1858) and SRRC 506 (= NRRL 4736), and Em. astellata SRRC 503 (= NRRL 2396). Given the tropical habitat of A. ochraceoroseus and Emericella SRRC 2520, two other species occurring predominately in tropical forest habitats also were screened; A. giganteus SRRC 52 (= NRRL 10) and A. unguis SRRC 271 (= NRRL 2393). Emericella astellata SRRC 512 (= NRRL 2397), 1470 (= CBS 469.88) and 1471 (= CBS 470.88) were obtained and screened after the initial screening.

Growth and culture.— – Methods of Klich (2002) were used to assess morphological characteristics. This included growth rates measured at 1 wk on a CYA media at 25 and 37 C and MEA and CY20S media at 25 C. Microscopic characters were measured using material grown on CYA25 and MEA plates.

For toxin analyses, isolates were grown on potato-dextrose agar (PDA) slants 7 d and spores collected in 0.001% Triton X-100. Spore suspensions were used to three-point inoculate YES and PDA agar plates followed by incubation in the dark 1 wk at 25 C. All experiments for HPLC and LC-MS were done in triplicate. For HPLC and LC-MS, fungal spores were three-point inoculated (2 µL per point) onto YES agar plates whose surface was covered with a sterile membrane (Spectra/Por Membrane 1, Spectrum, Gardena, California) to allow for easy removal and weighing of mycelia.

Extraction and analysis of toxins.— – For TLC, the entire contents of the plates were used for extraction of metabolites as described by Singh et al (1991). HPLC and LC-MS were used to quantify and confirm the identity of the toxins for strains found to be positive on TLC. Mycelia from YES plates were harvested by scraping the surface of the membrane to remove all growth. The mycelia were placed in a beaker, covered with mira cloth and dried 7 d (to a constant weight) in a 45 C drying oven. Toxins were extracted from agar media using the methods of Klich et al (2001). For aflatoxin and sterigmatocystin analyses, extracts were reconstituted in methanol (100–300 µL) and extracted by filter centrifugation (Costar, Corning, New York) at 14 000 rpm for 2 min. HPLC analyses were performed with a Waters 2690 HPLC combined with a Waters UV-VIS 996 detector. Sterigmatocystin was observed at a wavelength of 246 nm. Three µL of sample extract were injected for separation through a Nova-Pak C18 (3.9 x 150 mm, 5 µm) reverse-phase column. The analytical column was protected by a guard column containing the same packing. Column temperature was maintained at 38 C. Elution flow rate was 1.0 mL/min with an isocratic solvent system consisting of 60:40 acetonitrile : water. Retention time for sterigmatocystin was 6.0 min. Using a series of diluted standards, a calibration curve with high linearity (R² = 0.9896) was constructed for sterigmatocystin.

Aflatoxin analyses were performed similarly to a procedure developed by Sobolev and Dorner (2002). HPLC analyses were performed with a Waters 2690 HPLC combined with a Waters 2475 fluorescence detector and postcolumn derivatization was done with a Photochemical Reactor for Enhanced Detection (PHRED, Aura Industries, New York, New York) system. Aflatoxin B₁ detection wavelength was 365 nm (excitation) and 474 nm (emission). Sample extract (1 µL) was injected for separation through a Nova-Pak C18 (3.9 x 150 mm, 5 µm) reverse-phase column. The analytical column was protected by a guard column containing the same packing. Column temperature was maintained at 38 C. Elution flow rate was 0.8 mL/min with mobile-phase solvent consisting of water : methanol : n-butanol (1400:720: 15, v/v/v). Retention time for aflatoxin was 12.0 min. A calibration curve with high linearity (R² = 0.9953) was constructed for aflatoxin from a series of diluted standards. Each sample was injected three times.

For MS direct injection analysis, samples (20 µL) in methanol were injected directly into a ZMD (Waters Corp., Milford, Massachusetts) mass spectrometer using 3% methanol/ether eluant at 0.5 ml/min. An APCI source was used with the APCI heater set to 500 C; source block temp, 140 C; corona, 3.5 kV; cone, 30 V; positive ionization mode; m/z 300–375 scan range. For LC-MS analysis, samples were injected into an Alltech (Deerfield, Illinois) silica column (10 µm, 4.6 x 250 mm) using a 1 mL/min flow rate. Compound identification was based on the presence of the MH⁺ ions 313, 315 and 325 for aflatoxins B₁, B₂ and sterigmatocystin respectively. For LC-MS analysis, MH⁺ retention times of standards were also used (11.1 [B₁], 12.9 [B₂]; 3.8 min for sterigmatocystin).

DNA extraction, PCR and sequencing.— – Mycelia from 7 d old cultures in YES liquid media were frozen and ground into a fine powder in liquid nitrogen and DNA was extracted with the Qiagen DNeasy Plant Mini-Kit (Qiagen, Valencia, California). Regions of three genes were chosen for analysis, with the length of the amplified region being dependent on the host strain template DNA: a 648–918 bp region representing the partial aflR aflatoxin regulatory gene coding region; a 421–431 bp region representing the partial stcE sterigmatocystin biosynthetic gene coding region or its equivalent of the A. parasiticus nor-1 aflatoxin gene (for the purpose of clarity, all further references to the stcE/nor-1 genes will be designated as the stcE gene only); a 462–518 bp region representing the partial coding region of the beta tubulin (benA) gene, including portions of exons 6 and 7 and intron F. Using the numbering convention from GenBank for Em. nidulans FGSC 26 (accession No. U34740 [GenBank] ) and SRRC 273 (accession No. M17519 [GenBank] for benA) the nucleotides corresponding to the aligned regions of the above genes are: aflR (nt 16607 to nt 15968); stcE (nt 14371 to nt 14791); and benA (nt 1971 to nt 2480). Degenerate oligonucleotide primers designed from highly conserved regions of the A. parasiticus SRRC 143, A. nidulans FGSC 26 and A. ochraceoroseus SRRC 1432 for PCR were: aflR 5'- CGATGCGYMCGMCGTGGYCTSYCTTGCGAG and 3'-ACR TACTCATCCTSKGCGCAGCGGCA; stcE 5'-CGYCCCAAM AGYAYYGTSRTCGC and 3'-TTCTCAAAGTGGAWCTTGC GCAC; benA 5'-TGCGACTGCCTCCAGGGTTTCCAGAT and 3'-GTTCTTGGGGTCGAACATCTGCTGGGTC. PCR reaction mixtures contained in 25 µL: 200 µM dNTPs, 1 µM of each primer, 1 x Ex Taq buffer, 0.625 U Ex Taq HS polymerase (Takara Bio Inc, Shiga, Japan) and 50–100 ng genomic DNA template. Thermocycling parameters were: aflR, initial cycle of 94 C, 2 min; 94 C, 30 s; 68 C, 30 s; 72 C, 1 min; 34 cycles 94 C, 30 s; 68 C, 30 s; 72 C, 1 min; final 72 C, 7 min. stcE, thermocycling parameters were the same as aflR except the 72 C extension time was reduced to 30 s. benA, thermocycling parameters were the same as aflR except the annealing temperature was reduced to 55 C. The annealing temperature was increased to 60 C for Em. astellata SRRC 1470 and 1471 and to 65 C for A. parasiticus SRRC 143. Amplification reactions were performed in an iCycler thermocycler (Bio-Rad, Hercules, California). PCR products were analyzed on 1% agarose gels and directly sub-cloned into TOPO pCR 2.1 cloning vectors (Invitrogen, Carlsbad, California). If a number of PCR products were present in the gel the band of interest was cut from the gel and purified (QIAquick Gel Extraction Kit, Qiagen, Valencia, California) then subcloned into pCR 2.1 or re-amplified then subcloned. Attempts to subclone the Em. astellata SRRC 1470 and 1471 benA PCR products into pCR 2.1 did not yield vectors harboring the desired insert so the PCR products were directly sequenced. DNA sequencing was performed on a CEQ^®8000 Automated DNA Sequencer (Beckman Coulter, Fullerton, California) and the sequence data were edited using DNAMAN DNA analysis software (Lynon Biosoft, Quebec, Canada).

DNA sequence analysis.— – Sequences were aligned using Clustal X version 1.8 (Thompson et al 1997). Phylogenetic analyses were performed using PAUP* version 4.0b10 (Swofford 1998) for parsimony and bootstrap analysis. A. parasiticus SRRC 143 was used as outgroup for analyses of all three genes. Bootstrap values were generated by 1000 replications of the bootstrap procedure. Gaps were treated as missing data and thus were excluded from the analyses. Phylogenetic trees were generated from PAUP* and edited using Adobe Illustrator. The partition homogeneity test (PHT) in PAUP* was performed on parsimony informative sites only, with 1000 randomized datasets using heuristic search methods with stepwise sequence addition.

Southern hybridization analysis.— – Total genomic DNA was exhaustively digested with Eco RI, and the DNA was separated on a 0.8% agarose gel. Gels were transferred to a nylon membrane (Nytran SPC, Schleicher & Schuell, Keene, New Hampshire) by standard methods. Membranes were hybridized at 42 C in ULTRAhyb buffer (Ambion, Austin, Texas) with PCR probes (aflR and stcE) radiolabeled using the Rediprime^® II Random Prime Labelling System (Amersham Biosciences, Piscataway, New Jersey) and -P³²-dCTP. Membranes were washed once for 10 min at 42 C in 2 x SSPE/0.1% SDS (1x SSPE contains: 150 mM NaCl; 10 mM NaPO₄; 1 mM EDTA) followed by two washes for 20 min at 42 C then 50 C in 0.1x SSPE/0.1% SDS. Membranes then were exposed to autoradiography film overnight at room temperature before development.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Some of the morphological characteristics of the nonsection Flavi aflatoxin-producing species are listed (TABLE I). A characteristic of these species is that they do not grow at 37 C on the media tested. The two Emericella species also shared common characters of relatively slow growth at 25 C and large-flanged ascospores. All species were associated with woody plant biomes located near coastal areas in the tropics. Em. astellata SRRC 1470 and 1471 were morphologically distinct from the type strain (SRRC 503). Colony diameters of SRRC 1470 and 1471 were much larger than those of the type (33–34 mm on CYA and 33–38 mm on MEA), conidia were smaller (1.5–3 µm) and smooth-walled and the ascospores were slightly smaller (4–5 µm without flanges). These isolates were not warm tropical in origin. They were from Cadiz, Spain, at latitude of 37°N.

All toxin results obtained by thin layer chromatography also were confirmed and quantified by HPLC and LC-MS (TABLE I). Samples for HPLC and LC-MS were taken from extracts of fungi that were grown on YES agar because this medium consistently demonstrated the highest levels of aflatoxin and sterigmatocystin production. Aspergillus SRRC 1468 was shown to produce aflatoxins B₁, B₂ and sterigmatocystin. This represented about 2400x more B₁, 300x more B₂, and 2x more sterigmatocystin than produced by the closely related A. ochraceoroseus 1432. Aflatoxin B₁ production was only about twofold less than that observed for the section Flavi aflatoxin-producing species A. parasiticus 143 (228 087 ± 42 900 ng/g). Emericella SRRC 2520 produced approximately twofold less aflatoxin ^B1 than Aspergillus SRRC 1468 and about twofold more sterigmatocystin. No aflatoxin B₂ was detected from extracts of Emericella SRRC 2520. Emericella SRRC 2520 produced about 88 x more aflatoxin B₁ than the closely related species Em. astellata 503. Unlike Emericella SRRC 2520, Em. astellata 503 and 512 both produced aflatoxin B₂. Only sterigmatocystin was detected in extracts of Em. astellata 1470 and 1471. Production of aflatoxins and sterigmatocystin by each isolate, as determined by TLC and HPLC, was confirmed by LC-MS.

The molecular relationships of the aflatoxigenic, nonsection Flavi isolates to one another were determined by phylogenetic analysis of DNA sequences of two sterigmatocystin/aflatoxin pathway genes, aflR and stcE, and the beta tubulin (benA) gene. Examples of single most parsimonious trees obtained using PAUP* parsimony analysis are shown for these three genes (FIG. 1). Aspergillus parasiticus SRRC 143 was used as outgroup species to root all trees.

View larger version (22K): [in this window] [in a new window]   FIG. 1. One most-parsimonious (M-P) tree from each of the three gene regions (benA; aflR; stcE) and from the combined data. Calculations were performed using PAUP* version 4.0b10 in a heuristic search of the gene sequence data. Aspergillus parasiticus is used as outgroup species to root the tree on the basis of comprehensive trees of the genus Aspergillus (Peterson 2000). Numbers at internodes are bootstrap values based on 1000 replicate samples. Only values $50% are shown. DNA sequences are deposited in GenBank as accessions AY613932–613939, L49386 [GenBank] and M17519 [GenBank] , benA; AY616025–616027, AY590879–590883, AF526534 [GenBank] , L27801 [GenBank] and U34740 [GenBank] , stcE; AY616028–616029, AY596457–596460, AF489112 [GenBank] , L26220 [GenBank] and U34740 [GenBank] , aflR. Sequence alignment data has been deposited at TreeBASE under study accession number S1226. Matrix accession numbers: benA, M2129; stcE, M2127; aflR, M2130; combined tree, M2128. Abbreviations: CI, consistency index; RI, retention index; RC, rescaled consistency index; HI, homoplasy index; A, aflatoxin producer; S, sterigmatocystin producer.

Degenerate primers were designed from highly conserved regions within exon 2 of the norsolorinic acid reductase (stcE) gene. Regions to be analyzed varied 421–430 bp in length, with an aligned length of 432 bases. Of the aligned nucleotides, 36 characters were variable but parsimony uninformative, 202 characters were parsimony informative and 194 characters were constant. Two most parsimonious trees of 347 steps each were generated (FIG. 1). Tree topology and bootstrap values again indicated that Em. astellata SRRC 1470 and 1471 were not closely related to Em. astellata SRRC 503 and 512 sharing only 63% identity at the nucleotide level and only 61% identity at the amino acid level. As seen with the benA analysis, SRRC 503 and 512 grouped closely with Emericella SRRC 2520. Also Aspergillus SRRC 1468 demonstrated a high degree of homology with A. ochraceoroseus SRRC 1432 with 142 out of 143 amino acids of the stcE coding region identical.

All templates were amplified successfully with degenerate primers designed to a region just downstream of the alfR zinc-finger domain and upstream of the C-terminal activation domain. The resultant PCR products span the putative PEST domain that is highly variable between A. ochraceoroseus SRRC 1432 and A. parasiticus and Em. nidulans aflR genes (Klich et al 2003). AflR sequences to be aligned varied in length from 648 to 918 bp with a final aligned length of 983 bases. The sequences produced 175 characters that were variable but parsimony uninformative, 355 were parsimony informative and 453 characters were constant. Two most parsimonious trees of 857 steps were generated (FIG. 1). As observed for the other two trees Em. astellata SRRC 1470 and 1471 did not appear closely related to Em. astellata SRRC 503 and 512 demonstrating only 53% amino acid identity. Aspergillus SRRC 1468 and A. ochraceoroseus SRRC 1432 were closely related with 98% amino acid identity. Emericella SRRC 2520 was closely related to Em. astellata SRRC 503 and 512 sharing 88% amino acid identity.

A combined parsimony analysis of the three data-sets produced a tree of 1469 steps, 110 steps longer than the minimum length, indicating incongruence among the three datasets. When partitions consisting of the three gene sequences were examined with the partition homogeneity test (PHT), these regions also were found to be significantly incongruent (PHT test = 0.018). The topologies of all four trees were identical where internal branches were strongly supported. Three strongly supported groups were defined in each tree: A. ochraceoroseus 1432 and Aspergillus SRRC 1468; Em. astellata 1470 and 1471; and Em. astellata 503, 512, and Emericella SRRC 2520. However weakly supported structure was evident in each tree with respect to Em. nidulans 273. Weak support is observed in the benA tree for relatedness of Em. nidulans 273 to A. ochraceoroseus 1432 and Aspergillus SRRC 1468. However in the aflR, stcE and combined gene trees, Em. nidulans 273 demonstrates closest, albeit weak identity, to the Em. astellata 503, 512 and Emericella SRRC 2520 group.

PCR products representing the Aspergillus SRRC 1468, Em. astellata SRRC 1470, Em. astellata SRRC 512 and Emericella SRRC 2520 aflR and stcE gene regions were used as probes in Southern hybridizations of restriction digested genomic DNA from these four isolates. All probes demonstrated strong hybridization signals with their respective genomic DNAs but weak to no signals with negative control DNAs. This indicated that the PCR products in fact were amplified from their respective template genomic DNA (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Until Frisvad and Samson (pers comm) reported aflatoxin production by A. ochraceoroseus and Emericella SRRC 2520 in 1997, all Aspergillus species that were observed to consistently produce aflatoxin belonged to section Flavi. However with the isolation, morphological and molecular phylogenetic analysis of the aflatoxin and sterigmatocystin-producing fungus A. ochraceoroseus (currently placed in section Circumdati, Bartoli and Maggi 1978) and toxin analysis of Em. astellata, it is now becoming evident that isolates outside section Flavi can produce aflatoxin (Klich et al 2003, Frisvad and Samson 2004). Aspergillus SRRC 1468 and Emericella SRRC 2520 have been identified as capable of making aflatoxin in addition to sterigmatocystin (Frisvad and Samson unpubl). The intent of this study was to define both morphologically and phylogenetically the relatedness of the newly identified aflatoxigenic strains of Aspergillus SRRC 1468, Em. astellata and Emericella SRRC 2520 with previously characterized aflatoxin and sterigmatocystin-producing isolates. This included determining the relatedness of the Em. astellata isolates SRRC 503 and 512 with Em. astellata isolates SRRC 1470 and 1471 that demonstrated significant differences in morphology and toxin production.

Molecular phylogenetic analyses using PCR amplified portions of two aflatoxin/sterigmatocystin bio-synthetic genes and the benA gene indicates that Em. astellata SRRC 1470 and 1471 are not closely related to Em. astellata SRRC 503 and 512. These results correlate well with both macro- and micromorphological data that indicate significant differences between these two sets of isolates of presumed Em. astellata. Further, while both SRRC 1470 and 1471 and SRRC 503 and 512 produce sterigmatocystin, only SRRC 503 and 512 appear to produce aflatoxin, albeit at low levels. Similar results were obtained by Frisvad et al (2004) although they did not detect aflatoxin B₁ or sterigmatocystin production on YES agar medium. The reason for this discrepancy is not clear at this time, but minor differences in media have been known to have a large influence on mycotoxin production. Frisvad et al (2004) reported that aflatoxin B₂ was produced by Em. astellata, although they did not report the levels obtained. Phylogenetic analyses also indicated that Em. astellata SRRC 1470 and 1471 were more distantly related to Em. astellata SRRC 503 and 512 than was Emericella SRRC 2520. This would indicate that Em. astellata SRRC 1470 and 1471 should be reclassified as a separate species, as suggested by Frisvad et al (2004).

Bootstrap analysis of all four trees provided strong support (98%) for a clade consisting of Emericella SRRC 2520 and Em. astellata SRRC 503 and 512 indicating that they shared a recent common ancestor. No aflatoxin B₂ was detected from Emericella SRRC 2520, and it produced significantly higher levels of aflatoxin B₁ than either Em. astellata SRRC 503 and 512. It is interesting to note that all of these strains were isolated from approximately the same latitude and geographic location. Perhaps radiation of the ancestral species to a geographically isolated region such as the Galapagos Islands and subsequent adaptation to the new environment resulted in the differences observed now between Em. astellata 503 and 512 and Emericella SRRC 2520.

Although slightly different morphologically, tree topology and bootstrap analysis indicated that Aspergillus SRRC 1468 is closely related to A. ochraceoroseus SRRC 1432 and may represent sibling species. This is supported by toxin analyses that showed both strains producing aflatoxins B₁, B₂ and sterigmatocystin although A. ochraceoroseus SRRC 1432 produces these toxins at much lower levels. Production of aflatoxin B₁ and B₂ by Aspergillus SRRC 1468 was approximately twofold and threefold less respectively than that measured for the section Flavi aflatoxin-producer, A. parasiticus 143. It is interesting to note that partial DNA sequence analysis of the A. ochraceoroseus SRRC 1432 aflatoxin gene cluster demonstrated that it was similar to that of the Em. nidulans sterigmatocystin gene cluster with respect to gene order and direction of gene transcription (Klich et al 2003, Cary unpubl data). However the genes required for conversion of sterigmatocystin to aflatoxin, omtA and ordA, have not been identified during the process of cluster sequencing nor by Southern hybridization of A. ochraceoroseus SRRC 1432 genomic DNA with probes for these two genes. Continued analysis of the A. ochraceoroseus SRRC 1432 gene cluster and flanking regions might provide the identity of the genes responsible for conversion of sterigmatocystin to aflatoxins in this isolate. This information then can be used to better decipher the relationships of all nonsection Flavi aflatoxin producers to one another as well as to section Flavi and Nidulantes toxin producers.

	ACKNOWLEDGMENTS

 
We are grateful to P. Harris for providing technical assistance with sequencing and S. Boue, C. Carter-Wientjes and J. Bland for assistance with toxin analysis. We thank K.C. Ehrlich, J.W. Bennett and J.M. Dyer for helpful comments on drafts of the manuscript.

	FOOTNOTES

 
Accepted for publication November 5, 2004.

¹ Corresponding author. E-mail: jcary{at}srrc.ars.usda.gov

	LITERATURE CITED

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