Skip to main content
SpringerLink
Log in

A reference-gene-based quantitative PCR method as a tool to determine Fusarium resistance in wheat

  • Original Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

In recent years, plant breeders made great progress in breeding Fusarium-tolerant wheat lines. However, total resistance to this genus of plant pathogenic fungi has not yet been achieved as the resistance genes are located on several distinct genetic regions. Visual scoring of disease symptoms in combination with the analysis of mycotoxins is commonly applied to assess the tolerance of new lines. Both approaches are indirect methods and do not mandatorily determine the accumulated fungal biomass. Quantitative PCR is a useful tool to assess fungal biomass based on the abundance of organism-specific DNA. The aim of this study was the development of a quantitative PCR assay for trichothecene-producing Fusarium species and to adapt this method for resistance assessment of wheat lines artificially infected with Fusarium graminearum and Fusarium culmorum. Several DNA-extraction methods for wheat samples were evaluated and optimized for downstream real-time PCR analysis and furthermore, a new reference-gene-based approach for more accurate quantification of Fusarium biomass in cereals is presented. The co-determination of a plant gene was used to compensate for unequal DNA-extraction efficiencies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Parry DW, Jenkinson P, Mcleod L (1995) Fusarium ear blight (scab) in small grain cereals—a review. Plant Pathol 44:207–238

    Article  Google Scholar 

  2. Dill-Macky R, Jones RK (2000) The effects of previous crop residues and tillage on Fusarium head blight of wheat. Plant Dis 84:71–76

    Article  Google Scholar 

  3. Teich AH (1989) In: Chelkowski J (ed) Epidemiology of wheat (Triticum aestivum L.) scab caused by Fusarium spp, in Fusarium mycotoxins, taxonomy and pathogenicity. Elsevier, Amsterdam, pp 269–282

    Google Scholar 

  4. Teich AH, Hamilton JR (1985) Effect of cultural practices, soil phosphorus, potassium, and pH on the incidence of Fusarium head blight and deoxynivalenol levels in wheat. Appl Environ Microb 49:1429–1431

    CAS  Google Scholar 

  5. Bai GH, Shaner G (1994) Scab of wheat: prospects for control. Plant Dis 78:760–766

    Google Scholar 

  6. Buerstmayr H, Lemmens M, Hartl L, Doldi L, Steiner B, Stierschneider M, Ruckenbauer P (2002) Molecular mapping of QTLs for Fusarium head blight resistance in spring wheat. I. Resistance to fungal spread (Type II resistance). Theor Appl Genet 104(1):84–91

    Article  CAS  Google Scholar 

  7. Buerstmayr H, Steiner B, Hartl L, Griesser M, Angerer N, Lengauer D, Miedaner T, Schneider B, Lemmens M (2003) Molecular mapping of QTLs for Fusarium head blight resistance in spring wheat. II. Resistance to fungal penetration and spread. Theor Appl Genet 107(3):503–508

    Article  CAS  Google Scholar 

  8. Draeger R, Gosman N, Steed A, Chandler E, Thomsett M, Srinivasachary SJ, Buerstmayr H, Lemmens M, Schmolke M, Mesterhazy A, Nicholson P (2007) Identification of QTLs for resistance to Fusarium head blight, DON accumulation and associated traits in the winter wheat variety Arina. Theor Appl Genet 115(5):617–625

    Article  CAS  Google Scholar 

  9. Miedaner T (2006) Breeding wheat and rye for resistance to Fusarium diseases. Plant Breeding 116(3):201–220

    Article  Google Scholar 

  10. Bai GH, Plattner R, Desjardins A, Kolb F (2001) Resistance to Fusarium head blight and deoxynivalenol accumulation in wheat. Plant Breed 120:1–6

    Article  CAS  Google Scholar 

  11. Liu WZ, Langseth W, Skinnes H, Elen ON, Sundheim E (1997) Comparison of visual head blight ratings—seed infection levels and deoxynivalenol production for assessment of resistance in cereals inoculated with Fusarium culmorum. Eur J Plant Pathol 103:589–595

    Article  CAS  Google Scholar 

  12. Miller JD, Young JC, Sampson DR (1985) Deoxynivalenol and Fusarium head blight resistance in spring cereals. Phytopathol Z 113(4):359–367

    CAS  Google Scholar 

  13. Mirocha CJ, Xie W, Xu Y, Wilcoxson RD, Woodward RP, Etebarian RH, Behele G (1994) Production of trichothecene mycotoxins by Fusarium graminearum and Fusarium culmorum on barley and wheat. Mycopathologia 128(1):19–23

    Article  CAS  Google Scholar 

  14. Hill NS, Neate SM, Cooper R, Horsley R, Schwarz P, Dahlen LS, Smith KP, O'Donnell K, Reeves J (2008) Comparison of ELISA for Fusarium, visual screening, and deoxinivalenol analysis of Fusarium head blight for barley field nurseries. Crop Sci 48:1389–1398

    Article  Google Scholar 

  15. Champeil A, Fourbet JF, Dore T (2004) Effects of grain sampling procedures on fusarium mycotoxin assays in wheat grains. J Agric Food Chem 52(20):6049–6054

    Article  CAS  Google Scholar 

  16. Dowell FE, Ram MS, Seitz LM (1999) Pedicting scab, vomitoxin, and ergosterol in single wheat kernels using near infrared spectroscopy. Cereal Chem 76:573–576

    Article  CAS  Google Scholar 

  17. Hill NS, Hiatt EE, Chanh TC (2006) ELISA analysis for Fusarium in barley. Crop Sci 46:2636–2642

    Article  CAS  Google Scholar 

  18. Abramson D, Gan Z, Clear RM, Gilbert J, Marquardt RR (1998) Relationships among deoxynivalenol, ergosterol and Fusarium exoantigens in Canadian hard and soft wheat. Int J Food Microbiol 45(3):217–224

    Article  CAS  Google Scholar 

  19. Muthomi JW, Oerke EC, De Wolf E, Dehne HW, Mutitu EW (2002) Susceptibility of Kenian wheat varieties to head blight, fungal invasion, and deoxynivalenol accumulation inoculated with Fusarium graminearum. J Phytopathol 150:30–36

    Article  CAS  Google Scholar 

  20. Reid LM, Nicol RW, Ouellet T, Savard M, Miller JD, Young JC, Stewart DW, Schaafsma AW (1999) Interaction of Fusarium graminearum and F. moniliforme in maize ears: disease progress, fungal biomass, and mycotoxin accumulation. Phytopathology 89(11):1028–1037

    Article  CAS  Google Scholar 

  21. Demeke T, Clear RM, Patrick SK, Gaba D (2005) Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis. Int J Food Microbiol 103(3):271–284

    Article  CAS  Google Scholar 

  22. Mach RL, Kullnig-Gradinger CM, Farnleitner AH, Reischer G, Adler A, Kubicek CP (2004) Specific detection of Fusarium langsethiae and related species by DGGE and ARMS-PCR of a beta-tubulin (tub1) gene fragment. Int J Food Microbiol 95(3):333–339

    Article  CAS  Google Scholar 

  23. Nicholson P, Sampson DR, Weston G, Rezanoor HN, Lees AK, Parry DW, Joyce D (1998) Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiol Mol Plant Pathol 53:17–37

    Article  CAS  Google Scholar 

  24. Yli-Mattila T, Paavanen-Huhtala S, Jestoi M, Parikka P, Hietaniemi V, Gagkaeva T, Sarlin T, Haikara A, Laaksonen S, Rizzo A (2008) Real-time PCR detection and quantification of Fusarium poae, F. graminearum, F. sporotrichioides and F. langsethiae in cereal grains in Finland and Russia. Arch Phoytopathol Plant Protection 41(4):243–260

    Article  CAS  Google Scholar 

  25. Bluhm BH, Flaherty JE, Cousin MA, Woloshuk CP (2002) Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisin-producing species of Fusarium in cornmeal. J Food Prot 65(12):1955–1961

    CAS  Google Scholar 

  26. Mirete S, Vazquez C, Mulè G, Jurande M, Gonzales-Jaen MT (2004) Differentiation of Fusarium verticillioides from banana fruits by IGS and EF-1α sequence analyses. Eur J Plant Pathol 110:515–523

    Article  CAS  Google Scholar 

  27. Mulfinger S, Niessen L, Vogel R (2000) PCR based quality control of toxigenic Fusarium spp. in brewing malt using ultrasonication for rapid sample preparation. Adv Food Sci 22(1/2):38–46

    CAS  Google Scholar 

  28. Ward TJ, Clear RM, Rooney AP, O'Donnell K, Gaba D, Patrick S, Starkey DE, Gilbert J, Geiser DM, Nowicki TW (2008) An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America. Fungal Genet Biol 45(4):473–484

    Article  Google Scholar 

  29. Reischer GH, Lemmens M, Farnleitner A, Adler A, Mach RL (2004) Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe. J Microbiol Methods 59(1):141–146

    Article  CAS  Google Scholar 

  30. Strausbaugh CA, Overturf K, Koehn AC (2005) Pathogenicity and real-time PCR detection of Fusarium spp. in wheat and barley roots. Can J Plant Pathol 27:430–438

    CAS  Google Scholar 

  31. Waalwijk C, van der Heide R, de Vries I, van der Lee T, Schoen C, Corainville GC, Häusler-Hahn I, Kastelein P, Köhl J, Lonnet P, Demarquet T, Kema GHJ (2004) Quantitative detection of Fusarium in wheat using TaqMan. Eur J Plant Pathol 110:481–494

    Article  CAS  Google Scholar 

  32. Schnerr H, Niessen L, Vogel RF (2001) Real time detection of the tri5 gene in Fusarium species by lightcycler-PCR using SYBR Green I for continuous fluorescence monitoring. Int J Food Microbiol 71(1):53–61

    Article  CAS  Google Scholar 

  33. Waalwijk C, Koch SH, Ncube E, Allwood J, Flett B, de Vries I, Kema GHJ (2008) Quantitative detection of Fusarium spp. and its correlation with fumonisin content in maize from South African subsistence farmers. World Mycotox 1(1):39–47

    Article  CAS  Google Scholar 

  34. Fredlund E, Gidlund A, Olsen M, Borjesson T, Spliid NH, Simonsson M (2008) Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels. J Microbiol Methods 73(1):33–40

    Article  CAS  Google Scholar 

  35. Petersen G, Seberg O, Yde M, Berthelsen K (2006) Phylogenetic relationships of Triticum and Aegilops and evidence for the origin of the A, B, and D genomes of common wheat (Triticum aestivum). Mol Phylogenet Evol 39(1):70–82

    Article  CAS  Google Scholar 

  36. Buerstmayr H, Steiner B, Lemmens M, Ruckenbauer P (2000) Resistance to Fusarium head blight in winter wheat: heritability and trait associations. Crop Science 40:1012–1018

    Article  Google Scholar 

  37. Berthiller F, Dall'Asta C, Schuhmacher R, Lemmens M, Adam G, Krska R (2005) Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry. J Agric Food Chem 53(9):3421–3425

    Article  CAS  Google Scholar 

  38. Joint Research Centre CRLfGFaF (2007) Maize seed sampling and DNA extraction. Document CRLVL04/05XP.

  39. Peterbauer CK, Lorito M, Hayes CK, Harman GE, Kubicek CP (1996) Molecular cloning and expression of the nag1 gene (N-acetyl-beta-d-glucosaminidase-encoding gene) from Trichoderma harzianum P1. Curr Genet 30(4):325–331

    Article  CAS  Google Scholar 

  40. Schnerr H, Vogel RF, Niessen L (2002) Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat-samples. Lett Appl Microbiol 35(2):121–125

    Article  CAS  Google Scholar 

  41. Lemmens M, Scholz U, Berthiller F, Dall'Asta C, Koutnik A, Schuhmacher R, Adam G, Buerstmayr H, Mesterhazy A, Krska R, Ruckenbauer P (2005) The ability to detoxify the mycotoxin deoxynivalenol colocalizes with a major quantitative trait locus for Fusarium head blight resistance in wheat. Mol Plant Microbe Interact 18(12):1318–1324

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was financed by the Austrian Ministry of Agriculture (Lebensministerium), Project 100053. We also thank the Lower Austrian Government for financial support of the mycotoxin analyses.

Author information

Authors and Affiliations

  1. Institute of Chemical Engineering, Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology, Getreidemarkt 9, 1060, Vienna, Austria

    Kurt Brunner, Maria P. Kovalsky Paris, Guadalupe Paolino & Robert L. Mach

  2. Department for Agrobiotechnology IFA-Tulln, Institute of Biotechnology in Plant Production, University of Natural Resources and Applied Life Sciences, Vienna, Austria

    Hermann Bürstmayr & Marc Lemmens

  3. Department for Agrobiotechnology IFA-Tulln, Institute of Analytical Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria

    Franz Berthiller, Rainer Schuhmacher & Rudolf Krska

Authors
  1. Kurt Brunner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria P. Kovalsky Paris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Guadalupe Paolino
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hermann Bürstmayr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marc Lemmens
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Franz Berthiller
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rainer Schuhmacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rudolf Krska
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Robert L. Mach
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kurt Brunner.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brunner, K., Kovalsky Paris, M.P., Paolino, G. et al. A reference-gene-based quantitative PCR method as a tool to determine Fusarium resistance in wheat. Anal Bioanal Chem 395, 1385–1394 (2009). https://doi.org/10.1007/s00216-009-3083-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-009-3083-3

Keywords

Search

Navigation