European Journal of Plant Pathology

, Volume 110, Issue 5–6, pp 481–494 | Cite as

Quantitative Detection of Fusarium Species in Wheat Using TaqMan

  • Cees Waalwijk
  • Ruth van der Heide
  • Ineke de Vries
  • Theo van der Lee
  • Cor Schoen
  • Guillaume Costrel-de Corainville
  • Isolde Häuser-Hahn
  • Pieter Kastelein
  • Jürgen Köhl
  • Philippe Lonnet
  • Thierry Demarquet
  • Gert H.J. Kema
Article

Abstract

Fusarium head blight (FHB) of wheat and other small-grain cereals is a disease complex caused by several fungal species. To monitor and quantify the major species in the FHB complex during the growing season, real-time PCR was developed. TaqMan primers and probes were designed that showed high specificity for Fusarium avenaceum, F. culmorum, F. graminearum, F. poae and Microdochium nivale var. majus. Inclusion of an internal PCR control and serial dilutions of pure genomic DNAs allowed accurate determination of the concentration of fungal DNA for each of these species in leaves, ears as well as harvested grains of winter wheat. The DNA concentration of F. graminearum in grain samples correlated (r2= 0.7917) with the incidence of this species on the grain as determined by isolation from individual kernels. Application of the TaqMan technology to field samples collected in 40 wheat crops in the Netherlands during the growing season of 2001 revealed that M. nivale var. majus predominated on leaves early in the season (GS 45-65). Ears and harvested grains from the same fields, however, showed F. graminearum as the major species. In 2002, grain samples from 40 Dutch fields showed a much wider range of species, whereas in ears from 29 wheat crops in France, F. graminearum was the predominant species. The concentration of DON correlated equally well with the incidence of the DON-producing species F. culmorum and F. graminearum in the grain samples (r2= 0.8232) as well as with total DNA of both these species (r2= 0.8259). The Fusarium TaqMan technology is an important tool to quantify and monitor the dynamics of individual species of the complex causing FHB in cereals during the growing season. This versatile tool has been applied in a comparison of different genotypes, but can also be applied to other disease management systems, e.g. fungicide treatments.

Fusarium head blight (FHB) Gibberella toxin 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Afonina IA, Reed MW, Lusby E, Shishkina IG and Belousov YS (2002) Minor Groove Binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence. BioTechniques 32: 940-949PubMedGoogle Scholar
  2. Bai G and Shaner G (1994) Scab of wheat: Prospects for control. Plant Disease 78: 760-766Google Scholar
  3. Bottalico A and Perrone G (2002) Toxigenic Fusarium species and mycotoxins associated with head blight in small-grain cereals in Europe. European Journal of Plant Pathology 108: 611-624CrossRefGoogle Scholar
  4. Brennan JM, Fagan B, van Maanen A, Cooke BM and Doohan FM (2003) Studies on in vitro growth and pathogenicity of European Fusarium fungi. European Journal of Plant Pathology 109: 577-587CrossRefGoogle Scholar
  5. Brown DW, Desjardins AE, Yun SH, Plattner R, Lee T, Dyer R and Turgeon BG (2001) Are Gibberella zeae sexual spores the critical inoculum to FHB epidemics? 2001 National Fusarium Head Blight Forum Proceedings, (p. 104) Cincinnati, USAGoogle Scholar
  6. Cotton TK and Munkvold GP (1998) Survival of Fusarium moniliforme, F. proliferatum, and F. subglutinans in maize stalk residue. Phytopathology 88: 550-555Google Scholar
  7. CWSCG (1984) Fusarium species, distribution and pathogenic-ity from scabby heads in China. Journal of Shanghai Normal College 3: 69-82Google Scholar
  8. Dill-Macky R and Jones RK (2000) The effect of previous crop residues and tillage on Fusarium head blight of wheat. Plant Disease 84: 71-76Google Scholar
  9. Doohan FM, Parry DW and Nicholson P (1999) Fusarium ear blight of wheat: The use of quantitative PCR and visual disease assessment in studies of disease control. Plant Pathology 48: 209-217CrossRefGoogle Scholar
  10. Edwards SG, Pirgozliev SR, Hare MC and Jenkinson P (2001) Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium Head Blight of winter wheat. Applied and Environmental Microbiology 67: 1575-1580CrossRefPubMedGoogle Scholar
  11. Fernando WGD, Paulitz TC, Seaman WL, Dutilleul P and Miller J (1997) Head blight gradients caused by Gibberella zeae from area sources of inoculum in wheat fields plots. Phytopathology 87: 414-421Google Scholar
  12. Gruber F, Falkner FG, Dorner F and Hämmerle T (2001) Quantification of viral DNA by real-time PCR applying duplex amplification. Internal standardization, and two-color fluorescence detection. Applied and Environmental Microbiology 67: 2837-2839CrossRefPubMedGoogle Scholar
  13. Inch S. and J Gilbert (2002). Survival of Gibberella zeae in Fusarium-damaged wheat kernels. Plant Disease 87: 282-287Google Scholar
  14. Kema GHJ, Goodwin SB, Hamza S, Verstappen ECP, Cavaletto JR, Van der Lee TAJ, de Weerdt M, Bonants PJM and Waalwijk C (2002) A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic linkage map of Mycosphaerella graminicola, the Septoria tritici leaf blotch pathogen of wheat. Genetics 161: 1497-1505PubMedGoogle Scholar
  15. Maldonado-Ramirez SL and Bergstrom GC (2000) Temporal patterns of ascospore discharge by Gibberella zeae from colonized corn stalks under natural conditions. 2001 National Fusarium Head Blight Forum Proceedings, (pp. 159-162) Cincinnati, USAGoogle Scholar
  16. Mulfinger S, Niessen L and Vogel RF (2000) PCR based quality control of toxigenic Fusarium spp. ?.Google Scholar
  17. Nganje WE, Bangsund DA, Leistritz FL, Wilson WW and Tiapo N (2001) National Fusarium Head Blight Forum Proceedings, (pp. 275-281) Cincinnati, USAGoogle Scholar
  18. Nicholson P, Lees AK, Maurin N, Parry DW and Rezanoor HN (1996) Development of a PCR assay to identify and quantify Microdochium nivale var. nivale and Microdochium nivale var. majus in wheat. Physiological and Molecular Plant Pathology 48: 257-271CrossRefGoogle Scholar
  19. Nicholson P, Simpson DR, Weston G, Rezanoor HN, Lees AK, Parry DW and Joyce D (1998) Detection and quantification of Fusarium culmorum and Fusarium graminearum in cereals using PCR assays. Physiological and Molecular Plant Pathology 53: 17-37CrossRefGoogle Scholar
  20. Obst A, Günther B, Beck R, Lepschy J and Tischner H (2002) Weather conditions conducive to Gibberella zeae and Fusarium graminearum head blight of wheat. Journal of Applied Genetics 43A: 185-192Google Scholar
  21. Oerke E-C, Meier A, Kienemann K, Meyer G, Muthomi J, Schade-Schütze A, Steiner U and Dehne H-W (2002) Incidence and control of Fusarium species causing head blight in the Rhineland, Germany. In: Fusarium-Befall und Mykotoxinbelasting von Getreide. Tagungsband der 13. Wissenschaftlichen Fachtagung der Landwirtschaftlichen Fakultät der Universität Bonn, pp. 32–44Google Scholar
  22. O'Donnell K, Kistler HC, Tacke BK and Casper HH (2000) Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proceeding of the National Academy of Sciences USA 97: 7905-7910Google Scholar
  23. Parry DW, Jenkinson P and MacLeod L (1995) Fusarium ear blight (scab) in small grain cereals-a review. Plant Pathology 44: 207-238Google Scholar
  24. Parry DW, Pettitt TR, Jenkinson P and Lees AK (1994) The cereal Fusarium complex. In: Blakeman JP and Williamson B (eds) Ecology of Plant Pathogens (pp. 301–320) CABI, WallingfordGoogle Scholar
  25. Schnerr H, Niessen L and Vogel RF (2001) Real time detection of the tri5 gene in Fusarium species by lightcycler-PCR using SYBR Green I for continuous fluorescence monitoring. International Journal of Food Microbiology 71: 53-61CrossRefPubMedGoogle Scholar
  26. Schnerr H, Vogel RF and Niessen L (2002) Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat samples. Letters of Applied Microbiology 35: 121-125Google Scholar
  27. van Eeuwijk FA, Mesterhazy A, Kling CI, Ruckenbauer P, Saur L, Buerstmayr H, Lemmens M, Keizer LCP, Maurin N and Snijders CHA (1995). Assessing non-specificity of resistance in wheat to head blight caused by inoculation with European strains of Fusarium culmorum, F. graminearum and F. nivale using a multiplicative model for interaction. Theoretical and Applied Genetics 90: 221-228Google Scholar
  28. Waalwijk C, Kastelein P, de Vries PhM, Kerényi Z, van der Lee TAJ, Hesselink T, Köhl J and Kema GHJ (2003) Major changes in Fusarium spp. in wheat in The Netherlands. European Journal of Plant Pathology 109: 743-754CrossRefGoogle Scholar
  29. Wang YZ (1997) Epidemiology and management of wheat scab in China. In: Dubin HJ, Gilchrist L, Reeves J and McNab A (eds) Fusarium Head Scab: Global Status and Future Prospects. (pp. 97–105) CYMMIT, MexicoGoogle Scholar
  30. Whitehead Institute (2003) The initial release of our high quality draft sequence of the Fusarium graminearum genome (http: //www.genome.wi.mit.edu/annotation/fungi/fusarium/whatsnew.html)Google Scholar
  31. Windels CE (2000) Economic and social impacts of Fusarium head blight: Changing farms and rural communities in the Northern great plains. Phytopathology 90: 17-21Google Scholar
  32. Winton LM, Stone JK, Watrud LS and Hansen EM (2002) Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction. Phytopathology 92: 112-116Google Scholar
  33. Zadoks JC, Chang TT and Konzak CF (1974) A decimal code for the growth stages of cereals. Weed Research 14: 415-421Google Scholar
  34. Zeller KA, Bowden RL and Leslie JF (2003) Diversity of epidemic populations of Gibberella zeae from small quadrats in Kansas and North Dakota. Phytopathology 93: 874-880Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Cees Waalwijk
    • 1
  • Ruth van der Heide
    • 1
  • Ineke de Vries
    • 1
  • Theo van der Lee
    • 1
  • Cor Schoen
    • 1
  • Guillaume Costrel-de Corainville
    • 2
  • Isolde Häuser-Hahn
    • 2
  • Pieter Kastelein
    • 1
  • Jürgen Köhl
    • 1
  • Philippe Lonnet
    • 3
  • Thierry Demarquet
    • 3
  • Gert H.J. Kema
    • 1
  1. 1.Plant Research International BV, Droevendaalsesteeg 1WageningenThe Netherlands
  2. 2.Global Biology FungicidesBayer CropScience AG ResearchMonheimGermany
  3. 3.Florimond DesprezCapelle-en-PévèleFrance

Personalised recommendations