Phytoplasma pp 283-289 | Cite as

Reverse Transcription-PCR for Phytoplasma Detection Utilizing Crude Sap Extractions

  • Paolo Margaria
  • Sabrina PalmanoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 938)


Phytoplasmas are routinely detected by nucleic acid-based techniques. These approaches rely on enriched phytoplasma DNA extracts of good quality, following labor intensive and time-consuming purification protocols. Here we describe a very rapid, specific, sensitive, and reliable method for flavescence dorée phytoplasma detection, based on real-time Taqman® reverse transcription-PCR of the 16S rRNA. The protocol is particularly useful for large-scale screening of vineyards and nurseries, pathogen surveys, and field epidemiological studies.

Key words

Crude sap Grapevine Real-time TaqMan® reverse transcription-PCR Ribosomal RNA 



P. M. was supported by the grant “Studi sui fattori che favoriscono le epidemie di flavescenza dorata in Piemonte e loro superamento,” sub-project C, from Regione Piemonte, Italy (Report number 12851).


  1. 1.
    Bertaccini A (2007) Phytoplasmas: diversity, taxonomy, and epidemiology. Front Biosci 12:673–689PubMedCrossRefGoogle Scholar
  2. 2.
    Firrao G, Garcia-Chapa M, Marzachì C (2007) Phytoplasmas: genetics, diagnosis and relationships with the plant and insect host. Front Biosci 12:1353–1375PubMedCrossRefGoogle Scholar
  3. 3.
    Lee I-M et al (1994) Use of mycoplasmalike organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathol 84:559–566CrossRefGoogle Scholar
  4. 4.
    Gundersen DE, Lee I-M (1996) Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol Mediterr 35:144–151Google Scholar
  5. 5.
    Clair D et al (2003) A multiplex nested-PCR assay for sensitive and simultaneous detection and direct identification of phytoplasma in the Elm yellows group and Stolbur group and its use in survey of grapevine yellows in France. Vitis 42:151–157Google Scholar
  6. 6.
    Galetto L, Marzachì C (2010) Real-time PCR diagnosis and quantification of phytoplasmas. In: Weintraub PG, Jones P (eds) Phytoplasmas: genomes, plant hosts and vectors. CAB International, Cambridge, pp 1–18Google Scholar
  7. 7.
    Schneider B, Seemüller E (1994) Presence of two sets of ribosomal genes in phytopathogenic mollicutes. Appl Environ Microbiol 60:3409–3412PubMedGoogle Scholar
  8. 8.
    Margaria P et al (2007) Detection of Flavescence doreé phytoplasma in grapevine by reverse-transcription PCR. Plant Dis 91:1495–1501CrossRefGoogle Scholar
  9. 9.
    Margaria P, Turina M, Palmano S (2009) Detection of Flavescence dorée and Bois noir phytoplasmas, Grapevine leafroll associated virus-1 and -3 and Grapevine virus A from the same crude extract by reverse transcription-RealTime Taqman assays. Plant Pathol 58:838–845CrossRefGoogle Scholar
  10. 10.
    The IRPCM Phytoplasma/Spiroplasma Working Team—Phytoplasma taxonomy group (2004) ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int J Syst Evol Microbiol 54:1243–1255CrossRefGoogle Scholar
  11. 11.
    Osman F, Rowhani A (2006) Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan). J Virol Methods 133:130–136PubMedCrossRefGoogle Scholar
  12. 12.
    Osman F et al (2007) Real-time RT-PCR (TaqMan®) assays for the detection of Grapevine Leafroll associated viruses 1–5 and 9. J Virol Methods 141:22–29PubMedCrossRefGoogle Scholar
  13. 13.
    Osman F, Rowhani A (2008) Real-time RT-PCR (TaqMan®) assays for the detection of viruses associated with Rugose wood complex of grapevine. J Virol Methods 154:69–75PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Istituto di Virologia Vegetale, CNRTorinoItaly

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