Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir



Bois noir is a grapevine yellows disease that is gaining importance in many regions of Europe. The Bois noir phytoplasma (“Candidatus Phytoplasma solani”) was shown to be transmitted by the planthopper Hyalesthes obsoletus, which normally feeds on herbaceous weeds, and occasionally also on grapevines. Three subtypes of the Bois noir phytoplasma have been described and were shown to be associated with distinctive host plants. In this study, we developed a novel and rapid real-time PCR allelic discrimination assay for the distinction of the two major Bois noir phytoplasma subtypes, VK type I and II. Two TaqMan probes carrying different fluorescent dyes were designed to specifically bind to a polymorphism characteristic for the two Bois noir phytoplasma subtypes, thereby allowing discriminative amplification in a single-tube and single-step assay. A total of 259 bois noir-positive grapevine samples collected over 5 years were analysed using the conventional PCR-RFLP method and our newly developed TaqMan allelic discrimination assay. 257 out of 259 samples could be typed with the TaqMan method, compared to 200 out of 259 samples when using the conventional method. The overall concordance of the two methods was 100%. Our newly developed TaqMan assay represents a useful tool for fast and reliable determination of Bois noir phytoplasma subtypes in infected grapevine, insect vector, and host plant samples. The test is suitable for high-throughput analysis and will thereby facilitate further characterisation of Bois noir epidemiology.


Candidatus Phytoplasma solani” Real-time PCR Vergilbungskrankheit Vitis vinifera 



Bois noir


threshold cycle

delta Rn

relative fluorescence intensity


Flavescence dorée


minor groove binder


restriction fragment length polymorphism


single nucleotide polymorphism


elongation factor TU





We thank M. Maixner for providing DNA extracts of the different VK types, K. Mair and A. Morandell for supplying grapevine samples and C. Kerschbamer for technical assistance. This study was funded by the Autonomous Province of Bozen/Bolzano, Italy.


  1. Angelini, E., Bianchi, G. L., Filippin, L., Morassutti, C., & Borgo, M. (2007). A new TaqMan method for the identification of phytoplasmas associated with grapevine yellows by real-time PCR assay. Journal of Microbiological Methods, 68, 613–622.PubMedCrossRefGoogle Scholar
  2. Baric, S., & Dalla Via, J. (2004). A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. Journal of Microbiological Methods, 57, 135–145.PubMedCrossRefGoogle Scholar
  3. Baric, S., & Dalla Via, J. (2007). Temporal shifts of Bois Noir phytoplasma types infecting grapevine in South Tyrol (Northern Italy). Vitis, 46, 101–102.Google Scholar
  4. Baric, S., Kerschbamer, C., & Dalla Via, J. (2006). TaqMan real-time PCR versus four conventional PCR assays for detection of apple proliferation phytoplasma. Plant Molecular Biology Reporter, 24, 169–184.CrossRefGoogle Scholar
  5. Boudon-Padieu, E. (2002). Flavescence dorée of the grapevine: knowledge and new developments in epidemiology, etiology and diagnosis. ATTI Giornate Fitopatologiche, 1, 15–34.Google Scholar
  6. Boudon-Padieu, E., Béjat, A., Clair, D., Larrue, J., Borgo, M., Bertotto, L., et al. (2003). Grapevine yellows: Comparison of different procedures for DNA extraction and amplification with PCR for routine diagnosis of phytoplasmas in grapevine. Vitis, 42, 141–149.Google Scholar
  7. Caudwell, A. (1990). Epidemiology and characterization of flavescence dorée (FD) and other grapevine yellows. Agronomie, 10, 655–663.CrossRefGoogle Scholar
  8. Galetto, L., Bosco, D., & Marzachi, C. (2005). Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors. Annals of Applied Biology, 147, 191–201.CrossRefGoogle Scholar
  9. Langer, M., & Maixner, M. (2004). Molecular characterisation of grapevine yellows associated phytoplasmas of the stolbur-group based on RFLP-analysis of non-ribosomal DNA. Vitis, 43, 191–199.Google Scholar
  10. Lee, I. M., Gundersen-Rindal, D. E., Davis, R. E., & Bartoszyk, I. M. (1998). Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic and Evolutionary Microbiology, 48, 1153–1169.Google Scholar
  11. Maixner, M. (1994). Transmission of German grapevine yellows (Vergilbungskrankheit) by the planthopper Hyalesthes obsoletus (Auchenorrhyncha: Cixiidae). Vitis, 33, 103–104.Google Scholar
  12. Maixner, M., Ahrens, U., & Seemüller, E. (1995). Detection of the German grapevine yellows (Vergilbungskrankheit) MLO in grapevine, alternative hosts and a vector by a specific PCR procedure. European Journal of Plant Pathology, 101, 241–250.CrossRefGoogle Scholar
  13. Mamotte, C. (2006). Genotyping of single nucleotide substitutions. Clinical Biochemist Reviews, 27, 63–75.Google Scholar
  14. Pasquini, G., Ferretti, L., Gentili, A., Bagnoli, B., Cavalieri, V., & Barba, M. (2007). Molecular characterization of stolbur isolates collected in grapevines, weeds and insects in central and southern Italy. Bulletin of Insectology, 60, 355–356.Google Scholar
  15. Riolo, P., Landi, L., Nardi, S., & Isidoro, N. (2007). Relationships among Hyalesthes obsoletus, its herbaceous host plants and “bois noir” phytoplasma strains in vineyard ecosystems in the Marche region (central-eastern Italy). Bulletin of Insectology, 60, 353–354.Google Scholar
  16. Schneider, B., Gibb, K. S., & Seemüller, E. (1997). Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas. Microbiology, 143, 3381–3389.PubMedCrossRefGoogle Scholar
  17. Sforza, R., Clair, D., Daire, X., Larrue, J., & Boudon-Padieu, E. (1998). The role of Hyalesthes obsoletus (Hemiptera: Cixiidae) in the occurrence of bois noir of grapevines in France. Journal of Phytopathology, 146, 549–556.CrossRefGoogle Scholar

Copyright information

© KNPV 2008

Authors and Affiliations

  1. 1.Research Centre for Agriculture and Forestry LaimburgAuer/Ora (BZ)Italy

Personalised recommendations