Phytoplasma pp 159-171 | Cite as

Nested PCR and RFLP Analysis Based on the 16S rRNA Gene

  • Bojan Duduk
  • Samanta Paltrinieri
  • Ing-Ming Lee
  • Assunta Bertaccini
Part of the Methods in Molecular Biology book series (MIMB, volume 938)


Current phytoplasma detection and identification methods are primarily based on nested polymerase chain reaction followed by restriction fragment length polymorphism analysis and gel electrophoresis. These methods can potentially detect and differentiate all phytoplasmas including those previously not described. The present protocol describes the application of this method for identification of phytoplasmas at 16S rRNA (16Sr) group and 16Sr subgroup levels.

Key words

Detection Identification Ribosomal group Ribosomal subgroup 


  1. 1.
    Lee I-M et al (1998) Revised classification scheme of phytoplasmas based an RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int J Syst Bacteriol 48:1153–1169CrossRefGoogle Scholar
  2. 2.
    Lee I-M, Gundersen-Rindal DE, Bertaccini A (1998) Phytoplasma: ecology and genomic diversity. Phytopathology 88:1359–1366PubMedCrossRefGoogle Scholar
  3. 3.
    Al-Saady NA et al (2008) ‘Candidatus Phytoplasma omanense’, a phytoplasma associated with witches’ broom of Cassia italica (Mill.) Lam. in Oman. Int J Syst Evol Microbiol 58:461–466PubMedCrossRefGoogle Scholar
  4. 4.
    Arocha Y et al (2005) ‘Candidatus Phytoplasma graminis’ and ‘Candidatus Phytoplasma caricae’, two novel phytoplasmas associated with diseases of sugarcane, weeds and papaya in Cuba. Int J Syst Evol Microbiol 55: 2451–2463PubMedCrossRefGoogle Scholar
  5. 5.
    Bertaccini A, Duduk B (2009) Phytoplasma and phytoplasma diseases: a review of recent research. Phytopathol Mediterr 48:355–378Google Scholar
  6. 6.
    Lee I-M et al (2004) ‘Candidatus Phytoplasma asteris’, a novel taxon associated with aster yellows and related diseases. Int J Syst Bacteriol 54:1037–1048Google Scholar
  7. 7.
    Lee I-M et al (2004) Classification of phytoplasma strains in the elm yellows group (16SrV) and proposal of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated with elm yellows. Int J Syst Evol Microbiol 54: 337–347PubMedCrossRefGoogle Scholar
  8. 8.
    Lee I-M, Zhao Y, Bottner KD (2006) SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group. Mol Cell Probes 20:87–91PubMedCrossRefGoogle Scholar
  9. 9.
    Montano HG et al (2001) ‘Candidatus Phytoplasma brasiliense’, a new phytoplasma taxon associated with hibiscus witches’ broom disease. Int J Syst Evol Microbiol 51: 1109–1118PubMedCrossRefGoogle Scholar
  10. 10.
    Berges R, Rott M, Seemüller E (2000) Range of phytoplasma concentration in various plant hosts as determined by competitive polymerase chain reaction. Phytopathology 90: 1145–1152PubMedCrossRefGoogle Scholar
  11. 11.
    Bertaccini A, Marani F (1982) Electron microscopy of two viruses and mycoplasma-like organisms in lilies with deformed flowers. Phytopathol Mediterr 21:8–14Google Scholar
  12. 12.
    Cousin MT, Sharma AK, Isra S (1986) Correlation between light and electron microscopic observations and identification of mycoplasma-like organisms using consecutive 350 nm think sections. J Phytopathol 115: 368–374CrossRefGoogle Scholar
  13. 13.
    Haggis GH, Sinha RC (1978) Scanning electron microscopy of mycoplasma-like organisms after freeze fracture of plant tissues affected with clover phyllody and aster yellows. Phytopathology 68:677–680CrossRefGoogle Scholar
  14. 14.
    Seemüller E (1976) Investigation to demonstrate mycoplasma-like organisms in diseases plants by fluorescence microscopy. Acta Hortic 67:109–112Google Scholar
  15. 15.
    Hobbs HA, Reddy DVR, Reddy AS (1987) Detection of a mycoplasma-like organism in peanut plants with witches’ broom using indirect enzyme-linked immunosorbent assay (ELISA). Plant Pathol 36:164–167CrossRefGoogle Scholar
  16. 16.
    Deng SJ, Hiruki C (1991) Amplification of 16S ribosomal-RNA genes from culturable and nonculturable mollicutes. J Microbiol Methods 14:53–61CrossRefGoogle Scholar
  17. 17.
    Firrao G, Gobbi E, Locci R (1993) Use of polymerase chain reaction to produce oligonucleotide probes for mycoplasma-like organism. Phytopathology 83:602–607CrossRefGoogle Scholar
  18. 18.
    Lee I-M et al (1993) Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasma-like organisms. Phytopathology 83:834–842CrossRefGoogle Scholar
  19. 19.
    Namba S et al (1993) Detection and differentiation of plant-pathogenic mycoplasma-like organisms using polymerase chain reaction. Phytopathology 83:786–791CrossRefGoogle Scholar
  20. 20.
    Schneider B et al (1993) Classification of plant-pathogenic mycoplasma-like organisms using restriction site analysis of PCR-amplified 16S rDNA. J Gen Microbiol 139:519–527CrossRefGoogle Scholar
  21. 21.
    Baric S, Dalla VJ (2004) A new approach to apple proliferation detection: a highly sensitive real-time PCR assay. J Microbiol Meth 57:135–145CrossRefGoogle Scholar
  22. 22.
    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 Dis 83:482–485CrossRefGoogle Scholar
  23. 23.
    Schaff DA, Lee I-M, Davis RE (1992) Sensitive detection and identification of mycoplasma-like organisms by polymerase chain reactions. Biochem Biophys Res Comm 186:1503–1509PubMedCrossRefGoogle Scholar
  24. 24.
    Zhang Y, Uyemoto JK, Kirkpatrick BC (1998) A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay. J Virol Meth 71:45–50CrossRefGoogle Scholar
  25. 25.
    Alvarez E et al (2009) Characterization of a phytoplasma associated with frogskin disease in cassava. Plant Dis 93:1139–1145CrossRefGoogle Scholar
  26. 26.
    Cozza R et al (2008) Molecular identification of ‘Candidatus Phytoplasma asteris’ inducing histological anomalies in Silene nicaeensis. Phytoparasitica 36:290–293CrossRefGoogle Scholar
  27. 27.
    Duduk B et al (2004) Identification of phytoplasmas associated with grapevine yellows in Serbia. J Phytopathol 152:575–579CrossRefGoogle Scholar
  28. 28.
    Lee I-M et al (1995) Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology 85: 728–735CrossRefGoogle Scholar
  29. 29.
    Lorenz KH et al (1995) Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. Phytopathology 85:771–776CrossRefGoogle Scholar
  30. 30.
    Montano HG et al (2011) Hibiscus witches’ broom disease associated with different phytoplasmas taxa in Brazil. Bull Insectol 64:S249–S250Google Scholar
  31. 31.
    Tolu G et al (2006) Identification of 16SrII-E phytoplasmas in Calendula arvensis L., Solanum nigrum L. and Chenopodium spp. Plant Dis 90:325–330CrossRefGoogle Scholar
  32. 32.
    Davis RE et al (2003) Differential amplification of sequence heterogenous ribosomal RNA genes and classification of the ‘Fragaria multicipita’ phytoplasma. Microbiol Res 158: 229–236PubMedCrossRefGoogle Scholar
  33. 33.
    Duduk B et al (2007) Identification of phytoplasmas belonging to aster yellows ribosomal group in vegetables in Serbia. Bull Insectol 60:341–342Google Scholar
  34. 34.
    Duduk B et al (2009) Multigene analysis for differentiation of aster yellows phytoplasmas infecting carrots in Serbia. Ann Appl Biol 154:219–229CrossRefGoogle Scholar
  35. 35.
    Jomantiene R et al (2002) New group 16SrIII phytoplasma lineages in Lithuania exhibit rRNA interoperon sequence heterogeneity. Eur J Plant Pathol 108:507–517CrossRefGoogle Scholar
  36. 36.
    Liefting LW et al (1996) Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma. Appl Environ Microbiol 62:3133–3139PubMedGoogle Scholar
  37. 37.
    Schneider B, Seemüller E (1994) Presence of two sets of ribosomal genes in phytopathogenic mollicutes. Appl Environ Microbiol 60:3409–3412PubMedGoogle Scholar
  38. 38.
    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
  39. 39.
    Lee I-M, Davis RE, Gundersen-Rindal DE (2000) Phytoplasmas: phytopathogenic mollicutes. Annu Rev Microbiol 56:1593–1597Google Scholar
  40. 40.
    Lee I-M et al (2010) Phylogenetic analysis and delineation of phytoplasmas based on SecY gene sequences. Int J Syst Evol Microbiol 60: 2887–2897PubMedCrossRefGoogle Scholar
  41. 41.
    Marcone C et al (2000) Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences. Int J Syst Evol Microbiol 50:1703–1713PubMedGoogle Scholar
  42. 42.
    Martini M et al (2002) Genetic variability among Flavescence dorée phytoplasmas from different origins in Italy and France. Mol Cell Probes 16:197–208PubMedCrossRefGoogle Scholar
  43. 43.
    Martini M et al (2007) Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas. Int J Syst Evol Microbiol 57:2037–2051PubMedCrossRefGoogle Scholar
  44. 44.
    Mitrović J et al (2011) The groEL gene as an additional marker for finer differentiation of ‘Candidatus Phytoplasma asteris’-related strains. Ann Appl Biol 159:41–48CrossRefGoogle Scholar
  45. 45.
    Schneider B, Gibb KS, Seemüller E (1997) Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas. Microbiol 143:3381–3389CrossRefGoogle Scholar
  46. 46.
    Wei W et al (2004) An antibody against the SecA membrane protein of one phytoplasma reacts with those of phylogenetically different phytoplasmas. Phytopathology 94: 683–686PubMedCrossRefGoogle Scholar
  47. 47.
    Cai H et al (2008) Genetic diversity among phytoplasmas infecting Opuntia species: virtual RFLP analysis identifies new subgroups in the peanut witches’-broom phytoplasma group. Int J Syst Evol Microbiol 58: 1448–1457PubMedCrossRefGoogle Scholar
  48. 48.
    Duduk B, Bertaccini A (2006) Corn with symptoms of reddening: new host of stolbur phytoplasma. Plant Dis 90:1313–1319CrossRefGoogle Scholar
  49. 49.
    Khan AJ et al (2002) Molecular identification of a new phytoplasma associated with alfalfa witches’-broom in Oman. Phytopathology 92:1038–1047PubMedCrossRefGoogle Scholar
  50. 50.
    Wei W et al (2007) Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. Int J Syst Evol Microbiol 57:1855–1867PubMedCrossRefGoogle Scholar
  51. 51.
    Wei W et al (2008) Automated RFLP pattern comparison and similarity coefficient calculation for rapid delineation of new and distinct phytoplasma 16Sr subgroup lineages. Int J Syst Evol Microbiol 58:2368–2377PubMedCrossRefGoogle Scholar
  52. 52.
    Schneider B et al (1995) Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Razin S, Tully JG (eds) Molecular and diagnostic procedures in mycoplasmology. Academic, San Diego, pp 369–380CrossRefGoogle Scholar
  53. 53.
    Skrzeczkowski L et al (2001) Bacterial sequences interferring in detection of phytoplasma by PCR using primers derived from the ribosomal RNA operon. Acta Hortic 550:417–424CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Bojan Duduk
    • 1
  • Samanta Paltrinieri
    • 2
  • Ing-Ming Lee
    • 3
  • Assunta Bertaccini
    • 2
  1. 1.Laboratory of Applied PhytopathologyInstitute of Pesticides and Environmental ProtectionBelgradeSerbia
  2. 2.DiSTA, Plant PathologyAlma Mater Studiorum—University of BolognaBolognaItaly
  3. 3.Molecular Plant Pathology LaboratoryUSDA, ARSBeltsvilleUSA

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