Skip to main content

A Species-Specific PCR Assay Based on the Calmodulin Partial Gene for Identification of Fusarium Verticillioides, F. Proliferatum and F. Subglutinans

Abstract

Fusarium proliferatum, F. subglutinans and F. verticillioides are the most important Fusarium species occurring on maize world-wide, capable of producing a wide range of mycotoxins which are a potential health hazard for animals and humans. The ribosomal internal transcribed spacer and a portion of the calmodulin gene were sequenced and analysed in order to design species-specific primers useful for diagnosis. The primer pairs were based on a partial calmodulin gene sequence. Three pairs of primers (PRO1/2, SUB1/2 and VER 1/2) produced PCR products of 585, 631 and 578bp for F. proliferatum, F. subglutinans and F. verticillioides, respectively. Primer specificity was confirmed by analyzing DNA of 150 strains of these species, mostly isolated from maize in Europe and USA. The sensitivity of primers was 12.5 pg when the pure total genomic DNA of each species was analyzed. The developed PCR assay should provide a powerful tool for the detection of toxigenic fungi in maize kernels.

This is a preview of subscription content, access via your institution.

References

  1. Amoah BK, MacDonald MV, Rezanoor N and Nicholson P (1996) The use of random amplified polymorphic DNA technique to identify mating groups in Fusarium section Liseola. Plant Pathology 45: 115-125

    Article  Google Scholar 

  2. Amoah BK, Rezanoor N, Nicholson P and MacDonald MV (1995) Variation in the Fusarium section Liseola: Pathogenicity and genetic studies of isolates Fusarium moniliforme Sheldon from different hosts in Ghana. Plant Pathology 44: 563-572

    Google Scholar 

  3. Bacon CW, Porter JK, Norred WP and Leslie JF (1996) Production of fusaric acid by Fusarium. Applied and Environmental Microbiology 62: 4039-4043

    CAS  PubMed  Google Scholar 

  4. Blakemore EJA, Jaccoud Filho DS and Reeves JC (1994) PCR for the detection of Pyrenophora species, Fusarium moniliforme, Stentocarpella maydis, and the Phomopsis/Diaporthe complex. In: Schots A, Dewey FM, and Oliver R (eds) Modern Assays for Plant Pathogenic Fungi: Identification, Detection, and Quantification (pp. 205–214) CAB International, Oxford, UK

    Google Scholar 

  5. Bottalico A (1998) Fusarium disease of cereals: Species complex and related mycotoxin profiles. European Journal of Plant Pathology 80: 85-103

    CAS  Google Scholar 

  6. Huss MJ, Campbell CL, Jennings DB and Leslie JF (1996) Isozyme variation among biological species in the Gibberella fujikuroi species complex (Fusarium section Liseola). Applied Environmental Microbiology 62: 3750-3756

    CAS  Google Scholar 

  7. Klittich CJR and Leslie JF (1988) Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuroi). Genetics 118: 417-423

    CAS  Google Scholar 

  8. Geiser DM, Harbinski FM and Taylor JW (2000) Molecular and analytical tools for characterising Aspergillus and Penicillium species at the intra-and interspecific levels. In: Samson RA and Pitt JI (eds) Integration of Modern Taxonomic Methods of Penicillium and Aspergillus Classiffication (pp. 381–394) Harwood Academic Publishers, Amsterdam, The Netherlands.

    Google Scholar 

  9. Leslie JF (1993) Fungal vegetative compatibility. Annual Review of Phytopathology 31: 127-150

    Google Scholar 

  10. Leslie JF (1995) Gibberella fujikuroi: available populations and variable traits. Canadian Journal of Botany 73(Suppl. 1): S282-S291

    Google Scholar 

  11. Leslie JF, Pearson CA, Nelson PA and Toussoun TA (1990) Fusarium spp. from maize, sorghum, and soyabean fields in the central and eastern United States. Phytopathology 86: 343-350

    Google Scholar 

  12. Logrieco A, Moretti A, Ritieni A, Bottalico A and Corda P (1995) Occurrence and toxigenicity of Fusarium proliferatum from preharvest maize ear rot and associated mycotoxins in Italy. Plant Disease 79: 727-731

    Google Scholar 

  13. Logrieco A, Moretti A, Ritieni A, Chelkowski J, Altomare C, Bottalico A and Randazzo G (1993) Natural occurrence of beauvericin in preharvest Fusarium subglutinans infected maize ears in Poland. Journal of Agricultural and Food Chemistry 41: 2149-2152

    Article  CAS  Google Scholar 

  14. Marasas WFO, Nelson PE and Toussoun TA (1984) Toxigenic Fusarium species: Identity and mycotoxicology. The Pennsylvania State University Press, University Park, PA, USA

    Google Scholar 

  15. Moretti A, Logrieco A, Bottalico A, Ritieni A, Fogliano V and Randazzo G (1996) Diversity in beauvericin and fusaproliferin production by different populations of Gibberella fujikuroi (Fusarium section Liseola). Sydowia 48: 45-56

    Google Scholar 

  16. Moeller EM, Chelkowski J and Geiger HH (1999) Species-specific PCR assays for the fungal pathogens Fusarium moniliforme and Fusarium subglutinans and their application to diagnose maize ear rot. Journal of Phytopathology 147: 497-508

    Google Scholar 

  17. Munkvold G, Stahr HM, Logrieco A, Moretti A and Ritieni A (1998) Occurrence of fusaproliferin and beauvericin in Fusarium-contaminated livestock feed in Iowa. Applied Environmental Microbiology 64: 3923-3926

    CAS  Google Scholar 

  18. Murillo I, Cavallarin L and San Segundo B (1998) The development of a rapid assay for the detection of Fusarium moniliforme. European Journal of Plant Pathology 104: 301-311

    Article  CAS  Google Scholar 

  19. Nelson PE, Desjardins AE and Plattner RD (1993) Fumonisins and mycotoxins produced by Fusarium species: Biology, chemistry, and significance. Annual Review of Phytopathology 31: 233-252

    Article  Google Scholar 

  20. Nelson PE, Toussoun TA and Cook RJ (1981) Fusarium, Diseases, Biology, and Taxonomy. The Pennsylvania State University Press, University Park, PA, USA

    Google Scholar 

  21. Nelson PE, Toussoun A and Marasas W (1983) Fusarium species: An illustrated manual for identification. The Pennsylvania State University Press. University Park, PA, USA

    Google Scholar 

  22. O'Donnell K, Cigelnik E and Nirenberg HI (1998) Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90: 465-493

    Google Scholar 

  23. O'Donnell K, Nirenberg HI, Aoki T and Cigelnik E (2000) A multigene phylogeny of the Gibberella fujikuroi species complex: Detection of additional phylogenetically distinct species. Mycoscience 41: 61-78

    Google Scholar 

  24. Schlacht UF, Moller EM and Geiger HG (1997) Genetic diversity of Gibberella fugikuroi isolates from different geographic origins. Proceeding of the Fifth European Fusarium Seminar. Cereal Research Communication 25: 557-600

    Google Scholar 

  25. Simpson DR, Rezanoor HN, Parry DW and Nicholson P (2000) Evidence for differential host preference in Microdochium nivale var. majus and Microdochium nivale var. nivale. Plant Pathology 49: 261-268

    Article  Google Scholar 

  26. Srobarova A, Moretti A, Ferracane R, Ritieni A and Logrieco A (2002) Toxigenic Fusarium species of Liseola section in preharvest maize ear rot, and associated mycotoxins in Slovakia. European Journal of Plant Pathology 108: 299-306

    Article  CAS  Google Scholar 

  27. Summerell BA, Leslie FJ, Backhouse D, Bryden WL and Burgess LW (2001) Fusarium, Paul E. Nelson Memorial Symposium APS Press, The American Phytopathological Society St. Paul, Minnesota, USA

    Google Scholar 

  28. Xu JR and Leslie JF (1996) A genetic map of Gibberella fujikuroi mating population A (Fusarium moniliforme). Genetics 143: 175-189

    CAS  PubMed  Google Scholar 

  29. White TJ, Burns T, Lee S and Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfald DH, Sninsky JJ and White TJ (eds) PCR Protocol: A Guide to Methods and Applications (pp. 315–322) Academic press, New York, USA

    Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to G. Mulè.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mulè, G., Susca, A., Stea, G. et al. A Species-Specific PCR Assay Based on the Calmodulin Partial Gene for Identification of Fusarium Verticillioides, F. Proliferatum and F. Subglutinans . European Journal of Plant Pathology 110, 495–502 (2004). https://doi.org/10.1023/B:EJPP.0000032389.84048.71

Download citation

  • calmodulin gene
  • Fusarium proliferatum
  • Fusarium subglutinans
  • Fusarium verticillioides
  • maize
  • PCR-assay