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Phytoplasma pp 173-188 | Cite as

PCR and RFLP Analyses Based on the Ribosomal Protein Operon

  • Marta MartiniEmail author
  • Ing-Ming Lee
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 938)

Abstract

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16S rRNA gene. RFLP analysis of 16S rRNA gene sequences has identified 31 16S rRNA (16Sr) groups and more than 100 16Sr subgroups. Classification of phytoplasma strains can, however, become more refined and specific if moderately conserved genes, such as the ribosomal protein (rp) genes, are used as genetic markers. The use of additional genetic markers enhances the resolving power of phytoplasma classification. This chapter describes the methodology of detection, differentiation, and classification of phytoplasma strains based on rp gene sequences. RFLP analysis of amplicons obtained by group- or subgroup-specific rp gene-based primers is used for finer differentiation of phytoplasma strains within a given group or subgroup. The rp gene-based classification not only readily resolves 16Sr subgroups within a given 16Sr group, but also provides finer differentiation of closely related phytoplasma strains. Many individual 16Sr subgroups can be further differentiated into two or more distinct rp subgroups.

Key words

rplV (rpl22rpsC (rps3Semiuniversal primers 16Sr group-specific primers 16Sr subgroup-specific primers Restriction enzymes Phytoplasma strain differentiation 

References

  1. 1.
    Zhao Y et al (2010) Recent advances in 16S rRNA gene-based phytoplasma differentiation, classification and taxonomy. In: Weintraub PG, Jones P (eds) Phytoplasmas: genomes, plant hosts and vectors. CAB International, Wallingford, pp 64–92Google Scholar
  2. 2.
    Lee I-M et al (2011) ‘Candidatus Phytoplasma costaricanum’ a new phytoplasma associated with a newly emerging disease in soybean in Costa Rica. Int J Syst Evol Microbiol. doi: 10.1099/ijs.0.029041-0
  3. 3.
    Lee I-M, Zhao Y, Davis RE (2010) Prospects of multiple gene-based systems for differentiation and classification of phytoplasmas. In: Weintraub PG, Jones P (eds) Phytoplasmas: genomes, plant hosts and vectors. CAB International, Wallingford, pp 51–63Google Scholar
  4. 4.
    Lee I-M et al (1998) Revised classification scheme of phytoplasmas based on RFLP analyses of 16S rRNA and ribosomal protein gene sequences. Int J Syst Bacteriol 48:1153–1169CrossRefGoogle Scholar
  5. 5.
    Lee I-M et al (2004) ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases. Int J Syst Evol Microbiol 54:1037–1048PubMedCrossRefGoogle Scholar
  6. 6.
    Lee I-M et al (2004) Classification of phytoplasma strains in the elm yellows group (16SrV) and proposition of ‘Candidatus Phytoplasma ulmi’ for the phytoplasma associated with elm yellows. Int J Syst Evol Microbiol 54:337–347PubMedCrossRefGoogle Scholar
  7. 7.
    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
  8. 8.
    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
  9. 9.
    Lim P-O, Sears BB (1992) Evolutionary relationships of a plant-pathogenic mycoplasmalike organism and Acholeplasma laidlawii deduced from two ribosomal protein gene sequences. J Bacteriol 174:2606–2611PubMedGoogle Scholar
  10. 10.
    Lee I-M et al (2003) Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas. Phytopathology 93:1368–1377PubMedCrossRefGoogle Scholar
  11. 11.
    Martini M (2004) Ribosomal protein gene-based phylogeny: a basis for phytoplasma classification. PhD dissertation, University of Udine, Udine, Italy, p 106Google Scholar
  12. 12.
    Martini M et al (2008) Molecular differentiation of ‘Candidatus Phytoplasma mali’ and its spreading in Friuli Venezia Giulia region (north-east Italy). Acta Hortic 781:395–402Google Scholar
  13. 13.
    Gundersen DE et al (1996) Genomic diversity and differentiation among phytoplasma strains in 16S rRNA group I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). Int J Syst Bacteriol 46:64–75PubMedCrossRefGoogle Scholar
  14. 14.
    Griffiths HM et al (1999) The phytoplasma associated with ash yellows and lilac witches’-broom: ‘Candidatus Phytoplasma fraxini’. Int J Syst Bacteriol 49:1605–1614PubMedCrossRefGoogle Scholar
  15. 15.
    Durante G et al (2008) Bois noir in Lombardy (northern Italy): identification of molecular markers for diagnosis and characterization of 16SrXII-A phytoplasmas. Proceedings of the: 4th National Workshop on Phytoplasma Diseases. Rome, Italy 28–30 May 2008, p 300–303Google Scholar
  16. 16.
    Streten C, Gibb KS (2005) Genetic variation in Candidatus Phytoplasma australiense. Plant Pathol 54:8–14CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of Agriculture and Environmental SciencesUniversity of UdineUdineItaly
  2. 2.Molecular Plant Pathology LaboratoryUSDA, ARSBeltsvilleUSA

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