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Phylogenetic and morphological analysis of Aspergillus ochraceoroseus

     U.S.D.A., A.R.S., Southern Regional Research Center, 1100 Robert E. Lee Blvd., New Orleans, Louisiana 70124

	ABSTRACT

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Aspergillus ochraceoroseus produces the yellow-gold conidia and other characteristics of Aspergillus subgenus Circumdati section Circumdati. However, this species produces aflatoxin, a secondary metabolite characteristic of some members of subgenus Circumdati section Flavi and sterigmatocystin, a related secondary metabolite usually associated with subgenus Nidulantes sections Nidulantes and Versicolores, as well as members of several other genera. Our morphological data support the placement of A. ochraceoroseus in subgenus Circumdati. Sequence data from A. ochraceoroseus aflatoxin and sterigmatocystin genes aflR and nor-1/stcE, as well as 5.8S ITS and beta tubulin genes, were compared to those of aspergilli in sections Circumdati, Flavi, Nidulantes and Versicolores. In the sequence comparisons, A. ochraceoroseus was related more closely to the species in subgenus Nidulantes than to species from subgenus Circumdati.

Key words: aflatoxin, Aspergillus section Circumdati, Aspergillus section Flavi, Aspergillus section Nidulantes, Aspergillus section Versicolores, sterigmatocystin

	INTRODUCTION

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Aspergillus ochraceoroseus first was described in 1978 by Bartoli and Maggi. It was discovered in the Tai National Forest in the Ivory Coast and described as belonging to what is now subgenus Circumdati, section Circumdati (= Aspergillus ochraceus group). Unfortunately, only one valid isolate exists in culture collections. An isolate thought to be another strain of A. ochraceoroseus, ATCC 42001, is apparently yet another new species that produces aflatoxin (our unpublished observations; JC Frisvad and RA Samson pers comm). Aspergillus ochraceoroseus drew our attention when it was reported to produce both aflatoxin B₁ and sterigmatocystin (Jens Frisvad, Robert Samson pers comm). Neither of these toxins had been reported before from a member of section Circumdati. The aflatoxin producers all belong to section Flavi and, within Aspergillus, sterigmatocystin usually is associated with subgenus Nidulantes sections Nidulantes and Versicolores.

Overall, molecular studies of Aspergillus have supported the taxonomic status of most members within the various sections of the genus (See Samson and Pitt 2000). However, the relationship of the sections to one another according to their subgeneric classification has not been supported always. For instance, in his study of rDNA sequences, Peterson (2000) retained 15 of the 18 sections (three of which were modified) but deleted three of the six subgenera.

Aflatoxin and sterigmatocystin biosynthetic pathways have been studied extensively in Aspergillus flavus/parasiticus and A. nidulans, respectively. The pathways are related; in fact, sterigmatocystin is an intermediate in the aflatoxin biosynthetic pathway. The genes of both pathways are clustered, and the pathway gene products have similar structure and function. However, the arrangement of the genes in the cluster is different and the nucleotide sequence of the individual genes can be quite different (for recent reviews see Payne and Brown 1998, Cary et al 2001).

In earlier work (Klich et al 2000) it was observed that the conditions conducive to aflatoxin/sterigmatocystin production by A. ochraceoroseus were similar to those for sterigmatocystin production by A. nidulans and quite different from those for aflatoxin production by A. flavus/parasiticus. In Southern blots of A. ochraceoroseus DNA, using probes from aflatoxin and sterigmatocystin biosynthesis genes from A. parasiticus and A. nidulans, respectively, no strong hybridization signals were observed. In contrast, DNA from other aflatoxin-producing fungi, such as the species now called A. pseudotamarii (Ito et al 2001), hybridized strongly with the aflatoxin biosynthesis genes from A. flavus (Klich et al 2000).

In this study we examined morphological data and compared A. ochraceoroseus aflatoxin/sterigmatocystin and housekeeping gene sequences with those of other aspergilli to assess the taxonomic placement of this unusual species.

	MATERIALS AND METHODS

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Growth and culturing of Aspergillus strains – Morphological data were obtained using the media and methods described by Klich (2002). For genomic DNA isolation, fungal conidia were inoculated into liquid YES media (Filtenborg et al 1990) and incubated with agitation (150 rpm) at 28 C for 48 h. Mycelia were harvested by filtering through Miracloth, quickly frozen in liquid nitrogen and stored at -80 C. Some of the gene sequence data used in this study were obtained from GenBank submissions from previous reports on phylogenetic analyses of Aspergillus strains. These strains and the accession numbers are listed in the figure legends. Aspergillus strains and the GenBank accession numbers for the genes sequenced in this study also are listed. Aspergillus ochraceoroseus SRRC 1432 (= CBS 550.77) can be obtained from M. Klich at the Southern Regional Research Center.

DNA extraction, PCR and sequencing – Frozen mycelia were ground into a fine powder in liquid nitrogen and DNA was extracted using the Qiagen DNeasy Plant Mini-Kit (Qiagen, Valencia, California). Four gene regions were chosen for analysis, with the length of the amplified region being dependent on the host strain template DNA: a 402–438 bp region representing the complete 5.8S ribosomal RNA gene coding sequence and partial sequence of the flanking internal transcribed spacer (ITS) 1 and 2 regions (hereon termed 5.8S-ITS); a 367–479 bp region representing the partial coding region of the beta tubulin gene including introns 3, 4 and 5 and exons 4, 5 and part of 6; a 297–312 bp region representing the partial nor-1 aflatoxin biosynthetic gene coding region or its equivalent in the stergimatocystin pathway, stcE; a 507–1029 bp region representing the partial aflR aflatoxin regulatory gene coding region. Using the numbering convention from GenBank for A. flavus NRRL 1957 ex type, the nucleotides corresponding to the aligned regions of the above genes are: 5.8S-ITS regions (nt 35 to nt 470); beta tubulin (nt 244 to nt 683); nor-1 (nt 1 to nt 300); and aflR (nt 355 to nt 1135). Oligonucleotide primers for PCR were as follows: beta tubulin 5'-GTTCACCTTCAGACCGGCCAGTGTGTAAG and 3'-AGTGACCCTTGGCCCAGTTGTTACCAGCA; nor-1, 5'-CTSCWTCGTCCCAACAGYATCGTSRTCGC and 3'-AGCCAYTTRTTCTCAAAGTGGAACTTGC; aflR, 5'-CCAGTCCCCTTGAGCCAACT and 3'-ACAGGTGGTGGGACTGTTG. Degenerate primers based on the A. nidulans and A. ochraceoroseus aflR gene sequence were required to PCR amplify an internal region of the A. versicolor SRRC 108 aflR gene. These primers were Av aflR 5'-TCCAAGGTYAARTGCAATAAGGARAAGCC and 3'-ACSACRTACTCATCCTSKGCGCAGCGGCA. All sequences for the 5.8S-ITS gene sequences were obtained from GenBank. PCR reaction mixtures contained in 50 µL: 200 µM dNTPs, 1 µM of each primer, 2 mM MgCl₂, 15 mM Tris-HCl pH 8.0, 50 mM KCl, 1.25 U AmpliTaq Gold polymerase (Perkin-Elmer, Foster City, California) and 100–500 ng genomic DNA template. Thermocycling parameters were: beta tubulin, initial cycle of 95 C, 10 min; 65 C, 1 min; 72 C, 30 s; 34 cycles 95 C, 30 s; 65 C, 1 min; 72 C, 30 s; final 72 C, 7 min incubation; nor-1/stcE, initial cycle of 95 C, 10 min; 60 C, 1 min; 72 C, 30 s; 34 cycles 95 C, 30 s; 60 C, 1 min; 72 C, 30 s; final 72 C, 7 min incubation; aflR, initial cycle of 95 C, 10 min; 55 C, 3 min; 72 C, 3 min; 30 cycles 95 C, 45 s; 55 C, 1 min; 72 C, 1 min; final 72 C, 7 min incubation. Amplification reactions were performed in a PTC100 thermocycler (MJ Research). PCR reaction conditions for amplification of the A. versicolor aflR gene region contained in 50 µL: 22U/mL Taq DNA polymerase with Platinum Taq antibody (Invitrogen), 22mM Tris-HCl (pH 8.4), 55 mM KCl, 1.65 mM MgCl₂, 220 µM dNTPs, 1 µM each primer and 100 ng genomic DNA. Thermocycling parameters were: 94 C, 2 min; 34 cycles 94 C, 30 s; 55 C, 30 s; 72 C, 30 s; final 72 C, 7 min incubation. PCR products were analyzed on 1% agarose gels and subcloned into TOPO pCR 2.1 cloning vectors (Invitrogen) for sequencing. DNA sequencing was performed on an ABI Prizm 377 Automated DNA Sequencer (Applied Biosystems) and the sequence data were edited using DNAMAN DNA analysis software (Lynon Biosoft, Quebec, Canada).

DNA sequence analysis – Sequences were aligned using ClustalX version 1.8 (Thompson et al 1997). Phylogenetic analyses were performed using PAUP* version 4.0ß10 (Swofford 1998) for parsimony and bootstrap analysis. A. fumigatus ATCC 36607 was used as outgroup for analyses of the 5.8S-ITS and beta tubulin gene sequences. No suitable outgroup was available for analysis of the nor-1 and aflR sequences, so these data are presented as unrooted trees. 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 with Adobe Illustrator.

	RESULTS

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Morphological features showing variability between A. ochraceoroseus and potentially related sections of the genus are summarized in Table I. The subgenus and section data reflect ranges for only the common species of those groups, as indicated in the footnote. For the most part, colony colors also differentiate the groups: members of subgenus Nidulantes having white, blue green, green, pink or brown conidia; section Flavi having yellow-green to bronze conidia (except for A. alliaceus); and section Circumdati having yellow to golden conidia. Based on color, A. ochraceoroseus, with its yellow-gold conidia, would be placed in section Circumdati. However, A. alliaceus, which has yellow-gold conidia, recently has been placed in section Flavi based on other morphological and molecular characters (Peterson 1995, Frisvad and Samson 2000, Klich 2002). The data in Table I indicate that A. ochraceoroseus has colony diameters that are somewhat smaller than those of members of section Flavi and stipe lengths and vesicle diameters that are much larger than those of members of sections Nidulantes and Versicolores.

View larger version (19K): [in this window] [in a new window]   FIG. 1. One of the five most-parsimonious trees calculated using PAUP* version 4.0b10 in an heuristic search of the 5.8S rRNA gene and ITS sequence data. Tree length is 138 steps, consistency index (CI) = 0.9130, homoplasy index (HI) = 0.0870, retention index (RI) = 0.8983 and a rescaled consistency index (RC) = 0.8202. Aspergillus fumigatus is used as outgroup species to root the tree on the basis of comprehensive trees of the genus Aspergillus (Peterson 2000). Numbers at the internodes are bootstrap values based on 1000 replicate samples. DNA sequences are deposited in GenBank as accessions AF128852, L76745, L76746, AF203809, AF027863, AF027862, AF272576, AF004929, AF272574, AF004931, AF338641, AF027860, AF027864, AF149755 and AF138288. Sequence alignment data has been deposited at TreeBASE under study accession number S873, matrix accession number M1418

View larger version (19K): [in this window] [in a new window]   FIG. 4. Single most-parsimonious tree calculated using PAUP* version 4.0b10 in an heuristic search of the aflR sequence data. Tree length is 1060 steps, consistency index (CI) = 0.8670, homoplasy index (HI) = 0.1330, retention index (RI) = 0.8353 and a rescaled consistency index (RC) = 0.7242. DNA sequences are deposited in GenBank as accessions AF441422–23, AF441427–28, AF441415, AF441437, L26220, U34740, AF489112, AF547172 and AY197608–09. Sequence alignment data has been deposited at TreeBASE under study accession number S873, matrix accession number M1415

Sequences of the beta tubulin gene to be aligned varied from 367–479 bp in length, with an aligned length of 496 bases. One hundred twenty-one characters were variable but parsimony uninformative, 147 characters were parsimony informative and 228 characters were constant. One most-parsimonious tree of 474 steps was generated. At the amino acid level, all of the strains were identical except A. nidulans SRRC 273 (two residues), A. ochraceus (three residues), A. pseudotamarii NRRL 443 (one residue), A. versicolor (one residue) and A. fumigatus (three residues). None of the amino acid differences were shared in common by these isolates. The greatest differences were found in the intron sequences of the beta tubulin gene-coding region. A. ochraceoroseus, A. versicolor and A. nidulans demonstrated a high degree of divergence in intron 3 compared to the other strains analyzed. This correlated well with the bootstrap analysis and topology of the beta tubulin gene parsimony tree (Fig. 2), indicating that A. ochraceoroseus was related more closely to A. nidulans and A. versicolor. It is interesting to note that A. versicolor did not have an intron 5 resulting in exons 5 and 6 being fused. Aspergillus ochraceus showed little homology in the intron sequences with any of the other strains, and this was supported by the low bootstrap value.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Single most-parsimonious tree calculated using PAUP* version 4.0b10 in an heuristic search of the beta tubulin sequence data. Tree length is 474 steps, consistency index (CI) = 0.7932, homoplasy index (HI) = 0.2068, retention index (RI) = 0.7143 and a rescaled consistency index (RC) = 0.5666. Aspergillus fumigatus is used as outgroup species to root the tree. DNA sequences are deposited in GenBank as accessions M17519, AF525292, AY017538, AF255066–69, AF255071, AF255063–64, AF048754, AY017575 and AY160978–80. Sequence alignment data has been deposited at TreeBASE under study accession number S873, matrix accession number M1417.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Single most-parsimonious tree calculated using PAUP* version 4.0b10 in an heuristic search of the nor-1 sequence data. Tree length is 262 steps, consistency index (CI) = 0.8321, homoplasy index (HI) = 0.1679, retention index (RI) = 0.7833 and a rescaled consistency index (RC) = 0.6517. DNA sequences are deposited in GenBank as accessions AY017630–35, AF526534, U34740, AY017657, AY017659, AY017639 and AF547170–71. Sequence alignment data has been deposited at TreeBASE under study accession number S873, matrix accession number M1416

	DISCUSSION

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The taxonomic placement of A. ochraceoroseus is uncertain. Our data and those of others indicate that this species morphologically fits well into subgenus Cirumdati section Circumdati (Samson 1979, Kozakiewicz 1989). Based on the constriction below the vesicle, a character we found to be inconsistent in this species, Christensen (1982) placed it in what is now subgenus Circumdati section Cremei. A recent analysis of morphological and physiological characteristics of members of section Circumdati placed A. ochraceoroseus away from other members of the section (Varga et al 2000).

Production of aflatoxin by A. ochraceoroseus suggests that this species might have affinities with Section Flavi. Other recently identified aflatoxigenic species, A. pseudotamarii and A. bombycis are inarguably members of section Flavi on both the morphological and molecular level (Ito et al 2001, Peterson et al 2001). Morphologically, A. ochraceoroseus produces the large, predominantly globose vesicles and long stipes that are the features which characterize the species in subgenus Circumdati (Kozakeiwicz 1989, Frisvad and Samson 2000, Klich 2002).

Some physiological evidence suggests that A. ochraceoroseus might belong to subgenus Nidulantes rather than subgenus Circumdati. First, it produces sterigmatocystin, as do many members of subgenus Nidulantes. Second, conditions conducive to both aflatoxin and sterigmatocystin production by A. ochraceoroseus are more similar to those of A. nidulans than to those of A. flavus (Klich et al 2000). Finally, partial sequence analysis of the A. ochraceoroseus aflatoxin/sterigmatocystin gene cluster in our lab has shown that the organization and direction of transcription of the genes within the cluster are homologous to that of the A. nidulans sterigmatocystin gene cluster (unpubl data).

Molecular evidence from studies of large-subunit rRNA gene sequence (Peterson 2000) and 5.8S rRNA gene sequence (Varga et al 2000) suggests that A. ochraceoroseus does not belong in section Circumdati. These studies did not include direct comparisons of A. ochraceoroseus to strains from subgenus Nidulantes. Molecular data herein and previously published data using Southern blots with aflatoxin/sterigmatocystin biosynthesis genes as probes (Klich et al 2000) suggest that A. ochraceoroseus has affinities with subgenus Nidulantes.

Elucidating the morphological, ecological and genetic relationships among aflatoxin-producing species might help us understand why aflatoxin is produced. Another concern for clustered pathways, such as that of aflatoxin, is that the gene cluster (or part thereof) could transfer horizontally into new strains or species. Our data indicate that this is not the case with A. ochraceoroseus. If lateral transfer of the gene cluster had occurred, one would expect to see noncongruence among the four gene trees with respect to the two aflatoxin pathway genes and the unlinked beta tubulin and 5.8S-ITS genes. All the trees were congruent.

More evidence needs to be gathered before making a final taxonomic decision regarding the placement of A. ochraceoroseus. The apparent discrepancy between the morphological, physiological and phylogenetic data needs to be rectified. A. ochraceoroseus might be related to species we have not examined or represent a new section or subgenus within Aspergillus; additional isolates of A. ochraceoroseus need to be collected and studied. Unfortunately, civil war in the Ivory Coast is preventing further collections in that area at this time.

	ACKNOWLEDGMENTS

 
We thank S. Rogers for advice on phylogenetic analyses; K. Ehrlich, P. Cotty and B. Montalbano for providing some of the isolates and help with PCR and sequencing some of the genes; and P. Harris for technical assistance in sequencing. We are grateful to D. Bhatnagar, S. Ireland and S. Peterson for helpful comments on earlier drafts of the manuscript.

	FOOTNOTES

 
¹ Corresponding authors: E-mail: mklich{at}srrc.ars.usda.gov and jcary{at}srrc.ars.usda.gov

Accepted for publication March 24, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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Cary JW, Chang P-K, Bhatnagar D., 2001 Clustered metabolic pathway genes in filamentous fungi. In: Khachatourians GG, Arora DK, eds. Applied mycology and biotechnology. Vol. 1. Agriculture and food protection. Amsterdam,Netherlands: Elsevier Science BV. p 165–198

Christiansen M., 1982 The Aspergillus ochraceus group: two new species from western soils and a synoptic key. Mycologia 74:210-225

Filtenborg O, Frisvad JC, Thrane U., 1990 The significance of yeast extract composition on metabolite production in Penicillium. In: Samson RA, Pitt JI, eds. Modern concepts in Penicillium and Aspergillus classification.New York: Plenum. p 433–441

Frisvad JC, Samson RA., 2000 Neopetromyces gen. nov. and an overview of teleomorphs of Aspergillus subgenus Circumdati. Studies in Mycology 45:201-207

Ito Y, Peterson SW, Wicklow DT, Goto T., 2001 Aspergillus pseudotamarii, a new aflatoxin-producing species in Aspergillus section Flavi. Mycol Res 103:233-239

Klich MA., 2002 Identification of common Aspergillus species. Utrecht, Netherlands: Centraalbureau voor Schimmelcultures. 116 p

———, Mullaney EJ, Daly CB, Cary JW., 2000 Molecular and physiological aspects of aflatoxin and sterigmatocystin biosynthesis by Aspergillus tamarii and A. ochraceoroseus. Appl Microbiol Biotechnol 53:605-609[Medline]

Kozakiewicz Z., 1989 Aspergillus species on stored products. Mycological Papers 161

Payne GA, Brown MP., 1998 Genetics and physiology of aflatoxin biosynthesis. Ann Rev Phytopathol 36:329-362[Medline]

Peterson SW., 1995 Phylogenetic analysis of Aspergillus sections Cremei and Wentii, based on ribosomal DNA sequences. Mycol Res 99:1349-1355

———. 2000 Phylogenetic relationships in Aspergillus based on rDNA sequence analysis. In: Samson RA, Pitt JI, eds. Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Reading,United Kingdom: Harwood Academic Publishers. p 323–355

———, Ito Y, Horn BW, Goto T., 2001 Aspergillus bombycis, a new aflatoxigenic species and genetic variation in its sibling species, A. nomius. Mycologia 95:689-703

Samson RA., 1979 A compilation of the Aspergilli described since 1965. Studies in Mycology 18:1-38

———, Pitt JI, eds 2000 Integration of modern taxonomic methods for Penicillium and Aspergillus classification. Reading, United Kingdom: Harwood Academic Publishers. 510 p

Swofford DL., 1998 PAUP*. Phylogenetic analysis using parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG., 1997 The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24:4876-4882

Varga J, Toth B, Rigo K, Teren J, Hoekstra RF, Kozakeiwicz Z., 2000 Phylogenetic analysis of Aspergillus section Circumdati based on sequences of the internal transcribed spacer regions and the 5.8 S rRNA gene. Fungal Genetics and Biology 30:71-80[Medline]

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