Skip to main content

A Real-Time Multiplex PCR Assay Used in the Identification of Closely Related Fungal Pathogens at the Species Level

  • Protocol
Plant Pathology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1302))

Abstract

The advent of real-time PCR and new chemistries such as TaqMan™ and SYBR™green has been used in plant pathology to aid in the identification of fungal, bacterial and viral pathogens. These chemistries have provided another tool to be used in the identification of fungal pathogens that are hard to differentiate on the basis of morphology. This work describes an assay that was developed to identify five different species of the pathogen Tilletia that causes smuts and bunts in cereals.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Wiese MV (ed) (1987) Compendium of wheat diseases, 2nd edn. APS Press, St Paul, USA, 112 pp

    Google Scholar 

  2. USDA (2007) Karnal bunt manual. United States Department of Agriculture, Frederick, MD. 160 pp. http://www.aphis.usda.gov/import_export/plants/manuals/online_manuals.shtml. Accessed Feb 2012

  3. Fuentes-Davila G (1996) Karnal bunt. In: Wilcoxson RD, Saari EE (eds) Bunt and smut diseases of wheat: concepts and methods of disease management. CIMMYT, Mexico, DF, pp 26–32, 74 pp

    Google Scholar 

  4. Crous PW, van Jaarsveld AB, Castlebury LA, Carris LM, Frederick RD, Pretorius ZA (2001) Karnal bunt of wheat newly reported from the African continent. Plant Dis 85:561

    Article  Google Scholar 

  5. Sansford CE, Baker RHA, Brennan JP, Ewert F, Gioli B, Inman AJ, Kinsella A, Magnus H, Miglietta F, Murray GM, Porta-Puglia A, Porter JR, Rafoss T, Riccioni L, Thorne F (2008) The new pest risk analysis for Tilletia indica, the cause of Karnal bunt of wheat, continues to support the quarantine status of the pathogen in Europe. Plant Pathol 57:603–611

    Article  Google Scholar 

  6. Inman AJ, Hughes KJD, Bowyer R (2003) Protocol for extracting teliospores from untreated seed or grain by size-selective sieving. In: EU recommended protocol for the diagnosis of a quarantine organism: Tilletia indica. UK Department for Environment, Food and Rural Affairs, DIAGPRO (EU Project on Diagnostic Protocols), pp 21–26, 38 pp. www.fera.defra.gov.uk/plants/planthealth/pestsdiseases/documents/protocols/tipro.pdf

  7. Castlebury LA (1998) Morphological characterisation of Tilletia indica and similar fungi. In: Malik VS, Mathre DE (eds) Bunts and smuts of wheat: an international symposium. North American Plant Protection Organization, Ottawa, pp 97–105, 445 + xv pp

    Google Scholar 

  8. Castlebury LA, Carris LM (1999) Tilletia walkeri, a new species on Lolium multiflorum and L. perenne. Mycologia 91:121–131

    Article  Google Scholar 

  9. Pascoe IG, Priest MJ, Shivas RG, Cunnington JH (2005) Ustilospores of Tilletia ehrhartae, a smut of Ehrharta calycina, are common contaminants of Australian wheat grain, and a potential source of confusion with Tilletia indica, the cause of Karnal bunt of wheat. Plant Pathol 54:161–168

    Article  Google Scholar 

  10. Okubura PA, Schroeder KL, Paulitz TC (2005) Real-time polymerase chain reaction: applications to studies on soilborne pathogens. Can J Plant Pathol 27:300–313

    Article  Google Scholar 

  11. BIO-RAD (2006) Real-time PCR applications guide Bulletin 5279. http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5279.pdf. Accessed 22 May 2014

  12. Strategene (2014) Introduction to quantitative PCR methods and application guide. http://www.strategene.com. Accessed 22 May 2014

  13. Agilent Technologies (2102) Introduction to quantitative PCR. www.agilent.com/genomics/pcr. Accessed 30 May 2014

  14. Mayer L (ed) (2011) Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. http://www.cdc.gov/meningitis/lab-manual/chpt10-pcr.html. Accessed 22 May 2014

  15. Tan M-K, Ghalayini A, Sharma I, Yi J, Shivas R, Priest M, Wright D (2009) A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat. Australas Plant Pathol 38:101–109

    Article  Google Scholar 

  16. Levy L, Meyer RJ, Carris L, Peterson G, Tschanz AT (1998) Differentiation of Tilletia indica from the undescribed Tilletia species on ryegrass by ITS sequence differences. In: Proceedings of the XI Biennial workshop on smut fungi, p 29

    Google Scholar 

  17. Pimentel G, Carris LM, Levy L, Meyer R (1998) Genetic variability among isolates of Tilletia barclayana, T. indica and allied species. Mycologia 90:1017–1027

    Article  Google Scholar 

  18. Levy L, Castlebury LA, Carris LM, Meyer RJ, Pimentel G (2001) Internal transcribed spacer sequence-based phylogeny and polymerase chain reaction-restriction fragment length polymorphism differentiation of Tilletia walkeri and T. indica. Phytopathology 91:935–940

    Article  CAS  PubMed  Google Scholar 

  19. Tan M-K, Murray GM (2006) A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia indica, the causal agent of Karnal bunt of wheat. Mycol Res 110:203–210

    Article  CAS  PubMed  Google Scholar 

  20. Qiagen (2013) PCR protocols and applications. http://www.qiagen.com/resources/molecular-biology-methods/pcr/?Print=1#Real-time%20PCR. Accessed 23 May 2014

  21. Thompson JD, Higgins DG, Gibson TI (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Peterson GL, Bonde MR, Phillips JG (2000) Size-selective sieving for detecting teliospores of Tilletia indica in wheat seed samples. Plant Dis 84:999–1007

    Article  CAS  Google Scholar 

  23. Tan M-K, Wright DG (2009) Enhancing the detection of Tilletia indica, the cause of Karnal bunt. Final report. CRC20004: Karnal bunt detection. CRC National Plant Biosecurity, Canberra, 63 pp

    Google Scholar 

  24. Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. BioTechniques 39:1–11

    Article  Google Scholar 

  25. Wright DG, Murray GM, Tan M-K (2012) National diagnostic protocol for the identification of Tilletia indica, the cause of Karnal bunt. Department of Agriculture and Food, Western Australia

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dominie G. Wright .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Wright, D.G. (2015). A Real-Time Multiplex PCR Assay Used in the Identification of Closely Related Fungal Pathogens at the Species Level. In: Lacomme, C. (eds) Plant Pathology. Methods in Molecular Biology, vol 1302. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2620-6_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2620-6_4

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2619-0

  • Online ISBN: 978-1-4939-2620-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics