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Real-Time PCR for Specific Detection of Three Phytoplasmas from the Apple Proliferation Group

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Phytoplasma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 938))

Abstract

In this chapter, we describe a real-time PCR detection system for fast, reliable, specific, and sensitive detection and discrimination of ‘Candidatus Phytoplasma mali’, ‘Ca. P. prunorum’, and ‘Ca. P. pyri’ from the 16SrX (apple proliferation-AP) group. These phytoplasmas are causal agents of fruit tree diseases within the Rosaceae family, namely apple proliferation, European stone fruit yellows, and pear decline. The assays use (hydrolysis) TaqMan® minor groove binder probes. The panel of assays comprises the same set of primers and specific probes for species-specific amplification, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The assays described can be used in routine phytoplasma surveys and in certification programmes.

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References

  1. Seemüller E, Schneider B (2004) ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, the causal agents of apple proliferation, pear decline and European stone fruit yellows, respectively. Int J Syst Evol Microbiol 54:1217–1226

    Article  PubMed  Google Scholar 

  2. EPPO (2011) EPPO A2 List of pests recommended for regulation as quarantine pests. http://www.eppo.org/QUARANTINE/listA2.html. Last Accessed 10 June 2011

  3. Torres E et al (2005) Real-time PCR for simultaneous and quantitative detection of quarantine phytoplasmas from apple proliferation (16SrX) group. Mol Cell Probes 19:334–340

    Article  PubMed  CAS  Google Scholar 

  4. Jarausch W et al (2004) Establishment of a quantitative real-time PCR assay for the quantification of apple proliferation phytoplasmas in plants and insects. Acta Hortic 657:415–420

    CAS  Google Scholar 

  5. Galetto L, Bosco D, Marzachì C (2005) Universal and group-specific real-time PCR diagnosis of flavescence doreé (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors. Ann Appl Biol 147:191–201

    Article  CAS  Google Scholar 

  6. Yvon M et al (2009) Specific detection and quantification of the phytopathogenic agent ‘Candidatus Phytoplasma prunorum’. Mol Cell Probes 23:227–234

    Article  PubMed  CAS  Google Scholar 

  7. Pignatta D et al (2008) A real-time PCR assay for the detection of European stone fruit yellows phytoplasma (ESFYP) in plant propagation material. Acta Hortic 781:499–504

    CAS  Google Scholar 

  8. Babini AR et al (2008) Investigations with real-time PCR assay on the transmissibility of pear decline phytoplasma (PDP) with dormant buds. Acta Hortic 781:495–498

    CAS  Google Scholar 

  9. Baric S, Dalla VJ (2004) A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. J Microbiol Meth 57:135–145

    Article  CAS  Google Scholar 

  10. Kutyavin IV et al (2000) 3′-Minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucl Acids Res 28:655–661

    Article  PubMed  CAS  Google Scholar 

  11. Yao Y, Nellåker C, Karlsson H (2006) Evaluation of minor groove binding probe and Taqman probe PCR assays: Influence of mismatches and template complexity on quantification. Mol Cell Probes 20:311–316

    Article  PubMed  CAS  Google Scholar 

  12. Aldaghi M et al (2007) Development of a new probe for specific and sensitive detection of ‘Candidatus Phytoplasma mali’ in inoculated apple trees. Ann Appl Biol 151:251–258

    Article  CAS  Google Scholar 

  13. Nikolić P et al (2010) A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group. Mol Cell Probes 24:303–309

    Article  PubMed  CAS  Google Scholar 

  14. Weller SA et al (2000) Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Appl Environ Microbiol 66:2853–2858

    Article  PubMed  CAS  Google Scholar 

  15. Bustin SA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622

    Article  PubMed  CAS  Google Scholar 

  16. Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954–962

    PubMed  CAS  Google Scholar 

  17. Ellison SLR et al (2006) Routes to improving the reliability of low level DNA analysis using real-time PCR. BMC Biotechnol 6:33

    Article  PubMed  Google Scholar 

  18. Mehle N, Nikolić P, Rupar M, Boben J, Ravnikar M, Dermastia M (2012) Automated DNA extraction for large numbers of plant samples. In: Dickinson M, Hodgetts J (eds) Methods in molecular biology. Springer, New York

    Google Scholar 

  19. Lee I-M et al (1995) Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology 85:728–735

    Article  CAS  Google Scholar 

  20. Del Serrone P et al (1998) Occurrence of apple proliferation and pear decline phytoplasmas in diseased pear trees in Hungary. J Plant Pathol 80:53–58

    Google Scholar 

  21. Mehle N et al (2007) First report of ‘Candidatus Phytoplasma mali’ in Prunus avium, P. armeniaca and P. domestica. Plant Pathol 56:721

    Article  Google Scholar 

  22. Christensen NM, Nyskjold H, Nicolaisen M (2012) Real-time PCR for universal phytoplasma detection and quantification. In: Dickinson M, Hodgetts J (eds) Methods in molecular biology. Springer, New York

    Google Scholar 

  23. Hren M et al (2007) Real-time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics. Plant Pathol 56:785–796

    Article  CAS  Google Scholar 

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Correspondence to Nataša Mehle .

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Mehle, N., Nikolić, P., Gruden, K., Ravnikar, M., Dermastia, M. (2013). Real-Time PCR for Specific Detection of Three Phytoplasmas from the Apple Proliferation Group. In: Dickinson, M., Hodgetts, J. (eds) Phytoplasma. Methods in Molecular Biology, vol 938. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-089-2_23

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  • DOI: https://doi.org/10.1007/978-1-62703-089-2_23

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-088-5

  • Online ISBN: 978-1-62703-089-2

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