Phytoplasmas are cell wall-less bacteria that cause numerous diseases in several hundred plant species. During adaptation to transkingdom parasitism in diverse plant and insect hosts, phytoplasma evolution has given rise to widely divergent lineages. Since phytoplasmas cannot be cultured in a cell-free medium, measurable phenotypic characters suitable for conventional microbial classification are mostly inaccessible. Currently, phytoplasma differentiation and classification are mainly dependent on restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene sequences. Extending our recent efforts in the exploitation of computer-simulated 16S rRNA gene RFLP analysis and virtual gel plotting for rapid classification of phytoplasmas, we have developed a Perl program for automated RFLP pattern comparison and similarity coefficient calculation. This program streamlines virtual RFLP pattern analysis and has led to the establishment of a criterion for phytoplasma 16Sr subgroup classification and to the delineation of new and distinct subgroup lineages in the clover proliferation phytoplasma group (16SrVI).


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  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef] [Google Scholar]
  2. Bai, X., Zhang, J. H., 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]
  3. Cai, H., Wei, W., Davis, R. E., Chen, H. & Zhao, Y.(2008). Genetic diversity among phytoplasmas infecting Opuntia spp.: virtual RFLP analysis identifies new subgroups in the peanut witches'-broom phytoplasma group. Int J Syst Evol Microbiol 58, 1448–1457.[CrossRef] [Google Scholar]
  4. Chiykowski, L. N.(1962). Clover phyllody virus in Canada and its transmission. Can J Bot 40, 397–404.[CrossRef] [Google Scholar]
  5. Chiykowski, L. N. & Sinha, R. C.(1989). Differentiation of MLO disease by means of symptomatology and vector transmission. Zentralbl Bakteriol Hyg (Suppl) 20, 280–287. [Google Scholar]
  6. Davis, R. E. & Dally, E. L.(2001). Nonfunctional tRNA gene in an unusual example of rRNA interoperon sequence heterogeneity in phytoplasma. Phytopathology 91, S21 [Google Scholar]
  7. Davis, R. E. & Sinclair, W. A.(1998). Phytoplasma identity and disease etiology. Phytopathology 88, 1372–1376.[CrossRef] [Google Scholar]
  8. Faggioli, F., Pasquini, G., Lumia, V., Campobasso, G., Widmer, T. L. & Quimby, P. C., Jr(2004). Molecular identification of a new member of the clover proliferation phytoplasma group (16SrVI) associated with Centaurea solstitialis virescence in Italy. Eur J Plant Pathol 110, 353–360.[CrossRef] [Google Scholar]
  9. Firrao, G., Carraro, L., Gobbi, E. & Locci, R.(1996). Molecular characterization of a phytoplasma causing phyllody in clover and other herbaceous hosts in northern Italy. Eur J Plant Pathol 102, 817–822.[CrossRef] [Google Scholar]
  10. Freitag, J. H.(1964). Interaction and mutual suppression among three strains of aster yellows virus. Virology 24, 401–413.[CrossRef] [Google Scholar]
  11. Granados, R. R. & Chapman, R. K.(1968). Identification of some new aster yellows virus strains and their transmission by the aster leafhopper Macrosteles fascifrons (Stahl). Phytopathology 58, 1685–1692. [Google Scholar]
  12. Gundersen, D. E., Lee, I. M., Rehner, S. A., Davis, R. E. & Kingsbury, D. T.(1994). Phylogeny of mycoplasmalike organisms (phytoplasmas): a basis for their classification. J Bacteriol 176, 5244–5254. [Google Scholar]
  13. Hiruki, C. & Wang, K. R.(2004). Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’. Int J Syst Evol Microbiol 54, 1349–1353.[CrossRef] [Google Scholar]
  14. Jomantiene, R., Davis, R. E., Valiunas, D. & Alminaite, A.(2002). New group 16SrIII phytoplasma lineages in Lithuania exhibit rRNA interoperon sequence heterogeneity. Eur J Plant Pathol 108, 507–517.[CrossRef] [Google Scholar]
  15. Lauer, U. & Seemüller, E.(2000). Physical map of the chromosome of the apple proliferation phytoplasma. J Bacteriol 182, 1415–1418.[CrossRef] [Google Scholar]
  16. Lee, I.-M. & Davis, R. E.(1988). Detection and investigation of genetic relatedness among aster yellows and other mycoplasma-like organisms by using cloned DNA and RNA probes. Mol Plant Microbe Interact 1, 303–310.[CrossRef] [Google Scholar]
  17. Lee, I.-M., Davis, R. E., Chen, T.-A., Chiykowski, L. N., Fletcher, J., Hiruki, C. & Schaff, D. A.(1992a). A genotype-based system for identification and classification of mycoplasmalike organisms (MLOs) in the aster yellows MLO strain cluster. Phytopathology 82, 977–986.[CrossRef] [Google Scholar]
  18. Lee, I.-M., Gundersen, D. E., Davis, R. E. & Chiykowske, L. N.(1992b). Identification and analysis of a genomic strain cluster of mycoplasmalike organisms associated with Canadian peach (eastern) X-disease, Western X-disease, and clover yellow edge. J Bacteriol 174, 6694–6698. [Google Scholar]
  19. Lee, I.-M., Davis, R. E. & Hsu, H.-T.(1993a). Differentiation of strains in the aster yellows mycoplasmalike organism strain cluster by serological assays with monoclonal antibodies. Plant Dis 77, 815–817.[CrossRef] [Google Scholar]
  20. Lee, I.-M., Hammond, R. W., Davis, R. E. & Gundersen, D. E.(1993b). Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology 83, 834–842.[CrossRef] [Google Scholar]
  21. 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. Int J Syst Bacteriol 48, 1153–1169.[CrossRef] [Google Scholar]
  22. Lee, I.-M., Davis, R. E. & Gundersen-Rindal, D. E.(2000). Phytoplasma: phytopathogenic mollicutes. Annu Rev Microbiol 54, 221–255.[CrossRef] [Google Scholar]
  23. Lee, I.-M., Gundersen-Rindal, D. E., Davis, R. E., Bottner, K. D., Marcone, C. & Seemüller, E.(2004a).Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases. Int J Syst Evol Microbiol 54, 1037–1048.[CrossRef] [Google Scholar]
  24. Lee, I.-M., Martini, M., Macone, C. & Zhu, S. F.(2004b). 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, 337–347.[CrossRef] [Google Scholar]
  25. Lee, I.-M., Zhao, Y. & Bottner, K. D.(2006).SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma groups. Mol Cell Probes 20, 87–91.[CrossRef] [Google Scholar]
  26. Lee, I.-M., Zhao, Y., Davis, R. E., Wei, W. & Martini, M.(2007). Prospects of DNA-based systems for differentiation and classification of phytoplasmas. Bull Insectol 60, 239–244. [Google Scholar]
  27. Liefting, L. W., Andersen, M. T., Beever, R. E., Gardner, R. C. & Foster, L. S.(1996). Sequence heterogeneity in the two 16S rRNA genes of Phormium yellow leaf phytoplasma. Appl Environ Microbiol 62, 3133–3139. [Google Scholar]
  28. Lin, C.-P. & Chen, T.-A.(1985). Monoclonal antibodies against the aster yellows agent. Science 227, 1233–1235.[CrossRef] [Google Scholar]
  29. Martini, M., Lee, I.-M., Bottner, K. D., Zhao, Y., Botti, S., Bertaccini, A., Harrison, N. A., Carraro, L., Marcone, C. & Osler, R.(2007). Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas. Int J Syst Evol Microbiol 57, 2037–2051.[CrossRef] [Google Scholar]
  30. McCoy, R. E., Caudwell, A., Chang, C. J., Chen, T. A., Chiykowski, L. N., Cousin, M. T., Dale, J. L., de Leeuw, G. T. N., Golino, D. A. & other authors(1989). Plant diseases associated with mycoplasmalike organisms. In The Mycoplasmas, vol 5, pp. 545–560. Edited by R. F. Whitcomb & J. G. Tully. New York: Academic Press.
  31. Nei, M. & Li, W.-H.(1979). Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci U S A 76, 5269–5273.[CrossRef] [Google Scholar]
  32. Oshima, K., Kakizawa, S., Nishigawa, H., Jung, H.-Y., Wei, W., Suzuki, S., Arashida, R., Nakata, D., Miyata, S. & other authors(2004). Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma. Nat Genet 36, 27–29.[CrossRef] [Google Scholar]
  33. Padovan, A. C., Firrao, G., Schneider, B. & Gibb, K. S.(2000). Chromosome mapping of the sweet potato little leaf phytoplasma reveals genome heterogeneity within the phytoplasmas. Microbiology 146, 893–902. [Google Scholar]
  34. Pearson, W. R. & Lipman, D. J.(1988). Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85, 2444–2448.[CrossRef] [Google Scholar]
  35. Razin, S., Yogev, D. & Naot, Y.(1998). Molecular biology and pathology of mycoplasmas. Microbiol Mol Biol Rev 62, 1094–1156. [Google Scholar]
  36. Schneider, B. & Seemüller, E.(1994). Presence of two sets of ribosomal genes in phytopathogenic mollicutes. Appl Environ Microbiol 60, 3409–3412. [Google Scholar]
  37. 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.[CrossRef] [Google Scholar]
  38. Shao, J. Y., Jomantiene, R., Dally, E. L., Zhao, Y., Lee, I.-M., Nuss, D. L. & Davis, R. E.(2006). NusA: comparative properties, phylogeny, and use in detection of group 16Srl phytoplasmas. J Plant Pathol 88, 193–201. [Google Scholar]
  39. Siddique, A., Agrawal, G. K., Alam, N. & Krishnareddy, M.(2001). Electron microscopy and molecular characterization of phytoplasmas associated with little leaf disease of brinjal (Solanum melongena L.) and periwinkle (Catharanthus roseus) in Bangladesh. J Phytopathol 149, 237–244.[CrossRef] [Google Scholar]
  40. Smart, C. D., Schneider, B., Blomquist, C. L., Guerra, L. J., Harrison, N. A., Ahrens, U., Lorenz, K.-H., Seemüller, E. & Kirkpatrick, B. C.(1996). Phytoplasma-specific PCR primers based on sequences of 16S–23S rRNA spacer region. Appl Environ Microbiol 62, 2988–2993. [Google Scholar]
  41. Tamura, K., Dudley, J., Nei, M. & Kumar, S.(2007).mega4: molecular evolutionary genetics analysis (mega) software version 4.0. Mol Biol Evol 24, 1596–1599.[CrossRef] [Google Scholar]
  42. Tsai, J. H.(1979). Vector transmission of mycoplasmal agents of plant diseases. In The Mycoplasmas III, Plant and Insect Mycoplasmas, pp. 265–307. Edited by R. F. Whitcomb & J. G. Tully. New York: Academic Press.
  43. 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]
  44. Weisburg, W. G., Tully, J. G., Rose, D. L., Petzel, J. P., Oyaizu, H., Yang, D., Mandelco, L., Sechrest, J., Lawrence, T. G. & other authors(1989). A phylogenetic analysis of the mycoplasmas: basis for their classification. J Bacteriol 171, 6455–6467. [Google Scholar]

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Virtual 16S rRNA gene RFLP pattern similarity coefficients between pairs of phytoplasma strains. [ PDF] 137 KB


Perl program (RFLP_pattern_comparison.pl). [ Perl program] 22 KB Accessory file (restriction_enzyme_list.txt). [ Text file] 1 KB Example of an input file (sample_input_file.fas). [ Input file] 38 KB

Perl program (RFLP_pattern_comparison.pl). [ Perl program] 22 KB Accessory file (restriction_enzyme_list.txt). [ Text file] 1 KB Example of an input file (sample_input_file.fas). [ Input file] 38 KB


Perl program (RFLP_pattern_comparison.pl). [ Perl program] 22 KB Accessory file (restriction_enzyme_list.txt). [ Text file] 1 KB Example of an input file (sample_input_file.fas). [ Input file] 38 KB

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