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Field Assessment and Diagnostic Methods for Detection of Grapevine Phytoplasmas

  • Elisa Angelini
Chapter

Abstract

Grapevine phytoplasma occurring in Europe belong to two different species and are associated with two grapevine yellows diseases: Flavescence dorée and Bois noir. The diagnosis of the phytoplasma infections is usually carried out by both visual observation of the symptoms and molecular assay. The symptom observation directly in the field is the prerequisite for a correct diagnosis; however, only the molecular test allows the exact identification of the phytoplasma species, which is fundamental for disease monitoring. Presently, in routine mass screening of grapevine samples it is advisable to use the real time PCR approach, which is fast, robust and sensitive, while nested PCR/RFLP or sequencing of the amplicons are required for a finer differentiation of the phytoplasma strains.

Keywords

Polymerase Chain Reaction Protocol Phytoplasma Infection Direct Polymerase Chain Reaction Phytoplasma Strain Bois Noir 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

GY

Grapevine yellows

FD

Flavescence dorée

BN

Bois noir

Notes

Acknowledgments

All photos are courtesy of Dr. Michele Borgo, CRA-VIT, Conegliano (TV, Italy).

References

  1. Ahrens U, Seemüller E (1992) Detection of DNA of plant pathogenic mycoplasmalike organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82:828–832CrossRefGoogle Scholar
  2. Angelini E, Clair D, Borgo M, Bertaccini A, Boudon-Padieu E (2001) Flavescence dorée in France and Italy – Occurrence of closely related phytoplasma isolates and their near relationships to Palatinate grapevine yellows and an alder yellows phytoplasma. Vitis 40:79–86Google Scholar
  3. Angelini E, Filippin L, Michielini C, Bellotto D, Borgo M (2006) High occurrence of Flavescence dorée phytoplasma early in the season on grapevines infected with grapevine yellows. Vitis 45:151–152Google Scholar
  4. Angelini E, Bianchi GL, 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. J Microbiol Methods 68:613–622PubMedCrossRefGoogle Scholar
  5. Arnaud G, Malembic-Maher S, Salar P, Bonnet P, Maixner M, Marcone C, Boudon-Padieu E, Foissac X (2007) Multilocus sequence typing confirms the close genetic interrelatedness of three distinct Flavescence dorée phytoplasma strain clusters and group 16SrV phytoplasmas infecting grapevine and alder in Europe. Appl Environ Microbiol 73:4001–4010PubMedCrossRefGoogle Scholar
  6. Berger J, Dalla Via J, Baric S (2009) Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir. Eur J Plant Pathol 124:521–526CrossRefGoogle Scholar
  7. Bertaccini A, Vibio M, Stefani E (1995) Detection and molecular characterization of phytoplasmas infecting grapevine in Liguria (Italy). Phytopath Medit 34:137–141Google Scholar
  8. Bianco PA, Casati P, Marziliano N (2004) Detection of phytoplasmas associated with grapevine Flavescence dorée disease using real-time PCR. J Plant Pathol 86:259–264Google Scholar
  9. Botti S, Bertaccini A (2007) Grapevine yellows in Northern Italy: molecular identification of Flavescence dorée phytoplasma strains and of Bois noir phytoplasmas. J Appl Microbiol 103:2325–2330PubMedCrossRefGoogle Scholar
  10. Boudon-Padieu E 2002. Flavescence dorée of the grapevine: knowledge and new developments in epidemiology, etiology and diagnosis. In: Atti Giornate Fitopatologiche. Baselga di Pinè, Italy, 1:15–34Google Scholar
  11. Boudon-Padieu E, Béjat A, Clair D, Larrue J, Borgo M, Bertotto L, Angelini E (2003) Grapevine yellows: comparison of different procedures for DNA extraction and amplification with PCR for routine diagnosis of phytoplasmas in grapevine. Vitis 42:141–149Google Scholar
  12. Caudwell A, Kuszala C, Bachelier JC, Larrue J (1970) Transmission de la Flavescence dorée de la vigne aux plantes herbacées par l’allongement du temps d’utilisation de la cicadelle Scaphoideus littoralis Ball et l’étude de sa survie sur un grand nombre d’espèces végétales. Ann Phytopathol 2:415–428Google Scholar
  13. Caudwell A, Larrue J, Tassart V (1994). Caractère « porteur de la flavescence dorée » chez les vignes porte-greffes, en particulier le 3309 Couderc et le Fercal. Agronomie 2:83–94CrossRefGoogle Scholar
  14. Christensen NM, Nicolaisen M, Hansen M, Schulz A (2004) Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging. Mol Plant Microbe Interact 17:1175–1184PubMedCrossRefGoogle Scholar
  15. Cimerman A, Pacifico D, Salar P, Marzachì C, Foissac X (2009) Striking diversity of vmp1, a variable gene encoding a putative membrane protein of the stolbur phytoplasma. Appl Environ Microbiol 75:2951–2957PubMedCrossRefGoogle Scholar
  16. Clair D, Larrue J, Aubert G, Gillet J, Cloquemin G, Boudon-Padieu E (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
  17. Constable FE, Gibb KS, Symons RH (2003) Seasonal distribution of phytoplasmas in Australian grapevines. Plant Pathol 52:267–276CrossRefGoogle Scholar
  18. Credi R, Terlizzi F, Stimilli G, Nardi S, Lagnese R (2002) Flavescenza dorata della vite nelle Marche. L’Inf Agrario 22:61–63Google Scholar
  19. Daire X, Boudon-Padieu E, Berville A, Schneider B, Caudwell A (1992) Cloned DNA probes for detection of grapevine Flavescence dorée mycoplasma-like organism (MLO). Ann Appl Biol 121:95–103CrossRefGoogle Scholar
  20. Daire X, Clair D, Reinert W, Boudon-Padieu E (1997) Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA. Eur J Plant Pathol 103:507–514CrossRefGoogle Scholar
  21. Davis RE, Lee IM (1993) Cluster-specific polymerase chain reaction amplification of 16S rDNA sequences for detection and identification of mycoplasmalike organisms. Phytopathology 83:1008–1011CrossRefGoogle Scholar
  22. Davis RE, Dally EL, Bertaccini A, Credi R, Lee IM, Osler R, Carraro L, Barba M (1992) Cloned DNA probes for specific detection of Italian periwinkle virescence mycoplasmalike organism (MLO) and investigation of genetic relatedness with other MLOs. Phytopath Medit 31:5–12Google Scholar
  23. Davis RE, Dally EL, Bertaccini A, Lee IM, Credi L, Osler R, Savino V, Carraro L, Di Terlizzi B, Barba M (1993) Restriction fragment length polymorphism analyses and dot hybridizations distinguish mycoplasmalike organisms associated with Flavescence dorée and southern European grapevine yellows disease in Italy. Phytopathology 83:772–776CrossRefGoogle Scholar
  24. Deng S, Hiruki C (1991) Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes. J Microbiol Methods 14:53–61CrossRefGoogle Scholar
  25. Filippin L, Jović J, Cvrković T, Forte V, Clair D, Toševski I, Boudon-Padieu E, Borgo M, Angelini E (2009) Molecular characteristics of phytoplasmas associated with Flavescence dorée in clematis and grapevine and preliminary results on the role of Dictyophara europaea as a vector. Plant Pathol 58:826–837CrossRefGoogle Scholar
  26. Firrao G, Moretti M, Rosquete MR, Gobbi E, Locci R (2005) Nanobiotransducer for detecting flavescence dorée phytoplasma. J Plant Pathol 87:101–107Google Scholar
  27. Frosini A, Casati P, Bianco PA, Bordoni R, Consolandi C, Castiglioni B, Mezzelani A, Rizzi E, Battaglia C, Belli G, Rossi Bernardi L, De Bellis G (2002). Ligase detection reaction and universal array as a tool to detect grapevine infecting phytoplasmas. Minerva Biotec 14:265–267Google Scholar
  28. Galetto L, Bosco D, Marzachí 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. Ann Appl Biol 147:191–201CrossRefGoogle Scholar
  29. Gibb K, Padovan AC, Mogen BD (1995) Studies on sweet potato little-leaf phytoplasma detected in sweet potato and other plant species growing in northern Australia. Phytopathology 85:169–174CrossRefGoogle Scholar
  30. Gibb KS, Constable FE, Moran JR, Padovan AC (1999) Phytoplasmas in Australian grapevines – detection, differentiation and associated diseases. Vitis 38:107–114Google Scholar
  31. Green MJ, Thompson DA, MacKenzie DJ (1999) Easy and efficient DNA extraction from woody plants for the detection of phytoplasmas by polymerase chain reaction. Plant Disease 83:482–485CrossRefGoogle Scholar
  32. Gundersen DE, Lee IM (1996) Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol Medit 35:144–151Google Scholar
  33. Hren M, Boben J, Rotter A, Kralj P, Gruden K, Ravnikar M (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 56:785–796CrossRefGoogle Scholar
  34. IRPCM (2004). “Candidatus Phytoplasma”, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int J System Evol Microbiol 54:1243–1255CrossRefGoogle Scholar
  35. Kirkpatrick BC, Stenger DC, Morris TJ, Purcell AH (1987) Cloning and detection of DNA from a nonculturable plant pathogenic mycoplasma-like organism. Science 238:197–200PubMedCrossRefGoogle Scholar
  36. Kuzmanović S, Martini M, Ermacora P, Ferrini F, Starović M, Tosić M, Carraro L, Osler R (2008) Incidence and molecular characterization of Flavescence dorée and stolbur phytoplasmas in grapevine cultivars from different viticultural areas of Serbia. Vitis 47:105–111Google Scholar
  37. 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 43:191–199Google Scholar
  38. Lee IM, Hammond RW, Davis RE, Gundersen DE (1993) Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology 83:834–842CrossRefGoogle Scholar
  39. Lee IM, Gundersen DE, Hammond RW, Davis RE (1994) Use of mycoplasmalike organism (MLO) group-specific oligonucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathology 84:559–566CrossRefGoogle Scholar
  40. Lee IM, Gundersen–Rindal DE, Davis RE, Bartoszyk IM (1998) Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int J System Evol Microbiol 48:1153–1169Google Scholar
  41. Lorenz KH, Schneider B, Ahrens U, Seemüller E (1995) Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. Phytopathology 85:771–776CrossRefGoogle Scholar
  42. Maixner M (2006) Grapevine yellows – current developments and unsolved questions. In: Extended Abstracts 15th Meeting ICVG. Stellenbosch, South Africa, addendum. http://www.icvg.ch/data/maixner.pdf
  43. 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. Eur J Plant Pathol 101:241–250CrossRefGoogle Scholar
  44. Marcone C, Ragozzino A, Schneider B, Lauer U, Smart CD, Seemüller E (1996) Genetic characterization and classification of two phytoplasmas associated with spartium witches’-broom disease. Plant Dis 80:365–371CrossRefGoogle Scholar
  45. Margaria P, Rosa C, Marzachì C, Turina M, Palmano S (2007) Detection of flavescence dorée phytoplasma in grapevine by reverse-transcription PCR. Plant Dis 91:1496–1501CrossRefGoogle Scholar
  46. Martini M, Murari E, Mori N, Bertaccini A (1999) Identification and epidemic distribution of two Flavescence dorée-related phytoplasmas in Veneto (Italy). Plant Dis 83:925–930CrossRefGoogle Scholar
  47. Martini M, Botti S, Marcone C, Marzachì C, Casati P, Bianco PA, Benedetti R, Bertaccini A (2002) Genetic variability among flavescence dorée phytoplasmas from different origins in Italy and France. Mol Cell Probes 16:197–208PubMedCrossRefGoogle Scholar
  48. Marzachì C, Veratti F, d’Aquilio M, Vischi A, Conti M, Boccardo G (2000) Molecular hybridization and PCR amplification of non-ribosomal DNA to detect and differentiate stolbur phytoplasma isolates from Italy. J Plant Pathol 82:201–212Google Scholar
  49. Marzachì C, Palermo S, Boarino A, Veratti F, d’Aquilio M, Loria A, Boccardo G (2001) Optimisation of a one-step PCR assay for the diagnosis of Flavescence dorée-related phytoplasmas in field-grown grapevines and vector populations. Vitis 40:213–217Google Scholar
  50. Moutous G (1977) Définitions des symptômes de la “flavescence dorée” sur les variétés porte-greffes. Rev Zool Agr Pathol Végétale 76:90–98Google Scholar
  51. Nicolaisen M, Bertaccini A (2007) An oligonucleotide microarray-based assay for identification of phtytoplasma 16S ribosomal groups. Plant Pathol 56:332–336CrossRefGoogle Scholar
  52. Pacifico D, Alma A, Bagnoli B, Foissac X, Pasquini G, Tessitori M, Marzachi C (2009) Characterization of Bois noir isolates by restriction fragment length polymorphism of a stolbur-specific putative membrane protein gene. Phytopathology 99:711–715PubMedCrossRefGoogle Scholar
  53. Padovan AC, Gibb KS, Bertaccini A, Vibio M, Bonfiglioli RE, Magarey PA, Sears BB (1995) Molecular detection of the Australian grapevine yellows phytoplasma and comparison with grapevine yellows phytoplasmas from Italy. Aust J Grape Wine Res 1:25–31CrossRefGoogle Scholar
  54. Prince JP, Davis RE, Wolf TK, Lee IM, Mogen BD, Dally EL, Bertaccini A, Credi R, Barba M (1993) Molecular detection of diverse mycoplasmalike organisms (MLOs) associated with grapevine yellows and their classification with aster yellows, X-disease, and elm yellows MLOs. Phytopathology 83:1130–1137CrossRefGoogle Scholar
  55. Schaff D, Lee IM, Davis RE (1992) Sensitive detection and identification of mycoplasmalike organisms in plants by polymerase chain reactions. Biochem Biophys Res Comm 186:1503–1509PubMedCrossRefGoogle Scholar
  56. Schneider B, Gibb KS, Seemüller E (1997) Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas. Microbiology 143:3381–3389PubMedCrossRefGoogle Scholar
  57. Seddas A, Meignoz R, Daire X, Boudon-Padieu E (1996) Generation and characterization of monoclonal antibodies to Flavescence dorée phytoplasma: serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows phytoplasmas. Eur J Plant Pathol 102:757–764CrossRefGoogle Scholar
  58. Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, Lorenz KH, Seemüller E, Kirkpatrick BC (1996) Phytoplasma-specific PCR primers based on sequences of the 16S–23S rRNA spacer region. Appl Environ Microbiol 62:2988–2993PubMedGoogle Scholar
  59. Wei W, Davis RE, Lee IM, Zhao Y (2007) Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. Int J System Evol Microbiol 57:1855–1867CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.C.R.A. VIT Centro di Ricerca per la ViticolturaConeglianoItaly

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