European Journal of Plant Pathology

, Volume 132, Issue 2, pp 203–216 | Cite as

Specific characters of 16S rRNA gene and 16S–23S rRNA internal transcribed spacer sequences of Xylella fastidiosa pear leaf scorch strains

  • Chiou-Chu Su
  • Chung-Jan Chang
  • Wen-Jen Yang
  • Shih-Tien Hsu
  • Kuo-Ching Tzeng
  • Fuh-Jyh JanEmail author
  • Wen-Ling DengEmail author


Pear leaf scorch, the only Xylella fastidiosa-induced disease reported from Taiwan, was found in area where the variety Hengshan (Pyrus pyrifolia) was grown. Strains of pear leaf scorch Xyl. fastidiosa (XF-PLS) shared similarities to strains of other host origins in the requirement of complex medium and the exhibition of rippled cell walls, however, recent serological and molecular biology studies showed difference among them. Five strains of XF-PLS were compared with 20 other strains originally isolated from almond, oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee, and sycamore by sequence analyses of the 16S rRNA gene and 16S–23S rRNA internal transcribed spacer region (ITS). When sequences of 16S rRNA gene based on fragment size of 1,537–1,540 bp were compared, the similarity index among 5 XF-PLS strains was 99.3–99.8%, whereas it was 97.8–98.6% between XF-PLS strains and strains from other hosts. When sequences of 16S–23S rRNA ITS based on fragment size of 510–540 bp were compared, the similarity index among 5 XF-PLS strains was 99.0–100%, whereas it was 80.7–82% between XF-PLS strains and strains from other hosts. Multiple sequence alignments led to the identification of 5 polymorphic nucleotides in the 16S rRNA gene among the 25 Xyl. fastidiosa strains, and there were considerable variations in the nucleotide sequences of 16S–23S rRNA ITS between XF-PLS and the other 20 Xyl. fastidiosa strains. The phylogenetic trees revealed that XF-PLS strains were separated from strains of other hosts. Strains of other hosts were divided into four subgroups: strains from (1) oleander, (2) grape, almond M23 and mulberry, (3) citrus and coffee, and (4) pecan, peach, plum, sycamore and almond M12. Results indicate that XF-PLS strains were not closely related to the above-mentioned strains from other hosts and could possibly belong to a new subspecies of Xyl. fastidiosa.


16S rRNA gene Internal transcribed spacer region Pear disease Pear leaf scorch Phylogenetic analysis 



The authors would like to thank Mr. Che-Ming Chang for his assistance in phylogenetic analysis presented in Figs. 3 and 4. The research was funded by the Council of Agriculture grant 99AS-9.3.1-BQ-B2 to C.C.S. and W.L.D. and the National Science Council grants NSC 98-2811-B-005-044 and NSC100-2811-B-005-001to F.J.J. and C.J.C.


  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.PubMedGoogle Scholar
  2. Berisha, B., Chen, Y. D., Zhang, G. Y., Xu, B. Y., & Chen, T. A. (1998). Isolation of Pierce’s disease bacteria from grapevines in Europe. European Journal of Plant Pathology, 104, 427–433.CrossRefGoogle Scholar
  3. Chang, C. J., & Walker, J. T. (1988). Bacterial leaf scorch of northern red oak: isolation, cultivation, and pathogenicity of a xylem-limited bacterium. Plant Disease, 72, 730–733.CrossRefGoogle Scholar
  4. Chang, C. J., Ganier, M., Zreik, L., Rossetti, V., & Bove, J. M. (1993). Culture and serological detection of xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain of Xylella fastidiosa. Current Microbiology, 27, 137–142.CrossRefGoogle Scholar
  5. Chang, C. J., Donaldson, R., Brennen, P. M., Krewer, G., & Boland, B. (2009). Bacterial leaf scorch, a new blueberry disease caused by Xylella fastidiosa. Hortscience, 44, 413–417.Google Scholar
  6. Chen, J., Chang, C. J., Jarret, R. L., & Gawel, N. (1992). Genetic variation among Xylella fastidiosa strains. Phytopathology, 82, 973–977.CrossRefGoogle Scholar
  7. Chen, J., Bank, D., Jarret, R. L., Chang, C. J., & Smith, B. J. (2000a). Use of 16S rDNA sequences as signature characters to identify Xylella fastidiosa. Current Microbiology, 40, 29–33.CrossRefGoogle Scholar
  8. Chen, J., Banks, D., Jarret, R. L., & Jones, J. B. (2000b). Evidence for conserved tRNA genes in the 16S–23S rDNA spacer sequence and two rrn operons of Xylella fastidiosa. Canadian Journal of Microbiology, 46, 1171–1175.Google Scholar
  9. Chen, J., Jarret, R. L., Qin, X., Hartung, J. S., Banks, D., Chang, C. J., et al. (2000c). 16S rDNA sequence analysis of Xylella fastidiosa strains. Systematic and Applied Microbiology, 23, 349–354.CrossRefGoogle Scholar
  10. Chen, J., Hartung, J. S., Chang, C. J., & Vidaver, A. K. (2002). An evolutionary perspective of Pierce’s disease of grapevine, citrus variegated chlorosis, and mulberry leaf scorch diseases. Current Microbiology, 45, 423–428.PubMedCrossRefGoogle Scholar
  11. Chen, J., Groves, R., Civerolo, E. L., Viveros, M., Freeman, M., & Zheng, Y. (2005). Two Xylella fastidiosa genotypes associated with almond leaf scorch disease on the same location in California. Phytopathology, 95, 708–714.PubMedCrossRefGoogle Scholar
  12. Chen, J., Xie, G., Han, S., Chertkov, O., Sims, D., & Civerolo, E. L. (2010). Whole genome sequences of two Xylella fastidiosa strains (M12 and M23) causing almond leaf scorch disease in California. Journal of Bacteriology, 192, 4534.PubMedCrossRefGoogle Scholar
  13. Davis, M. J., Purcell, A. H., & Thomson, S. V. (1980). Isolation medium for the Pierce’s disease bacterium. Phytopatholgy, 70, 425–429.CrossRefGoogle Scholar
  14. Davis, M. J., French, W. J., & Schaad, N. W. (1981). Axenic culture of the bacteria associated with phony disease of peach and plum leaf scald. Current Microbiology, 6, 309–314.CrossRefGoogle Scholar
  15. Felsenstein, J. (2004). PHYLIP: Phylogeny inference package. Seattle: Department of Genome Sciences and Department of Biology, University of Washington.Google Scholar
  16. Gendel, S. M. (1996). Computational analysis of the specificity of 16S rRNA-derived signature sequences for identifying food-related microbes. Food Microbiology, 13, 1–15.CrossRefGoogle Scholar
  17. Goncalves, E. R., & Rosato, Y. B. (2002). Phylogenetic analysis of Xanthomonas species based upon 16S–23S rDNA intergenic spacer sequences. International Journal of Systematic and Evolutionary Microbiology, 52, 355–361.PubMedGoogle Scholar
  18. Hartung, J. S., Beretta, J., Brlansky, R. H., Spisso, J., & Lee, R. F. (1994). Citrus variegated chlorosis bacterium: axenic culture, pathogenicity, and serological relationships with other strains of Xylella fastidiosa. Phytopathology, 84, 591–597.CrossRefGoogle Scholar
  19. Hauben, L., Vauterin, L., Swings, J., & Moore, E. R. B. (1997). Comparison of 16S ribosomal DNA sequences of all Xanthomonas species. International Journal of Systematic Bacteriology, 47, 328–335.PubMedCrossRefGoogle Scholar
  20. Hendson, M., Purcell, A. H., Chen, D., Smart, C., Guilhabert, M., & Kirkpatrick, B. (2001). Genetic diversity of Pierce’s disease strains and other pathotypes of Xylella fastidiosa. Applied and Environmental Microbiology, 67, 895–903.PubMedCrossRefGoogle Scholar
  21. Hernandez-Martinez, R., de la Cerda, K., Costa, H. S., Cooksey, D. A., & Wong, F. P. (2007). Phylogenetic relationships of Xylella fastidiosa strains isolated from landscape ornamentals in southern California. Phytopathology, 97, 857–864.PubMedCrossRefGoogle Scholar
  22. Hillis, M. D., & Dixon, M. T. (1991). Ribosomal DNA: molecular evolution and phylogenetic inference. Quarterly Review of Biology, 66, 411–453.PubMedCrossRefGoogle Scholar
  23. Honeycutt, R. J., Sobral, B. W. S., & McClelland, M. (1995). tRNA intergenic spacers reveal polymorphisms diagnostic for Xanthomonas albilineans. Microbiology, 141, 3229–3239.PubMedCrossRefGoogle Scholar
  24. Hopkins, D. L. (1989). Xylella fastidiosa: xylem-limited bacterial pathogen of plants. Annual Review of Phytopathology, 27, 271–290.CrossRefGoogle Scholar
  25. Hopkins, D. L., & Adlerz, W. C. (1988). Natural hosts of Xylella fastidiosa in Florida. Plant Disease, 72, 429–431.CrossRefGoogle Scholar
  26. Hopkins, D. L., & Purcell, A. H. (2002). Xylella fastidiosa: cause of Pierce’s disease of grapevine and other emergent diseases. Plant Disease, 86, 1056–1066.CrossRefGoogle Scholar
  27. Jeannmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G., & Gibson, T. J. (1998). Multiple sequence alignment with Clustal X. Trends in Biochemical Sciences, 23, 403–405.CrossRefGoogle Scholar
  28. Leu, L. S., & Su, C. C. (1993). Isolation, cultivation, and pathogenicity of Xylella fastidiosa, the causal bacterium of pear leaf scorch disease in Taiwan. Plant Disease, 77, 642–646.CrossRefGoogle Scholar
  29. Mehta, A., & Rosato, Y. B. (2001). Phylogenetic relationships of Xylella fastidiosa strains from different hosts, based on 16S rDNA and 16–23S intergenic spacer sequences. International Journal of Systematic and Evolutionary Microbiology, 51, 311–318.PubMedGoogle Scholar
  30. Nathalie, L. B., Philippe, H., Vangin, I., & Decaris, B. (1996). 16S rRNA and 16S to 23S internal transcribed spacer sequence analyses reveal inter- and intra-specific Bifidobacterium phylogeny. International Journal of Systematic Bacteriology, 46, 102–111.CrossRefGoogle Scholar
  31. Neefs, J. M., Van de Peer, Y., De Rijk, P., Goris, A., & De Wachter, R. (1991). Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Research, 19(Suppl), 1987–2015.PubMedGoogle Scholar
  32. Page, R. D. (1996). TreeView: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences, 12, 357–358.PubMedGoogle Scholar
  33. Pooler, M. R., & Hartung, J. S. (1995). Genetic relationships among strains of Xylella fastidiosa from RAPD-PCR data. Current Microbiology, 31, 134–137.PubMedCrossRefGoogle Scholar
  34. Purcell, A. H., & Hopkins, D. L. (1996). Fastidious xylem-limited bacterial plant pathogens. Annual Review of Phytopathology, 34, 131–151.PubMedCrossRefGoogle Scholar
  35. Qin, X., Miranda, V. S., Machado, M. A., Lemos, E. G. M., & Hartung, J. S. (2001). An evaluation of the genetic diversity of Xylella fastidiosa isolated from diseased citrus and coffee in São Paulo, Brazil. Phytopathology, 91, 599–605.PubMedCrossRefGoogle Scholar
  36. Randall, J. J., Goldberg, N. P., Kemp, J. D., Radionenko, M., French, J. M., Olsen, M. W., et al. (2009). Genetic analysis of a novel Xylella fastidiosa subspecies found in the southwestern United States. Applied and Environmental Microbiology, 75, 5631–5638.PubMedCrossRefGoogle Scholar
  37. Rosato, Y. B., Neto, J. B., Miranda, V. S., Carlos, E. F., & Manfio, C. P. (1998). Diversity of a Xylella fastidiosa population isolated from Citrus sinensis affected by citrus variegated chlorosis in Brazil. Systematic and Applied Microbiology, 21, 593–598.CrossRefGoogle Scholar
  38. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.Google Scholar
  39. Schaad, N. W., Postnikova, E., Lacy, G., Fatmi, M., & Chang, C. J. (2004). Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei, subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp. nov. Systematic and Applied Microbiology, 27, 290–300.PubMedCrossRefGoogle Scholar
  40. Schreiber, H. L., IV, Koirala, M., Lara, A., Ojeda, M., Dowd, S. E., Bextine, B., et al. (2010). Unraveling the first Xylella fastidiosa subsp. fastidiosa genome from Texas. Southwestern Entomologist, 35, 479–483.CrossRefGoogle Scholar
  41. Schuenzel, E. L., Scally, M., Stouthamer, R., & Nunney, L. (2005). A multigene phylogenetic study of clonal diversity and divergence in north American strains of the plant pathogen Xylella fastidiosa. Applied and Environmental Microbiology, 71, 3832–3839.PubMedCrossRefGoogle Scholar
  42. Simpson, A. J., Reinach, F. C., Arruda, P., Abreu, F. A., Acencio, M., Alvarenga, R., et al. (2000). The genome sequence of the plant pathogen Xylella fastidiosa. Nature, 406, 151–157.PubMedCrossRefGoogle Scholar
  43. Smart, C. D., Schneider, B., Biomquist, C. L., Guerra, L. J., Harrison, N. A., Ahrens, U., et al. (1996). Phytoplasma-specific PCR primers based on sequences of the 16S–23S rRNA spacer region. Applied and Environmental Microbiology, 63, 2988–2993.Google Scholar
  44. Stackebrandt, E., & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA Sequence analysis in the present species definition in bacteriology. International Journal of Systematic Bacteriology, 44, 846–849.CrossRefGoogle Scholar
  45. Su, C. C., Yang, W. J., Feng, C. Y., Hsu, S. T., & Tzeng, K. C. (2008). The application of DNA fingerprintings amplified by arbitrary primers in differentiating pear leaf scorch bacterium from other Xylella fastidiosa strains. Plant Pathology Bulletin, 17, 261–269 (Chinese with English abstract).Google Scholar
  46. Toth, I. K., Avrova, A. O., & Hyman, L. J. (2001). Rapid identification and differentiation of soft rot Erwinias by 16S–23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses. Applied and Environmental Microbiology, 67, 4070–4076.PubMedCrossRefGoogle Scholar
  47. Van Sluys, M. A., de Oliveira, M. C., Monteiro-Vitorello, C. B., Miyaki, C. Y., Furlan, L. R., Camargo, L. E., et al. (2003). Comparative analyses of the complete genome sequences of Pierce’s disease and citrus variegated chlorosis strains of Xylella fastidiosa. Journal of Bacteriology, 185, 1018–1026.PubMedCrossRefGoogle Scholar
  48. Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, K., & Swings, J. (1996). Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiological Reviews, 60, 407–438.PubMedGoogle Scholar
  49. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173, 697–703.PubMedGoogle Scholar
  50. Wells, J. M., Raju, B. C., Hung, H.-Y., Weisburg, W. G., Mandelco-Paul, L., & Brenner, D. J. (1987). Xylella fastidiosa gen. nov., sp. nov: gram-negative, xylem-limited, fastidious plant bacteria related to Xanthomonas spp. International Journal of Systematic Bacteriology, 37, 136–143.CrossRefGoogle Scholar

Copyright information

© KNPV 2011

Authors and Affiliations

  • Chiou-Chu Su
    • 1
  • Chung-Jan Chang
    • 2
    • 3
  • Wen-Jen Yang
    • 1
  • Shih-Tien Hsu
    • 3
  • Kuo-Ching Tzeng
    • 3
  • Fuh-Jyh Jan
    • 3
    Email author
  • Wen-Ling Deng
    • 3
    Email author
  1. 1.Taiwan Agricultural Chemicals and Toxic Substances Research InstituteWufongTaiwan
  2. 2.Department of Plant PathologyUniversity of Georgia at GriffinGriffinUSA
  3. 3.Department of Plant PathologyNational Chung Hsing UniversityTaichungTaiwan

Personalised recommendations