Phytoplasma phylogenetics has focused primarily on sequences of the non-coding 16S rRNA gene and the 16S–23S rRNA intergenic spacer region (16–23S ISR), and primers that enable amplification of these regions from all phytoplasmas by PCR are well established. In this study, primers based on the gene have been developed into a semi-nested PCR assay that results in a sequence of the expected size (about 480 bp) from all 34 phytoplasmas examined, including strains representative of 12 16Sr groups. Phylogenetic analysis of gene sequences showed similar clustering of phytoplasmas when compared with clusters resolved by similar sequence analyses of a 16–23S ISR–23S rRNA gene contig or of the 16S rRNA gene alone. The main differences between trees were in the branch lengths, which were elongated in the 16–23S ISR–23S rRNA gene tree when compared with the 16S rRNA gene tree and elongated still further in the gene tree, despite this being a shorter sequence. The improved resolution in the gene-derived phylogenetic tree resulted in the 16SrII group splitting into two distinct clusters, while phytoplasmas associated with coconut lethal yellowing-type diseases split into three distinct groups, thereby supporting past proposals that they represent different candidate species within ‘ Phytoplasma’. The ability to differentiate 16Sr groups and subgroups by virtual RFLP analysis of gene sequences suggests that this gene may provide an informative alternative molecular marker for pathogen identification and diagnosis of phytoplasma diseases.


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  1. Altschul, S. F., Gush, W., Miller, W., Myers, W. & Lipman, D. J.(1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef] [Google Scholar]
  2. 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, 4001–4010.[CrossRef] [Google Scholar]
  3. Bai, X., Zhang, J., Holford, I. R. & Hogenhout, S. A.(2004). Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes. FEMS Microbiol Lett 235, 249–258.[CrossRef] [Google Scholar]
  4. Bai, X., Zhang, J., Ewing, A., Miller, S. A., Radek, A. J., Shevchenko, D. V., Tsukerman, K., Walunas, T., Lapidus, A. & other authors(2006). Living with genome instability: the adaptation of phytoplasmas to diverse environments of their insect and plant hosts. J Bacteriol 188, 3682–3696.[CrossRef] [Google Scholar]
  5. Bertaccini, A., Franova, J., Botti, S. & Tabanelli, D.(2005). Molecular characterization of phytoplasmas in lilies with fasciation in the Czech Republic. FEMS Microbiol Lett 249, 79–85.[CrossRef] [Google Scholar]
  6. Botti, S. & Bertaccini, A.(2003). Variability and functional role of chromosomal sequences in 16SrI-B subgroup phytoplasmas including aster yellows and related strains. J Appl Microbiol 94, 103–110.[CrossRef] [Google Scholar]
  7. De La Rue, S., Padovan, A. & Gibb, K.(2001).Stylosanthes is a host for several phytoplasmas, one of which shows unique 16S–23S intergenic spacer region heterogeneity. J Phytopathol 149, 613–619.[CrossRef] [Google Scholar]
  8. Deng, S. & Hiruki, C.(1991). Amplification of 16S rRNA genes from culturable and non-culturable mollicutes. J Microbiol Methods 14, 53–61.[CrossRef] [Google Scholar]
  9. Doyle, J. J. & Doyle, J. L.(1990). Isolation of plant DNA from fresh tissue. Focus 12, 13–15. [Google Scholar]
  10. Economou, A.(1999). Follow the leader: bacterial protein export through the Sec translocase. Trends Microbiol 7, 315–319.[CrossRef] [Google Scholar]
  11. Firrao, G., Gibb, K. & Streten, C.(2005). Short taxonomic guide to the genus ‘Candidatus phytoplasma’. J Plant Pathol 87, 249–263. [Google Scholar]
  12. Gundersen, D. E. & Lee, I.-M.(1996). Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol Mediterr 35, 144–151. [Google Scholar]
  13. Harrison, N. A., Myrie, W., Jones, P., Carpio, M. L., Castillo, M., Doyle, M. M. & Oropeza, C.(2002). 16S rRNA interoperon sequence heterogeneity distinguishes strain populations of the palm lethal yellowing phytoplasma in the Caribbean region. Ann Appl Biol 141, 183–193.[CrossRef] [Google Scholar]
  14. Hodgetts, J., Ball, T., Boonham, N., Mumford, R. & Dickinson, M.(2007). Use of terminal restriction fragment length polymorphism (T-RFLP) for identification of phytoplasmas in plants. Plant Pathol 56, 357–365. [Google Scholar]
  15. 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, 1243–1255.[CrossRef] [Google Scholar]
  16. Jomantiene, R., Zhao, Y. & Davis, R. E.(2007). Sequence-variable mosaics: composites of recurrent transposition characterizing the genomes of phylogenetically diverse phytoplasmas. DNA Cell Biol 26, 557–564.[CrossRef] [Google Scholar]
  17. Kumar, S., Tamura, K. & Nei, M.(2004).mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.[CrossRef] [Google Scholar]
  18. Lee, I.-M., Hammond, R. W., Davis, R. E. & Gundersen, D. E.(1993). Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology 83, 834–842.[CrossRef] [Google Scholar]
  19. Lee, I.-M., Davis, R. E. & Gundersen, D. E.(2000). Phytoplasma: phytopathogenic mollicutes. Annu Rev Microbiol 54, 221–255.[CrossRef] [Google Scholar]
  20. Lee, M. E., Grau, C. R., Lukaesko, L. A. & Lee, I.-M.(2002). Identification of aster yellows phytoplasmas in soybean in Wisconsin based on RFLP analysis of PCR-amplified products (16S rDNAs). Can J Plant Pathol 24, 125–130.[CrossRef] [Google Scholar]
  21. Lee, I.-M., Zhao, Y. & Bottner, K. D.(2006). SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group. Mol Cell Probes 20, 87–91.[CrossRef] [Google Scholar]
  22. Liefting, L. W., Shaw, M. & Kirkpatrick, B. C.(2004). Sequence analysis of two plasmids from the phytoplasma beet leafhopper-transmitted virescence agent. Microbiology 150, 1809–1817.[CrossRef] [Google Scholar]
  23. Marcone, C., Lee, I.-M., Davis, R. E., Ragozzino, A. & Seemüller, E.(2000). Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences. Int J Syst Evol Microbiol 50, 1703–1713. [Google Scholar]
  24. Martini, M., Lee, I.-M., Bottner, K. D., Zhao, Y., Botti, S., Bertaccini, A., Harrison, N. A., Carraro, L., Marcone, C. & other authors(2007). Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas. Int J Syst Evol Microbiol 57, 2037–2051.[CrossRef] [Google Scholar]
  25. Montano, H. G., Davis, R. E., Dally, E. L., Hogenhout, S., Pimentel, J. P. & Brioso, P. S. T.(2001).Candidatus Phytoplasma brasiliense’, a new phytoplasma taxon associated with hibiscus witches'-broom disease. Int J Syst Evol Microbiol 51, 1109–1118.[CrossRef] [Google Scholar]
  26. Mpunami, A. A., Tymon, A., Jones, P. & Dickinson, M. J.(1999). Genetic diversity in the coconut lethal yellowing disease phytoplasmas of East Africa. Plant Pathol 48, 109–114.[CrossRef] [Google Scholar]
  27. Nipah, J. O., Jones, P. & Dickinson, M. J.(2007). Detection of lethal yellowing phytoplasma in embryos from coconut palms infected with Cape St Paul wilt disease in Ghana. Plant Pathol 56, 777–784.[CrossRef] [Google Scholar]
  28. Schneider, B., Torres, M. P., Martin, M. P., Schroder, M., Behnke, H. D. & Seemüller, E.(2005).Candidatus Phytoplasma pini’, a novel taxon from Pinus silvestris and Pinus halepensis. Int J Syst Evol Microbiol 55, 303–307.[CrossRef] [Google Scholar]
  29. Seemüller, E., Garnier, M. & Schneider, B.(2002). Mycoplasmas of plants and insects. In Molecular Biology and Pathogenicity of Mycoplasmas, pp. 91–116. Edited by S. Razin & R. Herrmann. Dordrecht, Netherlands: Kluwer Academic/Plenum.
  30. Shao, J., Jomantiene, R., Dally, E. L., Zhao, Y., Lee, I.-M., Nuss, D. L. & Davis, R. E.(2006). Phylogeny and characterization of phytoplasmal NusA and use of the nusA gene in detection of group 16SrI strains. J Plant Pathol 88, 193–201. [Google Scholar]
  31. Smart, C. D., Schneider, B., Blomquist, C. L., Guerra, L. J., Harrison, N. A., Ahrens, U., Lorenz, K.-H., Seemüller, E. & Kirkpatrick, B.(1996). Phytoplasma-specific PCR primers based on sequence of the 16S–23S rRNA spacer region. Appl Environ Microbiol 62, 2988–2993. [Google Scholar]
  32. Streten, C. & Gibb, K. S.(2005). Genetic variation in Candidatus Phytoplasma australiense. Plant Pathol 54, 8–14.[CrossRef] [Google Scholar]
  33. Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994).clustalw: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[CrossRef] [Google Scholar]
  34. Wang, K. & Hiruki, C.(2005). Distinctions between phytoplasmas at the subgroup level detected by heteroduplex mobility assay. Plant Pathol 54, 625–633.[CrossRef] [Google Scholar]
  35. Wang, K., Hiruki, C. & Yeh, F.(2003). Molecular evolution of phytoplasmas based on polymorphisms in the 16S rRNA genes and the 16/23S spacer regions. Proc Jpn Acad Ser B 79B, 155–162.[CrossRef] [Google Scholar]
  36. Wei, W., Davis, R. E., Lee, I.-M. & Zhao, Y.(2007). Computer-simulated RFLP analysis of 16S rRNA genes: identification of ten new phytoplasma groups. Int J Syst Evol Microbiol 57, 1855–1867.[CrossRef] [Google Scholar]

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vol. , part 8, pp. 1826 - 1837


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