Antonie van Leeuwenhoek

, Volume 83, Issue 2, pp 191–200 | Cite as

Mycotoxin production and evolutionary relationships among species of Aspergillus section Clavati.

  • János VargaEmail author
  • Krisztina Rigó
  • János Molnár
  • Beáta Tóth
  • Szilvia Szencz
  • József Téren
  • Zofia Kozakiewicz


Aspergillusclavatus is a commonly encountered fungus in the environment, producing a number of mycotoxins including patulin, kojic acid, cytochalasins and tremorgenic mycotoxins. A. clavatus belongs to Aspergillus section Clavati together with six other species, all of which possess clavate-shaped vesicles. Patulin production was analysed by thin layer chromatography and high performance liquid chromatography, while a primer pair developed for the detection of an iso-epoxydon dehydrogenase gene involved in the biosynthesis of patulin in penicillia was used to detect the ability of patulin production in the isolates examined. A good correlation was observed between patulin producing properties, and the presence of an iso-epoxydon dehydrogenase gene fragment among the isolates tested. A. longivesica was found for the first time to produce patulin. Ribotoxin production was also examined using a PCR-based approach. Ribotoxins were detected for the first time in an A. pallidus and a Hemicarpenteles acanthosporus isolate. A phylogenetic analysis of intergenic transcribed spacer sequence data indicated that most isolates belong to two main clades that have also been identified earlier based on 26 S rDNA sequence data. A. pallidus isolates clustered together with A. clavatus strains. Although A. clavatus isolates produced highly homogeneous random amplified polymorphic DNA profiles, phylogenetic analysis of these data let us cluster A. clavatus isolates into distinct clades. Correlations were not observed between either patulin or ribotoxin production, and the taxonomic position of the isolates tested, indicating that patulin and ribotoxin producing abilities were lost several times during evolution of Aspergillus section Clavati. Although patulin was earlier found to inhibit mycovirus replication, one of the mycovirus carrying isolates also produced patulin, and both carried the iso-epoxydon dehydrogenase gene.

Aspergillus ITS region Patulin Phylogenetic analysis RAPD Ribotoxin Section Clavati 


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  1. Auvauvre-Brown A., Cohen J. and Holden D.W. 1992. Use of randomly amplified polymorphic DNA markers to distinguish isolates of Aspergillus fumigatus. J. Clin. Microbiol. 30: 2991–2993.Google Scholar
  2. Beretta B., Gaiaschi A., Galli C.L. and Restani P. 2000. Patulin in apple-based foods: occurrence and safety evaluation. Food Addit. Contam. 17: 399–406.PubMedCrossRefGoogle Scholar
  3. Bogaerts R. and Wolf F. 1980. A standardized method for the Midetection of residues of antibacterial substances in fresh meat. Fleischwirtschaft 60: 667–675.Google Scholar
  4. Detroy R.W. and Still P.E. 1976. Patulin inhibition of mycovirus replication in Penicillium stoloniferum. J. Gen. Microbiol. 92: 167–174.PubMedGoogle Scholar
  5. Fekete C., Giczey G., Papp I., Szabó L. and Hornok L. 1995. High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae. FEMS Microbiol. Lett. 131: 295–299.PubMedCrossRefGoogle Scholar
  6. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.CrossRefGoogle Scholar
  7. Felsenstein J. 1995. PHYLIP (Phylogeny Inference Package). Version 3.57c. Distributed by the author. Department of Genetics, University of Washington, Seattle.Google Scholar
  8. Flannigan B. and Pearce A.R. 1994. Aspergillus spoilage: spoilage of cereals and cereal products by the hazardous species Aspergillus clavatus. In: Powell K.A., Renwick A. and Peberdy J.F. (eds), The genus Aspergillus. From taxonomy and genetics to industrial application (pp 115–127). Plenum Press, New York.Google Scholar
  9. Gams W., Christensen M., Onions A.H.S., Pitt J.I. and Samson R.A. 1985. Infrageneric taxa of Aspergillus. In: Samson R.A. and Pitt J.I. (eds), Advances in Aspergillus and Penicillium systematics (pp 55–62). Plenum Press, New York.Google Scholar
  10. Geiser D.M., Frisvad J.C. and Taylor J.W. 1998. Evolutionary relationships in Aspergillus section Fumigati inferred from partial γ-tubulin and hydrophobin DNA sequences. Mycologia 90: 831–845.Google Scholar
  11. Ghabrial S.A., Bruenn J.A., Buck K.W., Wickner R.B., Patterswon J.L., Stuart K.D. et al. 1995. Totiviridae. In: Murphy F.A., Fauquet C.M., Bishop D.H.L., Ghabrial S.A., Jarvis A.W., Martelli G.P. et al. (eds), Virus taxonomy. Sixth report of the International Committee on Taxonomy of Viruses (pp 245–252). Springer Verlag, Wien.Google Scholar
  12. Harrison M.A. 1989. Presence and stability of patulin in apple products: a review. J Food Prot. 9: 147–153.Google Scholar
  13. Huang L.H. and Raper K.B. 1971. Aspergillus longivesica, a new species from Nigerian soil. Mycologia 63: 50–57.PubMedGoogle Scholar
  14. Kimura M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies on nucleotide sequences. J. Mol. Evol. 2: 87–90.CrossRefGoogle Scholar
  15. Kuraishi H., Itoh M., Tsuzaki N., Katayama Y., Yokoyama T. and Sugiyama J. 1990. The ubiquinone system as a taxonomic aid in Aspergillus and its teleomorphs. In: Samson R.A. and Pitt J.I. (eds), Modern concepts in Penicillium and Aspergillus classification (pp 407–421). Plenum Press, New York.Google Scholar
  16. Leach J., Finkelstein D.B. and Rambosek J.A. 1986. Rapid miniprep of DNA from filamentous fungi. Fungal Genet. Newslett 33: 32–32.Google Scholar
  17. Lin A., Huang K., Hwu L. and Tzean S.S. 1994. Production of type II ribotoxins by Aspergillus species and related fungi in Taiwan. Toxicon 33: 105–110.CrossRefGoogle Scholar
  18. Lopez-Diaz T.M. and Flannigan B. 1997. Production of patulin and cytochalasin E by Aspergillus clavatus during malting of barley and wheat. Int. J. Food Microbiol. 35: 129–136.PubMedCrossRefGoogle Scholar
  19. Martínez-Ruiz A., Kao R., Davies J. and Martïnez del Pozo A. 1999. Ribotoxins are a more widespread group of proteins within the filamentous fungi than previously believed. Toxicon 37: 1549–1563.PubMedCrossRefGoogle Scholar
  20. Mondon P., Thelu J., Lebeau B., Ambroise-Thomas P. and Grillot R. 1995. Virulence of Aspergillus fumigatus strains investigated by random amplified polymorphic DNA analysis. J. Med. Microbiol. 42: 299–303.PubMedCrossRefGoogle Scholar
  21. Morris T.J. and Dodds J.A. 1979. Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69: 854–858.CrossRefGoogle Scholar
  22. Paterson R.R.M., Archer S., Kozakiewicz Z., Lea A., Locke T. and O'Grady E. 2000. A gene probe for the patulin metabolic pathway with potential for use in patulin and novel disease control. Biocontrol Sci. Technol. 10: 509–512.CrossRefGoogle Scholar
  23. Peterson S.W. 2000. Phylogenetic relationships in Aspergillus based on rDNA sequence analysis. In: Samson R.A. and Pitt J.I. (eds), Integration of modern taxonomic methods for Penicillium and Aspergillus classification (pp 323–355). Harwood Academic Publishers, Amsterdam.Google Scholar
  24. Pontecorvo G., Roper J.A., Hemmons L.M., MacDonald K.D. and Bufton A.W.J. 1953. The genetics of Aspergillus nidulans. Adv. Genet. 5: 141–238.PubMedCrossRefGoogle Scholar
  25. Raper K.B. and Fennell D.I. 1965. The genus Aspergillus. Williams & Wilkins, Baltimore.Google Scholar
  26. Rinyu E., Varga J. and Ferenczy L. 1995. Phenotypic and genotypic analysis of variability in Aspergillus fumigatus. J. Clin. Microbiol. 33: 2567–2575.PubMedGoogle Scholar
  27. Saitou N. and Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425.PubMedGoogle Scholar
  28. Sambrook J., Fritsch E.F. and Maniatis T. 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  29. Scott R.E., Jones A., Lam K.S. and Gaucher G.M. 1986. Manganese and antibiotic biosynthesis. I. A specific manganese requirement for patulin production in Penicillium urticae. Can. J. Microbiol. 32: 259–267.PubMedCrossRefGoogle Scholar
  30. Scott P.M. 1974. Patulin. In: Purchase I.F.H. (ed.), Mycotodins. Elsevier, Amsterdam, pp. 383–403.Google Scholar
  31. Shindia A.A. 1997. Mevinolin production by some fungi. Folia Microbiol. (Praha) 42: 477–480.Google Scholar
  32. Smith J.E. and Moss M.O. 1985. Mycotoxins. Formation, analysis and significance. John Wiley & Sons, Chichester.Google Scholar
  33. Steiman R., Seiglle-Murandi F., Sage L. and Krivobok S. 1989. Production of patulin by Micromycetes. Mycopathologia 105: 129–133.PubMedCrossRefGoogle Scholar
  34. Tamura M., Hamamoto M., Canete-Gibas C., Sugiyama J. and Nakase T. 1999. Genetic relatedness among species in Aspergillus section Clavati as measured by electrophoretic comparison of enzymes, DNA base composition, and DNA-DNA hybridization. J. Gen. Appl. Microbiol. 45: 77–83.PubMedCrossRefGoogle Scholar
  35. Téren J., Varga J., Hamari Z., Rinyu E. and Kevei F. 1996. Immunochemical detection of ochratoxin A in black Aspergillus strains. Mycopathologia 34: 171–176.CrossRefGoogle Scholar
  36. Udagawa S. and Takada M. 1971. Soil and coprophilous microfungi. Bull. Nat. Sci. Mus. Tokyo 14: 501–544.Google Scholar
  37. Udagawa S. and Uchiyama S. 2002. Neocarpenteles: a new asganese comycete genus to accomodate Hemicarpenteles acanthosporus. Mycoscience 43: 3–6.CrossRefGoogle Scholar
  38. Varga J., Tóth B., Rigó K., Téren J., Hoekstra R.F. and Kozakiewicz 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 Genet. Biol. 30: 71–80.PubMedCrossRefGoogle Scholar
  39. Varga J., Tóth B., Szencz S., Molnár J., Fekete C. and Szabó L. 2001. Double-stranded RNA elements and virus-like particles in Aspergilli. Acta Biol. Hung. 52: 355–363.PubMedCrossRefGoogle Scholar
  40. White T.J., Bruns T., Lee S. and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M.A., Gelfand D.H., Sninsky J.J. and White T.J. (eds), PCR Protocols: A guide to methods and applications (pp 315–322). Academic Press, San Diego.Google Scholar
  41. Williams J.G.K., Kubelik A.R., Livak K.J., Rafalski J.A. and Tingey S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18: 6531–6535.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2003

Authors and Affiliations

  • János Varga
    • 1
    Email author
  • Krisztina Rigó
    • 1
  • János Molnár
    • 1
  • Beáta Tóth
    • 1
  • Szilvia Szencz
    • 1
  • József Téren
    • 2
  • Zofia Kozakiewicz
    • 3
  1. 1.Department of Microbiology, Faculty of SciencesUniversity of SzegedSzegedHungary
  2. 2.Animal Health and Food Control StationSzegedHungary
  3. 3.CABI Bioscience UK CentreBakeham Lane, EghamUK

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